Methods in Enzymology Volume 256 SMALL GTPases AND THEIR REGULATORS Part B Rho Family
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Methods in Enzymology Volume 256 SMALL GTPases AND THEIR REGULATORS Part B Rho Family
METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF
John N. Abelson
Melvin I. Simon
DIVISION Of BIOLOGY CALIfORNIA INSTrntrE Of TECHNOLOGY PASADENA. CALIFORNIA
FOUNDING EDITORS
Sidney P. Colowick and Nathan O. Kaplan
This book is printed on add-free paper.
Copyright
e
© 1995 by ACADEMIC PRESS, INC.
All Rights Reserved. No pan of this publication may be reproduced or transmilled in any form or by any means, electronic or mechanical, including photocopy. recording. or any information storage and retrieval system, without permission in writing rrom the publisher. Academic
Press. Inc.
& Company 525 B Street. Suite 1900. San Diego. California 92101-4495
A Division of Harcoun Brace
UnitetJ Kingdom Edition published by Academic Press Limited
24-28 Oval Road. London NWI 7DX
Intcrnational Standard Scrial Number:
0076-6879
International Standard Book Number:
0-12-182157-9
PRlNfED IN THE UNITEDSfATES OF AMERICA
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Table of Contents
C01'o'TRlJltn'OMS 1'0 VOI.UMI;
PREFACE
•
VOLUMES
.
•
•
IN S,,-KI ES
;,
256.
xiii
•
•
Section J. Expression. Purtfication. and Posttranslattonal Modification I. I'urification of Recombinant RhoiRaclG25K from ASNE1TE J. SELl' and Esdwricllill coli 2. Purification of Dacu!ovirus.Exprcsscd Cdc421'ls
At...\N HALL
3
RICllAltIl A. CmuoNE. DAVlJ) LeoNAll.I), "I'll) "
VI ZI-IF.NG 3. Purification and Propcrlics of Rac2 from Human Leukemia Cells
TAKAKAZU MIZUNO, HIMOYUKl
N ..."ANISIIi. ANI) 15
YOSIlIMI TAKAI 4. PUritiC-dlioo of Rac2 from Human Ncutrophils
ULiA G. KN....us
ANI)
GARY M. Bo"OC'u
25 33
S. Purification of Rae-GOP Dissociation Inhibitor ARIE Allo Complex hom Phagocyte Cytosol 6. Purification and propcnics of Recombinant RhoGOP Dissociation Inhibitor
KAZUMA TANAKA. T....KUV.... S...SAKI, YOSHIMI
....1'11)
TAKA'
41
7. Prcnykystcinc.Directed Carboxyl Mcthyhransrer- MARK R. Pmups ANO asc Activity in Human Neutrophil Membranes
MICUAEL H. PU.LINOER
49
Section II. Guanine Nucleotide Exchange and HydrolysIs 8. Measuremcnt of Intrinsic Nucleotide Exchange and ANNETrE J. Seu' "1'011> GTP Hydrolysis Rates
ALAN H"u.
67
9. Guanine Nucleotide Exchange CatalYled by dbl YI ZIIENG. Oncogene Product
MATTIIEW J. ]-tAItT, AND RlctiAItI) A. CEItIONE
77
10. Stimulation of Nucleotide Exchange on Ras· and EMIUO POItFiItI AND Rho-Related '''otcins by Small GTP.8inding JOHN F. HANCQCX Protein GDP Dissociation Slimulator
85
vi
TABLE OF CONTENTS
11. Interaction of Eell and Obi with Rho-related TORU MIKI
90
GTPases 12. Solubilization of Cdc42Hs from Membranes by D"vlI) A. LEONARD ASD Rho-GOP Dissociation Inhibitor
RIOI.... RU A. CERIONE
13. Purification and GTPase.Activating Prolein Activo JEFFREY SETI1...I!MAN AND itl' of Baculovirus Expressed pl90
ROSEMARY FOSTER
105
14. QTPase·Activated Protein Activity of n(al)·Chi· SOllAll Am.IED, maerin and Effect of Lipids
ROBERT KOZMA, CURISTINE HALL, AND loUiS LIM
114
15. Characterization of Breakpoint Ouster Region Ki- DANIEl. E. H. A""R AND nase and 5H2-Binding Activities
DWEll N. WITI-e
125
16. Identification of GTPase-Activ8ting Proteins by Ni- EDWARD MANSEll., troccllulose Overlay Assay
THOMAS LEUNG, AND LoUIS LIM
17. Idcnlificalionof)BP·1 in eDNA Expn:ssion Library
by SH3 Domain Screening
IJ
PU;'RA CICCHErrI ANI) DAVID BALTIMORE
140
Section Ill. Cen Expression and In Vitro Analysis 18. Serum Induction of RhoG Expression
PHILIPPE FORT AND SYLVIE VINCENT
151
19. Microinjection of Epitope-Tagged Rho Family HUGH PAlV-RSON. cDNAs and Analysis by Immunolabeling
PETER ADAMSON. AND DAVID RonERTSON
161
20. Purification and Assay of Recombinant C3 Trans SIMON T. DILLON ASO ferase 21. In Vitro ADP-Rihosylation of Rho by Bacterial
ADP-Rihosyltransfcrases
LARMY A. FEIG
1 74
KLAUS AItTORlf.s AND INOO JuST
184
22. Preparation of Native and Recombinant Clus/ria- NAIUTO MORll AND illm bowlinlln/ C3 ADP-Rihosyltransferase and SI�U" NARUMIVA
196
Identitication of Rho Proteins by ADP-Ribosylation 23. It. Vi/ro Binding Assay for Inlcractions of Rho and DAGMAR DIEKMANN AND Rac with GTPasc-Activating Proteins and Ef- ALAN HALL fe<:tors
207
24. Purification and Assay of Kinascs That Intcract with EDWARD MANSER. RacJCdc42 TIIDMAS LEUNG. AND LoUIS lJM
lIS
TABLE OF CONTENTS 25. Yeast T.... .o-Hybrid System to Detect
Prolein
Protein Interactions ....ith . Rho GTPases
VII
PONTIJS ASPENSTROM AND MIOtAEL F. OUON
26. Assay for Rho·Dependent Phosphoinositide 3-Ki SUSAN Erika RtlTI!.NttOUSE
228 241
nase Activity in Platelet Cytosol 27. Neutrophil Phospholipase D: Inhibition by Rho EOWAMO P. BOWMAN, GPD Dissociation Inhibitor and Stimulation by DAVit) J. UIIUNGEM. ANI) Small GTPase GDP Dissocation Stimulator
l. DAVID LAMIIETII
246
28. Measurement of Rac Translocation from Cytosol MARK T. QUINN AND to Membranes in Activated Neutrophils
GARY M. BOKOOI
29. Reconstitution of Cell·Free NADPH Oxidase Ac ARI£ ABO AND tivity by Purified Components
A"'"l1tOSY W. SEGAL
268
Section IV. Biological Activity 30. Genetic and Biochemical Analysis ofCdc42p Func· JAMES POSAOA. tion in SlIcdlllrolll),CI'S cered.filll! and SclIi:oslIc, PIo'ER 1. MILU'R. clllIromyces pombe
JANIo. MC.:CULLOUGIt. MICHAF.L ZIMAN. ANO DouOi-"s I. JOIINSON
281
31. Lymphocyte Aggregation Assay and Inhibition by TOMOK(I TO,-HNAOA ASI) Clostridillm ootlllitlllm C3 ADP-Ribosyltrans· SHUtt NARUMI"....
290
ferase 32. Inhibition ofp21 Rho in IntactCells by C3 Diphthe· PATRICE BOOUET, ria Toxin Chimera Proteins
MICIII,L R. POl'Off', MURIEI.I.E GIRY, EMMANUEL LEMICHEZ, AND PATRICIA BERGEZ-AULLO
33. Growth Factor·lnduced Actin Reorganization in ANNE J. RJl)L.EY
297 306
Swiss 31'3 Cells 34. Microinjection of Rhoand Rac into Quiesccnt Swiss
ANNE J. RIDL'£Y
313
3T3 Cells 35. Inhibition of Lymphocyte. MediatedCytotoxicity by Cfoslri(Jillm b{Jwlinum C3 Transferase
PAUL LANO AND JACOUES BEfl.TOOUO
320
36. Neutrophil Chemotaxis Assay and Inhibition by C3 MARIE-lost! STASi.... AND ADP.Ribosyltransferase
PIERRE V. VIGNAIS
327
37. Cell Motility Assay and Inhibition by Rho-GOP KENJI TAKAISIII, Dissociation Inhibitor
TAK UYA SAS....KI. ANI) YOStUMI TAK AI
38. Cell Transformation by dbf Oncogene
336
DANIELA z...N . GMILU AND ALESSANDRA EVA
347
viii
TABLE OF CONTENTS
39. Inhibition of Rac Function Using Antisense Oligo. OUVIER OoRSEUIL nuclcotidcs
GERAI.D LEeA, AIME VAZQUEZ, ANI) GEII.AII.I) GACON
358
AUTltOR INDEX
367
SUBJECT I/IIOEX
385
C o n t r i b u t o r s to V o l u m e 2 5 6 Article numbers arc in parentheses following the names of contributors. Affiliations listed are currenl.
Biochemistry, Emory University Medical School, Atlanta, Georgia 30322 RICHARD A. CERIONE (2, 9, 12), Department of Pharmacology, Cornell University, Ithaca, New York 14853 DANIEL E. H. AFAR (15), Department of Mi- PIERA CICCHETTI (17), Institute for Genetics, University of Cologne, Cologne D-50674, crobiology and Molecular Genetics, UniverGermany sity of California-Los Angeles, Los Angeles, DAGMAR DIEKMANN (23), CRC Oncogene California 90024 and Signal Transduction Group, MRC LabSOHAIL AHMED (14), Department of Neurooratory for Molecular Cell Biology and chemistry, Institute of Neurology, London Department of Biochemistry, University WC1N 1PJ, United Kingdom, and Institute College London, London WC1E 6BT, of Molecular and Cell Biology, National United Kingdom University of Singapore, Singapore 0511 SIMON T. DILLON (20), Department of MicroKLAUS AKTORIES (21), Institute of Pharmabiology and Molecular Biology, Tufts Unicology and Toxicology, Albert-Ludwigs versity School qfl Medicine, Boston, MassaUniversity, D-79104 Freiburg, Germany chusetts 02111 PONTUS ASPENSTROM (25), Department of OLIVIER DORSEUIL (39), Institut Cochin de Zoological Cell Biology, Arrhenius LaboG4n4tique Mol&ulaire, 1NSERM UnitO ratories E5, The Wenner-Gren Institute, 257, 75014 Paris, France Stockholm University, S106-91, Sweden ALESSANDRAEVA (38), Laboratory of CelluDAVID BALTIMORE (17), Massachusetts Instilar and Molecular Biology, National Cancer tute of Technology, Cambridge, MassachuInstitute, National Institute of Health, setts 02139 Bethesda, Maryland 20892 PATR1C1A BEROEZ-AULLO (32), Laboratoire LARRY a . FEIG (20), Department of Biochemde Biologie Mol(culaire et S4quencage, istry, Tufts University, School of Medicine, Universit~ Bordeaux II, 33076 Bordeaux, Boston, Massachusetts 02111 France PHILIPPE FORT (18), Institute of Molecular GeJACQUES BERTOGLIO (35), INSERM CJF 93netics, University Montipellier, F 340.33 01, Facultd de Pharmacie-Universit~ ParisMontpellier, France Sud, 92296 Chatenay Malabry Cedex, ROSEMARY FOSTER (13), MGM Cancer CenFrance ter and Department of Medicine, Harvard GARY M. BOKOCH (4, 28), Departments of Medical School, Charlestown, MassachuImmunology and Cell Biology, The Scripps setts 02129 Research Institute, La Jolla, California GERARD GACON (39), Institut Cochin de G~n92037 (ique Mol~culaire, 1NSERM Unit4 257, PATR1CE BOQUET (32), Unit~des Toxines Mi75014 Paris, France crobiennes, Institut Pasteur, 75724 Paris, MURIELLE GIRY (32), Unit~ des Toxines MiFrance crobiennes, Institut Pasteur, 75724 Paris, EDWARD P. BOWMAN (27), Department of. France IX
ARIE ABO (5, 29), Onyx Pharmaceuticals, Richmond, California 94806 PETER ADAMSON (19), Vascular Biology Research Centre, Kings College London, London, W8 7AH, United Kingdom
X
CONTRIBUTORS TO VOLUME 256
ALAN HALL (1, 8, 23), MRC Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, London WC1E 6BT, England CHRISTINE HALL (14), Institute of Neurology, London WC1N 1PJ, United Kingdom JOHN F. HANCOCK (10), Onyx Pharmaceuticals, Richmond, California 94806 MATTHEW J. HART (9), Department of Pharmacology, Ithaca, New York 14853 DOUGLAS I. JOHNSON(30), Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont05405 INGO JUST (21), Institute of Pharmacology and Toxicology, Albert-Ludwigs University, D-79104 Freiburg, Germany ULLA G. KNAUS (4), Department oflmmunolDAy, The Scripps Research Institute, La Jolla, California 92037 ROBERT KOZMA (14), Institute of Neurology, London WC1N IPJ, United Kingdom, and Institute of Molecular and Cell Biology, National University of Singapore, Singapore 0511 J. DAVID LAMBETH (27), Department of BiDchemistry, Emory University MediCal School, Atlanta, Georgia 30322 PAUL LANG (35), INSERM CJF93-O1,Facultd de Pharmacie-Universit~ Paris-Sud, 92296 Chatenay Malabry Cedex, France GI~RALD LECA (39), INSERM Unit~131, Association Chlude Bernard, Institute d'Hematologie-HOpital Saint-Louis, Paris, France EMMAUEL LEMICHEZ (32), Unit~ des Toxines Microbiennes, Institut Pasteur, 75724 Paris, France DAVID LEONARD (2,12), Department of Pharmacology, Cornell University, Ithaca, New York 14853 THOMAS LEUNG (16, 24), Institute of Molecular and Cell Biology, National University of Singapore, Singapore 0511 Louis LIM (14, 16, 24), Institute of Neurology, London WC1N 1PJ, United Kindgom, and Institute of Molecular and Cell Biology, National University of Singapore, Singapore 0511
EDWARD MANSER (16, 24), Institute of Molecular and Cell Biology, National University of Singapore, Singapore 0511 JANET MCCULLOUGH (30), Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405 TORU MIKI (11), Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 PETER J. MILLER (30), Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405 TAKAKAZU MIZUNO (3), Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Osaka 565, Japan NARITO MORII (22), Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606, Japan HIROYUKI NAKANISHI(3), Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Osaka 565, Japan SHUH NARUMIYA (22, 31), Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto University, Kyoto 606, Japan MICHAEL F. OLSON (25), CRC Oncogene and Signal Transduction Group, MRC Laboratory for Molecular Cell Biology, University College London, London WCIE 6BT, United Kingdom Huort PATERSON (19), Section of Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6BJ, United Kingdom MARK R. PHILIPS (7), Departments of Medicine and Cell Biology, New York University School of Medicine, New York, New York 10016 MICHAEL H. PILLINGER (7), Department of Medicine, New York University School of Medicine, New York, New York 10016 MICHEL R. POPOFF (32), Unit~ des Toxines Microbiennes, Institut Pasteur, 75724 Paris, France
CONTRIBUTORS TO VOLUME 256 EMILIO PORFIRI (10), Onyx Pharmaceuticals, Richmond, California 94806 JAMES POSADA (30), Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405 MARK T. QUINN (28), Veterinary Molecular Biology, Department of Microbiology, Montana State University, Bozeman, Montana 59717 ANNE J. RIDLEY (33, 3,4), Ludwig Institute for Cancer Research, London WCIP 8BT, United Kingdom SUSAN E. RITTENHOUSE (26), Jefferson Cancer Institute and Cardeza Foundation for Hematologic Research, Philadelphia, Pennsylvania 19107 DAVID ROBERTSON (19), Haddow Laboratories, Institute of Cancer Research, Sutton, Surrey, SM2 5NG, United Kingdom TAKUYA SASAKI (6, 37), Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Osaka 565, Japan ANTHONY W. SEGAL (29), Division of Molecular Medicine, University College London, London WCIE 6JJ, United Kingdom ANNETTE J. SELF (1, 8), MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom JEFFREY SETTLEMAN(13), MGH Cancer Center and Department of Medicine, Harvard Medical School, Charlestown, Massachusetts 02129 MARIE-JOSE STASlA (36), Laboratoire d'Enzymologie, Centre Hospitalier Universitaire de Grenoble, Grenoble, France YOSHIMI TAKAI (3, 6, 37), Department of Molecular Biology and Biochemistry, Osaka University Medical School, Osaka 565, Ja-
xi
pan, and Department of Cell Physiology, National Institute for Physiological Sciences, Okagaki 444, Japan KENJI TAKAISHI (37), Department of Molecular Biology and Biochemistry, Osaka University Medical School Suita 565, Japan KAZUMA TANAKA (6), Department of Molecular Biology and Biochemistry, Osaka University Medical School Suita, Osaka 565, Japan TOMOKO TOMINAGA (31), Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan DAVID J. UHLINGER (27), Department of Biochemistry, Emory University Medical School Atlanta, Georgia 30322 A1ME VASQUEZ (39), 1NSERM Unit 131, Association Claude Bernard Research Center, 92140 Clamart, France PIERRE V. VIGNAIS (36), Laboratoire de Biochimie, Departement de Biologie Moleculaire et Structurale, CEA CEN-Grenoble, F-38054 Grenoble, France SYLVIE VINCENT (18), Institute of Molecular Genetics, University Montipellier, F 34033 Montpellier, France OWEN N. WITrE (15), Molecular Biology Institute and Howard Hughes Medical Institute, University of California-Los Angeles, Los Angeles, California 90024 DANIELA ZANGR1LLI(38), Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Y1 ZHENG (2, 9), Department of Pharmacology, Cornell University, Ithaca, New York 14853 MICHAEL ZIMAN (30), Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720
Preface Rho-related GTP-binding proteins constitute a functionally distinct group in the small GTPase superfamily. Like Ras, they control intracellular signal transduction pathways, and it is now firmly established that Rhorelated GTPases regulate the organization of the actin cytoskeleton of all eukaryotic cells. Accordingly, this family of GTPases controls cell adhesion, cell movement, and cytokinesis. This volume describes a wide range of experimental approaches that have been used to study the function of Rho-related GTPases both in vitro and in vivo. The availability of recombinant proteins has been of enormous benefit in characterizing the biochemical and biological activities of the GTPases and of the proteins with which they interact. The first part of this volume deals with expression systems used both in Escherichia coli and in insect cells. The driving force for the enormous interest now being taken in the Rho family of GTPases stems from their demonstrated biological roles, particularly as regulators of adhesion and movement. Thus many of the cellular assays that have been used to establish these effects are included in this volume. The ultimate test for any cellular activity attributed to a GTPase is the ability to reconstitute that activity in vitro. To date, this has been achieved only for Rac-dependent activation of phagocytic NADPH oxidase, and several chapters are devoted to this topic. Although the area has already generated an enormous amount of general interest, the functional analysis of small GTPases is still in its infancy. There are many more surprises to come as the biochemical details of the pathways controlled by small GTPases are elucidated. The prize is a molecular explanation of many aspects of contemporary cell biology. We are extremely grateful to all the contributors who have taken the time to commit their expertise to paper, and are confident that their efforts will be greatly appreciated by the scientific community. Dr. Hall thanks the Cancer Research Campaign (UK), the Wellcome Trust, and the Medical Research Council (UK) for providing the funds and environment that have allowed him to work in this very exciting area. ALAN HALL W. E. BALCH CHANNING J. DER
xiii
Section I Expression. Purification. and Posttranslational Modification
Rho/Rac/G25K FROME. coli
[ 1]
[ 1]
3
Purification of Recombinant Rho / Rac / G25K f r o m E s c h e r i c h i a coli
By
ANNETTE J. SELF a n d A L A N H A L L
Introduction The purification of Ras-related GTP-binding proteins from recombinant sources has proved to be invaluable for studying their biochemical properties and biological effects. The simplest expression systems have made use of Escherichia coli, although Ras-like GTPases produced in this way are not posttranslationally modified. Yeast and baculovirus-Sf9 (Spodaptera frugiperda, full armyworm ovary) insect cells have also been used and since they are eukaryotic hosts, the GTPases expressed are at least partially modified.1'2 A wide range of expression levels has been reported for Rasrelated proteins in E. coli; in the case of Ras, yields of 7.5 mg/liter of culture have been obtained, 3 whereas others such as Rap1, for example, have proved much more difficult to make in a stable form. Members of the Rho family have been relatively difficult to express in E. coli in large amounts; as described below, we obtain yields of around 0.1-1 rag/liter. The mammalian Rho subfamily consists of RhoA, B, and C, Racl and 2, G25K/CDC42, RhoG, and TC10. 4-9 These proteins are 30% identical to Ras in amino acid sequence and 55% identical to each other, and their overall three-dimensional structure is expected to be very similar to that of Ras. 1° RhoA, B, and C are 85% identical to each other, with almost all x S. G. Clark, J. P. McGrath, and A. D. Levinson, Mol. Cell Biol. 5, 2726 (1985). 2 M. J. Page, A. Hall, S. Rhodes, R. H. Skinner, V. Murphy, M. Sydenham, and P. N. Lowe, J. Biol. Chem. 264, 19147 (1989). 3 A. M. De Vos, L. Tong, M. V. Milburn, P. M. Matias, J. Jancarik, S. Noguchi, S. Nishimura, K. Mitra, E. Ohtsuka, and S. Kim, Science 239, 888 (1988). 4 p. Madaule and R. Axel, Cell 41, 31 (1985). 5 j. Didsbury, R. F. Weber, G. M. Bocock, T. Evans, and R. Synderman, J. Biol. Chem. 264, 16378 (1989). 6 K. Shinjo, J. G. Koland, M. J. Hart, V. Naraismham, D. J. Johnson, T. Evans, and R. A. Cerione, Proc. Natl. Acad. Sci. U.S.A. 87, 9853 (1990). 7 S. Munemitsu, M. A. Innis, R. Clark, F. McCormick, A. Ullrich, and P. Polakis, Mol. Cell. Biol. 10, 5977 (1990). s G. T. Drivas, A. Shih, E. Coutavas, M. G. Rush, and P. D' Eustachio, Mol. Cell. Biol. 10, 1793 (1990). 9 S. Vincent, P. Jeanteur, and P. Fort, MoL Cell Biol. 12, 3138 (1992). 10E. F. Pai, W. Kabsch, U. Krengal, K. C. Holmes, J. John, and A. Wittinghofer, Nature 341, 209 (1989).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
4
EXPRESSION AND PURIFICATION
[ l]
of the divergence being at the carboxy-terminal end of the proteins; R a c l and 2 are 92% identical to each other with 15 amino acids different; and G25K and C D C 4 2 H s are the closest related isoforms with only 9 amino acid differences between them. All R h o family m e m b e r s contain a Cterminal C A A X box motif (A = aliphatic amino acid; X = L for R h o and Rac; X = F for CDC42/G25K), and all are posttranslationally modified in vivo by the addition of a C 20 geranylgeranyl isoprenoid, u 13 Interestingly, R h o B also appears to be a substrate for the farnesyltransferaseJ 4 Like all small GTPases, the Rho-related proteins are regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and the characterization of these regulatory proteins has relied on a source of recombinant protein. All G A P s and most G E F s are active in vitro on E. coli-produced, nonmodified Rho-related GTPases. E. coliproduced recombinant proteins are also very useful for studying the biological function of the R h o subfamily by microinjection because the G T P a s e s b e c o m e posttranslationally modified and functionally active after injectionJ 5 To characterize the function of Rho-related proteins, we have purified R h o A , R a c l , and G 2 5 K from E. coli using the glutathione S-transferase (GST) gene fusion vector p G E X - 2 T (Pharmacia L K B Biotechnology, Inc.). 16 As described in the following section, the yields of these proteins f r o m this vector are not as high as have been reported for other proteins expressed using this system, but purification is extremely rapid and the final preparations are of high purity.
C o n s t r u c t i o n of V e c t o r s c D N A s generated by the polymerase chain reaction (PCR) and encoding h u m a n R h o A , R a c l , and G 2 5 K were fused to the carboxy-terminal end of the S c h i s t o s o m a ] a p o n i c u m glutathione S-transferase gene by cloning into the B a m H I / E c o R I sites of p G E X - 2 T (see Fig. 1). Expression of the fusion protein is under the control of the tac promoter, and the nucleotide sequences across the fusion junctions are shown in Fig. lb. After cleavage 11M. Katayama, M. Kawata, Y. Yoshida, H. Horiuchi, T. Yamamoto, Y. Matsuura, and Y. Takai, J. Cell. Biol. 266, 12639 (1991). 12B. T. Kinsella, R. A. Erdman, and W. A. Maltese, J. Biol. Chem. 15, 9786 (1991). 13H. Yamane, C. C. Farnsworth, H. Xiec, T. Evans, W. N. Howald, M. H. Gelb, J. A. Glomset, S. Clarke, and B. K. K. Fung, Proc. Natl. Acad. Sci. U.S.A. 88, 286 (1991). a4p. Adamson, C. J. Marshall, A. Hall, and P. A. Tilbrook, J. Biol. Chem. 267, 20033 (1992). 15H. F. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell Biol. 111, 1001 (1990). 16D. B. Smith and K. S. Johnson, Gene 67, 31 (1988).
[1]
Rho/Rac/G25K FROM E. coli
5
THROMBIN
b
ILeu
Val Pro Arg~Gly serlpro GIy lie His Arg Asp GST..........CTG G-I-F CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACG I I I I BamHl [ _ _ ] EcoRl Stop codons Smal GST.......... CTG G-I-r CCG CGT GGA TCC CCG GCT....rhoA GST..........CTG GTT CCG CGT GGA TCC CCG CAG.,..racl GST.......... CTG GTT CCG CGT GGS TCC CCG CAG.,..GZSK codon 2
FIG. 1. Structure of the glutathione S-transferase vector pGEX-2T. (a) Schematic representation of pGEX-2T. (b) Nucleotide sequence of pGEX-2T and of pGEX-2T containing RhoA,
Racl, and G25K cDNAs across the fusion junction. with thrombin it is predicted that the GTPases will each have Gly-Ser-Pro fused to the second codon of the native sequence. The p G E X - 2 T vectors containing RhoA, Racl, and G25K were each introduced into the E. coli strain JM101 and stored as glycerol stocks at - 7 0 °. Purification of Wild-Type RhoA, Rac 1, a n d G 2 5 K Growth and Purification One hundred milliliters of L-broth containing 50/~g/ml ampicillin is inoculated with E. coli containing the expression plasmids taken from the
6
EXPRESSION AND PURIFICATION
[l ]
glycerol stock. After overnight incubation at 37°, the culture is diluted 1 : 10 into fresh, prewarmed (37 °) L-broth/ampicillin and is incubated for 1 hr in two 2-liter flasks in a bacterial shaker at 37°. To induce fusion protein expression, isopropyl-/3-D-thiogalactopyranoside (IPTG) is added to 0.1 mM (0.5 ml of a 0.1 M stock made in water and stored at -20°), and the culture is incubated with shaking for a further 3 hr. After induction, the cells are collected in l-liter buckets by centrifugation at 4000 rpm for 10 min at 4° and then resuspended (on ice) in 3 ml of cold lysis buffer [50 mM Tris-HC1, pH 7.6, 50 mM NaC1, 5 mM MgC12, 1 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. We have noted that many purification procedures for GST fusion proteins use buffers containing phosphate, a chelator of magnesium ions. 16 In low magnesium concentrations, Rho-related GTPases rapidly lose their bound guanine nucleotide (see [9] in this volume) and are unstable. It is therefore important that phosphate buffers or other chelators of magnesium such as EDTA are not used in the purification procedure and that there is an excess of free magnesium in all buffers used. Resuspended bacteria are lysed by sonication on ice (three times at 1 min each). We use a small probe on an MSE Soniprep 150 sonicator at an amplitude of 14 tzm, and the bacterial suspension is kept cool at all times. As lysis occurs the suspension turns from a light creamy color to a muddy brown and becomes somewhat more viscous. The sonicate is centrifuged at 10,000 rpm for 10 min at 4°, and the supernatant (4 ml) is carefully transferred to a 5-ml bijou tube (Sterillin). Some 30-50% of GST-RhoA, GST-Racl, and GST-G25K produced by this expression system in JM101 is found in the pellet after centrifugation of the sonicate. Glutathione-agarose beads (Sigma G4510) or glutathione-Sepharose 4B beads (Pharmacia) are prewashed with several volumes of lysis buffer and kept as a 1 : 1 suspension. One milliliter of this suspension is added to the supernatant and is incubated for 30 min on a rotating wheel at 4°. The beads are pelleted in a benchtop centrifuge at 4000 rpm for 1 min, and the supernatant is removed and discarded. The beads are then washed with 5 ml of cold lysis buffer (without DTT and PMSF) five times to remove unbound proteins. Recovery of bound protein can be achieved in one of two ways. a. Recovery of Fusion Protein. The GST fusion protein can be eluted from the beads by competition with free glutathione. An equal volume (0.5 ml) of freshly prepared release buffer [50 mM Tris-HC1, pH 8.0, 150 mM NaC1, 5 mM MgC12, 1 mM DTT + 5 mM reduced glutathione (Sigma G4251) (final pH 7.5)], is added to the washed beads and incubated for 2 min at 4° on a rotating wheel. The beads are pelleted and the supernatant
[11
Rho/Rac/G25K FROME. coli
7
is removed. The procedure is repeated, and the two supernatants are pooled (1 ml) and dialyzed overnight (see later). b. Recovery of Nonfused Rho/Rac/G25K. The washed beads (0.5 ml) are transferred to a 1.5-ml microcentrifuge tube and resuspended in 0.5 ml of thrombin digestion buffer (50 mM Tris-HC1, pH 8.0, 150 mM NaC1, 2.5 mM CaCI2,5 mM MgCI2, 1 mM DTT) containing 5 units of bovine thrombin (Sigma T6634). The suspension is incubated at 4 ° on a rotating wheel overnight. After thrombin digestion, the beads are pelleted in a microcentrifuge (1 min), and the supernatant is removed. Sometimes after thrombin digestion, the cleaved protein remains partly associated with the beads so we routinely incubate the beads with another 0.5 ml of high salt/DTY buffer (50 mM Tris-HC1, 7.6, 150 mM NaC1, 5 mM MgC12, 1 mM DTT) for 2 rain at 4 °. After centrifugation the two supernatants are pooled (1 ml). The efficiency of thrombin cleavage of G S T - R h o A and G S T - R a c l approaches 100%, but GST-G25K is more resistant and usually only 50% is cleaved by an overnight incubation with thrombin. Thrombin can be removed by adding 10 ~1 of a suspension of p-aminobenzamidine-agarose beads (Sigma) to the supernatant and incubating for a further 30 rain at 4° on a rotating wheel. Dialysis and Storage For microinjection purposes we dialyze against 2 liters of 10 mM TrisHC1, pH 7.6, 150 mM NaC1, 2 mM MgC12, and 0.1 mM DTT at 4 ° overnight with one buffer change. For GTPase assays where a low salt concentration is required (10 mM NaC1), we dialyze against 10 mM Tris-HCl, pH 7.6, 2 mM MgC12, and 0.1 mM DTT. Proteins are concentrated to approximately 150/xl in an Amicon Centricon 10 filter device by centrifugation in a fixed angle rotor at 7000 rpm. We routinely store the final protein preparations at approximately 1 mg/ml in 10-/zl aliquots, snap frozen in liquid nitrogen. The protein concentration is determined by a [3H]GTP/[3H]GDP binding assay as described below. The yield of wild-type proteins as determined by nucleotide binding is in the order of 0.1-0.2 rag/liter of bacterial culture. Figure 2 shows a Coomassie-stained gel of GST fusion and thrombincleaved RhoA, N25RhoA (see later), Racl, and G25K proteins. Determination of Protein Concentration Protein concentration is determined by a guanine nucleotide nitrocellulose filter binding assay. We use [3H]GTP or [3H]GDP but 32p-labeled nucleotides can also be used. Samples of concentrated protein (0.1, 0.2,
8
EXPRESSION AND PURIFICATION 1
2
3
4
5
6
7 t
qmlllP q l l l , tlllBP ~
~_..
~
~
8
9
[ 1] kD ' ~ 1 , - - 69 ~I--46
,~,..-3o g~21.5
FI6. 2. Purification of fusion and thrombin-cleaved proteins. Samples loaded are GST (lane 1), GST-wild-type RhoA (lane 2), GST-N25RhoA (lane 3), GST-Racl (lane 4), GSTG25K (lane 5), wild-type RhoA (lane 6), N25RhoA (lane 7), Racl (lane 8), and G25K (lane 9).
and 0.3/zl) are incubated in a total volume of 40/zl of assay buffer (50 mM Tris-HC1, pH 7.6, 50 mM NaC1, 5 mM MgC12, 5 mM DT]?) containing 10 mM E D T A and 0.5/zl [3H]GTP or [3H]GDP (Amersham, 10 Ci/mmol, 1 mCi/ml) for 10 min at 30°. Samples are diluted with 1 ml of cold assay buffer (without DTT) and are filtered through prewetted 25-ram nitrocellulose filters (NC45 Schleicher & Schuell 0.45/zm) using a Millipore filtration device. The filters are washed three times with 3 ml of cold assay buffer (without DTT) and are allowed to dry in air. Radioactivity is determined by scintillation counting. If 1 tool of Rho binds 1 mol of [3H]GTP, then 1 /zg Rho should yield 10 6 dpm (disintegrations per minute). The concentration of the protein sample (mg/ml) is calculated using Eq. (1): [Protein]
=
cpm//zl 100 106 x counting efficiency"
(1)
In our hands counting efficiency can be as low as 20%. Protein concentration can also be determined by comparing samples with bovine serum albumin (BSA) standards after electrophoresis on a 12% polyacrylamide gel and staining with Coomassie Brilliant Blue R (Sigma). The concentration of Rho proteins determined by this method is 3- to 5-fold higher than that determined by guanine nucleotide binding. The estimation of protein concentration by Bradford or Lowry methods gives values approximately 10-fold higher than those determined by guanine nucleotide binding. We do not understand the reason for the differences in the three assays, but a similar discrepancy has been found by others and also with Ras protein preparations. We use the guanine nucleotide binding assay as a measure of protein concentration. Protein Stability We previously reported that wild-type RhoA produced as a nonfusion protein in a trp promoter expression system was biologically inactive after
[ 11
Rho/Rac/G25K FROMg. coli
9
microinjection into cells. 15 The protein was, however, still able to bind guanine nucleotide and to hydrolyze GTP. Subsequent experiments revealed that the protein was substantially clipped at its C terminus during the relative long purification procedure required using this system. A similar observation is found with Ras expression plasmids; Ki-Ras in particular is highly susceptible to proteolysis at its C terminus in E. coli. We found, however, that Rho with an amino acid substitution of phenylalanine to asparagine at codon 25 (N25Rho), produced using the same expression system, is biologically active. Since N25Rho has a similar nucleotide exchange rate and GTP hydrolysis rate to wild-type Rho and is sensitive to R h o - G A P , we have used N25 versions of Rho proteins for many of our experiments. We have reexamined the problem of expressing wild-type RhoA using the pGEX-2T expression system described earlier. As can be seen from Fig. 2, N25Rho migrates slightly slower than wild-type RhoA and produces a much sharper band. We have found that all Ras and Rho-related GTPases are prone to smearing after electrophoresis, particularly if freshly prepared sample loading buffer is not used, and it is likely that the proteins are sensitive to oxidation. Even with fresh buffer, however, the smearing observed with wild-type RhoA could not be overcome. Despite this, wildtype RhoA purified from the pGEX expression system is only around twofold less active than N25RhoA in the microinjection assay.
TABLE I Y m L o s OF MUTANT R h o A , R a c l , AND G25K G T P a s e s Yield (/zg/liter bacteria) Mutant
Nucleotide binding"
Coomassie stain b
V14RhoA V12Rac1 V12G25K L63RhoA L61Racl N17Rac1 N17G25K
200 300 70 50 200 80 C 3c
800 1200 700 200 800 320 30
" Nitrocellulose filter binding assay using [3H]GDP or [3H]GTP. b Coomassie blue staining of electrophoresed proteins using B S A as standard. c Binding assays with N 1 7 R a c l and N17G25K carried out using [3H]GDP only.
10
EXPRESSIONAND PURIFICATION
[ 1]
M u t a n t Rho, Rac, and G25K Proteins We have purified a variety of Rho, Rac, and G25K proteins containing amino acid substitutions using the pGEX-2T vector and the protocol described earlier. These include constitutively activated protein with (i) glycine to valine substitutions at codon 14 in RhoA (V14Rho) or codon 12 in Rac (V12Rac) and G25K (V12G25K), equivalent to the oncogenic V12 mutation in Ras; and (ii) glutamine to leucine substitutions at codon 63 in Rho (L63Rho) or codon 61 in Rac (L61Rac), equivalent to the oncogenic L61 mutation in Ras. In addition, we have made dominant negative mutations with a threonine to asparagine substitution at codon 17 in Rac (N17Rac) and G25K (N17G25K), equivalent to the dominant negative N17 mutation in Ras. The yields of these mutant proteins as determined by nucleotide binding and Coomassie staining of acrylamide gels are shown in Table I. Table I shows that the yields, as judged by nucleotide binding of N17Racl, V12G25K, and particularly N17G25K, are very low but that the actual concentrations of the proteins, as determined by gel electrophoresis, are clearly much higher. In addition, we have found that N17Racl and N17G25K only bind [3H]GDP and not [3H]GTP in the guanine nucleotide filter binding assay. This appears to be a common feature of the N17 dominant negative proteins first observed by Cooper and Feig with Rasff Attempts to produce a dominant negative RhoA protein, N19RhoA, in E. coli have so far been unsuccessful. The fusion protein is expressed, but after sonication almost all of the protein is found in the pellet (A. Ridley, personal communication, 1994). Although N19RhoA can be solubilized from the pellet using detergent, the resulting protein has no detectable biological effect when microinjected into cells. Acknowledgments We thankSuzanneBrill,DagmarDiekmann,and AnneRidleyfor dataon mutantproteins; CatherineNobesfor comparingwild-typeand N25RhoAbymicroinjection;and MarkShipman for help with figures.This work was supported by the Cancer Research Campaignand the Medical Research Councilof Great Britain.
17L. A. Feig and G. M. Cooper, Mol. Cell, Biol. 8, 3235 (1988).
[2]
PURIFICATIONOF Cdc42Hs
[2]
Purification
of Baculovirus-Expressed
11
Cdc42Hs
By RICHARD A . CERIONE, D A V I D LEONARD, and YI Z H E N G Introduction The mammalian Cdc42 GTP-binding protein was initially identified through its ability to serve as a specific phosphosubstrate for the purified epidermal growth factor (EGF) receptor tyrosine kinase in reconstituted phospholipid vesicle systems. 1 This reconstitution assay enabled the purification of the 22-kDa GTP-binding protein from bovine brain membranes, following solubilization with 1% sodium cholate, by using a series of steps that included DEAE-Sephacel, Ultrogel AcA34, phenyl-Sepharose, hydroxyapatite, and Mono Q chromatographies. 1 Based on immunological cross-reactivity, the bovine brain 22-kDa GTP-binding protein/phosphosubstrate represents a form of the Gp (G25K) protein that was originally identified in human placenta and platelet plasma membranes. 2,3 Two cDNAs encoding this GTP-binding protein have been cloned from human cDNA libraries: one from a human placental library 4 and the other from a human fetal brain library. 5 These two cDNAs predicted amino acid sequences that were 95% identical. However, it was especially interesting that the amino acid sequences for the human GTP-binding protein were 80% identical and 90% similar to the sequence for the Saccharomyces cerevisiae cell division cycle protein, Cdc42 (designated Cdc42Sc), which had been shown to be essential for proper assembly of the bud site. 6 The human cDNAs complement fully temperature-sensitive mutations of the yeast cdc42. Thus, based on the high degree of sequence similarity as well as the functional complementation, it was concluded that the brain phosphosubstrate and the cloned human GTP-binding proteins represent the mammalian (or human) homologs of the yeast cell division cycle protein and so we have designated the human proteins as Cdc42Hs.
1 M. J. Hart, P. G. Polakis, T. Evans, and R. A. Cerione, J. Biol. Chem. 265, 5990 (1990). 2 T. Evans, M. L. Brown, E. D. Fraser, and J. K. Northup, J. Biol. Chem. 261, 7052 (1986). 3 p. G. Polakis, R. Snyderman, and T. Evans, Biochem. Biophys. Res. Commun. 160, 25 (1989). 4 K. Shinjo, J. G. Koland, M. J. Hart, V. Narasimhan, D. I. Johnson, T. Evans, and R. A. Cerione, Proc. Natl. Acad. Sci. U.S.A. 87, 9853 (1990). 5 S. Munemitsu, M. A. Innis, R. Clark, F. McCormick, A. Ullrich, and P. Polakis, Mol. Cell. Biol. 10, 5977 (1990). 6 D. I. Johnson and J. R. Pringle, J. Cell Biol. 111, 143 (1990).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
12
EXPRESSION AND PURIFICATION
[2]
Three classes of regulatory proteins for Cdc42Hs have been identified: a GTPase-activating protein (GAP), 7,8 a GDP-dissociation inhibitor (GDI), 9 and a guanine nucleotide exchange factor, the Dbl oncogene product, l°,u The purification of some of these regulatory proteins is considered in other chapters in this volume. In order to fully characterize the mechanisms that underly the regulation of the GTP-binding/GTPase cycle of Cdc42Hs by these regulatory proteins, it was desirable to develop systems for expressing recombinant forms of wild-type Cdc42Hs as well as different mutated forms of the GTP-binding protein. In addition, because of the possibility that the isoprenylation (geranylgeranylation) of the carboxyl-terminal cysteine of Cdc42Hs may be crucial to its recognition by other regulatory proteins, it seemed important to establish procedures for the expression of Cdc42Hs in Spodoptera frugiperda (fall armyworm) cells via baculovirus infection, because it has been well documented that insect cell-expressed proteins (unlike Escherichia coli-expressed proteins) are correctly posttranslationally modified. By comparing the purified E. coli- and insect cell-expressed Cdc42Hs proteins, we have shown that the C d c 4 2 H s - G A P as well as the Dbl oncogene product (i.e., the C d c 4 2 H s - G E F ) are able to interact functionally with the E. coli Cdc42Hs protein and do not appear to require the presence of an isoprenoid moiety on the GTP-binding protein. However, the G D I absolutely requires isoprenylated Cdc42Hs to bind and inhibit G D P dissociation as well as to elicit the removal of Cdc42Hs from membranes and inhibit its GTPase activity. The following sections describe the relatively straightforward methods that can be used to express Cdc42Hs in S. frugiperda cells and to purify the recombinant GTP-binding protein.
Purification of S p o d o p t e r a f r u g i p e r d a - E x p r e s s e d C d c 4 2 H s Expression o f Cdc42Hs in S. frugiperda Cells via Baculovirus Infection The Cdc42Hs protein was first expressed in S. frugiperda (Sf21) cells by subcloning a 660-bp DraI fragment from the full-length CDC42Hs c D N A into the SmaI site of pUC19 (designated p U C - C D C 4 2 ) . A 700-bp B a m H I / 7 M. J. Hart, K. Shinjo, A. Hall, T. Evans, and R. A. Cerione,J. Biol. Chem. 266,20840 (1991). 8 E. T. Barfod, Y. Zheng, W.-J. Kuang, M. J. Hart, T. Evans, R. A. Cerione, and A. Askenazi, J. Biol. Chem. 268, 26059 (1993). 9D. Leonard, M. J. Hart, J. V. Platko, A. Eva, W. Henzel, T. Evans, and R. A. Cerione, J. Biol. Chem. 267, 22860 (1992). x0M. J. Hart, A. Eva, T. Evans, S. A. Aaronson, and R. A. Cerione, Nature 354, 311 (1991). u M. J. Hart, A. Eva, D. Zangrilli, S. A. Aaronson, T. Evans, R. A. Cerione, and Y. Zheng, J. Biol. Chem. 269, 62 (1994).
[2]
PURIFICATION OF Cdc42Hs
13
EcoRI fragment from p U C - C D C 4 2 was then cloned into the SmaI/BamHI sites of the baculovirus transfer vector (pACYMP2). 12 Cotransfection of the wild-type baculovirus D N A with the transfer vector pACYMP2CDC42 into Sf21 cells was then performed using calcium phosphate 12 to generate recombinant virus that contained the full-length cDNA encoding Cdc42Hs. After 2-3 days, the extracellular viral supernatant was harvested from the cells and was then used to infect new Sf21 cells. High titer extracellular viral DNA was then harvested and assayed for effective recombination by Southern blot analysis using 32p-labeled CDC42Hs cDNA as a probe. The virus containing the CDC42Hs cDNA was then isolated using a dilution/hybridization method, i.e., the Sf21 cells in multiwell plates (-104 cells/well) were infected with different dilutions of the viral supernatant. 12 The Sf21 cells that were infected with the pure recombinant virus were used to purify recombinant Cdc42Hs from the particulate membrane fraction. Purification of Cdc42Hs from the Membrane Fractions of Sf21 Cells In order to ensure that the Cdc42Hs purified from S. frugiperda cells is the isoprenylated form, membrane fractions were first prepared. The cells from a l-liter spinner flask, grown in Grace's media and 10% fetal calf serum, are pelleted and then lysed by resuspending the pellet in 20 mM Tris-HC1, 100 mM NaC1, 6 mM EDTA, 1 mM dithiothreitol (DTT), pH 8.0, 0.5 mM phenylmethylsulfonyl fluoride, and 25 /xg/ml (each) of leupeptin and aprotinin. The lysate is then homogenized (in a 7-ml glass/ glass homogenizer) and the homogenates are split between two plastic 15ml centrifuge tubes and centrifuged at 2500 rpm (4 °) for 10 min in an IEC tabletop centrifuge. This step pellets the nucleus and any unbroken insect cells. The supernatant is removed and the membrane fraction from the insect cells is then pelleted by centrifugation for 15 min at 12,000 g in a Sorvall SS-34 rotor. The membranes are resuspended in 20 mM Tris-HC1, 100 mM NaC1, 1 mM EDTA, 3.75 mM MgCI2, 1 mM DTF, pH 8.0, 3/zM GDP, and 1 /xg/ml (each) of aprotinin and leupeptin, and recentrifuged for 15 min at 12,000 g. This step is repeated and then Cdc42Hs is solubilized by incubating the membranes with - 1 0 ml of 20 mM Tris-HC1, 100 mM NaC1, 1 mM EDTA, 1 mM DTT, 3.75 mM MgCI2, 3/xM GDP, pH 8.0, 1% sodium cholate, and 1 /xg/ml (each) of aprotinin and leupeptin (this is designated "solubilization buffer") for 90 min at 4 °. The particulate (membrane) fraction is removed by centrifugation at 100,000 g for 1 hr at 4°. The supernatant ( - 1 0 ml) is then applied to a 400-ml Ultrogel AcA34 column equilibrated with 20 mM Tris-HC1, 100 mM NaCI, 1 mM EDTA, 12 p. M. Guy, K. L. Carraway III, and R. A. Cerione, J. Biol. Chem. 267, 13851 (1992).
14
EXPRESSION AND PURIFICATION
[2]
1 mM DTT, 3.75 mM MgC12, and 1% sodium cholate, pH 8.0. The protein is eluted with the solubilization buffer at approximately 50 ml per hr. The fractions containing Cdc42Hs can be identified by Western blot analysis, using specific antipeptide Cdc42Hs antibodies (raised against peptides representing amino acid residues 167-183 and 180-191 of the Cdc42Hs protein 4) and by assaying [35S]GTPyS binding) The peak fractions containing Cdc42Hs activity are pooled and dialyzed against 20 mM Tris-HC1, 1 mM EDTA, 0.5% CHAPS, 1 t~g/ml leupeptin, and 5% glycerol, pH 8.0 (two changes of 500 ml each for at least 10 hr). The dialyzed Cdc42Hs is then applied to a Pharmacia Mono Q column, equilibrated with 20 mM TrisHC1, 1 mM EDTA, 1 mM DTT, pH 8.0, and 0.5% CHAPS. The purified Cdc42Hs is eluted from the column using a linear NaC1 gradient (0-300 raM). At this stage, the purity of the Cdc42Hs preparation can be assessed by SDS-PAGE and protein (Coomassie blue) staining. Typically, the sole band detected is the 22-kDa Cdc42Hs protein. If necessary, the peak fractions containing the Cdc42Hs can be concentrated by hydroxyapatite chromatography. In such cases, the Cdc42Hs is applied to a 3-ml hydroxyapatite
B
A
a~
16x10 3 _
25-
14-
20-
~12-
15.
~ 10-
o 8I11 a, 6-
10-
,-~ 45~
0
i-
o.o
;.
a
•
o'.=
o14 GDI (p,g)
2-
0-
JL
o'.6
o'.8
i i
i
i
i
i
i
i
1
2
3 Time
4
5
6
(rain)
FIG. 1. GDI activity on the S. frugiperda- and E. coli-recombinant Cdc42Hs. (A) The S. frugiperda-expressed Cdc42Hs ( 0 ) or the E. coli-recombinant Cdc42Hs (&) was preincubated with [c~-32p]GTP (7 tzM) for 25 rain at room temperature. This incubation period ensures that all of the bound GTP is converted to G D P as a result of the intrinsic GTPase activity of Cdc42Hs. The [c~-32p]GDP-bound Cdc42Hs proteins (~15 ng) were then incubated with the indicated amounts of the Mono S-purified GDI activity (in the presence of 2.5 m M E D T A ) as described in Leonard et aL 9 After 6 rain, the samples were then filtered on nitrocellulose (BA85) filters and the amount of [a-32p]GDP that remained bound to Cdc42Hs (relative to the amount of G D P bound at the start of the assay), as a function of the amount of GDI added to the assay incubation, was determined. (B) Cdc42Hs (20 ng) that was purified from the membrane fraction of S. frugiperda cells was preincubated with [3H]GDP (7/~M) for 25 min. The dissociation of the radiolabeled G D P was measured at the indicated times in the absence (A) and presence (O) of the GDI. Reproduced from Leonard et al. 9 with permission.
[3]
15
PURIFICATION AND PROPERTIES OF Rac2
column that was equilibrated in 20 mM Tris-HCl, pH 8.0, and 0.5% CHAPS. The Cdc42Hs is eluted from the column with 20 mM Tris-HC1, 1 mM EDTA, 1 mM DTT, pH 8.0, plus 100 mM potassium phosphate, 40% glycerol, and 0.5% CHAPS. The peak Cdc42Hs fractions can be identified by Western blot analysis using the anti-Cdc42Hs antibody or by Coomassie blue staining (i.e., as indicated by the presence of a single protein band at 22 kDa). We had previously demonstrated that the interactions of Cdc42Hs with its GAP (7) or GEF (11) were independent of the presence of an isoprenoid moiety on the GTP-binding protein. However, the interactions of the GDI with Cdc42Hs do appear to require that Cdc42Hs is geranylgeranylated. Figure 1A shows a comparison of the ability of the R h o - G D I to inhibit the dissociation of radiolabeled GDP from Cdc42Hs expressed and purified from insect cells and E. coli. Under the conditions of this experiment, 100% of the bound radiolabeled GDP is dissociated from the recombinant Cdc42Hs proteins within 5 min (in the presence of excess EDTA, i.e., no added MgC12 and [EDTA] = 2.5 mM). Likewise, complete dissociation of the bound GDP occurs when the E. coli Cdc42Hs is incubated with the brain GDI, whereas 25% to as much as 50% of the originally bound GDP remains associated when the insect cell-expressed Cdc42Hs is incubated with the brain GDI. The fact that only a percentage of the insect cellexpressed Cdc42Hs was sensitive to the GDI stems from the fact that the GTP-binding protein was prepared from whole cell lysates. When the same experiments were performed with Cdc42Hs that was purified from the membrane fractions of insect cells, as outlined earlier, the extents of inhibition by the R h o - G D I approached 100% (Fig. 1B). These results then indicate that when the Cdc42Hs is purified from insect cell membranes, virtually all of the Cdc42Hs is in the geranylgeranylated form.
[3] P u r i f i c a t i o n
By
TAKAKAZU
and Properties of Rac2 from Human Leukemia Cells
MIZUNO,
HIROYUKI
NAKANISHi,
and
YOSHIMI
TAKAI
Introduction The superoxide-generating N A D P H oxidase system in phagocytes, such as neutrophils and monocytes, consists of membrane-associated cytochrome b-558, composed of gp91-phox and p22-phox heterodimer, as a terminal METHODS IN ENZYMOLOGY,VOL. 256
Copyright © 1995by AcademicPress, Inc. All rights of reproductionin any form reserved.
16
EXPRESSION AND PURIFICATION
[3]
redox carrier and at least three cytosolic regulatory components. 1,2 Two of them, p47-phox and p67-phox, have been identified as the products of the genes causing autosomal recessive type of chronic granulomatous disease. 3,4 A series of studies from several laboratories, including our own, have revealed that the third cytosolic component is a member of the Rho-related small GTPases, Rac. 5-8 The Rac family consists of highly homologous Racl and Rac2, and our result indicates that both members stimulate the superoxide generation. 9 Smg-GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for a group of small GTPases including at least Ki-Ras, Rapl, Rho, and Rac, 1°-13 stimulates the conversion of GDP-Rac to GTPRac and thereby stimulates the NADPH oxidase activity. 7'9 In contrast, the R h o - G D P dissociation inhibitor (GDI), an inhibitory GDP/GTP exchange protein for a group of small GTPases including at least Rho, Rac, and mCdc42 (see [6], this volume), 13-t8 inhibits the conversion of GDP-Rac to 1 A. W. Segal, J. Clin. Invest. 83, 1785 (1989). z R. A. Clark, J. Infect. Dis. 161, 1140 (1990). 3 K. J. Lomax, T. L. Leto, H. Nunoi, J. I. Gallin, and H. L. Malech, Science 245, 409 (1989). 4 T. L. Leto, K. J. Lomax, B. D. Volpp, H. Nunoi, J. M. G. Sechler, W. M. Nauseef, R. A. Clark, J. I. Gallin, and H. L. Malech, Science 248, 727 (1990). s A. Abo, E. Pick, A. Hall, N. Totty, C. G. Teahan, and A. W. Segal, Nature 353, 668 (1991). 6 U. G. Knaus, P. G. Heyworth, T. Evans, J. T. Curnutte, and G. M. Bokoch, Science 254, 1512 (1991). 7 T. Mizuno, K. Kaibuchi, S. Ando, T. Musha, K. Hiraoka, K. Takaishi, M. Asada, H. Nunoi, I. Matsuda, and Y. Takai, J. Biol. Chem. 267, 10215 (1992). s C. H. Kwong, H. L. Malech, D. Rotrosen, and T. L. Leto, Biochemistry 32, 5711 (1993). 9 S. Ando, K. Kaibuchi, T. Sasaki, K. Hiraoka, T. Nishiyama, T. Mizuno, M. Asada, H. Nunoi, I. Matsuda, Y. Matsuura, P. Polakis, F. McCormick, and Y. Takai, Z Biol. Chem. 267, 25709 (1992). 10T. Yamamoto, K. Kaibuchi, T. Mizuno, H. Hiroyoshi, H. Shirataki, and Y. Takai, J. Biol. Chem. 265, 16626 (1990). 11 K. Kaibuchi, T. Mizuno, H. Fujioka, T. Yamamoto, K. Kishi, Y. Fukumoto, Y. Hori, and Y. Takai, 3,1ol. Cell. Biol. 11, 2873 (1991). 12 T. Mizuno, K. Kaibuchi, T. Yamamoto, M. Kawamura, T. Sakoda, H. Fujioka, Y. Matsuura, and Y. Takai, Proc. Natl. Acad. Sci. U.S.A. 88, 6442 (1991). 13 K. Hiraoka, K. Kaibuchi, S. Ando, T. Musha, K. Takaishi, T. Mizuno, M. Asada, L. Menard, E. Tomhave, J. Didsbury, R. Snyderman, and Y. Takai, Biochem. Biophys. Res. Commun. 182, 921 (1992). 14 N. Ohga, A. Kikuchi, T. Ueda, J. Yamamoto, and Y. Takai, Biochem. Biophys. Res. Commun. 163, 1523 (1989). is T. Ueda, A. Kikuchi, N. Ohga, J. Yamamoto, and Y. Takai, J. Biol. Chem. 265, 9373 (1990). 16 y. Fukumoto, K. Kaibuchi, Y. Hori, H. Fujioka, S. Araki, T. Ueda, A. Kikuchi, and Y. Takai, Oncogene 5, 1321 (1990). 17T. Sasaki, K. Kato, T. Nishiyama, and Y. Takai, Biochem. Biophys. Res. Commun. 194, 1188 (1993). 18 D. Leonard, M. J. Hart, J. V. Platko, E. Alessandra, W. Henzel, T. Evaans, and R. A. Cerione, J. Biol. Chem. 267, 22860 (1992).
[3]
PURIFICATIONAND PROPERTIESOF Rac2
17
G T P - R a c and thereby inhibits the N A D P H oxidase activity. 7'9 Therefore, once G D P - R a c is converted to G T P - R a c , S m g - G D S or R h o - G D I does not affect the superoxide generation in our assay system, although other groups have reported that the R a c / R h o - G D I complex stimulates the N A D P H oxidase activity in the absence of exogenous GTP. 5'8 The Rho family members, including Racl and Rac2, have a unique Cterminal amino acid structure of Cys-A-A-Leu (A, aliphatic amino acid), which undergoes postranslational modifications including geranylgeranylation followed by removal of three amino acids and the carboxylmethylation of the exposed cysteine. 19'2° The lipid modifications of Rac are essential for its interactions with S m g - G D S and R h o - G D I . 9 Moreover, lipid-modified Rac stimulates the N A D P H oxidase activity more efficiently than does a lipid-unmodified one, 9 although another group has reported that both forms stimulate the N A D P H oxidase activity with similar efficiency.21,22 This chapter describes the assay for the N A D P H oxidase activity, the procedures for the purification of Rac2 from the cytosol fraction of the differentiated HL-60 (human promyelocytic leukemia) cells, and the properties of Rac2.
Materials RPMI 1640 medium and fetal calf serum are purchased from G I B C O B R L (Gaithersburg, MD). Sodium cholate, sodium deoxycholate, and Lo~-dimyristoylphosphatidylcholine (DMPC) are from Wako Pure Chemicals (Osaka, Japan). N-2-Hydroxyethylpiperadine-N'-2-ethanesulfonic acid (HEPES), 3- [(3-cholamidopropyl)dimethylammonio]-l-propanesulfonic acid (CHAPS), and E D T A are from Dojindo Laboratories (Kumamoto, Japan). Dithiothreitol ( D T T ) and E G T A are from Nacalai Tesque (Kyoto, Japan). Phenylmethylsulfonyl fluoride (PMSF), 2-(N-morpholino)ethanesulfonic acid (MES), ferricytochrome c, N A D P H , FAD, catalase, arachidonic acid, and superoxide dismutase (SOD) are from Sigma (St. Louis, MO). G D P and guanosine 5'-(3-O-thio)triphosphate (GTPyS) are from Boehringer Mannheim (Indianapolis, IN). [35S]GTPyS is from Du PontNew England Nuclear (Boston, MA). Carboxymethyl (CM)-Sepharose and Mono Q HR5/5 are from Pharmacia P-L Biochemicals Inc. (Milwaukee, 19M. Katayama, K. Kawata, Y. Yoshida, H. Horiuchi, T. Yamamoto, Y. Matsuura, and Y. Takai, J. Biol. Chem. 266, 12639 (1991). 20p. Adamson, C. J. Marshall, A. Hall, and P. A. Tilbrook, J. Biol. Chem. 267, 20033 (1992). 21p. G. Heyworth, U. G. Knaus, X. Xu, D. J. Uhlinger, L. Conray, G. M. Bokoch, and J. T. Curnutte, Mol. Biol. Cell 4, 261 (1993). 22M. L. Kreck, D. J. Uhlinger, S. R. Tyagi, K. L. Inge, and J. D. Lambeth, J. Biol. Chem. 269, 4161 (1994).
18
EXPRESSION AND PURIFICATION
[3]
WI). Hydroxyapatite is from Seikagaku Kogyo Co. (Tokyo, Japan). All other chemicals are of reagent grade. HL-60 cells are obtained from the American Tissue Culture Center (Rockville, MD). Recombinant p47-phox and p67-phox are purified from Spodoptera frugiperda (Sf9) cells by use of a baculovirus system. 23 The baculoviruses carrying the cDNAs of p47-phox and p67-phox are from H. Nunoi and I. Matsuda (Kumamoto University School of Medicine, Kumamoto, Japan). Recombinant Smg-GDS is purified from Smg-GDS-overexpressing Escherichia coli.u R h o - G D I is purified from the cytosol fraction of bovine brain. 1~ Lipid-modified and lipid-unmodified recombinant Rac2s are purified from the membrane and cytosol fractions of Sf9 cells, respectively, using a baculovirus expression system. 9 The baculovirus carrying the cDNA of Rac2 is from P. Polakis and F. McCormick (Onyx Pharmaceuticals, Richmond, CA). GTPTS-Rac2 and GDP-Rac2 are prepared as described. 9
Methods
Purification of Rac2 from Differentiated HL-60 Cells The various buffers used in the purification of Rac2 are as follows: Buffer A: 10 mM KHzPO4/KzHPO4 at pH 7.5, 1 mM EGTA, 1 mM PMSF, 130 mM NaCI, 5 mM MgCI2, 340 mM sucrose Buffer B: 10 mM MES at pH 6.8, 1 mM EGTA, 1 mM PMSF, 5 mM 2-mercaptoethanol Buffer C: 20 mM Tris/HCl at pH 8.0, l mM EDTA, 1 mM DTT, 5 mM MgCI2, 100 mM NaC1, 1% sodium cholate Buffer D: 20 mM Tris/HC1 at pH 8.0, 0.1 mM EDTA, 1 mM DTT, 3 mM MgC12 Buffer E: 20 mM Tris/HC1 at pH 8.0, 1 mM EDTA, 1 mM DTT, 5 mM MgCI2, 0.5% sodium cholate Buffer F: 20 mM Tris/HC1 at pH 8.0, 1 mM EDTA, 1 mM DTT, 5 mM MgC12, 0.6% CHAPS The steps used in the purification of Rac2 are as follows: (1) preparation of differentiated HL-60 cells; (2) preparation of the cytosol and membrane fractions; (3) CM-Sepharose column chromatography; (4) Ultrogel AcA 44 column chromatography; (5) hydroxyapatite column chromatography; (6) Mono Q HR5/5 column chromatography; and (7) Mono Q HR5/5 column rechromatography. 23 T. L. Leto, M. C. Garrett, H. Fujii, and H. Nunoi, J. BioL Chem. 266, 19812 (1991).
[3]
PURIFICATION AND PROPERTIES OF R a c 2
19
Preparation of Differentiated HL-60 Cells HL-60 cells are grown in RPMI 1640 medium containing 10% fetal calf serum, 100 rag/liter streptomycin, and 100,000 units/liter penicillin at 37° in 5% COj95% air (v/v). The cells (5 x 109 cells) are differentiated into neutrophil-like cells by treatment with 3/zM retinoic acid for 4 days. The differentiation is estimated by measuring the expression of CDllb, a marker antigen of neutrophils.
Preparation of Cytosol and Membrane Fractions All the following procedures are carried out at 0-4 °. The differentiated HL-60 cells (5 x 109 cells) are washed twice with phosphate-buffered saline (PBS) at pH 7.4, suspended in 20 ml of buffer A, and then sonicated for 15 sec three times at 10-sec intervals. After removal of unbroken cells and nuclei, the sonicate is layered on 20 ml of buffer A containing 40% sucrose and is centrifuged at 140,000g for I hr. The supernatant is further centrifuged at 200,000g for 1 hr and is used as the cytosol fraction. The membrane fraction is collected from the surface of the 40% sucrose layer, rinsed with buffer A, and resuspended with 10 ml of buffer A. Both fractions are stored at -80 ° and are stable for at least several months.
CM-Sepharose Column Chromatography One-third of the cytosol fraction (10 ml, 30 mg of protein) is diluted fivefold with buffer B and applied to a CM-Sepharose column (1.5 x 20 cm) equilibrated with buffer B. Elution is performed with 50 ml of buffer B followed by buffer B containing 300 mM NaCI at a flow rate of 1 ml/ min. Fractions of 4 ml each are collected. When each fraction is assayed for [35S]GTPyS-binding activity, one broad peak and one sharp peak appear in fractions 6-25 and 31-40, respectively. These peaks correspond to those of absorbance at 280 nm. The active fractions of the first peak are collected.
Ultrogel AcA 44 Column Chromatography The active fractions of the CM-Sepharose column chromatography (80 ml, 16 mg of protein) are pooled and concentrated to approximately 10 ml by an ultrafiltration cell (Amicon) equipped with a PM-10 filter membrane. After the addition of MgC12 and sodium cholate at final concentrations of 5 mM and 1%, respectively, the concentrate is applied to an Ultrogel AcA44 column (2 X 80 cm) equilibrated with buffer C. Elution is performed with 210 ml of buffer C at a flow rate of 0.275 ml/min. Fractions of 2.2 ml each are collected. When each fraction is assayed for [35S]GTPyS-binding activity, two peaks appear in fractions 50-58 and 62-73. The first peak
20
eXVRZSSIONAND PURIFICATION
[31
contains heterotrimeric GTPases and the second peak contains small GTPases including Rac2. When each fraction is assayed for the NADPH oxidase activity, a single peak appears, which corresponds to the second peak of [35S]GTPyS-binding activity. The active fractions of the NADPH oxidase activity are pooled and purified further.
Hydroxyapatite Column Chromatography The pooled fractions of the Ultrogel AcA-44 column chromatography (26 ml, 10 mg of protein) are diluted to 40 ml with 20 mM Tris/HC1 at pH 8.0 containing 1 mM DTT and are applied to a hydroxyapatite column (1.5 × 6 cm) equilibrated with buffer D containing 10 mM KH2PO4. Elution is performed first with 160 ml of buffer D containing 10 mM KHzPO4 and 0.6% CHAPS and then with 80 ml of buffer D containing 100 mM KHePO4 and 0.6% CHAPS at a flow rate of 0.67 ml/min. Fractions of 4 ml each are collected. When each fraction is assayed for [35S]GTPyS-binding activity, two peaks appear in fractions 16-40 and 53-56. When each fraction is assayed for the NADPH oxidase activity, two peaks appear in fractions 17-23 as a major peak and in fractions 53-56 as a minor peak. The major peak of the NADPH oxidase activity contains Rac2 and the minor peak contains Rac! as estimated by Western blot analysis using their respective antibodies. The active fractions of the major peak are pooled and purified further.
Mono Q HR5/5 Column Chromatography The active fractions of the major peak of the hydroxyapatite column chromatography (28 ml, 2.5 mg of protein) are concentrated to approximately 1.5 ml by an ultrafiltration cell equipped with a YM5 filter membrane (Amicon). The concentrate is diluted 10-fold with buffer E and is applied to a Mono Q HR5/5 column equilibrated with buffer E containing 10 mM NaC1. After the column is washed with 15 ml of the same buffer, elution is performed with a 30-ml linear gradient of NaC1 (10-500 mM) in buffer E at a flow rate of 0.5 ml/min. Fractions of 0.5 ml each are collected. When each fraction is assayed for [35S]GTPyS-binding activity, two peaks appear in fractions 76-88 and 90-100 as shown in Fig. 1. When each fraction is assayed for the NADPH oxidase activity, a single peak appears in fractions 88-92. The active fractions of the NADPH oxidase activity are collected and stored at -80 °. The rest of the cytosol fraction is treated in the same way. The pooled fractions can be stored for at least 3 months at -80 ° without loss of activity.
[3]
21
PURIFICATION AND PROPERTIES OF R a c 2
L
A
'T
i
i
i
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~
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-
]
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-
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60 80 100 FractionNumber
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120
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FIG. 1. Mono Q column chromatography. A 5-/A aliquot of each fraction was assayed for NADPH oxidase and [35S]GTPyS-binding activities. (3, the NADPH oxidase activity; O, the [35S]GTPyS-binding activity.
Mono Q HR 5/5 Column Rechromatography The combined pools of the active fractions of the Mono Q HR5/5 column chromatography (7.5 ml, 120 mg of protein) are dialyzed against buffer E and further applied to a Mono Q HR 5/5 column equilibrated with buffer F. After the column is washed with 15 ml of the same buffer, elution is performed with a 30-ml linear gradient of NaC1 (0-500 mM) in buffer F at a flow rate of 0.5 ml/min. Fractions of 0.5 ml each are collected. When each fraction is assayed for [35S]GTPyS-binding and NADPH oxidase activities, a single peak appears in fractions 50-58 as shown in Fig. 2. This A ¢
o o. ' ~
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/
~
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!
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o 2 m
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~-
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FractionNumber FIG. 2. Mono Q column rechromatography. A 5-/xl aliquot of each fraction was assayed for NADPH oxidase and [35S]GTPyS-binding activities. (3, the NADPH oxidase activity; e , the [35S]GTPyS-binding activity. (Adapted from Mizuno et al. 7 with permission from the Journal o f Biological Chemistry and the American Society for Biochemistry and Molecular Biology.)
22
EXPRESSIONAND PURIFICATION
[3]
GTPase is a nearly homogeneous protein with a Mr of about 22,000 and is identified to be Rac2 by the partial amino acid sequences.
Assay for Cell-Free NADPH Oxidase Activity The cell-free NADPH oxidase activity is assayed by measuring the arachidonic acid-elicited superoxide generation, which is determined by the SOD-inhibitable ferricytochrome c reduction by use of Rac2, p47-phox, p67-phox, GTPyS, and the solubilized membrane components including membrane-associated cytochrome b - 5 5 8 . 24
Preparation of Solubilized Membrane Components The solubilized membrane components including membrane-associated cytochrome b-558 are prepared as follows. The membrane fraction from the differentiated HL-60 cells (10 ml, 36 mg of protein) described earlier is incubated for 30 min at 4° with the same volume of 20 mM glycine/ NaOH at pH 8.0 containing 50% glycerol, 1 mM NAN3, 1.7/xM CaC12, and 2.3% sodium deoxycholate. After centrifugation at 100,000g for 1 hr, the extract is diluted 20-fold by water and is used as the solubilized membrane components. These membrane components can be stored for at least 3 months at -80 ° without loss of activity. Repeated freezing and thawing of this sample should be avoided. This sample is free from p47-phox p67phox, Smg-GDS, and R h o - G D I but is contaminated by a 10 -8 M level of unidentified endogenous GTPases.
Assay Fourteen nanomolar p47-phox, 24 nM p67-phox, and 2 nM Rac2 or a 5-~1 aliquot of each fraction of the column chromatographies are first incubated in a reaction mixture (300/xl) containing 10 mM MES at pH 6.8, 1 mM EGTA, 1 mM PMSF, 5 mM 2-mercaptoethanol, and 10/xM GTPTS. After a 5-rain incubation at 25 °, the mixture is cooled on ice. The second reaction mixture (200/zl) containing 30 mM HEPES/NaOH at pH 7.3, 30 mM KH2POa/K2HPO4 at pH 7.0, 240/~M ferricytochrome c, 750 /zM NADPH, 3/zM FAD, 75/zg/ml catalase, 1.5 mM EDTA, 9 mM MgC12, and 6 mM NaN3 is added, and then the solubilized membrane components (60/zl, 5.4/xg of protein) are added to the mixture. The second reaction is initiated by the additional of 10/zl of 700/xM arachidonic acid to give a final concentration of 12.5 nM and is performed for 15 min at 25 °. The reaction is stopped by the addition of 30/xl of 500/zg/ml SOD. The rates 24E. Pick, Y. Bromberg, S. Shpungin,and R. Gadba,J. Biol. Chem.262, 16476 (1987).
[3]
PURIFICATION AND PROPERTIES OF R a c 2
23
of the superoxide production are calculated from the absorbance at 550 nm as [/xmol superoxide/min/mg membrane protein], based on Ae550 = 2.1 × 104 M-acm -1 (reduced minus oxidized cytochrome).24 The reference reaction is performed in the presence of 25/~g/ml SOD. During the purification procedures of Rac2, the NADPH oxidase activity is assayed in the presence of 150 nM Smg-GDS. When the properties of Rac2 are studied, the NADPH oxidase activity is assayed in the presence of various combinations of 2 nM Rac2, 14 nM p47-phox, 24 nM p67-phox, 10 mM GTPyS, 150 nM Smg-GDS, and 300 nM Rho-GDI.
Assay for f~SS]GTPyS-BindingActivity The [35S]GTPyS-binding activity is assayed by measuring the radioactivity of [35S]GTPTS bound to a small GTPase trapped on nitrocellulose filters (BA-85, Schleicher & Schuell). A 20-/A aliquot of each fraction of the column chromatographies described earlier is incubated for 20 min at 30° in a reaction mixture (40/zl) containing 20 mM Tris-HCl at pH 7.5, 10 mM EDTA, 5 mM MgC12, 1 mM DTT, 1 mM DMPC, and 1/zM [35S]GTPTS (3-6 x 103 cpm/pmol). The reaction is stopped by the addition of about 2 ml of an ice-cold stopping solution containing 20 mM Tris/HC1 at pH 7.5, 25 mM MgC12, and 100 mM NaC1, followed by rapid filtration on nitrocellulose filters. Filters are washed five times with the same ice-cold stopping solution. After filtration, the radioactivity is counted.
Properties of Rac2 Activation of NADPH oxidase by the Rac2 purified from differentiated HL-60 cells is summarized in Fig. 3A. Rac2 stimulates the cell-free NADPH oxidase activity in the presence of p47-phox, p67-phox, and GTPyS. Smg-GDS enhances the Rac2-induced NADPH oxidase activity, whereas R h o - G D I counteracts this stimulatory effect of Smg-GDS. A removal of each component completely abolishes the NADPH oxidase activity. The recombinant Rac2, produced in insect ceils using a baculovirus system, shows a similar effect (Fig. 3B). Moreover, lipid-modified Rac2 is far more effective than lipid-unmodified Rac2. When the first incubation is performed with GTPTS-Rac2, Smg-GDS is not required for the NADPH oxidase activation. R h o - G D I is unable to inhibit the GTPyS-Rac2-induced NADPH oxidase activation. Similar results are also obtained using recombinant Racl. 9 Rapl, RhoA, or Ki-Ras do not affect the NADPH oxidase activity.7
24
[3]
EXPRESSION AND PURIFICATION A
"0-~
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.
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FIG. 3. Effect of Smg-GDS and R h o - G D I on NADPH oxidase activity. (A) Effect of Rac2 purified from HL-60 cells. The first incubation was performed in the presence of various combinations of 2 nM Rac2, 14 nM p47-phox, 24 nM p67-phox, 150 nM Smg-GDS, 300 nM Rho-GDI, and 10/zM GTPyS. (B) Effect of recombinant Rac2. The first incubation was peformed in the presence of 14 nM p47-phox, 24 nM p67-phox, 10/xM GTPyS, and various combinations of 12 nM lipid-modified recombinant G D P - or GTPyS-Rac2 (mRac2), 12 nM lipid-unmodified recombinant G D P - or GTPyS-Rac2 (cRac2), 150 nM Smg-GDS, and 300 nM Rho-GDI.
Comments Several points need to be considered for the NADPH oxidase assay. First, an optimal concentration of arachidonic acid varies depending on preparations of the solubilized membrane components. It also varies depending on the concentrations of the proteins, including Rac, p47-phox, and p67-phox. Therefore, it is recommended to determine the optimal concentration of arachidonic acid used for each assay system. Second, an excess amount of sodium cholate or CHAPS suppresses the NADPH oxidase activity. Therefore, when an aliquot of each fraction of the column chromatographies is assayed for NADPH oxidase activity, the volume of the aliquot should be less than 5/zl. We have purified Rac2 free from R h o - G D I from the differentiated HL-60 cells as an activator of superoxide generation, and have shown that GTPyS-recombinant Rac2 stimulates NADPH oxidase activity irrespective of the presence or absence of Smg-GDS and Rho-GDI. These regulatory proteins just regulate the conversion of GDP-Rac2 to GTP-Rac2 and do not affect the NADPH oxidase activity itself. However, other groups have purified Racl and Rac2 as a heterodimer with R h o - G D I from the cytosol fractions of macrophages and neutrophils, respectively, and have shown that the R a c / R h o - G D I complex stimulates the NADPH oxidase activity in the absence of exogenous GTP, 5's although it has been also shown that
[41
PURIFICATIONOF Rac2 FROM NEUTROPHILS
25
the superoxide-producing activity of the purified R a c / R h o - G D I complex is increased three-fold by G T P and completely inhibited by GDP. 25 The discrepancy between the results of ours and other groups concerning the purification of Rac as a monomeric form free from R h o - G D I and a heterodimeric form complexed with R h o - G D I may be due to the fact that we use detergents during the purification procedures and that other groups do not. The reason why Racl or Rac2 complexed with R h o - G D I is active on the N A D P H oxidase is currently unknown, but we have found that R h o G D I preferentially forms a complex with G D P - R a c but weakly forms a complex with G T P - R a c . The efficiency of R h o - G D I to form a complex with G T P - R a c is about 10% that of R h o - G D I to form a complex with G D P - R a c . 26 The R h o - G D l / R a c complex purified from other groups may contain G T P - R a c . We have shown that lipid-modified Rac is far more effective on the activation of N A D P H oxidase than lipid-unmodified Rac. However, another group has shown that both forms of Rac are equally effective.21'= The reason for this discrepancy is currently unknown. 25A. Abo, M. R. Webb, A. Grogan, and A. Segal, Biochem. J. 298, 585 (1994). 26T. Sasaki, M. Kato, and Y. Takai, J. Biol. Chem. 268, 23959 (1993).
[4] P u r i f i c a t i o n
of Rac2 from Human
Neutrophils
By ULLA G. KNAUS and GARY M. BOKOCH The Rac2 protein belongs to the Rho family of GTP-binding proteins and is closely related to the Racl protein (92% identity). Whereas Racl is ubiquitously expressed, Rac2 is only found in cells of myeloid origin. Studies with a human leukemia cell line (HL-60) showed a seven- to ninefold increase of Rac2 expression on differentiation to a neutrophiMike type, whereas R a c l levels rose only slightly. 1 Analysis of Rac protein levels by immunoblotting with specific antibodies revealed also the predominance of Rac2 in human neutrophils. 2 These data indicate that neutrophils are an excellent source for the purification of biologically active Rac2, if recombinant sources that allow post-translational processing of GTP-binding pro1J. Didsbury, R. F. Weber, G.M. Bokoch, T. Evans, and R. Snyderman,J. BioL Chem. 264, 16378 (1989). 2M. T. Quinn, T. Evans, L. R. Loetterle, A.J. Jesaitis, and G. M. Bokoch, Z BioL Chem. 268, 20983 (1993).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
26
EXPRESSIONAND PURIFICATION
[4]
teins, such as the baculovirus expression system in Sf9 ( S p o d o p t e r a frugip e r d a falls armyworm ovary) cells, are not available. The Rac2 protein is localized in the cytosolic compartment of unstimulated neutrophils, where it forms a stable complex with the GDP dissociation inhibitor [rho]GDI. 3 Upon stimulation with the chemoattractant fMLP or with phorbol myristate acetate, this complex partially dissociates and Rac2 translocates to the plasma membrane. 2 This underscores the importance of avoiding any step during the isolation procedure of neutrophils that could lead to activation and loss of cytosolic Rac2 protein. Biologically active Rac2 protein can be readily detected by its ability to bind GTP and G D P with high affinity. Screening of column fractions for Rac and several related GTP-binding proteins is achieved by [32S]GTPTS binding to the protein under low magnesium conditions, followed by a rapid filtration assay, as described later. Western blotting with specific peptide antibodies, directed to the carboxyl terminus of the GTP-binding proteins Racl, Rac2, and Cdc42Hs (i.e., Santa Cruz Biotech., CA), allows further identification. To distinguish Rac2 from the Rho-GTP-binding protein, the specific ability of botulinum toxin C3 transferase to ADP-ribosylate Rho can be utilized. 4 A specialized biological role for Rac2 in phagocyte function has been reported. Rac2 has been shown to be absolutely required for activation of the N A D P H oxidase 5,6and for the subsequent generation of reactive oxygen species. This function provides another means to detect Rac2 protein during purification by using a cell-free N A D P H oxidase assay system consisting of neutrophil cytosol and membranes 7 or recombinant proteins as described. 8 The following purification protocol implements foremost the use of the GTPyS-binding assay to detect active GTP-binding protein, assisted by the more specific methods for Rac2. The procedure is carried out at 4 ° unless otherwise stated. P r e p a r a t i o n of Neutrophil Cytosol As a source for human neutrophils, blood products from healthy, normal donors are typically obtained by leukapheresis, a procedure that leads to 3T.-H. Chuang, B. P. Bohl, and G. M. Bokoch, J. Biol. Chem. 268, 26206 (1993). 4This volume [21]. 5U. G. Knaus, P. G. Heyworth, T. Evans, J. T. Curnutte, and G. M. Bokoch, Science 254, 1512 (1991). 6 This v o l u m e [39]. 7j. T. Curnutte, P. J. Scott, and L. A. Mayo, Proc. Natl. Acad. Sci. U.S.A. 86, 825 (1989). 8This volume [29].
[4]
PURIFICATION OF Rac2 FROM NEUTROPHILS
27
a 30- to 40-fold enrichment in white blood cells. After removal of erythrocytes by hypotonic lysis, neutrophils are separated from monocytes and platelets by differential centrifugation through Ficoll-Hypaque. 9 In a different procedure, whole human blood can also be used as starting material. 1° The neutrophils are treated with 3 m M diisopropyl fluorophosphate for 15 min on ice, washed several times with phosphate-buffered saline (PBS), and subjected to nitrogen cavitation (450 psi, 20 min) in a buffer consisting of 100 m M KCI, 3 m M NaCI, 1 m M ATP, 3.5 m M MgCI2, 10 mM PIPES (pH 7.3), 1 m M phenylmethylsulfonyl fluoride (PMSF), and 100 kallikrein inhibitory units of aprotinin/ml. Cavitated cells are collected into sufficient E G T A to give a final concentration of i mM, centrifuged at low speed (1000 rpm, 10 rain) to remove unbroken cells and nuclei, and then fractionated on discontinuous, 15/40/60% sucrose gradients in 25 m M H E P E S (pH 8.0), 1 m M E G T A , 1 m M E D T A buffer. The cytosol overlay is collected and immediately frozen at - 7 0 °, whereas the 15 to 40% interface containing plasma membranes is washed, repelleted, and stored in aliquots at - 7 0 ° in 25 m M H E P E S , p H 8.0, and 20 m M sucrose.
Analysis of G u a n i n e Nucleotide Binding Binding of [35S]G T P y S to GTP-binding proteins during all chromatography steps is determined with the rapid filtration technique as described, u For the standard assay, 10/xl of column fraction is incubated for 5 rain at 30 ° in 90/xl of reaction mixture containing 50 m M H E P E S (pH 8.0), 1 m M dithiothreitol (DTT), 2 m M E D T A , and 1 /xM [35S]GTPyS (1-2 × 104 cpm/pmol, Du Pont-NEN). The reaction is terminated by addition of 2 ml of ice-cold stop mixture [25 m M Tris-HC1 (pH 8.0), 100 m M NaC1, 30 m M MgCle, 2 m M D T T , 1 mg/ml bovine serum albumin] and binding is quantitated by vacuum filtration on BA-85 nitrocellulose filters (Schleicher and Schuell) and liquid scintillation counting (Scint-A XF, Packard). The free magnesium ion concentration under the conditions described is calculated to be 950 nM. 12
9j. T. Curnutte, R. Kuver, and P. J. Scott, J. Biol. Chem. 262, 5563 (1987). 10A. J. Jesaitis, J. R. Naemura, R. G. Painter, L. A. Sklar, and C. G. Cochrane, Biochim. Biophys. Acta 719, 556 (1982). 11U. G. Knaus, P. G. Heyworth, B. T. Kinsella, J. T. Curnutte, and G. M. Bokoch, J. BioL Chem. 267, 23575 (1992). 12T. Higashijima, K. M. Ferguson, P. C. Sternweis, M. D. Smigel, and A. G. Gilman, J. Biol. Chem. 262, 762 (1987).
28
EXPRESSION AND PURIFICATION
[4]
Purification Procedure
Buffers TEDMPM: 25 mM Tris-HC1 (pH 7.5), 1 mM EDTA, 0.1 mM DTT, 5 mM MgCI2, 0.5 mM PMSF, 1 mM 2-mercaptoethanol TEDMM: 25 mM Tris-HC1 (pH 7.5), 1 mM EDTA, 0.1 mM DTT, 5 mM MgCI2, 1 mM 2-mercaptoethanol TEDM: 25 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM DTT, 5 mM MgCI2
DEAE-Sephacel Anion-Exchange Chromatography Neutrophil cytosol (200-250 ml) from different donors (4 x 10 l° cell equivalents) is pooled and concentrated 10-fold by Amicon filtration using a 10,000 MW cut-off filtration membrane. The concentrated cytosol is then supplemented to final concentrations of 1 mM 2-mercaptoethanol, 0.1 mM DTT, 0.5 mM PMSF, 1/xM leupeptin, 1 ~M pepstatin, 100 kallikrein inhibitory units of aprotinin per ml, and 100/~M 1-chloro-3-tosylamido-7-amino2-heptanone (TLCK). After a subsequent 10-fold dilution in TEDMPM buffer containing 1.5 mM ATP, the cytosol is applied at a flow rate of 25 ml/hr to a column (2.5 x 25 cm) of DEAE-Sephacel (Pharmacia LKB Biotechnology Inc.) equilibrated with the same buffer. The column is washed with TEDMPM/1.5 ATP buffer until the monitored absorbance at 280 nm reaches the baseline and eluted with a linear gradient of NaC1 (280 ml, 0 --~ 180 mM NaC1) in TEDMPM/0.1 mM ATP. After completion of the gradient, the column is further eluted with 150 ml of 1 M NaC1 in TEDMPM buffer. Fractions of 3.5 ml are collected and assayed for GTPySbinding activity. The flow through and two overlapping peaks in the salt gradient (100-170 mM NaC1) show GTPyS binding (Fig. 1). When assayed for N A D P H oxidase activity in a cell-free system, as mentioned earlier, the first part of the GTPyS-binding peak (100-130 mM NaC1) correlates with oxidase stimulatory activity. The second part of the biphasic GTPySbinding peak represents the Rac-related proteins Rho (detected by ADPribosylation with botulinum toxin C3 ADP-ribosyltransferase) and Cdc42Hs (detected by Western blotting). To assure clean separation of Rac2 from these proteins, only the early fractions of the first peak should be pooled for further purification.
Sephacryl S-200 HR Gel Filtration The pooled DEAE-Sephacel fractions are concentrated to 600/~1 and applied to a Sephacryl S-200 H R column (1.5 x 120 cm, Pharmacia LKB
[4]
PURIFICATION OF R a c 2 FROM NEUTROPHILS
o000 g O
29
I ~
O
~-aoo
~ 1.o
(/) lOO
0.5
0
,
0
20
,
40
,
,
,
60
80
100
,
120
,
140
,
v
0.0
160
Fraction Number F1G. 1. DEAE-Sephacel chromatography. Human neutrophil cytosol is fractionated on a DEAE-Sephacel anion-exchange column as described. Every second fraction in the flow through and NaCI gradient is assayed for active GTP-bindingprotein in a 5-min, low magnesium [35S]GTPyS-bindingassay (A). Fractions of the first part of the biphasic GTPyS-binding peak in the salt gradient are pooled and further purified. Biotechnology Inc.) equilibrated with 100 m M NaCI in T E D M P M buffer/ 0.15 m M ATP. The column is eluted at a flow rate of 13 ml/hr, and fractions of 2 ml are collected. The column should be preevaluated with molecular weight standards under the same gel filtration conditions. On this column GTPTS binding and oxidase stimulatory activity coelute in a single peak with a relative molecular weight of 160,000.
Mono Q Anion-Exchange Chromatography The peak fractions of the gel filtration column are pooled, concentrated to 2 ml, adjusted to 10 m M NaC1 with T E D M M , and injected onto a Mono Q H R 5/5 column connected to a FPLC system (Pharmacia LKB Biotechnology, Inc.). The column is then washed with T E D M M and eluted with a shallow linear NaCI gradient (25 ml, 0 ~ 120 m M NaCI in T E D M M ) , followed by a steeper linear NaC1 gradient (10 ml, 120 ~ 250 mM NaC1 in T E D M M ) and a 1 M NaC1 wash (5 ml, in T E D M M ) at a flow rate of 0.5 ml/min. Fractions (1 ml) are collected and assayed for GTPTS binding. A single peak can be detected at 90-110 m M NaC1 in the salt gradient.
Heptylamine-Sepharose Chromatography The peak Mono Q fractions are pooled, adjusted to 0.2% cholate, and injected onto a heptylamine-Sepharose column (1.5 x 20 cm, FPLC sys-
30
EXPRESSION
__ooo
AND
[4]
PURIFICATION
2 . 0
t
,ot °
m O
E D.
1.0
(.9 200
f
. . , ...... ="*~'-"'-~i'ik=~.. ] 0 0
10
o= 0
" ~ ,
~oo
t'-
1.5
0.2/ o
'400 e,. :D 0 t~ 300
E
0.5
-""'"'"'"'"'""'"'"'"'",
20
30
, 40
, 50
, 60
0.0
Fraction Number
FIG.2. Heptylamine-Sepharosechromatography.The pooled peak fractions obtained from the Mono Q column are adjusted to 0.2% sodium cholate, applied to a heptylamine-Sepharose column, and eluted with a sodium cholate gradient as described. The GTPTS-bindingfractions in the gradient are combined, dialyzed, and subjected to phenyl-Superosechromatography.
tern), 13 equilibrated with T E D M M containing 100 m M NaCl, and 0.2% sodium cholate (v/v). The column is then washed with 50 ml of equilibration buffer at a flow rate of 0.2 ml/min. The elution is performed with two successive linear cholate gradients in T E D M M buffer (10 ml, 0.2 ~ 1.0% sodium cholate (v/v); 25 ml, 1.0 ~ 1.6% sodium cholate), followed by two cholate step gradients (15 ml 1.6% sodium cholate and 30 ml 2.0% sodium cholate in T E D M M buffer). At the same time a negative NaC1 gradient in T E D M M buffer (250 ~ 0 m M NaCI) is performed. Fractions of 2 ml are collected and assayed for [35S]GTPTS-binding. Active fractions, detected at 1.2-1.6% sodium cholate (Fig. 2), are combined and concentrated. After extensive dialysis in T E D M M buffer, the pool exhibits stimulatory activity in the cell-free N A D P H oxidase assay.
Phenyl-Superose Chromatography The dialyzed heptylamine-Sepharose pool is diluted with an equal amount of T E D M M buffer containing 1.5 M ammonium sulfate and 5% ethylene glycol. After injection onto a phenyl-Superose H R 5/5 F P L C column (Pharmacia LKB Biotechnology Inc.) equilibrated with the same 13s. Shaltiel, in "Methods in Enzymology" (W. B. Jakoby and M. Wilchek, eds.), Vol. 34, p. 126. Academic Press, New York, 1974.
[41
PURIFICATION OF R a c 2 FROM NEUTROPHILS
31
buffer, the column is washed with 8 ml of equilibration buffer until the absorbance baseline is obtained. The elution is performed with a simultaneous reverse linear gradient of ammonium sulfate in TEDMM (12 ml, 1.5 ~ 0 M ammonium sulfate) and a linear ethylene glycol gradient (12 ml, 5 ~ 50% ethylene glycol). This is immediately followed by a 3-ml wash with TEDMM buffer/50% ethylene glycol and 5 ml TEDMM/60% ethylene glycol. The elution is performed at a flow rate of 0.1 ml/min, and fractions of 0.5 ml are collected. A major peak of GTPyS binding is detectable in fractions eluted with 50-60% ethylene glycol. These fractions are combined, concentrated, and dialyzed extensively in TEDM and are stored in aliquots at - 7 0 °. The amount of active, GTP-binding Rac2 can be determined by [3SS]GTPyS binding using the rapid filtration assay. Results and Comments The purity of the Rac2 preparation achieved with the just outlined purification procedure is shown in Fig. 3. Each chromatography pool and the final concentrated Rac2 preparation are electrophoresed and silver
kDa 97-66-45-31-21-14--
1
2 3 4 5 6
FIG. 3. SDS-PAGE of Rac2 purification from human neutrophil cytosol at each step of chromatography. Samples containing pooled peak fractions were electrophoresed on a 13% acrylamide gel and silver stained. Lane I, molecular mass markers (kDa, Bio-Rad); land 2, first part of GTPyS binding in salt gradient (100-130 mM NaC1), DEAE-Sephacel; lane 3, Sephacryl S-200 HR; lane 4, Mono Q HR 5/5; lane 5, heptylamine-Sepharose; and lane 6, concentrated peak, phenyl-Superose HR 5/5.
32
EXPRESSION AND PURIFICATION
[4]
stained. Rac2 is obtained with greater than 90% purity and migrates with a relative molecular weight of 22,000. In some preparations, a second band, migrating at 21,000, is visible. Tryptic peptide sequences of this band show identity with Rac2. A possible explanation is the occurrence of proteolytic breakdown or different post-translational processing of this lower band. The actual yield of pure Rac2 from the starting material is difficult to calculate exactly because of the presence of multiple GTP-binding proteins in the cytosol and an overlap in binding activity. During later chromatography steps, an increase in the apparent recovery of Rac2 can be observed. This is because of the separation of Rac2 from its complex with the cytosolic protein [rho]GDI, which has the property of inhibiting GDP dissociation and thus guanine nucleotide exchange. Western blot analysis with an affinity-purified peptide antibody directed against [rho]GDI (generated to aa 17-28) shows complete dissociation of this complex after the fourth chromatographic step. u Most detergents, as well as several lipids, are able to disrupt the interaction of Rac2 with [rho]GDI. 3 Therefore it might be useful to include l% sodium cholate in the reaction mixture for [35S]GTPySbinding assays. After release from the [rho]GDI protein, the hydrophobicity of Rac2 results in high unspecific binding of the protein. Therefore the protein should be kept in detergents as long as possible. Judged by Western blot analysis with Rac2 antibodies, cytosol of 1 × 10 l° cell equivalents contains 0.5-0.6 mg Rac2. Each chromatographic step will result in protein loss in the range of 30-45%, with the last phenyl-Superose step being significantly higher. The Rac2 protein is very labile at higher temperatures, low magnesium concentrations, and repeated freeze-thaw cycles. This purification protocol should be easily applicable to isolate Rac from other cellular sources. It should also be noted that two slightly different procedures for Rac2 purification from differentiated HL-60 cells as well as neutrophils have been reported. 14,15 Acknowledgments We acknowledge Drs. Paul Heyworth and John Curnutte (Department of Molecular and Experimental Medicine, The Scripps Research Institute) for collaborative studies. We thank Dr. Tony Evans (Onyx Pharmaceuticals, Richmond, CA) for kindly supplying the [rho]GDI antibody. Benjamin B. Bohl provided excellent technical assistance. This work was supported by USPHS Grant HL 48008 and by a fellowship of the American Arthritis Foundation (to U. K.).
17T. Mizuno, K. Kaibuchi, S. Ando, T. Musha, K. Hiraoka, K. Takaishi, M. Asada, H. Nunoi, I. Matsuda, and Y. Takai, J. Biol. Chem. 267, 10215 (1992). 15 C. H. Kwong, H. L. Malech, D. Rotrosen, and T. L. Leto, Biochemistry 32, 5711 (1993).
PURIFICATION OF R a c - G D I
[5]
33
[5] Purification of Rac-GDP Dissociation Inhibitor Complex from Phagocyte Cytosol By ARIE ABO Introduction Several small molecular mass G T P - b i n d i n g proteins have b e e n isolated by sequential c o l u m n c h r o m a t o g r a p h y p r o c e d u r e s and have shown to resolve into molecular masses of 2 0 - 3 0 kDa. 1 T h e G T P a s e R a c has b e e n isolated f r o m platelets, differentiated H L - 6 0 cells, and neutrophils as a 2 1 - k D a m o n o m e r . 1-3 H o w e v e r , in contrast, attempts to reconstitute the N A D P H oxidase activity d e m o n s t r a t e d a r e q u i r e m e n t of a novel cytosolic c o m p o n e n t which u p o n purification resolves into a h e t e r o d i m e r i c c o m p l e x of proteins identified as p21 R a c l and R h o - G D I ( G D P dissociation inhibit o r ) Y M o r e o v e r , studies have shown that the entire p21 R a c pool is in a c o m p l e x with R h o - G D I of a molecular mass of 4 5 - 5 0 k D a which m o s t likely represents the physiological f o r m of R a c and R h o proteins. 6 O n neutrophil stimulation, p21 R a c dissociates f r o m R h o - G D I and subsequently translocates to the plasma m e m b r a n e . 6'7 Interestingly, o t h e r m e m bers of the R a c / R h o family including R h o , Rac2, and C D C 4 2 H copurified with R h o - G D I f r o m neutrophil cytosol as a heterodimer. 8'9 Pick and cow o r k e r s 1° have shown that R a c - G D I h e t e r o d i m e r s can be readily disrupted by d e t e r g e n t to f o r m a m o n o m e r i c R a c and can reassociate with R h o - G D I w h e n the detergent is r e m o v e d . A detergent that was included in the early purification p r o c e d u r e s is most likely responsible for the disruption of the 1 K. Hiraoka, K. Kaibuchi, S. Ando, T. Musha, K. Takaishi, T. Mizuno, M. Asada, L. Menard, E. Tomhav, J. Didsbury, R. Snyderman, and Y. Takai, Biochem. Biophys. Res. Commun. 182, 921 (1992). 2 p. G. Polakis, R. F. Weber, B. Nevins, J. R. Didsbury, T. Evans, and R. Snyderman, J. BioL Chem. 264, 16383 (1989). 3 U. G. Knaus, P. G. Heyworth, T. Evans, J. T. Curnutte, and G. M. Bokoch, Science 254, 1512 (1991). 4 A. Abo and E. Pick, J. BioL Chem. 266, 23577 (1991). 5 A. Abo, E. Pick, A. Hall, N. Totty, C. G. Teahan, and A. W. Segal, Nature 353, 668 (1991). 6 A. Abo, M. R. Webb, A. Grogan, and A. W. Segal, Biochem. J. 298, 585 (1994). 7 M. T. Quinn, T. Evans, L. R. Loetterle, A. J. Jesaitis, and G. M. Bokoch, J. Biol. Chem. 268, 20983 (1993). 8 C. H. Kwong, H. L. Malech, D. Rotrosen, and T. L. Leto, Biochemistry 32, 5711 (1993). 9 N. Bourmeyster, M. J. Stasia, J. Garin, J. Gagnon, P. Boquet, and P. V. Vignais, Biochemistry 31, 12863 (1992). 10E. Pick, Y. Gorzalczany, and S. Engel, Eur. J. Biochem. 217, 441 (1993).
METHODS 1N ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
34
EXPRESSION AND PURIFICATION
[5]
R a c - G D I complex which led to the isolation of the monomeric form of p21 Rac. R h o - G D I inhibits the GDP dissociation from post-translationally modified Rho and Rac proteins and forms a complex only with the GDP-bound form of these proteins. 11In contrast, the isolated Rac/GDI complex purified from macrophage cytosol could activate the oxidase in the absence of GTP, suggesting that the Rac is in the GTP-bound form when complexed in GDI. This is in agreement with reports demonstrating that R h o - G D I can inhibit the intrinsic and GAP-stimulated GTPase activity of Rac and Rho proteinsJ 2-14 It is not clear what is the physiological relevance of these multitude roles which R h o - G D I plays in regulating Rac and Rho proteins. This chapter describes a rapid method for purification of R a c - G D I complex from phagocyte cytosol.
Source of Phagocyte Cytosol We used peritoneal macrophages from guinea pigs as the initial source of cytosol; however, cytosol can also be prepared from HL-60 cells or neutrophils as described in this series.
Isolation of Peritoneal Macrophages from Guinea Pigs The isolation of macrophages from guinea pigs is a reliable procedure and can serve as a continuous source of relatively large amounts of cells (200 × 106 macrophages/animal); however, it requires animal facilities and can be often unpleasant. Hartley strain guinea pigs (250-300 g) are injected with 10 ml of sterilized mineral oil into the peritoneum cavity. The animal is held vertically by one person and is injected by another person with great caution to avoid internal organ damage. The animals are sacrificed after 6 days and the peritoneum exudate is harvested by washing the peritoneum cavity with phosphate-buffered saline (PBS). The macrophages are suspended at a concentration of 10s cells/ml in ice-cold sonication buffer consisting of 8 mM Na phosphate buffer, pH 7.0, 130 mM NaCI, 340 mM sucrose, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2/zM leupeptin. The cells are 11 y . Hori, A. Kikuchi, M. Isomura, M. Katayama, Y. Miura, H. Fujioka, K. Kaibuchi, and Y. Takai, Oncogene 6, 515 (1991). 12 M. J. Hart, Y. Maru, D. Leonard, O. W. Witte, T. Evans, and R. A. Cerione, Science 258, 812 (1992). 13T. H. Chuang, X. Xu, U. G. Knaus, M. J. Hart, and G. M. Bokoch, J. Biol. Chem. 268, 775 (1993). 14j. F. Hancock and A. Hall, E M B O J. 12, 1915 (1993).
[5]
PURIFICATION OF Rac-GDI
35
sonicated and cell debris and nuclei are removed by centrifugation at 200g at 4° for 10 rain. The particulate fraction is removed from the cytosol by centrifugation at 165,000g for 1 hr at 4° in a Type 65 fixed-angle rotor (Beckman). The resulting supernatant is what I will refer to throughout the chapter as cytosol and the sediment represents the membrane. Membranes and cytosol are stored at - 7 0 °, and the cytosol serves as a source for the purification of Rac-GDI proteins.
HL-60 Cell line HL-60 cells are a leukemic cell line derived from patients with promyelocytic leukemia and can be obtained from the American Type Culture Collection (Rockville, MD). These cells can be grown in large batches in suspension and are readily harvested by centrifugation. In addition, the cells can be differentiated into neutrophils or macrophages by simply treating them with an agent such as dimethyl sulfoxide (DMSO). HL-60 cells are grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine (Irvine Scientific, Santa Ana, CA) at 0.5-2 x 106 cells/ml in 5% CO2/air. The cells can be differentiated by various agents to differentiate the cells to neutrophil like. The addition of 1.25% DMSO causes 80-90% of the cells to differentiate into neutrophillike cells within 4-5 days. Cells are harvested by centrifugation, washed twice with PBS, and resuspended in sonication buffer at 1-2 × l0 s cells/ ml. Cytosol and membrane can be prepared in exactly the same manner as described earlier for macrophages or for neutrophils as described in this series.
Neutrophils Preparation of neutrophils and subcellular fractionation of these cells are described in detail in this series.
Detection Assays for Rac-GDI The most important preliminary step is to develop an appropriate assay which will facilitate the detection of the Rac-GDI complex during the purification procedure. A simple way to follow these two proteins is by Western blot analysis with antibodies against Rac proteins which are available commercially (Santa Cruz Biotechnology, Inc.) and from groups active in the field. The dilution, incubation time, and cross-reactivity should be determined and optimized prior to the purification. In addition, it is important to note that neutrophils have a high level of expression of a R h o -
36
EXPRESSION AND PURIFICATION
[5]
GDI homolog 15 (70% homology, known also as D4) which cross-reacts with the polyclonal antibody derived from the recombinant Rho-GDI. Thus, it is advisable to probe the fractions only with p21rac antibodies during the first few purification steps. Partially purified proteins should be also analyzed on SDS-PAGE and visualized by silver staining. An alternative assay is the reconstitution of cell-free NADPH oxidase which is described in detail in this series. To follow this activity, each fraction should be tested for the ability to activate the NADPH oxidase when supplemented with the other oxidase components: p47-phox, p67phox, and the membrane-bound cytochrome b. Purification of Rac-GDI Complex All chromatographic procedures can be performed in a system containing two pumps, controller, injector, detector used to follow the absorbance at 280 nm, and fraction collector. We have used the HPLC system (Waters Milford, MA) and the FPLC system (Pharmacia LKB Biotechnology Inc.). In addition, all purification steps are conducted at 4°; the columns are immersed in ice during the separations. The purification scheme is outlined in Fig. 1.
a. Ammonium Sulfate Precipitation Ammonium sulfate precipitation is a convenient first step; it allows the processing of a large volume of extract and can be directly applied to phenylSepharose with no buffer exchange. The ammonium sulfate conditions are first optimized for the required protein by the precipitation of small amounts of cytosol under different concentrations of ammonium sulfate. The optimization of Rac-GDI precipitation is described in detail elsewhere. 16 We found that a 37% saturation of ammonium sulfate is sufficient to separate about 30% of the protein and to recover Rac-GDI in the supernatant. Ammonium sulfate (37% saturation) is added to 10-20 ml of phagocyte cytosol (1-2 × 108 cell equivalents/ml) in small amounts over a period of 30 rain. The mixture is stirred on ice for 1 hr and is centrifuged for 15 rain at 200,000g in a TLX Beckman ultracentrifuge. The pellet should contain the p47-phox and p67-phox which can be used for the oxidase assay as described in this series. The supernatant containing the Rac-GDI proteins is concentrated by reprecipitating with 85% saturation of ammonium sulfate. The mixture is stirred for an additional hour on ice and is sedimented by is j. M. Lelias, C. N. Adra, G. M. Wulf, J. C. Guillemot, M. Khagad, D. Caput, and B. Lira, Proc. Natl. Acad. Sci. U.S.A. 911, 1479 (1993). 16 E. Pick, T. Kroizman, and A. Abo, J. lmmunoL 143, 4180 (1989).
[5]
PURIFICATION OF Rac-GDI
37
Phagocyte Cytosol (Macrophages, HL-60 Cells, Neutrophils) Ammonium Sulfate Precipitation (37% saturation)
Centrifugation
/ Pellet
",,, Supernatant
Phenyl - Superose CM -Sepharose
DEAE- Sepharose
Gel Filtration Superose 12 FIG. 1. Flow scheme for the isolationof Rac-GDI heterodimersfrom phagocytes.
centrifugation as mentioned earlier. The pellet is redissolved in buffer A containing 100 mM sodium phosphate, pH 7.4, 1 mM MgC12, 1 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM PMSF, and 2/xM leupeptin. The second concentration step is not essential; the diluted sample can be applied to the column by a 50-ml superloop (Pharmacia LKB Biotechnology Inc.).
b. Hydrophobic Interaction on Phenyl-Superose The concentrated supernatant is adjusted to 1 M ammonium sulfate and the insoluble material is removed by centrifugation. Three to 5 ml (2040 mg protein) of the mixture is applied to a phenyl-Superose HR 5/5 column (Pharmacia) equilibrated with 20 ml of buffer A containing 1.4 M ammonium sulfate. Large amounts of protein (300-500 mg proteins) can also be applied and eluted faster from the Hiload phenyl-Sepharose 16/10 column (Pharmacia LKB Biotechnology Inc.) with no loss in resolution. Proteins are eluted from the column by reducing the amount of ammonium
38
EXPRESSION AND PURIFICATION
[5]
sulfate from 1.4 M to 0 in buffer A over a period of 150 min. Fractions (1 ml) are collected and analyzed for oxidase activity or by immunodetection for the presence of R a c - G D I by Western blotting. Fractions containing R a c - G D I activity are pooled, and the buffer is exchanged by dialysis or on a PD 10 desalting column (Pharmacia LKB Biotechnology Inc.) to buffer B: 20 mM Tris-HC1, pH 7.0, 1 mM EGTA, 2/zM leupeptin, and 0.5 mM PMSF. c. Absorption with CM-Sepharose
This step is used to remove contaminating proteins which would otherwise be copurified with Rac-GDI. Two milliliters of CM-Sepharose beads (Pharmacia LKB Biotechnology Inc.) is equilibrated with 20 ml of buffer B and are then mixed with a 4- to 8-ml pool of fractions containing the RacGDI protein (in buffer B) obtained from phenyl-Superose. The mixture is rotated end over end for i hr at 4°. The beads are sedimented by centrifugation at 300g for 5 rain and the supernatant is collected. The beads are then washed twice with 2 ml of buffer B and the supernatants are collected and pooled together. The bound proteins are eluted with 0.5 M NaC1 with buffer B. Fractions containing Rac-GDI proteins are pooled and directly applied to the next step in the purification. d. DEAE-Sepharose
The advantage in using the CM-Sepharose step is that no buffer exchange is required because all the Rac-GDI complex is found in the unbound material and can be applied directly to an anion-exchange column. The best separation is obtained on a weak anion-exchange column DEAESepharose, other anion exchange columns such as the Mono Q are perfectly suitable, however, the resolution will differ from one colunn to another. Five to 10 ml of DEAE-Sepharose is packed in the C 10/10 column (Pharmacia LKB Biotechnology Inc.) and is equilibrated with 50 ml of buffer B. The unbound material from the CM-Sepharose step (30-50 mg proteins) is applied to the column at the flow rate of 0.2 ml/min. The bound proteins are eluted from the column with 50-ml linear gradient (0-0.5 M NaCI, and 0.5-ml fractions are collected and analyzed. e. Gel Filtration on Superose 12
This is an ideal final purification step since it can be used to remove minor contaminants and can also provide some information concerning the size of native proteins. Superose 12 and 6 (Pharmacia LKB, Biotechnology Inc.) are suitable, however, but will not separate proteins very well in the
PURIFICATION OF Rac-GDI
[51
39
range of 30-70 kDa. An alternative gel filtration column, the Superdex 75 H R 10/30 (Pharmacia Biotechnology Inc.), has been used to isolate the R a c - G D I complex. 1° Pooled fractions containing the R a c - G D I complex are concentrated on a Centricon 10 (Amicon), and 250/xl is injected into a Superose 12 column equilibrated with buffer A. Fractions (300-500/xl) are collected and analyzed. For molecular size estimation, molecular standards ranging from 12 to 669 kDa (Pharmacia LKB Biotechnology Inc.) are applied under the same conditions. Approximately 30-70/xg of pure R a c - G D I proteins is recovered from 200 to 400 mg of phagocyte cytosol. During all of the chromatographic steps it is suggested that the purity of the fractions be analyzed on SDSP A G E and visualized by Coomassie and silver staining, as shown in
"13
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,
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~
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~ - p21 rac 20
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- -
~..,m.
FIG. 2. SDS-PAGE of Rac-GDI fractions from successive purification steps. Peak fractions containing Rac-GDI proteins are from a typical separation sequence from guinea pig macrophage cytosol. Proteins (1-10 p,g) were applied per lane and visualized by silver staining.
E3 73
"-r
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~- R h o - G D I
-4 p21 rac
Fl6.3. S D S - P A G E analysis of purified R a c - G D I heterodimers isolated from differentiated HL-60 cells and macrophages. One microgram of purified R a c - G D I proteins was applied per lane and visualized by silver staining. The analyzed proteins were obtained from the last purification step on Superose 12.
1.0
1000-
66KDa
45KDa
25KDa
Y
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T
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~
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i
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45 Fraction n u m b e r
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|
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Fraction n u m b e r
FIG. 4. Elution profile of R a c - G D I heterodimers on a gel filtration Superose 12 column. (A) Thirty to 50/zg of a partially purified macrophage R a c - G D I complex obtained from the peak fraction of D E A F was separated on Superose 12. Collected fractions were tested for the reconstituted activity of NADPH oxidase (as described in detail in this series), analyzed on SDS-PAGE, and visualized by silver staining. (B) Partially purified neutrophil R a c - G D I heterodimers obtained from peak fractions on a Mono Q column were further separated on Superose 12. Fractions were analyzed for R a c - G D I by Western blotting with specific antibodies against R a c - G D I and R h o - G D I .
[61
Rho-GDP
DISSOCIATION INHIBITOR
41
Figs. 2 and 3. The elution profile from the Superose 12 gel filtration column is shown in Fig. 4.
Acknowledgments Thanks to Edgar Pick for allowing me to present data and methodology originated in his laboratory. I thank David Stokoe and Amy Brodo for comments on the manuscript.
[6] P u r i f i c a t i o n a n d P r o p e r t i e s of R e c o m b i n a n t R h o - G D P Dissociation Inhibitor B y KAZUMA TANAKA, TAKUYA SASAKI, a n d YOSHIMI TAKAI
Introduction R h o - G D I ( G D P d i s s o c i a t i o n i n h i b i t o r ) has b e e n o r i g i n a l l y i s o l a t e d as a c y t o s o l i c p r o t e i n t h a t i n t e r a c t s with G D P - R h o A a n d G D P - R h o B a n d t h e r e b y inhibits t h e d i s s o c i a t i o n o f G D P f r o m a n d the s u b s e q u e n t b i n d i n g to G T P to R h o A a n d R h o B . 1'2 Its c D N A has b e e n c l o n e d f r o m a b o v i n e b r a i n c D N A library. 3 B o v i n e R h o - G D I is a p r o t e i n h a v i n g Mr o f 23,421 with 204 a m i n o acids. B y N o r t h e r n b l o t a n d i m m u n o b l o t a n a l y s e s , R h o G D I is u b i q u i t o u s l y e x p r e s s e d . 3,4 A c o u n t e r p a r t a n d h o m o l o g s of b o v i n e R h o - G D I h a v e b e e n c l o n e d f r o m h u m a n s 5 a n d h u m a n ( L y / D 4 G D I ) , 6,7 a n d y e a s t ( R D I 1 ) , 8 r e s p e c t i v e l y . R h o - G D I is active o n all t h e R h o f a m i l y small G T P a s e s e x a m i n e d so far, i n c l u d i n g R h o A , R h o B , R a c l , Rac2, a n d Cdc42.1,2'9-13 R h o - G D I a n t a g o n i z e s t h e a c t i o n s of S m g - G D S , R h o - G D S , 1 N. Ohga, A. Kikuchi, T. Ueda, J. Yamamoto, and Y. Takai, Biochem. Biophys. Res. Commun. 163, 1523 (1989). 2 T. Ueda, A. Kikuchi, N. Ohga, J. Yamamoto, and Y. Takai, J. Biol. Chem. 265, 9373 (1990). 3 y. Fukumoto, K. Kaibuchi, Y. Hori, H. Fujioka, S. Araki, T. Ueda, A. Kikuchi, and Y. Takai, Oncogene 5, 1321 (1990). 4 K. Shimizu, K. Kaibuchi, H. Nonaka, T. Yamamoto, and Y. Takai, Biochem. Biophys. Res. Commun. 175, 199 (1991). A, Maeda, K. Kaibuchi, and Y. Takai, unpublished results. 6 J.-M. Lelias, C. N. Adra, G. M. Wulf, J.-C. Guillemot, M. Khagad, D. Caput, and B. Lira, Proc. Natl. Acad. Sci. U.S.A. 90, 1479 (1993). 7 p. Scherle, T. Behrens, and L. M. Staudt, Proc. NatL Acad. Sci. U.S.A. 90, 7568 (1993). 8 T. Masuda, K. Tanaka, H. Nonaka, W. Yamochi, A. Maeda, and Y. Takai, J. Biol. Chem. 269, 19713 (1994). 9 T. Mizuno, K. Kaibuchi, S. Ando, T. Musha, K. Hiraoka, K. Takaishi, M. Asada, H. Nunoi, I. Matsuda, and Y. Takai, J. BioL Chem. 267, 10215 (1992).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
42
EXPRESSION AND PURIFICATION
[6]
a n d D b l to s t i m u l a t e the G D P / G T P e x c h a n g e r e a c t i o n o f R h o A . 14-16 I n a d d i t i o n to i n h i b i t i n g t h e G D P / G T P e x c h a n g e r e a c t i o n , R h o - G D I i n d u c e s the dissociation of these Rho family small GTPases from membranes and inhibits t h e i r a s s o c i a t i o n with m e m b r a n e s , 13,17'18 suggesting t h a t R h o - G D I r e g u l a t e s t h e cycling o f t h e R h o f a m i l y small G T P a s e s b e t w e e n t h e m e m b r a n e a n d t h e cytosol. T h e R h o s u b f a m i l y small G T P a s e s consist o f R h o A , R h o B , a n d R h o C a n d t h e y a r e i n v o l v e d in the r e g u l a t i o n o f t h e a c t o m y o s i n d e p e n d e n t cell functions. T h e r e f o r e , R h o - G D I m a y also s e r v e as a k e y r e g u l a t o r o f t h e a c t o m y o s i n - d e p e n d e n t cell functions. A c t u a l l y , m i c r o i n j e c t i o n o f R h o - G D I c h a n g e s t h e m o r p h o l o g y in Swiss 3T3 cells 19 a n d inhibits c y t o p l a s m i c division in X e n o p u s e m b r y o , 2° cell m o t i l i t y in Swiss 3T3 cells, el cell s c a t t e r i n g in m o u s e k e r a t i n o c y t e 308R cells, 22 a n d m e m b r a n e ruffling in h u m a n K B cells. 23 The Rho family small GTPases have a unique C-terminal structure o f C y s - A - A - L (A, a l i p h a t i c a m i n o acid; L, l e u c i n e ) t h a t u n d e r g o e s p o s t translational modifications including the geranylgeranylation or farnesylation of the cysteine residue followed by proteolytic removal of the A-A-L portion and the subsequent carboxyl methylation of the exposed cysteine
10S. Ando, K. Kaibuchi, T. Sasaki, K. Hiraoka, T. Nishiyama, T. Mizuno, M. Asada, H. Nunoi, I. Matsuda, Y. Matsuura, P. Polakis, F. McCormick, and Y. Takai, J. Biol. Chem. 267, 25709 (1992). la K. Hiraoka, K. Kaibuchi, S. Ando, T. Musha, K. Takaishi, T. Mizuno, M. Asada, L. Menard, E. Tomhave, J. Didsbury, R. Snyderman, and Y. Takai, Biochem. Biophys. Res. Commun. 182, 921 (1992). 12T. Sasaki, M. Kato, T. Nishiyama, and Y. Takai, Biochem, Biophys. Res. Commun. 194, 1188 (1993). 13D. Leonard, M. J, Hart, J. V. Platko, E. Alessandra, W. Henzel, T. Evans, and R. A. Cerione, J. Biol. Chem. 267, 22860 (1992). 14m. Kikuchi, S. Kuroda, T. Sasaki, K. Kotani, K. Hirata, M. Katayama, and Y. Takai, J. Biol. Chem. 267, 14611 (1992). 15S. Kuroda, A. Kikuchi, K. Hirata, T. Masuda, K. Kishi, T. Sasaki, and Y. Takai, Biochem. Biophys. Res. Commun. 185, 473 (1992). 16H. Yaku, T. Sasaki, and Y. Takai, Biochem. Biophys. Res. Commun. 198, 811 (1994). 17M. Isomura, A. Kikuchi, N. Ohga, and Y. Takai, Oncogene 6, 119 (1991). 18T. Sasaki, M. Kato, and Y. Takai, Z Biol. Chem. 268, 23959 (1993). a9y. Miura, A. Kikuchi, T. Musha, S. Kuroda, H. Yaku, T. Sasaki, and Y. Takai, Z Biol. Chem. 268, 510 (1993). z0 K. Kishi, T. Sasaki, S. Kuroda, T. Itoh, and Y. Takai, J. Cell Biol. 120, 1187 (1993). 21 K. Takaishi, A. Kikuchi, S. Kuroda, K. Kotani, T. Sasaki, and Y. Takai, Mol. Cell. Biol. 13, 72 (1993). 22 K. Takaishi, T. Sasaki, M. Kato, W. Yamochi, S. Kuroda, T. Nakamura, M. Takeichi, and Y. Takai, Oncogene 9, 273 (1993). 23T. Nishiyama, T. Sasaki, K. Takaishi, M. Kato, H. Yaku, K. Araki, Y. Matsuura, and Y. Takai, Mol. Cell. Biol. 14, 2447 (1994).
[6]
R h o - G D P DISSOCIATIONINHIBITOR
43
residue. 24'25 These C-terminal lipid modifications of the Rho family small GTPases are essential for their interaction with R h o - G D I . 26 This chapter describes the procedures for the purification of recombinant R h o - G D I from overexpressing Escherichia coli by use of the glutathione S-transferase (GST) fusion system and the properties of R h o - G D I .
Materials Sodium cholate and dithiothreitol (DTT) are from Nacalai Tesque (Kyoto, Japan). E D T A and 3-[(3-cholamidopropyl)-dimethylammonio]-lpropanesulfonic acid (CHAPS) are from Dojindo Laboratories (Kumamoto, Japan). Human thrombin (T3010) and reduced glutathione are from Sigma (St. Louis, MO). (p-Amidinophenyl)methanesulfonyl fluoride (APMSF), isopropyl-/~-D-thiogalactopyranoside (IPTG), and L-a-dimyristoylphosphatidylcholine are from Wako Pure Chemicals (Osaka, Japan). u-a-Dimyristoylphosphatidylcholine is stored as a stock solution of 100 mM in chloroform. To make liposome, aliquots are dried to a thin film under a stream of nitrogen at a room temperature, hydrated with 20 mM TrisHC1, pH 7.5, to a final concentration of 10 mM, and sonicated at a setting of 60 by Ultrasonic Processor (Taitec, Tokyo, Japan) on ice for 30 sec twice at 30-sec intervals. This solution is used within 3 hr. Guanosine 5'-(3-0thio)triphosphate (GTPyS) is from Boehringer-Mannheim (Indianapolis, IN). A GST expression vector, pGEX-2T, and glutathione-Sepharose 4B are from Pharmacia P-L Biochemicals Inc. (Milwaukee, WI). [3H]GDP (518 GBq/mmol) and [35S]GTPyS (40.7 TBq/mmol) are from Amersham Corp. (Buckinghamshire, England) and Du Pont-New England Nuclear (Boston, MA), respectively. BA-85 nitrocellulose filters (pore size, 0.45 /zm) are from Schleicher & Schuell (Dassel, Germany). All other chemicals are reagent grade. Native bovine R h o - G D I is purified from bovine brain as described previously. 2 Lipid-modified R h o A is purified from the membrane fraction of Spodoptera frugiperda cells (Sf9 cells) which are infected with baculovirus carrying the cDNA of RhoA, 27 whereas the lipid-unmodified form is purified from overexpressing E. coli. 26 Unless specified, lipid-modified R h o A 24M. Katayama, M. Kawata, Y. Yoshida, H. Horiuchi, T. Yamamoto, Y. Matsuura, and Y. Takai, J. Biol. Chem. 266, 12639 (1991). 25p. Adamson, C. J. Marshall,A. Hall, and P. A. Tilbrook,J. BioL Chem. 267, 20033 (1992). 26y. Hori, A. Kikuchi,M. Isomura, M. Katayama,Y. Miura, H. Fujioka, K. Kaibuchi, and Y. Takai, Oncogene 6, 515 (1991). z7T. Mizuno,K. Kaibuchi,T. Yamamoto,M. Kawamura,T. Sakoda, H. Fujioka,Y. Matsuura, and Y. Takai, Proc. Natl. Acad. Sci. U.S.A. 88, 6442 (1991).
44
EXPRESSION AND PURIFICATION
[6]
is used as a substrate. RhoA is dissolved in a buffer containing 20 mM TrisHCI at pH 7.5, 5 mM MgC12, 1 mM EDTA, 1 mM DTT, and 0.6% CHAPS. A plasmid for expression of GST-Rho GDI is constructed as follows. The 0.6-kbp fragment, containing the complete R h o - G D I coding region with the BamHI sites upstream of the initiator methionine codon and downstream of the termination codon, is synthesized by polymerase chain reaction. This fragment is digested with BamHI and inserted into the BamHI-cut pGEX-2T to construct pGEX-2T-Rho-GDI. The E. coli strain JM109 is transformed with this plasmid, and the resulting brain is used as a source for G S T - R h o - G D I .
Methods
Purification of G S T - R h o GDP Dissociation Inhibitor Buffers used in the purification of G S T - R h o - G D I are as follows: Buffer A: 25 mM Tris-HC1 at pH 7.5, 0.5 mM EDTA, 1 mM DTT, 10% sucrose, and 10/zM APMSF Buffer B: 25 mM Tris-HC1 at pH 7.5, 0.5 mM EDTA, and 1 mM DTT Buffer C: 50 mM Tris-HC1 at pH 8.0 and 5 mM reduced glutathione Recombinant R h o - G D I is purified by the following steps: (1) cultivation of E. coli and induction of GST-GDI; (2) preparation of crude supernatant; (3) affinity purification of G S T - R h o - G D I ; (4) cleavage of G S T - R h o - G D I with thrombin; and (5) purification of the thrombin-cleaved Rho-GDI.
Cultivation of E. coli and Induction of G S T - R h o - G D I JM109 transformed with pGEX-2T-Rho-GDI is cultured at 30° in 1 liter of LB medium containing 50/zg of ampicillin per ml to an OD595 of 0.2. After the addition of IPTG at a final concentration of 0.5 mM, cells are further cultured for 5 hr. All procedures after this step should be performed at 0-4 °. Cells are harvested, suspended in 20 ml of phosphatebuffered saline (BPS), and washed with 20 ml of PBS. The cell pellet can be frozen at - 8 0 ° for at least 2 weeks.
Preparation of Crude Supernatant The cell pellet is quickly thawed at 37° and is suspended in 20 ml of ice-cold buffer A and the cell suspension is sonicated at a setting of 60 by Ultrasonic Processor (Taitec, Tokyo, Japan) on ice for 30 sec four times at 30-sec intervals. The homogenate is centrifuged at 100,000g for 1 hr. The supernatant is used for the affinity purification.
[6]
Rho-GDP DISSOCIATIONINHIBITOR
45
Affinity Purification of GST-Rho-GDI Glutathione-Sepharose 4B beads are packed onto 10-ml disposable syringe (bed volume, 2 ml). The beads are washed with 30 ml of buffer B and equilibrated with 20 ml of buffer B. Twenty milliliters of crude supernatant prepared as described earlier is applied to the column and the pass fraction is reapplied to the column. After the column is washed with 20 ml of buffer B, G S T - R h o - G D I is eluted with 5 ml of buffer C.
Cleavage of GST-Rho-GDI with Thrombin The eluate containing G S T - R h o - G D I (5 ml) is dialyzed against 500 ml of PBS three times. To 5 ml of the dialyzed sample, 1.4 ml of ice-cold H20 and 32.5 /zl of thrombin (5 units//A) are added in this order. The reaction mixture is incubated at 25 ° for 60 rain and is then kept on ice.
Purification of Cleaved Rho-GD1 Another glutathione-Sepharose 4B column (bed volume, 2 ml) is prepared and equilibrated with 40 ml of buffer B, and the sample containing cleaved G S T - R h o - G D I is applied to the column. The pass fraction in which GST is removed is reapplied to the column and the pass fraction is dialyzed against 500 ml of buffer B three times. The dialyzed sample can be kept as purified recombinant R h o - G D I at - 8 0 ° for at least 6 months without loss of activity. About 500 tzg of R h o - G D I can be purified to near homogeneity by use of this procedure.
Properties of Rho-GDI
1. Assay for Rho-GDI Activity to Regulate GDP/GTP Exchange Reaction of RhoA The activity of R h o - G D I to regulate the GDP/GTP exchange reaction of RhoA is assayed by measuring the dissociation of [3H]GDP from RhoA or the binding of [35S]GTPTS to GDP-RhoA.
Dissociation Assay RhoA (2 pmol) is incubated in a reaction mixture (24 txl) containing 20 mM Tris-HCl at pH 7.5, 5 mM MgC12, 10 mM EDTA, 1 mM DT]?, 1 mM L-ot-dimyristoylphosphatidylcholine, 0.25% CHAPS, and 1 /zM [3H]GDP (0.9-1.2 × 104 cpm/pmol) for 20 min at 30°. After the first incubation, 1 /~1 of 375 mM MgCI2 is added to give a final concentration
46
EXPRESSION AND PURIFICATION
[6]
of 20 mM to prevent the dissociation of [3H]GDP from RhoA, and the mixture is immediately cooled on ice. The dissociation of [3H]GDP from RhoA is started by adding a 75-/zl mixture containing an appropriate amount of Rho-GDI, 200/~M GTP, 20 mM Tris-HC1 at pH 7.5, 10 mM EDTA, and 1 mM DTT, and the mixture (100/~1) is incubated for various periods of time at 30°. The reaction is stopped by adding 2 ml of an icecold solution containing 20 mM Tris-HC1 at pH 7.5, 25 mM MgC12, and 100 mM NaC1 to the reaction mixture, followed by rapid filtration on BA-85 nitrocellulose filters and washing with 2 ml of the same solution four times. The radioactivity trapped on the filters is measured by liquid scintillation counting.
Binding Assay G D P - R h o A (2 pmol) is incubated with 1/~M [35S]GTPyS (6-8 × 103 cpm/pmol) in a 100-/zl reaction mixture containing an appropriate amount of Rho-GDI, 20 mM Tris-HC1 at pH 7.5, 5 mM MgC12, 10 mM EDTA, 1 mM DTT, 0.75 mM L-o~-dimyristoylphosphatidylcholine, and 0.06% CHAPS. The G D P - R h o A used is the RhoA purified from Sf9 cells because it is purified as the GDP-bound form. The reaction is started by adding [35S]GTPyS, and incubation is performed for various periods of time at 30°. The reaction is stopped and the radioactivity trapped on the filters is counted as described earlier. In these two types of experiments, R h o - G D I is active on lipid-modified RhoA but is inactive on the lipid-unmodified form. Purified recombinant R h o - G D I shows similar physical and kinetic properties with those of native R h o - G D I and recombinant G S T - R h o - G D I , which has not been cleaved with thrombin.
2. Assay for Rho-GDI Activity to Form Complex with GDP-RhoA [3H]GDP-RhoA (30 pmol) is prepared as described earlier and is incubated with 75 pmol of R h o - G D I for 10 min at 4° in a reaction mixture (350/A) containing 25 mM Tris-HC1 at pH 7.5, 10 mM MgCl2, 2.5 mM EDTA, and 1 mM DTT. This reaction mixture is subjected to 4.8 ml of a continuous sucrose density gradient (0.15-0.58 M sucrose in 20 mM TrisHC1 at pH 7.5 containing 1 mM DTT, 5 mM MgCI2, and 0.1% sodium cholate). The ultracentrifugation is performed at 220,000g for 13.8 hr at 4°. R h o - G D I or RhoA alone is separately subjected to the same centrifugation. After the centrifugation, fractions of 170/~1 each are collected from the bottom of the tube as described previously.2 The amount of R h o - G D I is
[6]
Rho-GDP DISSOCIATIONINHIBITOR
47
determined by SDS-PAGE analysis followed by protein staining with silver or immunoblot analysis. The amount of RhoA is determined in a similar method or by measuring the radioactivity of an aliquot of each fraction after the filtration through nitrocellulose filters. In these experiments, R h o - G D I forms a stable complex with lipidmodified G D P - R h o A at a molar ratio of 1:1. R h o - G D I also forms a stable complex with lipid-modified GTPTS-RhoA, but the efficiency of this complex formation with lipid-modified GTPTS-RhoA is about 10% of that with lipid-modified GDP-RhoA. R h o - G D I does not form a stable complex with lipid-unmodified G D P - R h o A or with lipid-unmodified GTPTS-RhoA. 18
3. Assay for R h o - G D I Activity to Regulate Translocation of RhoA Assay for R h o - G D 1 Activity to Inhibit Binding of RhoA to Membranes [3H]GDP-RhoA (30 pmol) is prepared as described earlier and is incubated for 5 min at 30° with various membranes (30/zg of protein), such as the synaptic plasma membrane and the synaptic vesicle, in the presence or absence of R h o - G D I (500 pmol) in a reaction mixture (400/zl) containing 25 mM Tris-HC1 at pH 7.5,200/zM GDP, 5 mM MgCI2, and 1 mM DTT. The synaptic plasma membrane and the synaptic vesicle are prepared as described. 28 After the incubation, the 400-/zl mixture is centrifuged on a discontinuous sucrose density gradient at 64,700g for 2 hr at 4°. The discontinuous sucrose density gradient consists of 1.4 ml of the same buffer containing 2 M sucrose, 2.8 ml of the buffer containing 0.5 M sucrose, 400 /zl of the sample containing 0.1 M sucrose, and 400/zl of the same buffer in a 5-ml tube from the bottom in this order. After centrifugation, fractions of 200/zl each are collected. The radioactivity of a 100-/zl aliquot of each fraction is counted after filtration through the nitrocellulose filters. In these experiments, R h o - G D I inhibits the binding of lipid-modified G D P - R h o A to the membrane. R h o - G D I also inhibits the binding of lipidmodified GTPyS-RhoA to the membrane, but the efficiency of this inhibition of the binding of lipid-modified GTPyS-RhoA is about 10% of that of lipid-modified GDP-RhoA. R h o - G D I does not inhibit the binding of lipid-unmodified G D P - R h o A or lipid-unmodified GTPTS-RhoA to the membrane.IS 28A. Mizoguchi,T. Ueda, K. Ikeda, H. Shiku,H. Mizoguchi,and Y. Takai, Mol. Brain Res. 5, 31 (1989).
48
EXPRESSION AND PURIFICATION
[6]
Assay for Rho-GDI Activity to Stimulate Dissociation of Exogenous RhoA from Membranes [3H]GDP-RhoA (30 pmol) is prepared as described earlier; is incubated for 5 min at 30° with various membranes (30/zg of protein), such as the synaptic plasma membrane and the synaptic vesicle; and is subjected to the discontinuous sucrose density gradient ultracentrifugation described earlier. The membrane fraction is collected, diluted with 3 vol of 25 mM Tris-HC1 at pH 7.5 containing 200/zM GDP, 5 mM MgC12, and 1 mM DTT, and centrifuged at 200,000g for 60 min at 4°. The membrane recovered in the precipitate is suspended in 400 /zl of the same buffer containing 0.2 M sucrose. The dissociation of the [3H]GDP-RhoA from the membrane is assayed in the presence or absence of R h o - G D I (500 pmol) by the discontinuous sucrose density gradient ultracentrifugation described earlier. After the centrifugation, fractions of 200/zl each are collected. The radioactivity of a 100-/zl aliquot of each fraction is counted after filtration through the nitrocellulose filters, In these experiments, R h o - G D I stimulates the dissociation of lipidmodified G D P - R h o A but not that of lipid-unmodified G D P - R h o A from the membrane. The effect of R h o - G D I on the dissociation of lipid-modified or lipid-unmodified GTPyS-RhoA from the membrane has not yet been examined. Although we use exogenously added RhoA in these experiments, it has been demonstrated that R h o - G D I induces the dissociation of endogenous Cdc42 from the membrane. 13
4. Assay for Rho-GDI Activity to Inhibit Actomyosin-Dependent Cell Functions of RhoA When microinjected into cells, R h o - G D I inhibits various actomyosindependent cell functions through the inhibition of RhoA activities as described earlier. 19-23 The assay protocol for inhibition of cell motility by Rho-GD1 is described (see [39] this volume).
Comments G S T - R h o - G D I is highly expressed in E. coli so that a large amount (about 250/zg) of R h o - G D I can be purified to near homogeneity from 500 ml of the E. coli culture by use of the procedures described earlier. Since G S T - R h o - G D I is equally active as the thrombin-cleaved Rho-GDI, G S T - R h o - G D I can also be used in various assays as a substitute for the thrombin-cleaved Rho-GDI.
[7]
CARBOXYL
METHYLTRANSFERASE
IN N E U T R O P H I L S
49
As to the R h o - G D I activity to regulate the GDP/GTP exchange reaction, the free Mg 2+ concentration is a critical factor since it greatly affects the rate of GDP dissociation from small GTPases. At the free Mg 2+ concentration of 0.5 ~M used in the procedures described here, the rate of GDP dissociation from RhoA is so fast that the R h o - G D I activity can be easily detected. This document is applicable to R h o - G D I assays employing other members of the Rho family small GTPases as substrates. When crude samples are used for detecting the R h o - G D I activity, it is better to use the [3H]GDP dissociation assay than to use the [35S]GTPT-S binding assay because the crude samples often contain other GTP-binding proteins. These GTP-binding proteins bind [35S]GTPTS and interfere with the assay.
[7] P r e n y l c y s t e i n e - D i r e c t e d Carboxyl Methyltransferase Activity in Human Neutrophil Membranes By M A R K R . PHILIPS a n d MICHAEL H . PILLINGER Introduction Ras-related GTP binding proteins of the Ras and Rho subfamilies are among a class of proteins that end with the sequence CXXX. This sequence directs a series of post-translational modifications that include prenylation with a farnesyl (C15) or geranylgeranyl (C20) group, XXX proteolysis, and methyl esterification of the ee-carboxyl group of the resulting C-terminal prenylcysteine. 1 The Rab family of Ras-related proteins is also geranylgeranylated on the cysteine residues of their C-terminal CXC or CC sequences, but only proteins translated with the CXC sequence are carboxyl methylated. 1 These modifications render Ras-related proteins hydrophobic and target them to membranes.: Prenylation of CXXX-containing proteins is catalyzed by a family of heterodimeric cytosolic prenyltransferases designated farnesyltransferase 3 and geranylgeranyltransferase I, 4,5 whereas Rab proteins are prenylated by heterotrimeric geranylgeranyltransferase II which is also cytosolic. 6 Prenylcysteine-XXX protease and prenylcysteine1 S. Clarke, Annu. Rev. Biochem. 61, 355 (1992). 2 j. F. Hancock, K. Cadwallader, and C. J. Marshall, E M B O J. 10, 641 (1991). 3 y. Reiss, J. L. Goldstein, M. C. Seabra, P. J. Casey, and M. S. Brown, Cell 62, 81 (1990). 4 M. C. Seabra, Y. Reiss, P. J. Casey, M. S. Brown, and J. L. Goldstein, Cell 65, 429 (1991). 5 S. L. Moores, M. D. Schaber, S. D. Mosser, E. Rands, M. B. O'Hara, V. M. Garsky, M. S. Marshall, D. L. Pompliano, and J. B. Gibbs, J. Biol. Chem. 266, 14603 (1991). t, M. C. Seabra, J. L. Goldstein, T. C. Stidhof, and M. S. Brown, J. Biol. Chem. 267,14497 (1992).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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EXPRESSION AND PURIFICATION
[7]
directed carboxyl methyltransferase (pcCMT) activities are associated with membrane fractions I and have not been fully characterized. Because of the stable thioether bond linking the prenyl group to the cysteine and the proteolytic loss of the sequence that directs C X X X prenyltransferase activity, protein prenylation is irreversible. In contrast, protein carboxyl methylation is readily reversible under physiologic conditions. 7'8 We have shown that carboxyl methylation of Ras-related proteins is associated with signal transduction in human neutrophils and that specific inhibitors of pcCMT block discrete neutrophil functions. 9 We have also reported that neutrophils are among the richest sources of pcCMT and that the neutrophil enzyme is plasma membrane-associated and phospholipiddependent. 1° This chapter reviews these observations and details the methods we employed to study human neutrophil pcCMT.
Methods Neutrophil Isolation and Subcellular Fractionation Neutrophils are isolated from heparinized venous blood of normal volunteers using a modification of the method of Boyum. n Fifty to 150 ml of blood from a single donor is layered on top of equal volumes of leukocyte separation medium [10% (w/v) Ficoll-400 (Pharmacia) : Hypaque (Winthrop) : distilled H20, 105 : 35 : 10 (v/v) or a commercially available density medium, e.g., Accu-Prep (Accurate Chemical & Scientific Corp.)] in 50-ml tubes and spun at 1500 rpm for 40 min. The platelet-rich plasma, peripheral blood mononuclear cell layers, and clear Ficoll/Hypaque are removed by aspiration and discarded. The neutrophil layers formed on the surfaces of the packed erythrocytes are poured into a 250-ml graduated cylinder along with the erythrocytes and are diluted 1 : 1 with room temperature cell buffer (150 mM NaC1, 5 mM KOH, 1.3 mM CaCI2, 1.2 mM MgC12, 10 mM HEPES, pH 7.4). One-fifth volume of 6% (w/v) dextran T500 (Pharmacia) in 150 mM NaCI is added, and the cylinder is inverted several times to mix well and is then left undisturbed at room temperature. When the erythrocytes have sedimented half of the distance of the fluid column ( - 4 0 min), the clear, neutrophil-rich upper layer is aspirated into 50-ml 7 D. Chelsky, B. Ruskin, and D. E. Koshland, Biochemistry 24, 651 (1985). 8 D. P6rez-Sala, E. W. Tan, F. J, Cafiada, and R. R. Rando, Proc. NatL Acad. Sci. U.S.A. 88, 3043 (1991). 9 M. R. Philips, M. H. Pillinger, R. Staud, C. Volker, M. G. Rosenfeld, G. Weissmann, and J. B. Stock, Science 259, 977 (1993). 10M. H. Pillinger, C. Volker, J. B. Stock, G. Weissmann, and M. R. Philips, Z Biol. Chem. 269, 1486 (1994). al A. Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl 97), 77 (1968).
[7]
C A R B O X Y L M E T H Y L T R A N S F E R A S E IN N E U T R O P H I L S
51
conical tubes and the cells are pelleted (800 rpm × 10 min). Neutrophils are brought up in 8 ml of cell buffer and then subjected to two cycles of hypotonic lysis of residual erythrocytes by adding 24 ml of distilled H20 for 45 sec followed by 8 ml of 0.6 M NaC1 to restore isotonicity. Isolated neutrophils are washed, resuspended, and counted in cell buffer. This procedure yields >98% neutrophils. Neutrophil pcCMT is localized to the plasma membrane (see below). Crude membrane preparations of human neutrophils contain abundant proteases. It is therefore important to start with highly purified plasma membrane preparations as a source of pcCMT. Neutrophils to be fractionated (1-2 × 109 cells) are treated with 5 mM diisopropyl fluorophosphate for 10 min at room temperature, washed twice with cell buffer, and then brought up in 18 ml of ice-cold relaxation buffer (100 mM KC1, 3 mM NaC1, 3.5 mM MgCI2, 1 mM ATP, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 ~g/ml leupeptin, 10 /~g/ml chymostatin, 10/~g/ml pepstatin A, 100 KIU/ml aproteinin, 10 mM HEPES, pH 7.3). These neutrophil suspensions are stirred at 350 psi nitrogen in a bomb (Parr Instruments) for 20 min at 4 ° followed by dropwise cavitation. The cavitate is centrifuged at 3000 rpm to remove nuclei and unbroken cells. The postnuclear supernatant is layered on discontinuous sucrose gradients composed of 3.5/3.5/1.0ml steps of 40%/50%/60% (w:v) sucrose 0?D = 1.389, 1.404, and 1.418 at 20 °, respectively) in 20 mM Tris, pH 7.4, containing 100 KIU/ml aprotinin formed in 12-ml Ultra-Clear (Beckman) polycarbonate ultracentrifuge tubes. When neutrophil cytosol and plasma membrane are to be harvested without granules the cavitate can be layered on top of 40% sucrose alone. The gradients are spun in a SW-41 rotor (Beckman) for 2 hr at 4 ° at 35,000 rpm. Cytosol is collected from above the sucrose layers. Plasma membranes are collected as an opalescent band just below the cytosol-40% sucrose interface. Specific granules appear as a milky band between the 40 and 50% sucrose, and azurophilic granules can be harvested as a granular, yellow-green material layered on top of the 60% sucrose. Plasma membrane fractions are washed in 20 mM Tris-HCl, pH 7.4, containing 100 KIU/ml aprotinin, freeze-thawed in the same buffer five to seven times to remove cytosol contaminating the reclosed membrane vesicles, and stored in aliquots at - 7 0 °. Cytosol is dialyzed overnight against 20 mM Tris-HC1, pH 7.4, containing 10 KIU/ml aprotinin, concentrated three- to fivefold by vacuum centrifugation, centrifuged to remove precipitates, and stored at - 7 0 °.
Carboxyl Methylation of Ras-related Proteins in Intact Neutrophils Studying carboxyl methylation of endogenous Ras-related proteins in intact human neutrophils affords the opportunity to observe the effect of
52
EXPRESSION AND PURIFICATION
[7]
inflammatory agonists and other agents on this process. Because the methyl donor for the a-carboxyl methylesterification reaction, S-adenosyl-L-methionine (AdoMet), is impermeant to cells, carboxyl methylation is best monitored in intact cells by metabolically labeling with the precursor of AdoMet, L-[methyl-3H]methionine. Neutrophils (1.5 × 107) are suspended in 0.2 ml cell buffer containing 350-500 /zCi/ml [methyl-3H]methionine and incubated at 37 °. After 1 hr the cells are washed and lysed with 2 vol of icecold 20% trichloroacetic acid (TCA). The resulting precipitate is washed with ice-cold acetone, dissolved in electrophoresis sample buffer, and subjected to 12% glycine SDS-PAGE. Radiolabeled proteins are visualized by fluorography [adequate exposures range from 1 to 3 days using EnHance (Dupont-NEN)]. Because L-[methyl-3H]methionine will metabolically label newly synthesized proteins and because AdoMet is the methyl donor for other transmethylation reactions, carboxyl methylation of labeled proteins must be confirmed by alkaline hydrolysis using the method of Stock et al. 12 This method is specific for o~-carboxyl methylation of proteins since the methyl ester group that can modify aspartic acid side chains is hydrolyzed during standard SDS-PAGE at pH 8.8 whereas a-carboxyl methyl esters require pH >10 for hydrolysis. The gels dried onto filter paper are sliced into strips corresponding to --1-kDa bands from regions of interest. The dried polyacrylamide slices are peeled away from the filter paper backing and placed in uncapped 1.5-ml microcentrifuge tubes and hydrated with 0.05 ml 1 N NaOH to hydrolyze methyl ester bonds. The tubes are immediately placed inside capped scintillation vials containing 8 ml of scintillation fluid which cannot enter the 1.5-ml tubes but serves as a sink for volatile [3H]methanol. These vials are left at room temperature for 24 hr before counting. Counting alkaline labile [3H]methanol not only qualitatively confirms carboxyl methylation but also allows for a reliable and reproducible quantitation of carboxyl methylation of Ras-related proteins. In these neutrophil lysates the only significant peak of protein carboxyl methylation below the 35-kDa region (position of carboxyl methylated protein phosphatase 2A 13) is observed in the 20- to 25-kDa region corresponding to the Mr of Ras-related proteins. As reagents become available to reliably immunoprecipitate Ras-related proteins other than p2F aS, this method can be adapted to analyze carboxyl methylation of specific Ras-related proteins by employing lysis by a nonionic detergent instead of by TCA, followed by immunoprecipitation and alkaline hydrolysis of immunoprecipitates with or without SDS-PAGE analysis. 12 j. B. Stock, S. Clarke, and D. E. Koshland, in "Methods in Enzymology" (F. Wold and K. Moldave, eds.), Vol. 106, p 310. Academic Press, San Diego, 1984. 13 j. Lee and J. Stock, J. BioL Chem. 268, 19192 (1993).
[7]
CARBOXYL METHYLTRANSFERASE IN, NEUTROPHILS + + +
+
+ +
+ + +
3
4
53
Cytosol Plasma Membrane GTPyS
29-
181
2
FIG. 1. Carboxyl methylation of endogenous neutrophil Ras-related proteins. Cytosol (Ras-related protein substrates), plasma membrane (pcCMT), or both were incubated with [3H]AdoMet in the presence or absence of GTPTS (100/xM) as described. Carboxyl-methylated Ras-related proteins were analyzed by 12% glycine SDS-PAGE and fluorography 0-week exposure).
pcCMT Activity in Neutrophil Membranes Endogenous Ras-related Protein Substrates. Neutrophil Ras-related proteins can be carboxyl methylated in a GTPyS-dependent fashion using a cell-free system consisting of plasma membrane as a source of pcCMT, cytosol as a source of Rho family Ras-related proteins, and [3H]AdoMet as the methyl donor (Fig. 1). Neutrophil plasma membranes (25/~g) and cytosol (100/~g) are suspended in 50/.d 20 mM Tris, pH 8.0, containing 10-100 /~M GTPyS, 1 mM EDTA (methylation buffer), and 85 /~Ci/ml [3H]AdoMet (76 Ci/mmol, Dupont-NEN) and incubated at 37°. After 1 to 60 min the reaction is stopped by the addition of 25/~1 of 3x electrophoresis sample buffer and the proteins are analyzed by 12% glycine SDS-PAGE. Labeled proteins are visualized by fluorography (EnHance, Dupont-NEN; adequate exposure 1-10 days). Carboxyl methylation of labeled proteins can be confirmed by excising bands from the dried gels, hydrolyzing methyl esters, and quantitating [3H]methanol as described earlier. Using this method, few methylated proteins outside the 20- to 25-kDa range of Ras-related proteins are detected. These include several unidentified high molecular weight proteins, cytosolic protein phosphatase 2A (35 kDa) which is not prenylated but carboxyl methylated on a C-terminal leucine, 13two cytosolic proteins of 8 and 14 kDa, and membrane-associated neutrophil G~.TM Carboxyl-methylated G~ (apparent Mr 6000) can be resolved with 15% tricine SDS-PAGE (Novex precast) (Fig. 2). The cell-free neutrophil carboxyl methylation assay is extremely sensitive to detergents (Table I). This is consistent with the pcCMT activities 14M. R. Philips, R. Staud, M. H. Pillinger, A. Feoktistov, C. Volker, J. B. Stock, and G. Weissmann, Proc. Natl. Acad. Sci. USA 92, 2283 (1995).
54
EXPRESSIONAND PURIFICATION -
-I--
43-
[7]
GTP'yS
<--PP2A
29} ras-related
1814-
6.2 -
<--G~, 1
2
FIG.2. Neutrophil Ras-related proteins and G~are the predominant substrates for pcCMT. Cytosol and plasma membrane were incubated in the absence or presence of GTPyS (100 /xM) with [3H]AdoMetas described and analyzedby 15%tricine SDS-PAGE and fluorography (2-day exposure). In addition to Ras-related proteins and Gr, several unidentified high and two low molecular weight proteins and PP2A (35 kDa) are faintly labeled.
described in other mammalian tissues 8,t°'aS-t7 and in yeast. 18'19 Since the deduced sequence of a S a c c h a r o m y c e s cerevisiae pcCMT, the product of the S T E 1 4 gene, predicts several hydrophobic membrane-spanning helices, 2° the detergent sensitivity of p c C M T suggests that enzymatic activity depends on the tertiary structure conferred by insertion(s) through the phospholipid bilayer. R e c o m b i n a n t Ras-related Proteins. Recombinant, prenylated Ras-related proteins can be substituted for neutrophil cytosol in the cell-free assay described earlier in order to study carboxyl methylation of specific substrates. Several methods for preparing prenylated, recombinant Rasrelated proteins have been described (see preceding chapters). The baculovirus system produces both processed and unprocessed recombinant Rasrelated proteins, and the former can be isolated by Triton X-114 extraction. Bacterially produced or in vitro-translated recombinant Ras-related proteins can be prenylated with crude cell extracts or partially purified prenyltransferase and farnesyl or geranylgeranyl pyrophosphate. We have used 15R. C. Stephenson and S. Clarke, J. Biol. Chem. 265, 16248 (1990). 16C. Volker, R. A. Miller, W. R. McCleary,A. Rao, M. Poenie, J. M. Backer, and J. B. Stock, Z Biol. Chem. 266, 21515 (1991). 17R. C. Stephenson and S. Clarke, J. Biol. Chem. 267, 13314 (1992). 18C. A. Hrycyna and S. Clarke, Mol. Cell. Biol. 10, 5071 (1990). 19C. Volker, P. Lane, C. Kwee, M. Johnson, and J. B. Stock, FEBS Lett. 295, 189 (1991). 20M. N. Ashby, P. R. Errada, V. L. Boyartchuk, and J. Rine, Yeast 9, 907 (1993).
[7]
CARBOXYL METHYLTRANSFERASE IN NEUTROPHILS
55
TABLE I EFFECTSOF DETERGENTSON pcCMT Acrlv[Tva
Detergent
Methylation (%)
None 0.05% CHAPS 0.5% CHAPS 0.5% n-Octylglucoside 1% Nonidet P-40 1% Digitonin 1% Lubrol PX 1% Sodium cholate 1% Triton-X 100
100 72.6 1.5 1.3 0.4 0.3 1.5 0.7 0.6
"
Cell-free [3H]AdoMet labeling of endogenous neutrophil Ras-related proteins quantitated by SDS-PAGE, fluorography, and densitometry.
E. coli-expressed p21K-rasBthat we prenylated with partially purified bovine brain farnesyltransferase as a substrate for neutrophil pcCMT (Fig. 3). Prenylcysteine Analogs. Unlike prenyltransferases that require at least the CXXX tetrapeptide sequence for modification, pcCMT requires nothing 70000 c
~
60000.
50000 •. ~ 4 0 0 0 0 = E , 30000
=
2000010000 0
. . . .
0
q
5
. . . .
I
10
. . . .
I
'
'
'
1
15
. . . .
20
I
25
. . . .
I
'
30
T i m e (min)
FIG. 3. Carboxyl methylation of recombinant p21K-RasBby neutrophil plasma membranes. p21K-RasBwas expressed in E. coli and prenylated with partially purified bovine brain farnesyltransferase and farnesylpyrophosphate. Recombinant p21K-RaSBwas incubated with neutrophil plasma membranes and [3H]AdoMet as described. Carboxyl methylation was assayed by TCA precipitation, alkaline hydrolysis, and quantitation of volatilized [3H]methanol.
56
EXPRESSION AND PURIFICATION
[71
more than the prenylcysteine moiety for activity. Consequently, low molecular weight analogs of the prenylcysteine C-terminal residue of Ras-related proteins such as N-acetyl-S-trans, trans-L-farnesylcysteine (AFC) and Nacetyl-S-all-trans-L-geranylgeranylcysteine (AGGC) are excellent substrates for pcCMTs, including the enzyme expressed in neutrophils. 1°,19 In fact, even the amino group is not required since S-farnesylthiopropionic acid (FTP) also serves as a good substrate. 19 These prenylcysteine compounds are also competitive inhibitors of Ras-related protein carboxyl methylation. Interestingly, removal of the methylene group from FTP to form S-farnesylthioacetic acid results in a pure inhibitor that is not itself methylated] 9 AFC and A G G C (synthesis and purification described elsewhere 2l) have proved to be very useful for analyzing pcCMT activity in several tissues, including neutrophil membranes. 9'1°Among the advantages of these low molecular weight surrogate pcCMT substrates is that they can be easily separated from cellular proteins by trichloroacetic acid (TCA) precipitation and heptane extraction to facilitate quantitation of prenylcysteine o~-carboxyl methylesterification without the background of nonspecific protein carboxyl methylation (e.g., on aspartic acid residues). The reaction studied is then prenylcysteine + S-adenosylmethione ~ prenylcysteine methyl ester + S-adenosylhomocysteine. For a standard assay using saturating concentrations of substrate, neutrophil membranes (25/~g), prepared as described earlier, are incubated in 50/xl of TE buffer (50 mM Tris, pH 8.0, 1 mM EDTA) containing 56 /zCi/ml [3H]AdoMet (700 nM) and 100/zM AFC or 20/zM A G G C at 37 ° for 1-120 min in 1.5-ml microcentrifuge tubes. The reaction rate can be increased by adding unlabeled AdoMet (Km 1.3/xM), but the loss in specific activity of the methylated product is usually undesirable unless the kinetics of pcCMT with respect to the methyl acceptor are to be studied. Concentrations of AFC exceeding 200/zM and of A G G C exceeding 100/zM have detergent-like effects (e.g., cause leakage of the neutrophil cytosolic enzyme lactate dehydrogenase) and consequently inhibit the pcCMT activity of membrane preparations. AFC or A G G C methylesterification is quantitated by a modification of the method of Volker et al.21 which measures volatilized [3H]methanol generated by the alkaline hydrolysis of AFC or A G G C methyl ester (AFCME or AGGCME): at pH >10 AFCME + H20 AFC + methanol. Methylation reactions are stopped by addition of an equal volume of ice-cold 20% TCA, followed by vigorous mixing by vortex. Heptane (0.4 ml) is added directly to the TCA/protein suspensions. The samples are again vortexed × 10 sec followed by centrifugation (3 rain at 21 C. Volker, R. A. Miller, and J. B. Stock, Methods 1, 283-287.
[7]
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C A R B O X Y L M E T H Y L T R A N S F E R A S E IN N E U T R O P H I L S
15,000 × rpm). Unreacted AFC or AGGC and [3H]AFCME or [3H]AGGCME, but not [3H]AdoMet, are partitioned into the heptane layer (top). Portions (0.3 ml) of the heptane layers are transferred to 1.5-ml microcentrifuge tubes and dried by rotary vacuum evaporation. NaOH (0.1 ml of 1 N) is added to each tube, which is immediately placed uncapped into a scintillation vial containing 8 ml of an organic scintillation fluid (e.g., EcoScint), taking care not to allow any scintillation fluid to enter the microcentrifuge tube. Scintillation vials are capped and allowed to stand for at least 24 hr before counting. Although alkaline hydrolysis of AFCME or AGGCME is complete within minutes, scintillation counts rise for the 72 hr required to reach an equilibrium partitioning of [3H]methanol among the aqueous, vapor, and organic phases. The efficiency of partition of [3H]methanol from the aqueous sample to the organic scintillation fluid is 0.8. 21 Using saturating concentrations of AdoMet (15 /xM), 10 /zg plasma membrane protein, and incubating for 30 min, the rate of AFC (0.1-100 tzM) methylation can then be calculated from the following equation pmol AFC methylated/mg plasma membrane protein- min cpmi3Hlmethanol/(cpmi3HlAdoMet/pmo1AdoMet) = (fraction heptane recovered) (efficiency of MeOH partition) (mg plasma membrane) (min reaction) which, using the parameters described earlier, becomes cpm[3Hlmethanol/(cpm[3HlAdoMet/pmo1AdoMet) (0.75)(0.8)(0.01 rag)(30 min) With these methods we have determined that neutrophil plasma membrane methylates AFC and AGGC with Vmaxand Km comparable to that of rat brain microsomes, ~9 the tissue fraction with the highest pcCMT activity previously recorded (Table II). The pH optima for carboxyl methylation T A B L E II KINETICS OF p c C M T : NEUTROPHIL PLASMA MEMBRANE VERSUS RAT BRAIN AND RAT LIVER
Wmax (pmol/ m g . min)
K m (/zM)
pcCMT source
AFC a
AGGC a
AFC"
AGGC"
Human neutrophil, p l a s m a m e m b r a n e Rat brain, m i c r o s o m e s Rat liver, m i c r o s o m e s
16.4 18 2.8
22.1 16 2.4
11.6 25 --
1.4 7 --
a Substrate.
58
[7]
EXPRESSION AND PURIFICATION E 120000 .~_~10000080000600001 40000 ~., 20000 0
,
,
,
[
7.5
I
8 pH
.
.
.
.
.
.
.
815
.
9
FIG. 4. pH dependence of neutrophil pcCMT. AFC was methylated by neutrophil plasma membranes and [3H]AdoMet as described using Tris-HC1 buffered to the pH indicated. Carboxyl methylation was assayed by heptane extraction, alkaline hydrolysis, and quantitation of volatilized [3H]methanol.
of AFC by neutrophil plasma membranes is 8.0 (Fig. 4), identical to that for rat brain microsomal pcCMT activity. 21
Enrichment for Neutrophil Surface Membrane and Subcellular Localization of pcCMT Neutrophil subcellular fractionation, as described earlier, produces a light membrane fraction referred to by most authors as plasma membrane. However, the light membrane fraction is contaminated with other membranes such as smooth endoplasmic reticulum (ER, microsomes), Golgi, and endosomes. Since neutrophils are postmitotic, biosynthetically quiescent cells with very little ER, the vast majority of light membrane vesicles are indeed derived from the plasma membrane. Nevertheless, to assign pcCMT activity definitively to the plasma membrane we adapted a method for biotinylating surface membrane which could then be isolated on streptavidin-conjugated agarose. Neutrophil surface membrane is biotinylated by a modification of the method of Rosen et aL22Neutrophils maintained at 4 ° to prevent endocytosis are washed twice in Dulbecco's phosphate-buffered saline (PBS). These cells are incubated twice for 15 min in PBS containing 0.5 mg/ml sulfoNHS-biotin (Pierce) and are then washed successively in Dulbecco's modified Eagle's medium and Dulbecco's PBS. Biotinylated neutrophils are 22 C. L. Rosen, M. P. Lisanti, and J. L. Salzer, Z Cell Biol. 117(3), 617 (1992).
[7]
CARBOXYLMETHYLTRANSFERASEIN NEUTROPHILS
59
subjected to subcellular fractionation as described earlier. The copurification of the surface membranes with the light (40% sucrose) membrane fraction can be confirmed by dot blots of subcellular fractions onto nitrocellulose.* T o enrich for biotinylated surface membranes, streptavidin-conjugated agarose beads (Sigma) hydrated and washed with T E buffer are used. Biotinylated light membranes (20/xg) in 50/zl T E buffer at 4 ° are incubated with 25/zl (1 : 1 suspension in TE) streptavidin-conjugated agarose beads with vigorous mixing. Agarose beads conjugated to an irrelevant ligand [e.g., cellobiose-conjugated agarose beads (Sigma)] may be used in parallel to streptavidin-conjugated agarose to control for nonspecific adsorption of membrane vesicles. After 30 min the beads are allowed to sediment without centrifugation and the supernatants are removed to separate tubes. Each pellet is washed twice and resuspended in 50/zl TE. Cellobiose-conjugated agarose beads (25/zl of a 1 : 1 suspension in T E ) are added to each supernatant to control for nonspecific effects of agarose beads on the pcCMT assay. pcCMT activity is measured using A F C as the substrate as described earlier. Whereas the bulk of light membrane pcCMT activity in rat liver homogenates has been reported to be microsomal, 17 we have used this surface membrane enrichment technique to demonstrate that neutrophil lightmembrane p c C M T activity is associated with surface plasma membrane where it may be involved in GTP-binding protein-mediated signal transduction. 1°
Reconstitution and Partial Purification of Detergent-Extracted Neutrophil pcCMT Reconstitution in Liposomes. The detergent sensitivity of pcCMTs, including the activity associated with neutrophil plasma membrane (Table I), has hindered efforts to extract and purify the enzyme. We have therefore developed a m e t h o d of reconstituting detergent-extracted pcCMT activity in phospholipid vesicles (liposomes). Neutrophil plasma membranes (20 /xg) are extracted in 50/~1 T E buffer containing 0.1% C H A P S (TEC) for 30 min at 4 °. Extracts are incubated for 30 min at room temperature in the presence of 5 mg/ml diacylphosphatidylcholine (PC, egg lecithin, Sigma) with or without varying concentrations of anionic phospholipids such as/3* Samples of subcellular fractions (2-200 ng) are blotted onto nitrocellulose, which is then blocked for 1 hr at room temperature in PBS, 0.5% (v/v) Tween 20, 1 M glucose, 10% (v/v) glycerol (TGG buffer) containing 3% (w/v) BSA and 2% (w/v) nonfat dry milk. The nitrocellulose is then washed twice on PBS/0.5%Tween 20 and incubated for 2 hr in TGG buffer containing 0.3% BSA and 500,000 cpm/ml 1251labeled streptavidin(80 Ci/mmol, Amersham). The nitrocellulose is washed three times with PBS/0.5% Tween 20 and once with water, dried, and visualized by autoradiography and/or phosphorimaging.
60
EXPRESSION AND PURIFICATION
[7]
arachidonoyl-y-stearoyl-L-ot-phosphatidic acid (PA, Sigma). Phospholipid stocks (100 mg/ml PC and 10 mg/ml PA) are maintained in chloroform, mixed in the desired proportion, blown dry under nitrogen, and dissolved in the TEC buffer containing the solubilized plasma membrane. Samples are then added to 50/zl (1 : 1 suspension in TE buffer) of Exractigel detergent removing gel (Pierce, prewashed x 2 in TE buffer) and incubated for 1 hr at 4° with frequent mixing. Extractigel beads are removed by centrifugation (500g), and supernatants are assayed for pcCMT activity using the AFC methylation method described earlier. Although little pcCMT activity can be reconstituted with PC alone, the addition of an anionic phospholipid markedly improves the efficiency of reconstitution (Fig. 5A). Of the phospholipids tested, the most anionic, PA, has the highest activity in the reconstitution assay when added to PC (Fig. 5B). The optimal ratio of PC to PA for pcCMT reconstitution is 10 : 1 (w : w) (Fig. 5A). Our standard reconstitution system therefore contains 5 mg/ml PC and 0.5 mg/ml PA. This method ~30 o25
L
~ 30
A
O 25 o~
.e
B
~2o ~15-
~15-
~10
~10
7
~
~o-
Contol
PC 10 mg/ml
PC:PA 10:0,1
C
PC:PA 10:1
+ _
5
E 0-
PC:PA 10:10
L,ontrol
I"L,:~'~ 10:1
P'L;:I"~ 10:1
+ +
+
+ +
Chaps Detergent Removal
_
+
+
PC
P'L~:fft 10:1
10:1
ras-related proteins
1
2
3
4
5
FIG. 5. Reconstitution of detergent-extracted neutrophil pcCMT in phospholipid vesicles. Neutrophil plasma membrane was solubilized with 0.1% CHAPS and used as a source of pcCMT to methylate AFC (A and B) or endogenous neutrophil cytosolic Ras-related proteins (C). Phosphatidylcholine (10 mg/ml) alone or in combination with varying amounts ofphosphatidic acid (A) or other phospholipids (B) were added prior to detergent removal by ExtractiGel to promote liposome formation. [3H]AdoMet was added and the carboxyl methylation of AFC was measured as described. Reconstitution of carboxyl methylation of neutrophil Rasrelated proteins (C) was analyzed by 12% glycine SDS-PAGE and fluorography.
[7]
CARBOXYL METHYLTRANSFERASE IN NEUTROPHILS
61
not only reconstitutes detergent-extracted pcCMT activity toward the low molecular weight prenylcysteine analog, AFC, but also reconstitutes the carboxyl methylation of endogenous neutrophil Ras-related proteins (Fig. 5C). We have tested several other methods of reconstitution of detergentextracted pcCMT activity with liposomes. Microdialysis of 0.1% CHAPSextracted plasma membrane in the presence of PC/PA results in poor recovery of activity. Racker 23 has suggested that, in some instances, removal of detergent from solubilized membranes prior to addition of phospholipids, followed by addition of phospholipids and sonication (to disturb lipid structures and allow incorporation of extracted proteins), may be more effective than detergent removal in the presence of phospholipids for restoring function to integral membrane proteins. However, we found that this method did not reconstitute detergent-extracted pcCMT activity. More successful is the dilution of samples such that the detergent concentration falls below the critical micellar concentration in the presence of sufficient PC/PA (10:1) to achieve a final PC/PA concentrations of 5 and 0.5 mg/ml, respectively. This method is faster and technically simpler than detergent removal with Extractigel but has the disadvantage of diluting the pcCMT. We have found that a 1:4 dilution with TE of CHAPS 0.1%-extracted plasma membrane results in recovery of pcCMT activity equivalent to that of the Extractigel method. Ion-Exchange Purification. Using the liposomal pcCMT reconstitution method described earlier, neutrophil plasma membrane pcCMT can be partially purified with an ion-exchange column. Because large quantities of neutrophil membrane are required for this purification, we have found it convenient to use, when available, plasma membrane preparations from neutrophils of patients with chronic myelogenous leukemia (CML) undergoing therapeutic leukaphoresis. These leukemic cells are terminally differentiated, morphologically identical to normal neutrophils and have equivalent pcCMT activity. The neutrophil membrane is solubilized in TEC and loaded onto a D E A E column. The column is washed with two or more column volumes of TEC and is then eluted with a linear gradient of 0 to 0.5 M NaC1 in TEC followed by a step to 1 M. Whereas concentrations of NaC1 below 0.5 M do not affect the pcCMT activity of nonextracted neutrophil plasma membrane, concentrations above 0.5 M inhibit 25-50%, reducing the sensitivity of the assay in these samples. Eluted fractions are reconstituted by the addition of PC/PA followed by detergent removal using Extractigel as described earlier and assayed for pcCMT activity using AFC 23 E. Racker, in "Methods in Enzymology" (S. Fleischer and L. Packer, eds.), Vol. LV, p. 699. Academic Press, San Diego, 1979.
62
[7]
EXPRESSION A N D P U R I F I C A T I O N
as the substrate. A single peak of pcCMT activity is eluted from the column at 0.275 M NaC1 (Fig. 6). This peak corresponds to one of several protein peaks. Neutrophil pcCMT activity is eluted exclusively in the void volume of a Sephacryl S-200 (Pharmacia) column affecting a purification from extracted membrane proteins that enter the gel. Hydrophobic interaction chromatography using octyl-/3-Sepharose (Pharmacia) appeared to bind solubilized neutrophil pcCMT but failed to release the activity even at high detergent and low salt concentrations, suggesting a strong interaction between pcCMT and octyl-/3-Sepharose. Discussion Signal transduction in human neutrophils is associated with carboxyl methylation of Ras-related GTP-binding proteins. The enzyme that catalyzes this reaction, pcCMT, is, therefore, potentially regulatory. Although circulating neutrophils are short-lived, terminally differentiated, and biosynthetically quiescent, their plasma membranes are a particularly rich source of pcCMT, suggesting a role for this enzyme in the inflammatory response. Because of the absolute detergent sensitivity of pcCMT, characterization of the mammalian enzyme has been difficult. The methods described in this chapter allow analysis of neutrophil pcCMT activity both in
18000
16000-
pccM
14ooo~
12000:
!.aC!! .............
1
/ \ [protein]/ ~ /J,_\ ,I;
10000"
v §;.
I /i
- -
8000.
-o.s~
~"20004°°°6°°°~~.......:~ ...:;.-~,,~ .......... 0~
0
....
20
"~
i ....
30
~ ........
40
~ ....
~ ....
50 60 70 Fraction number
~.~
~ ' ' ''-~ . . . .
80
90
0
1O0
FIG. 6. D E A E - S e p h a r o s e c o l u m n p u r i f i c a t i o n of n e u t r o p h i l p l a s m a m e m b r a n e p c C M T . N e u t r o p h i l p l a s m a m e m b r a n e s w e r e s o l u b i l i z e d w i t h 0.1% C H A P S , p a s s e d o v e r a D E A E S e p h a r o s e c o l u m n , a n d e l u t e d w i t h a l i n e a r g r a d i e n t of NaCI. p c C M T activity was a s s a y e d b y r e c o n s t i t u t i o n i n t o P C : P A (10 : 1) l i p o s o m e s a n d m e t h y l a t i o n of A F C as d e s c r i b e d . P r o t e i n w a s a s s a y e d as &Ae80.
[7]
CARBOXYL METHYLTRANSFERASE IN NEUTROPHILS
63
intact cells capable of activation with inflammatory agonists and in cellfree systems. Reconstitution of detergent-extracted pcCMT in phospholipid vesicles has allowed further characterization and partial purification of the enzyme and may ultimately permit sequencing and molecular analysis.
Section II Guanine Nucleotide Exchange and Hydrolysis
[8]
[8]
EXCHANGE
Measurement
AND GTPase
RATES
of Intrinsic Nucleotide GTP Hydrolysis Rates
67
Exchange
and
By ANNETTE J. SELF and ALAN HALL Introduction Like all GTP-binding proteins, Rho-related GTPases exist in two conformational states, an inactive G D P - b o u n d form and an active G T P - b o u n d form, and their interconversion occurs through a cycle of guanine nucleotide exchange and G T P hydrolysis. The intrinsic nucleotide exchange and GTPase activities of all members of the Ras superfamily are relatively slow and in vivo they are stimulated by guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs). Most detailed information on exchange rates and G T P hydrolysis rates is currently available for Ras. 1 The exchange rate of G D P bound to Ras with exogenous G T P is relatively low in vitro, and the rate-limiting step has been shown to be the loss of bound nucleotide, making the reaction pseudo first-order. The half-life 0-1/2) at 30 ° is around 60 rain. 2 We have shown that the off rate can be dramatically increased by lowering the free magnesium concentration to 0.5 txM 0-1/2 <1 min) and have suggested that removal of a bound magnesium ion from the protein might induce an open conformation allowing free exchange with exogenous nucleotides. It appears that magnesium depletion in vitro mimics the effect of G E F in vivo. Mammalian G E F s for Ras have now been identified and one of these, Sos, dramatically speeds up the nucleotide exchange rate on Ras after stimulation of plasma membrane receptors. 3 The intrinsic GTPase rate of Ras, also low 0-1/2 at 30 ° = 30 min), is stimulated in vivo by at least two GAPs, p120 R a s - G A P and neurofibromin, the product of the NF1 gene lOCUS.4'5 These factors act as in vivo downregulators of Ras, although there is some evidence that they may also play an additional positive role in signal transduction in their own right. 6-s The
i S. E. Neal, J. F. Eccleston, and M. R. Webb, J. Biol. Chem. 263, 19718 (1989). A. Hall and A. J. Self, J. BioL Chem. 261, 10963 (1986). 3L. Buday and J. Downward, Cell 73, 611 (1993). 4M. Trahey and F. McCormick, Science 238, 542 (1987). 5 R. Ballester, D. Marchuk, M. Boguski, A. Saulino, R. Letcher, M. Wigler, and F. Collins, Cell 63, 851 (1990). 6 A. Yatani, K. Okabe, P. Polakis, R, Halenbeck, F. McCormick, and A. M. Brown, Cell 61, 769 (1990). METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
68
GUANINE NUCLEOTIDE EXCHANGE
[8]
identification of GEFs and GAPs is clearly, therefore, an essential step forward in understanding the regulation of other small GTPases. GEFs and GAPs for members of the Rho subfamily have also been identified, although their cellular roles and exact specificities are still unclear. The first GAP described for this family (Rho-GAP) was purified biochemically from human spleen and was shown to be active on Rho, but it is also active on Rac and C D C 4 2 / G 2 5 K . 9-11 Cloning of the cDNA for R h o - G A P has revealed that this 50-kDa protein is in fact most active on CDC42/G25K in vitro. 12,13 With the amino acid sequence of R h o - G A P available, other GAPs for the Rho subfamily have subsequently been identified. The breakpoint cluster region gene, bcr, and n-chimerin, a brainspecific cDNA of unknown function, are GAPs for Rac and CDC42/G25K but not for Rho. 1°'11The Ras-GAP-associated protein, p190, has significant homology to R h o - G A P in its C terminus and indeed p190 has been shown to be a GAP for Rho, Rac and G25K (see [13]). 14'15 Other members of the R h o - G A P family so far identified are Abr, 3BP-1, and p85 (the regulatory subunit of PI 3-kinase), 16-18although no GAP activity has yet been reported for 3BP-1 or p85. It has proved difficult to purify nucleotide exchange factors for Rholike GTPases from mammalian tissues using standard biochemical procedures. A breakthrough occurred, however, through the observation that the product of CDC24, a gene lying upstream of CDC42 in Saccharomyces cerevisiae and therefore a candidate exchange factor, has significant se-
7 G. A. Martin, A. Yantani, R. Clark, L. Conroy, P. Polakis, A. M. Brown, and F. McCormick, Science 255, 192 (1992). s G. Bollag and F. McCormick, Nature 356, 663 (1992). 9 M. D. Garrett, G. N. Major, N. Totty, and A. Hall, J. BioL Chem. 276, 833 (1991). 10 D. Diekmann, S. Brill, M. D. Garrett, N. Totty, J. Hsuan, C. Monfries, C. Hall, L. Lim, and A. Hall, Nature 351, 400 (1991). 11 A. J. Ridley, A. J. Self, F. Kasmi, H. F. Paterson, A. Hall, C. J. Marshall, and C. Ellis, EMBO J. 12, 5151 (1993). 12 C. A. Lancastor, P. M. Taylor-Harris, A. J. Self, S. Brill, H. E. van Erp, and A. Hall, J. BioL Chem. 269, 1137 (1994). 13E. T. Barfod, Y. Zheng, W. Kuang, M. J. Hart, T. Evans, R. A. Cerione, and A. Ashkenazi, J. Biol. Chem. 268, 26056 (1993). 14j. Senleman, V. Narasimhan, L. C. Foster, and R. A. Weinberg, Cell 69, 539 (1992). 15j. Settleman, C. F. Albright, L. C. Foster, and R. A. Weinberg, Nature 359, 153 (1992). 16 N. Heisterkamp, V. Kaartinen, S. van Soest, G. M. Bokoch, and J. Groffen, J. Biol. Chem. 268, 16903 (1993). 17 p. Cicchetti, B. J. Mayer, G. Thiel, and D. Baltimore, Science 257, 803 (1992), 18M. Otsu, I. Hiles, I. Gout, M. J. Fry, F. Ruiz-Larrea, G. Panayotou, A. Thompson, R. Dhand, J. Hsuan, N. Totty, A. D. Smith, S. J. Morgan, S. A. Courtneidge, P. J. Parker, and M. D. Waterfield, Cell 65, 91 (1991).
[8]
EXCHANGEAND GTPase RATES
69
quence homology to a mammalian oncogene d b L 19 It has since been shown that recombinant Dbl protein can act as an exchange factor for CDC42 (and Rho) in v i t r o (see [9]). 20 A family of proteins is emerging with homology to the exchange domain of Dbl, including two other oncogenes, v a v and ect2, but it is not yet clear how or if they all contribute to the regulation of the Rho-related GTPases (see [11]). 21'22 A n o t h e r important regulator of the R h o family of proteins is the R h o G D P dissociation inhibitor, R h o - G D I . 23 This protein inhibits the dissociation of G D P from Rho, Rac, and CDC42/G25K, but unlike G A P s and GEFs, it only interacts with the post-translationally modified forms of the GTPases (see [6]). 24 It also binds to the G T P - b o u n d form of the proteins and is capable of inhibiting the intrinsic and GAP-stimulated GTP hydrolysis ratesY -27 This suggests that R h o - G D I may be able to maintain the active state of R h o family proteins even in the presence of GAPs. A n o t h e r very interesting activity of R h o - G D I is that it can solubilize both G D P and G T P forms of Rho-related proteins from membranes, leading to the hypothesis that this family of GTPases may cycle on and off the plasma membrane (see [12]). 28 This chapter describes methods to measure the nucleotide exchange rates and G T P hydrolysis rates of Rho, Rac, and G25K.
G u a n i n e Nucleotide Off R a t e s For all Ras-related proteins that have so far been examined, the ratelimiting step in the exchange reaction is the nucleotide off rate, and the reaction [Rho. G D P + G T P --~ R h o - G T P + GDP] is, therefore, pseudo first-order. 19D. Ron, M. Zanini, M. Lewis, R. B. Wickner, L. T. Hunt, G. Graziani, S. R. Tronick, S. A. Aaronson, and A. Eva, New Biol. 3, 372 (1991). 20M. J. Hart, A. Eva, T. Evans, S. A. Aaronson, and R. A. Cerione, Nature 354, 311 (1991). 2~F. Galland, S. Katzav, and D. Birnbaum, Oncogene 7, 585 (1992). 22T. Miki, C. L. Smith, J. E. Long, A. Eva, and T. P. Fleming, Nature 362, 462 (1993). 23T. Ueda, A. Kikuchi, N. Ohga, J. Yamamoto, and Y. Takai, J. Biol. Chem. 267, 9373 (1990). 24y. Hori, A. Kikuchi, M. Isomura, M. Katayama, H. Fujioka, K. Kaibuchi, and Y. Takai, Oncogene 6, 515 (1991). 25T. H. Chuang, X. Xu, U. G. Knaus, M. J. Hart, and G. M. Bokoch, J. Biol. Chem. 268, 775 (1993). 26j. F. Hancock and A. Hall, E M B O J. 12, 1925 (1993). 27M. J. Hart, Y. Maru, D. Leonard, O. N. Witte, T. Evans, and R. A. Cerione, Science 258, 812 (1992). 28D. Leonard, M. J. Hart, J. V. Platko, A. Eva, W. Henzel, T. Evans, and R. A. Cerione, J. Biol. Chem. 267, 22860 (1992).
70
GUANINE NUCLEOTIDE EXCHANGE
[8]
Preloading GTPase Recombinant protein (2.5/xg) purified from Escherichia coli (see [1]) is first incubated in a total volume of 320/A assay buffer (50 mM TrisHC1, pH 7.6, 50 mM NaC1, 5 mM MgC12) containing 10 mM EDTA, 5 mM dithiothreitol (DTT), and 2/xCi [3H]GDP or [3H]GTP (10 Ci/mmol, 1 ~Ci/ ml Amersham) in a water bath for 10 min at 30°. The free Mg 2+ concentration under these conditions is very low (0.5/xM) and in this way the GTPase is loaded with tritiated guanine nucleotide.
Off Rates in Low Magnesium Concentrations A 40-/xl aliquot (time zero) is diluted with 1 ml of cold assay buffer and placed on ice. An excess of unlabeled GTP (to 2 mM) is added to the remainder of the reaction mixture which is then incubated at 30°. Aliquots (40 tzl) are removed at 1-, 2-, 5-, 10-, 15-, and 30-min intervals, each is diluted with 1 ml of cold assay, and all samples are filtered through prewetted 25mm nitrocellulose filters (NC45 Schleicher & Schuell 0.4/xm). The filters are washed three times with 3 ml of cold assay buffer and are allowed to dry in air, and the amount of radioactive nucleotide remaining on the GTPase is determined by scintillation counting.
Off Rates in High Magnesium Concentrations After preloading the GTPase with radioactive nucleotide, the mixture is placed on ice and brought to 10 mM MgCle. An excess of GTP (to 2 mM) is added and the reaction mixture is returned to 30°. Aliquots are removed and assayed as described earlier. The loss of bound radioactive nucleotide from the protein can be plotted against time, and half-lives for the pseudofirst-order exchange reaction are determined. Figures 1 and 2 show the [3H]GTP and [3H]GDP off rates in high and low magnesium for wild-type RhoA, Racl, and G25K. Figure 1 shows that the [3H]GTP and [3H]GDP off rates for RhoA in high magnesium are both slow with a half-life of approximately 50 min similar to that reported previously for the Ras protein. 2 Surprisingly the off rates for Racl and G25K appear to be much faster. However, the reactions are biphasic with a rapid initial rate of nucleotide loss followed by a slower rate comparable to Rho. It is likely that under these experimental conditions a proportion of Racl and G25K fails to undergo the conformational change after adding magnesium to the preloading reaction. Figure 2 shows the off rates in the presence of a low free magnesium concentration. For RhoA the [3H]GTP off rate is increased compared to high magnesium, although is still relatively slow with a half-life of 15 min
[8]
EXCHANGE AND G T P a s e RATES
71
40000 30000
","
20000 -
',, "~..
"~i'> 10000 -
""~': .......................... :.:.~
0
1;
2; TIME(min)
3;
50000. 4000o
3oooo- 't, l''k w
~b, . ,
20000 10000
~.,,
o
"b-..... o ...........
0
10
20 TIME(min)
30
Fro. 1. Off rates for wild-type RhoA ((2), Racl (O), and G25K (©) in high magnesium. Proteins were preloaded with [3H]GTP (a) or [3H]GDP (b) as described in the text, and an excess of MgCI2 and unlabeled GTP was added. Aliquots were removed from the reaction, and radioactivity bound to the protein was determined.
(Fig. 2a). T h e [3H]GTP off rates of R a c l and G 2 5 K are also increased. T h e m o s t dramatic effect of lowering the m a g n e s i u m concentration, however, is on the [ 3 H ] G D P off rates. Figure 2b (note different scale) shows that the [ 3 H ] G D P off rate of all three proteins is very fast with half-lives of less than 1 min. W e predict that the effect of the low m a g n e s i u m c o n c e n t r a t i o n mimics that of an exchange factor, speeding up the G D P off rate but having a m u c h less effect on the G T P off rate.
72
GUANINE NUCLEOTIDE EXCHANGE
[8]
60000 500OO-c 400003000020000100000 0
b
10
20 TIME (min)
5
10 TIME (min)
30
60000, 50000-' 40000-
30000¸
1000000
15
FIG. 2. Off rates for wild-type RhoA (N), Racl (©), and G25K (O) in low magnesium (0.5/xM). Proteins were preloaded with [3H]GTP (a) or [3H]GDP (b) as described in the text, and an excess of MgC12 and unlabeled GTP was added. Aliquots were removed from the reaction, and the amount of radioactivity bound to the protein was determined. Intrinsic and Rho-GAP-Stimulated
GTPase Activity
W e h a v e d e t e r m i n e d t h e intrinsic a n d R h o - G A P - s t i m u l a t e d G T P a s e activity o f t h e R h o f a m i l y p r o t e i n s at low G T P a s e c o n c e n t r a t i o n s (6 n M ) a n d at t h e l o w e r t e m p e r a t u r e o f 20 °. T h e R h o - G A P u s e d in t h e s e assays is t h e C - t e r m i n a l 3 0 - k D a p o r t i o n o f h u m a n R h o - G A P ( a m i n o acids 1 9 8 439) p r o d u c e d using t h e g l u t a t h i o n e S - t r a n s f e r a s e fusion v e c t o r p G E X - 2 T (see [1]). R h o - G A P was c l o n e d into t h e EcoRI site o f p G E X - 2 T a n d was
[8]
EXCHANGE AND G T P a s e RATES
73
purified in the same way as the Rho family proteins (see [1]) except that the fusion protein was cleaved using 0.5 units of thrombin for i hr at room temperature. 12
GTPase Assays Recombinant Rho, Rac, and G25K (30 ng) are preloaded with [y-3Zp]GTP (6000 Ci/mmol, 10/xCi//xl NEN) in 20/xl of 20 mM Tris-HC1, pH 7.6, 0.1 mM DTT, 25 mM NaCI, and 4 mM EDTA for 10 min at 30°. The mixture is placed on ice and MgClz is added to a final concentration of 17 mM. Three microliters (3.5 ng) of the preloaded protein (to give a final concentration of 6 nM) is diluted with buffer (20 mM Tris-HC1, pH 7.6, 0.1 mM DTT, 1 mM GTP, 1 mg/ml bovine serum albumin) to give a final volume of 30 txl. A 5-/xl sample is removed (time zero) and diluted into 1 ml of cold assay buffer (50 mM Tris-HC1, pH 7.6, 50 mM NaC1, and 5 mM MgCI2). For GAP-stimulated GTPase assays (GAP assays), an aliquot of GAP protein is added at this stage. The remainder of the reaction is then incubated at 20 °. Samples (5 ixl) are removed at 5-, 10-, and 15-rain intervals and diluted with cold assay buffer (1 ml). After filtration through prewetted nitrocellulose filters as described previously, the amount of radioactivity remaining bound to the protein is determined by scintillation counting. Figure 3 shows the intrinsic GTPase rates of RhoA, Racl, and G25K
80
60
~-
~"
"'""
40-
"...........o
20 ;
10
ll5
TIME (min) FIG. 3. Intrinsic GTPase of wild-type Rho A([~), Racl (O), and G25K (©). Proteins were preloaded with [y-32p]GTP as described in the text, and an excess of MgCI2 was added. Aliquots were removed, and GTP hydrolysis was determined by a filter-binding assay.
74
GUANINE NUCLEOTIDE EXCHANGE
[8]
100t
:ii",, :','
80. 60,~
.2 \
40-
~"
200
........~ ..... ".,
0
............
;
1'0
1;
TIME (rain) FIG. 4. Stimulation of GTPase activities of wild-type R h o A ([~), Racl (~), and G25K (O) by recombinant R h o - G A P . Proteins were preloaded with [y-32P]GTP as described in the text, and an excess of MgC12 was added followed by 2 n M R h o - G A P . Aliquots were removed, and the amount of radioactivity remaining bound to the protein was determined by a filterbinding assay.
100-
75-
50-
<
25-
10mM
150mM
NaC1 CONCENTRATION FIG. 5. Relative R h o - G A P activities for wild-type Rho A (D), and Racl ([]), and G25K (IB) at low and high ionic strength. Proteins were preloaded with [7-32p]GTP for 10 min, and an excess of MgCI2 was added. NaC1 was added to a final concentration of 10 or 150 mM, and the reactions were incubated with various concentrations of R h o - G A P protein. After 5 min the amount of radioactivity remaining bound to the GTPase was determined by filter binding. G A P activity was determined as the concentration of R h o - G A P protein required to stimulate the intrinsic GTPase rate by 50% in 5 rain under assay conditions. The activity of R h o - G A P toward the three GTPases is expressed relative to the maximum activity observed, i.e., toward G25K, at low ionic strength (= 100).
[8]
EXCHANGE AND GTPase RATES
75
TABLE I INHIBITIONOF GAP-STIMULATEDRho GTPase ACTIVITY BY COMPETINGPROTEINSa Competing protein b
50% inhibition of GAP activity (/xM)c
Rho GppNHp Rho GDP/3S Rac GppNHp Rac GDP/3S G25K GppNHp G25K GDP/3S L63Rho GppNHp
1.2 2.5 1.7 3.0 0.8 4.0 0.01
a Rho was preloaded with [y-32p]GTP,and sufficient RhoGAP was added to give 50% hydrolysis of GTP in 5 min. In the absence of Rho-GAP, only 10% GTP hydrolysis occurs under these conditions. b Rho, Rac, or G25K was preloaded with nonhydrolyzable GTP (GppNHp) or GDP (GDP/3S) analogs. c Increasing concentrations of Rho, Rac, or G25K preloaded with the nonhydrolyzable analogs were added to the GAP reaction. The concentration of competing protein (/xM) to inhibit GAP-stimulated GTP hydrolysis by 50% was determined.
at 20 °. T h e rate constant for the first-order G T P hydrolysis reaction (k) can be calculated using Eq. (1)
tl/2-
In 2
k'
(1)
w h e r e tl/2 is the time required to h y d r o l y z e half the G T P b o u n d to the protein. T h e half-lives for G T P hydrolysis are R h o A , 18 min (k = 0.039 rain-l); R a c l , 10 min (k = 0.069 rain-l); and G25K, 14 rain (k = 0.05 min 1). Figure 4 shows the G A P - s t i m u l a t e d hydrolysis of R h o A , R a c l , and G 2 5 K u n d e r the conditions described earlier using 2 n M r e c o m b i n a n t R h o G A P . A m o r e detailed analysis of G T P a s e rates in the presence of R h o G A P reveals that G 2 5 K is the preferred substrate: 5-fold m o r e active than R h o and 35-fold m o r e active than R a c at low ionic strength (10 m M NaC1) (see Fig. 5). 12 W e have also o b s e r v e d that the R h o - G A P - s t i m u l a t e d G T P a s e activity of R h o and G 2 5 K but not of R a c is inhibited at high ionic strength as shown in Fig. 5. Activating mutations in Ras, particularly Gly-12 to Val-12 (V12 Ras), inhibit the intrinsic G T P a s e rate as well as blocking G A P - s t i m u l a t e d G T P
76
GUANINE NUCLEOTIDE EXCHANGE
[8]
hydrolysis. 4,29 The intrinsic GTPase rates of V14RhoA, V12Racl, and V12G25K are also much slower than the wild-type proteins, having halflives of around 100, 55, and 60 min, respectively. Competition Assays The relative binding constants of R h o - G A P for the GDP- and GTPbound forms of Rho, Rac, and G25K can be determined using competition assays. In these assays, the effectiveness of each GTPase, bound to a nonhydrolyzable analog of G D P or GTP, to block R h o - G A P - s t i m u l a t e d loss of 32p from R h o . [y-32p]GTP is measured. 3° We use a concentration of R h o G A P sufficient to give 50% hydrolysis of bound G T P in 5 min. Twenty microliters of Rho, Rac, and G25K (1 mg/ml) in 10 m M Tris-HCl, p H 7.6, 1 m M DTT, and 2 m M MgC12 is preloaded with GDP/3S (1 raM) or G p p N H p (1 mM) in 5 m M E D T A for 10 rain at 30 °. MgC12 is added to 10 mM. Increasing amounts of these preloaded proteins are included in the GTPase reaction described earlier, and the extent of [T-32p]GTP hydrolysis is determined. The results of these assays are shown in Table I. Under these conditions all three proteins bind to R h o - G A P with apparently similar affinity, in the low micromolar range. Interestingly, R h o - G A P appears to have only a slightly higher affinity for the G T P - b o u n d form of these GTPases relative to the G D P - b o u n d form. This is in contrast to pl20RasG A P and neurofibromin, both of which have a 100-fold preference for the G T P form of Ras. 31 It has been reported that an activated mutant of Ras (L61Ras), with a Gin to Leu mutation at codon 61, binds to R a s G A P and neurofibromin 10- and 100-fold more tightly than does wild-type Ras. 3l We have tested the affrlity of the corresponding activated version of R h o A (L63RhoA). As shown in Table I, L63RhoA has an affinity for R h o - G A P of 10 nM, 100-fold greater than the wild-type protein. This increased affinity can also be observed in an in v i t r o bead-binding assay (see [23]) and in the yeast dual-hybrid system (see [25]). The reason for the increased affinity of this particular mutant R h o A protein for R h o - G A P is unclear. Acknowledgments We thank Dagmar Diekmann and Suzanne Brill for data on Rac and G25K mutants. This work was supported by the Cancer Research Campaign and the Medical Research Council of Great Britain. 29M. Barbacid, Annu. Rev. Biochem. 56, 779 (1987). 30M. Frech, J. John, V. Pizon, P. Chardin, A. Tavitian, R. Clark, E McCormick, and A. Wittinghofer, Science 249, 169 (1990). 3i G. Bollag and F. McCormick, Nature 351, 576 (1991).
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[9] G u a n i n e N u c l e o t i d e E x c h a n g e C a t a l y z e d by dbl Oncogene Product B y Y I ZHENG, MATTHEW J. H A R T , a n d RICHARD A . CERIONE
Introduction Guanine nucleotide exchange factors (GEFs) activate Ras family members of GTP-binding proteins by accelerating their rate of GDP release and therefore facilitating their GTP binding in cells) Unlike the case for Ras-GEFs, for which the in vivo activities can be examined by the extent of conversion of Ras-bound GDP to GTP by immunoprecipitation of Ras and thin-layer chromatography separation of the bound nucleotides (Gale et al. 2 and this series, Volume 255 [12]), the GEF activities for Rho-type GTPases are difficult to determine in this manner mostly because of the fast hydrolysis rates of members of this family (e.g., Rac and Cdc42Hs 3) and the presence of abundant R h o - G D I (GDP dissociation inhibitor) which renders most of the Rho-type proteins cytosolic and blocks the GEF action. 4 Thus, the in vitro reconstituted assay has been indispensable for detecting and quantitating the activities of potential Rho family GEF molecules. Oncogenic activation of dbl occurs as a result of an amino-terminal truncation of the proto-dbl product, resulting in a malignant transforming phenotype in NIH 3T3 cells (Ron et aL 5 and [38] in this volume). A region between residues 498 and 738 of proto-Dbl (designated the Dbl-homology domain or D H domain), retained by oncogenic Dbl, shares significant sequence similarity with the Saccharomyces cerevisiae cell division cycle protein Cdc24, 6 which acts together with the Rho-type GTP-binding protein Cdc42 to regulate bud site assembly in yeast: This clue has led to the discovery that Dbl acts as a potent GEF for Cdc42Hs. 8 The ability to purify the recombinant Dbl protein and to monitor the functional interactions i M. S. Boguski and F. McCormick, Nature 366, 643 (1993). 2 N. W. Gale, S. Kaplan, E. J. Lowenstein, J. Schlessinger, and D. Bar-Sagi, Nature 363, 88 (1993). 3 M. J. Hart, K. Shinjo, A. Hall, T. Evans, and R. A. Cerione, J. Biol. Chem. 266, 20840 (1991). 4 D. Leonard, M. J. Hart, J. V. Platko, A. Eva, W. Henzel, T. Evans, and R. A. Cerione, J. Biol. Chem. 267, 22860 (1992). 5 D. Ron, S. R. Tronick, S. A. Aaronson, and A. Eva, E M B O J. 7, 2465 (1988). 6 D. Ron, M. Zannini, M. Lewis, R. B. Wickner, L. T. Hunt, G. Graziani, S. R. Tronick, S. A. Aaronson, and A. Eva, N e w Biol. 3, 372 (1991). 7 D. G. Drubin, Cell 65, 1093 (1992). 8 M. J. Hart, A. Eva, T. Evans, S. A. Aaronson, and R. A. Cerione, Nature 354, 311 (1991).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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[9]
between Dbl and the Rho-type GTP-binding proteins by the in vitro GEF assay provides convincing biochemical evidence that the DH domain of the dbl oncogene product is sufficient in stimulating the GDP/GTP exchange for Rho-type proteins. 9 This chapter describes methods for the expression of Dbl and the DH domain in baculovirus expression systems and the in vitro assays to demonstrate that Dbl and its DH domain contain GEF activity for the Cdc42Hs and Rho GTP-binding proteins. Expression and Purification of dbl Oncogene Product in Insect S p o d o p t e r a f r u g i p e r d a Cells The entire coding sequence of the db! oncogene is removed from the mammalian expression vector p C l l ( p Z i p - n e o ) t° by BamHI digestion, and the released insert is ligated to the BamHI sites of the baculovirus transfer vector pAC373. The resulting construct pAC373dbl contains all but eight nucleotides of the Y-untranslated sequences of the baculovirus polyhedrin gene fused to the complete coding sequences of the db! oncogene, including the start ATG codon, pAC373dbl is cotransfected with the wild-type virus DNA into S. frugiperda (Sfg) insect cells to generate recombinant virus. The resulting virus is further purified according to the detailed procedure described by Piwnica-Worms. u In order to facilitate later purification steps, a version of Dbl joined at the N terminus to glutathione S-transferase (GST) is also expressed in Sf9 cells by fusion of cDNAs coding both GST and Dbl. The B a m H I insert, described earlier, that encodes oncogenic db! or the DH domain itself is first introduced into the BamHI site of the pGEX-2T vector to generate p G E X d b ! and pGEXDH. The cDNA encoding GST is digested between the X b a I - B a m H I sites and is then ligated together with the B a m H I - P s t I partial digestion fragments from p G E X d b ! or pGEXDH (which includes Dbl or the DH domain coding sequences) into the X b a I - P s t l sites of baculovirus transfer vector pVL1393. Recombinant viruses encoding GSTDbl and the G S T - D H domain are generated using the Baculogold kit from Pharmingen. Sf9 cells are grown to subconfluency and are infected with the respective recombinant baculovirus for 50-60 hr. Cells are then pelleted and resuspended in 5 ml of ice-cold buffer containing 20 mM TrIs-HC1, pH 8.0, 100 9 M. J. Hart, A. Eva, D. Zangrilli, S. A. Aaronson, T. Evans, R. A. Cerione, and Y. Zheng,
I. Biol. Chem. 269, 62 (1994). 10A. Eva, G. Vecchio,C. D. Rao, S. R. Tronick, and S. A. Aaronson,Proc. Natl. Acad. Sci. U.S.A. 85, 2061 (1988). 11H. Piwnica-Worms,in "Current Protocols in MolecularBiology,"(F. M. Ausubel et at, eds.), Wiley(Interscience),New York, Vol. 2, 16.8.1, 1994.
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79
mM NaC1, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.2% Triton X-100, plus 0.5 mM phenylmethylsulfonyl fluoride, 10 tzg/ml leupeptin, and 10/zg/ml aprotinin. The resuspended insect cells are homogenized with a Dounce homogenizer for 10 min and then centrifuged for 15 min at 4 ° in a microfuge. The supernatants for the control as well as Dbl-expressing cell lysates can be used directly in the guanine nucleotide exchange experiments. Alternatively, supernatants from the cells expressing GST-Dbl and the G S T - D H domain are further incubated with glutathione-agarose for 2 hr at 4 °. The glutathione-agarose beads are pelleted and washed four times in the cell suspension buffer. The GST fusion proteins are then eluted from the affinity beads in a buffer with 5 mM glutathione, 20 mM Tris-HC1, pH 8.0, 100 mM NaC1, 1 mM EDTA, and 0.5 mM DTT. The purified GSTDbl or G S T - D H domain can be dialyzed overnight at 4 ° to remove the free glutathione or used directly in a guanine nucleotide exchange assay.
G TP-Binding Assay To minimize the complications resulting from GTP hydrolysis during the time course of GTP binding, a nonhydrolyzable analog such as GTPyS is commonly used in place of GTP. Cdc42Hs or other GTP-binding proteins to be tested are first loaded with GDP in loading buffer supplemented with MgCI2. The guanine nucleotide exchange reaction is initiated by mixing the GDP-loaded G protein with Dbl or Dbl-containing lysates in the reaction buffer with [35S]GTPTS and is terminated at various time points by dilution of the reaction mixture in ice-cold termination buffer. The amount of [35S]GTPyS bound to the G protein is finally quantified by filtration of the terminated reactions through nitrocellulose filters. Loading buffer: 20 mM Tris-HCl, pH 8.0, 100 mM NaCI, 2 mM EDTA, 0.2 mM DTT, 100/xM AMP-PNP, and 10/xM GDP. Reaction buffer: 20 mM Tris-HC1, pH 8.0, 100 mM NaC1, 10 mM MgCI2, 100/xM AMP-PNP, 0.5 mg/ml bovine serum albumin, and 5/zM [35S]GTPTS (~11,000 cpm/pmol). Termination buffer: 20 mM Tris-HC1, pH 8.0, 100 mM NaC1, and 10 mM MgCI2. Figure 1 shows a time course of GTP binding to Cdc42Hs catalyzed by insect cell lysates in the presence or absence of Dbl. Approximately 2/zg of purified Cdc42Hs protein is first incubated in 60/zl loading buffer for 5 min at room temperature. MgC12 is then added to a final concentration of 5 mM and the loading incubation is continued for an additional 15 min. Aliquots (20/xl) of the GDP-loaded Cdc42Hs are mixed with 5/xl control cell lysates or lysates containing Dbl in the reaction buffer (total volume 100 tzl) to initiate the exchange reaction at room temperature. Aliquots
80
GUANINE NUCLEOTIDE EXCHANGE
[9]
4 o E e,
3
O. IO
2&
"O ,"
1
O ill
0 o
5
10
15
20
Time (min) FIG. 1. Effects of the Sf9 cell lysates containing Dbl on [35S]GTPyS binding to Cdc42Hs. Time course for the binding of [35S]GTPyS to Cdc42Hs. The Cdc42Hs was preloaded with GDP and then added to reaction incubations containing [35S]GTPyS together with aliquots from Sf9 control lysates ( 0 ) or from lysates expressing Dbl (A). From Hart e t al. 8 with permission.
(15/xl) of samples are taken at various time points from the reaction mixture and added to 10 ml ice-cold termination buffer. The terminated reactions are filtered immediately through the BA85 nitrocellulose filters, followed by one wash with 5 ml ice-cold termination buffer. The filters are dissolved completely in scintillation fluid, and the radioactivity detained by the filter is measured by scintillation counting. A significant stimulation by Dbl lysates of the binding of [35S]GTPyS to Cdc42HS is observed within 5 min of the reaction, indicating that Dbl accelerates the rate of the GTPyS/GDP exchange on Cdc42Hs.
GDP Dissociation Assay The GDP dissociation assay is a direct measurement of the guanine nucleotide exchange activity of Dbl since the rate-limiting step in the nucleotide exchange reaction is GDP dissociation. The protocol for assaying Dblcatalyzed GDP dissociation is similar to that used to assay GTP binding except that - 1 0 / z M radioactive [3H]GDP (10 Ci/mmol) or [a-32p]GDP is used in the loading buffer replacing GDP and 1 mM GTP is used in the reaction buffer instead of [35S]GTPTS. It is important for GTP to be present during the exchange reaction, under conditions where [Cdc42Hs] > > [Dbl].
[9]
EXPRESSIONOV Dbl AND DH DOMAIN
81
The presence of GTP results in a Dbl-catalyzed exchange of the medium GTP for the [3H]GDP bound to Cdc42Hs and enables Dbl to act catalytically in stimulating GDP dissociation from multiple Cdc42Hs proteins (i.e., because each newly formed Cdc42Hs-GTP species dissociates from Dbl and thereby frees Dbl to bind to other Cdc42Hs-[3H]GDP complexes). Data are typically plotted as the percentage of radiolabeled GDP that remains bound to Cdc42Hs as a function of time. Figure 2 shows that the insect cell lysates expressing oncogenic Dbl markedly catalyze the dissociation of [3H]GDP from Cdc42Hs purified from human platelet membranes. The half-time for GDP dissociation in the presence of Dbl occurs within 5 min, and GDP dissociation is essentially complete in 20 min. In contrast, the control lysates cause <20% dissociation of radiolabeled GDP from Cdc42Hs even after 15 min. To show that purified Dbl and the D H domain are sufficient for GEF activity, both oncogenic Dbl and the D H domain are expressed in Sf9 insect ceils as GST fusion proteins and are then highly purified by glutathione-agarose chromatography (Fig. 3A). Figure 3B shows that the GST-Dbl and G S T - D H domain are each fully capable of stimulating the dissociation of GDP from the Cdc42Hs. Purified GST-Dbl acts catalytically to stimulate the dissociation of [3H]GDP, causing - 6 5 % of the total Cdc42Hs to lose labeled GDP a. (3 .i.
100
or; i,.,..i
"o r= o .,Q
80 60
q,,,,
o 40
o
E m
20
o ~
m
0
I
0
5
I
10
I
15
20
Time (rain) FIG. 2. Effects of the Sf9 cell lysates containing Dbl on [3H]GDP dissociation from Cdc42Hs. Time course for the dissociation of [3H]GDP from Cdc42Hs. Aliquots of Sf9 control cell lysates (O) or lysates expressing Dbl (/k) were added to the reaction incubations together with [3H]GDP-preloaded Cdc42Hs. From Hart et aL 8 with permission.
82
[9]
GUANINE NUCLEOTIDE EXCHANGE
A 498
735
925 1
2
3
•-,I- GST-Dbl Oncogenic Dbl 498
757
GST-DbI-H -.,,I- GST-CDC42
DbI-H iiiiTiiiii!~7~ i
B
i
1.001 o Q.
(:3 (D 0.75
o m
3
A 0.50
0 . 2 .=
I 4
I 8
I 12
I 16
I 20
T i m e (rain)
FIG. 3. Effects of purified GST-Dbl and G S T - D H on [3H]GDP dissociation from Cdc42Hs. (A) Left: Schematic diagram of proto-Dbl and a minimal unit containing the Dbl homology domain (Dbl-H). Right: S D S - P A G E (10% polyacrylamide) of the purified Cdc42Hs, G S T Dbl, and G S T - D H domain (GST-DbI-H) used in the experiments. (B) Measurements of the dissociation of [3H]GDP from the E. coli-expressed GST-Cdc42Hs protein in the presence of purified oncogenic Dbl or the DH domain. The time course for [3H]GDP dissociation from Cdc42Hs ( - 2 tzg) was measured in the presence of 2/zg of GST alone (O), 0.2/zg of Dbl ( • ) , or 1.5/zg of the D H domain (A). From Hart et aL 9 with permission.
[91
EXPRESSION OF Dbl AND D H DOMAIN
83
lOO
0
%o FIG. 4. Effects of the purified GST-Dbl on [3H]GDP dissociations from Ras and various Rho family GTPases. Two micrograms of each GTP-binding protein tested was preloaded with [3H]GDP and then mixed in reaction buffer with 1/xg of GST alone (solid bars) or with 0.1/xg GST-Dbl (cross-hatched bars) for 15 min before termination of reactions.
within the time course of the experiment, under conditions where the ratio of [Cdc42Hs] : [Dbl] is 20 : 1. Using the G D P dissociation assay, the specificity of D b l - G E F activity toward other related GTP-binding proteins was tested as shown in Fig. 4. In addition to Cdc42Hs, Dbl exhibits potent G E F activities toward R h o A , R h o B , RhoC, to a lesser extent R h o G , but shows little or no G E F activity on the Ras, R a c l , and TC10 proteins.
Discussion The " D b l family" of potential G E F s for Rho-type GTP-binding proteins have b e e n expanding rapidly since the first discovery of D b I - G E F activity. All m e m b e r s of this family share the c o m m o n structural features of an - 2 3 0 amino acid stretch that is designated the D H domain and a pleckstrin homology (PH) domain, which is located C-terminally adjacent to the D H domain and is thought to mediate p r o t e i n - p r o t e i n interactions in analogy to SH2 and SH3 domains. 12 M e m b e r s of the Dbl family appar12A. Musacchio, T. Gibson, P. Rice, J. Thompson, and M. Saraste, Trends Biochem. Sci. 18, 343 (1993).
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ently play roles in cell growth-related pathways and include Bcr, 13 R a s G R F , 14 mSos, 15 Cdc24,16 and the dbl, 9 ect2, a7 vav, 18 and lbc 19 oncogene products. Dbl, as well as the Ect2, Vav, and Lbc oncoproteins, are activated by protein truncation. We found that proto-Dbl, which exhibits - 6 0 - f o l d less transforming activity than Dbl in N I H 3T3 cells, exhibits as potent exchange activity for Cdc42Hs as the oncogenic form of Dbl (unpublished observation), implying that a third protein factor may be involved in the regulation of the activities of p r o t o - D b l in vivo. The efficient expression of Dbl-like proteins is critical for the detection of their G E F activity. A t t e m p t s to express Dbl and its D H domain in E. coli have resulted in nonfunctional proteins, most likely because of insolubility and i m p r o p e r folding. We also have had similar experience with the expression of Cdc24 in E. coli. The baculovirus expression system has been proven to be an efficient way to generate milligram quantities of purified Dbl and Cdc24 proteins. 9,16 Alternatively, stable expression in m a m m a l i a n cells has also been used to provide functional Dbl, although at a much lower level of expression. 9 On the contrary, the E. coli-expressed recombinant Cdc42Hs and Rho can interact with Dbl indistinguishably from the insect cell-expressed Cdc42Hs and Rho, 9 suggesting that posttranslational geranylgeranylations of the carboxyl-terminal cysteine of these G T P a s e s are not essential for functional interactions with Dbl. The D b l - C d c 4 2 H s interaction can also be observed directly by the complex formation assay (Miki et al.17 and [11] in this volume). This binding interaction is highly dependent on the nucleotide-bound state of Cdc42Hs, i.e., Dbl binds tightest to the guanine nucleotide-free form of Cdc42Hs, to a lesser extent to the G D P - b o u n d form of Cdc42Hs, and to a much less extent to the G T P - b o u n d form of Cdc42Hs. This property is consistent with the catalytic mechanism of a conventional G E F such that Dbl binds to the G D P - b o u n d f o r m of Cdc42Hs and stimulates G D P release and consequently facilitates G T P binding to the nucleotide-free Cdc42Hs.
13N. Heisterkamp, J. R. Stephenson, J. Groffen, P. F. Hansen, A. de Klein, C. R. Bartram, and G. Grasveld, Nature 306, 239 (1983). 14C. Shou, C. L. Farnsworth, B. G. Neel, and L. A. Feig, Nature 358, 351 (1992). 15S. E. Egan, B. W. Giddings, M. W. Brooks, L. Buday, A. M. Sizeland, and R. A. Weinberg, Nature 363, 45 (1993). 16y. Zheng, R. A. Cerione, and A. Bender, J. BioL Chem. 269, 2369 (1994). 17T. Miki, C. L. Smith, J. E. Long, A. Eva, and T. Fleming, Nature 362, 462 (1993). 18E. Gulbins, K. M. Coggeshall, G. Baier, S. Katzav, P. Burn, and A. Altman, Science 260, 822 (1993). 19D. Toksoz and D. A. Williams, Oncogene 9, 621 (1994).
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[101 S t i m u l a t i o n o f N u c l e o t i d e E x c h a n g e o n R a s - a n d Rho-Related Proteins by Small GTP-Binding Protein GDP Dissociation Stimulator
By
EMILIO PORFIRI a n d JOHN F. HANCOCK
Introduction Guanine nucleotide exchange factors (GEFs), also known as guanine nucleotide dissociation stimulators (GDSs), are enzymes which, by catalyzing the exchange of bound GDP for GTP, trigger the activation of Ras and Ras-related proteins. 1 Several Ras GDSs have been identified, including CDC25, Drosophila SOS, mSOS1 and 2, hSOS1, SDC25, Ras-GRF, and H-GRF55.1 All of these GDSs contain a region of homology with the Cterminal 450 amino acids of CDC25 which is sufficient to promote guanine nucleotide exchange on Ras. 2 Efforts have been made to identify GDSs for Rho and Rac. At present, only the product of the dbl oncogene promotes guanine nucleotide exchange on Rho and CDC42Hs in vitro. 3 Another exchange factor, small GTP-binding protein GDS (smgGDS), has been identified. 4 smgGDS differs from the other GDSs in that it has no homology with CDC25 or Dbl, nevertheless, smgGDS shows exchange activity against a wide range of Ras-related proteins including Racl, RhoA, and RaplA, K-Ras4B, but not to H-Ras. 5'6 This is to some extent surprising since H-Ras is 85% homologous to K-Ras4B whereas RhoA and Racl are only 35% homologous to Ras. One explanation for this wide range of specificitycomes from the observation that smgGDS works only on isoprenylated K-Ras4B, RaplA, RhoA, and Racl, although K-Ras4B is modified with a Cls farnesyl group, whereas RaplA, Racl, and RhoA are modified with a C20 geranylgeranyl group. 5 Therefore, smgGDS must interact with the prenoid group at the C terminus of posttranslationally processed Ras and Ras-related proteins. 1 M. S. Boguski and F. McCormick, Nature 366, 643 (1993). 2 C. C. Lai, M. Boguski, D. Broek, and S. Powers, Mol, Cell, Biol. 13, 1345 (1993). 3 M. J. Hart, A. Eva, T. Evans, S. A. Aaronson, and R. A. Cerione, Nature 354, 311 (1991). 4 T. Yamamoto, K. Kaibuchi, T. Mizuno, M. Hiroyoshi, H. Shirataki, and Y. Takai, J. Biol. Chem. 265, 16626 (1990). 5 T. Mizuno, K. Kaibuchi, T. Yamamoto, M. Kawamura, T. Sakoda, H. Fujioka, Y. Matsuura, and Y. Takai, Proc. Natl. Acad. Sci. U.S.A. 88, 6442 (1991). 6 S. Ando, K. Kaibuchi, T. Sasaki, K. Hiraoka, T. Nishiyama, T. Mizuno, M. Asada, H. Nunoi, I. Matsuda, Y. Matsuura, P. Polakis, F. McCormick, and Y. Takai, J. Biol. Chem. 267, 25709 (1992).
METHODS IN ENZYMOLOOY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Assays for Estimation of smgGDS-Promoted Guanine Nucleotide Exchange in Vitro Expression and Purification of smgGDS smgGDS cDNA is cloned into the BamHI and EcoRI sites of the pGEX2T vector (Pharmacia) for expression in Escherichia coli as a glutathione S-transferase (GST) fusion protein. 7 A 50-ml bacterial culture, inoculated with a colony of the pGEX-2T smgGDS E. coli transformant, is grown to saturation overnight. The 50-ml culture is diluted 1:10 in prewarmed Lbroth and the expression of the fusion protein is induced with 0.15 mM isopropylthiogalactoside (IPTG) at 37° for 4-6 hr. Bacteria are collected by centrifugation for 15 min at 5000g, washed once in phosphate-buffered saline (PBS), and resuspended in 20 ml of PBS containing 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM Pefabloc, and 10/zg each of leupeptin, aprotinin, and soybean trypsin inhibitor. Following resuspension, bacteria are snap-frozen in 5-ml aliquots and stored at - 8 0 ° until use. To purify GST-smgGDS, bacteria are lysed by gentle sonication (2 x 30 sec) on ice, 1% Triton X-100 is then added, and the lysate is centrifuged at 10,000g for 20 min at 4° to remove cell debris. The cleared supernatant is applied onto a 1-ml column of glutathione-agarose. The column is washed with 100 vol of ice-cold PBS and GST-smgGDS is eluted in 6 x 1-ml fraction of 50 mM Tris-HC1, pH 8, 5 mM reduced glutathione. Following elution, an aliquot (1/100) of each fraction is analyzed by SDS-PAGE on a 10% polyacrylamide gel. GST-smgGDS is concentrated to approximately 2 mg/ml and stored at - 2 0 ° in 50% glycerol until use. The protein concentration is determined using the Bradford reaction. [3H] G D P Dissociation Assays Epitope (Glu-Glu)-tagged 8 Ras and Rho proteins are expressed in the baculovirus-insect cell system and purified by immunoaffinitychromatography on a protein G-Sepharose column conjugated to an anti-Glu-Glu monoclonal antibody as described in [2] in Volume 255 of this series. To study smgGDS-catalyzed [3H]GDP release, processed and unprocessed Ras and RhoA (400 nM, 8/xg/ml) are loaded with [3H]GDP in a buffer containing 20 mM Tris-HC1, pH 7.5, 50 mM NaC1, 5 mM MgC12, 1 mg/ml bovine serum albumin (BSA), 1 mM DTT, 10 mM EDTA, 1 tzM [3H]GDP (31.2 Ci/mmol), and 0.05% (w/v) sodium cholate. After 10 min 7D. B. Smith and K. S. Johnson, Gene 67, 31 (1988). 8T. Grussenmeyer,K. H. Scheidtmann,M. A. Hutchinson,W. Eckhart, and G. Walter,Proc. Natl. Acad. Sci. U.S.A. 82, 7952 (1985).
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87
at room temperature, 10 mM MgC12 is added and the tubes are kept at room temperature for a further 5 rain and then placed on ice until use. The dissociation assay is initiated by aliquoting 20/zl (8 pmol) of the [3H]GDP-Ras mixture into tubes containing 20/zl of 20 mM Tris-HCl, pH 7.5, 50 mM NaC1, 5 mM MgC12, 1 mg/ml BSA, 1 mM DTT, 0.05% (w/v) sodium cholate, 0.1 mM GTPTS, and the required amount of GSTsmgGDS. The final Ras concentration is 200 nM. Assays are incubated for the required time, typically for 30 min, at room temperature and are stopped by adding 1 ml of ice-cold 20 mM Tris-HC1, pH 7.5, 50 mM NaCI, and 5 mM MgC12. The resulting mixture is filtered through a nitrocellulose filter (pore size 45/.~m) using a 1225 sampling manifold apparatus (Millipore). Filters are washed with 10 ml of ice-cold 20 mM Tris-HC1, pH 7.5, 50 mM NaCI, and 5 mM MgC12, dried, and counted in a scintillation counter to determine the radioactivity remaining bound to Ras. The activity of smgGDS is expressed as a decrease in the radioactivity trapped on the filters compared to control assays without smgGDS. In the conditions of this assay the dissociation of prebound [3H]GDP is linear for 60 min and proportional to the amount of smgGDS up to 120 pmol (3/~M). Importantly, smgGDS fails to stimulate [3H]GDP release from K-Ras4B[3H]GDP when unlabeled GTPTS is omitted from the reaction. smgGDS appears to be extremely sensitive to the concentration and type of detergent used in the assay. We find that 0.05% sodium cholate, which is necessary for preventing prenylated Ras proteins from denaturing, does not affect smgGDS activity but that the addition of 0.1% sodium cholate can result in significant inhibition of the exchange reaction (Fig. 1).
[3H]GTP BindingAssay To study smgGDS-catalyzed [3H]GTP binding, Ras or Rho (8/.~g/ml, 400 nM) is equilibrated at room temperature for 10 min in a buffer including 20 mM Tris-HC1, pH 7.5, 50 mM NaC1, 5 mM MgCI2, 1 mg/ml BSA, 1 mM DTT, and 0.05% sodium cholate. The binding assay is initiated by aliquoting 20/~1 of the Ras mixture into tubes containing 20/~1 of 20 mM Tris-HC1, pH 7.5, 50 mM NaC1, 5 mM MgCI2, 1 mg/ml BSA, 1 mM DTT, 0.05% sodium cholate, 1 /~M [3H]GTP (31.5 Ci/mmol), and the required amount of smgGDS protein. The final Ras concentration in the assays is 200 nM, so that the concentration of [3H]GTP is fivefold higher than that of the guanine nucleotide-binding protein. The binding assays are carried out for the required times at room temperature and are stopped by adding 1 ml of ice-cold 20 mM Tris-HC1, pH 7.5, 50 mM NaC1, and 5 mM MgC12. The radioactivity bound to Ras is determined using a nitrocellulose filterbinding method identical to that described above for the [3H]GDP dissocia-
88
GUANINE NUCLEOTIDE EXCHANGE
[ 10]
120 100 &
:i 8o -~
6O
~
20
},o h.
~
o 0
10
20
30
40
50
60
Time (min)
Fro. 1. Effect of sodium cholate concentration on smgGDS promoted guanine nucleotide exchange on prenylated K-Ras4B. K-Ras4B (200 nM, 8 pmol) was loaded with [3H]GDP and incubated with or without smgGDS (600 nM, 24 pmol) for the indicated times at room temperature. At the end of the incubation the radioactivity remaining bound to Ras was determined by a nitrocellulose filter assay. Results are expressed as percentages of the radioactivity remaining bound to Ras at the end of the incubation compared to the radioactivity bound to Ras at zero time. Open symbols, K-Ras4B alone, solid symbols, K-Ras4B + smgGDS. ©, e : 0.03% sodium cholate; A, A: 0.05% sodium cholate; and [], I1: 0.1% sodium cholate.
tion assays. The activity of smgGDS is expressed as the increase in radioactivity trapped on the filters compared to control assays carried out without smgGDS. Whereas no significant [3H]GTP binding to prenylated K-Ras4B is detected in the absence of smgGDS, under the conditions of this assay, the amount of radioactivity bound to K-Ras4B in the presence of smgGDS increases in a time-dependent manner up to 1 hr. Analysis of smgGDS Activity in Vivo We use a modification of the method of Downward et aL 9 to study the activity of smgGDS on Ras transiently expressed in COS1 cells. COS1 Cell Transfection by Electroporation smgGDS and ras cDNAs are cloned into the pEXV3 mammalian expression vector. 1° COS1 cells, grown to 75% confluence in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) donor calf serum and 1% 9 j. Downward, J. D. Graves, P. H. Warne, S. Rayter, and D. A. Cantrell, Nature 346, 719 (1990). 10j. Miller and R. N. Germain, J. Exp. Med. 164, 1478 (1986).
[101
ANALYSISOVsmgGDS ACTIVITY
89
L-glutamine, are harvested by trypsinization, washed twice in electroporation buffer (HeBS: 20 mM Hepes, pH 7.05, 137 mM NaC1, 5 mM KC1, 0.7 mM NazHPO4, 6 mM dextrose), and counted. Cells (2 × 106) are resuspended in 0.24 ml HeBS, and 10 ~g of each plasmid D N A plus 100 p~g of sonicated salmon testes D N A are added to a total volume of 20/~1. The suspension (260 t~l, final volume) is dispensed into a 0.4-cm Bio-Rad electroporation cuvette and is pulsed at 250 V/125 txF (giving a time constant of approximately 0.6 msec) using a Gene Pulser (Bio-Rad) apparatus. Cells are allowed to rest for 10 min before seeding in a 6-cm plate.
Labeling and Extraction of Ras-Bound Guanine Nucleotides Following transfection (72 hr), the culture medium is aspirated and the plates are washed three times with phosphate-free minimum essential medium (MEM). Cells are labeled for 4 hr with 0.5 mCi/ml of ortho[3ZP] phosphate in phosphate-free MEM (2 ml/plate), then lysed using 1 ml of 50 mM HEPES, pH 7.4, 1% Triton X-100, 100 mM NaC1, 5 mM MgC12, 0.1% BSA, 10 mM benzamidine, 1 mM Pefabloc, and 10 tzg/ml of each leupeptin, aprotinin, and soybean trypsin inhibitor. Nuclei and insoluble material are removed by centrifugation at 15,000g at 4 ° for 2 min; the lysate is then cleared by adding 50 tzl of a 20% protein A-Sepharose (PAS) slurry followed by 100/xl of 5 M NaC1, 100 tzl of 5% (w/v) sodium deoxycholate, and 0.5% (w/v) sodium dodecyl sulfate. Tubes are rotated at 4° for 15 min and briefly centrifuged. The supernatant is transferred into fresh tubes containing 100 ~1 of a 10% slurry of PAS coupled to the Y13-259 anti-Ras monoclonal antibody by a rabbit anti-rat immunoglobulin (see below). Immunoprecipitations are rotated at 4 ° for 45 rain and are washed eight times with 1 ml of 50 mM HEPES, pH 7.4, 500 mM NaCI, 5 mM MgCI2, 0.1% Triton X-100, and 0.005% sodium dodecyl sulfate. After the final wash the nucleotide bound to Ras is eluted with 35 tzl of 2 mM EDTA, pH 8, 2 mM DTT, 0.2% sodium dodecyl sulfate, 0.5 mM GDP, and 0.5 mM GTP at 68° for 20 min. Ras-bound guanine nucleotides are resolved by analyzing 15 /~1 of the eluate by thin-layer chromatography on polyethyleneimine (PEI)-cellulose. Plates are developed for 2 hr in 1 M LiC1, dried, and autoradiographed. The radioactivity associated with each guanine nucleotide species is quantified using an AMBIS scanner. The activity of smgGDS is expressed as an increase of the ratio of the radioactivity detected in Rasbound GTP to the total radioactivity in Ras-bound guanine nucleotides.
Preparation of PAS-Coupled Y13-259 Monoclonal Antibody To couple Y13-259 to PAS, PAS (2 ml packed volume) is mixed with 0.5 ml of rabbit anti-rat immunoglobulin (1 mg/ml) in 10 ml of PBS for 4
90
GUANINE NUCLEOTIDE EXCHANGE
[1 1]
hr at 4 °. The beads are collected by centrifugation, washed, and then mixed with 0.5 ml of Y13-259 monoclonal antibody (7 mg/ml) for 12-18 hr at 4°. The PAS-Y13-259 beads are collected, washed with 250 vol of PBS, and stored at 4° as a 10% slurry until use.
[ 1 1] I n t e r a c t i o n o f E c t 2 a n d D b l w i t h Rho-Related GTPases By TORU MIKI Introduction The ect2 and dbl oncogenes encode proteins containing Dbl homology (DH) domains. The ect2 oncogene was isolated as a transforming gene from the mouse epithelial cell line BALB/MK, 1 by an expression cDNA cloning strategy,a Transfection of a BALB/MK cDNA library into NIH 3T3 cells led to the isolation of three morphologically distinct foci of transformed cells. Transforming cDNA clones rescued from cells of each focus were designated ectl, ect2, and ect3 (epithelial cell transforming genes). Structural analyses revealed that ectl encodes the keratinocyte growth factor receptor, 1,3 ect2 encodes a novel oncogene,4 and ect3 represents an unprocessed N-ras transcript containing three introns. The transforming ect2 cDNA encodes a truncated product relative to the ect2 protooncogene product. Thus, removal of the N-terminal domain of the proto-ect2 product activates its transforming activity.4 The dbl oncongene was originally isolated by transfection of NIH 3T3 cells with DNA of a human diffuse B-lymphoma.5 Dbl can be also activated by N-terminal truncation, although overexpression of proto-dbl results in weak transforming activity.6 When dbl was isolated, no related molecules 1 T. Miki, T. P. Fleming, D. P. Bottaro, J. S. Rubin, D. Ron, and S. A. Aaronson, Science 251, 72 (199l). 2 T. Miki, T. P. Fleming, M. Crescenzi, C. J. Molloy, S. B. Blam, S. H. Reynolds, and S. A. Aaronson, Proc. Natl. Acad. Sci. U.S.A. 88, 5167 (1991). 3 T. Miki, D. P. Bottaro, T. P. Fleming, C. Smith, W. H. Burgess, A. M.-L. Chan, and S. A. Aaronson, Proc. Natl. Acad. Sci. U.S.A. 89, 246 (1992). 4 T. Miki, C. L. Smith, J. E. Long, A. Eva, and T. P. Fleming, Nature 362, 462 (1993). 5 A. Eva and S. A. Aaronson, Nature 316, 273 (1985). 6 D. Ron, S. R. Tronick, S. A. Aaronson, and A. Eva, E M B O Z 7, 2465 (1988),
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
[1 1]
BINDING OF Ect2 AND D b l TO RhO-LIKE G T P a s e s
91
w e r e k n o w n . H o w e v e r , a c c u m u l a t i o n of a n u m b e r o f n e w s e q u e n c e s in t h e d a t a b a n k s finally a l l o w e d i d e n t i f i c a t i o n of t w o g e n e s with significant s i m i l a r i t y to dbl: a b r e a k p o i n t c l u s t e r gene, bcr, a n d a y e a s t cell cycle c o n t r o l gene, C D C 2 4 . 7 Y e a s t c d c 2 4 m u t a n t s can b e s u p p r e s s e d b y t h e o v e r e x p r e s s i o n of C D C 4 2 , w h i c h e n c o d e s a R h o f a m i l y m e m b e r o f s m a l l G T P a s e s . 8 B i o c h e m i c a l a n a l y s e s of t h e D b l p r o t e i n r e v e a l e d t h a t it c a t a l y z e s guanine nucleotide exchange on Cdc42Hs, the human homolog of the yeast C D C 4 2 g e n e p r o d u c t . 9 T h e s e results suggest t h a t D H d o m a i n s can i n t e r a c t with the Rho family GTPases. W e h a v e f o u n d t h a t D b l a n d E c t 2 can a s s o c i a t e with subsets o f R h o f a m i l y p r o t e i n s . A n u m b e r o f m o l e c u l e s , i n c l u d i n g Cdc24, Bcr, D b l , Vav, Ect2, Sos, R a s - G R F , A B R , L B C , T I M , N E T , T i a m - 1 , a n d Ost, c o n t a i n D H d o m a i n s b u t t h e s e s e q u e n c e s a r e o t h e r w i s e diverse. 4,7,1°-18 F o r instance, V a v also c o n t a i n s S H 2 a n d S H 3 d o m a i n s , 19'2° a n d B c r p o s s e s s e s d o m a i n s for s e r i n e / t h r e o n i n e k i n a s e a n d R a c - G A P activities as well as s e r i n e - r i c h s e q u e n c e s t h a t can b i n d to t h e A B L o n c o g e n e p r o d u c t . 21,22 T h e r e f o r e , D H c o n t a i n i n g m o l e c u l e s m a y p l a y r o l e s in v a r i o u s c e l l u l a r functions. Since D b l a n d Ect2 can a s s o c i a t e with c e r t a i n R h o f a m i l y p r o t e i n s b u t d o n o t c a t a l y z e n u c l e o t i d e e x c h a n g e in vitro, a s s o c i a t i o n o f s m a l l G T P a s e s with t h e i r r e g u l a t o r s m a y i n v o l v e m o r e g e n e r a l f u n c t i o n s as well. T h e m e t h o d s u s e d to a n a l y z e t h e s e i n t e r a c t i o n s a r e d e s c r i b e d h e r e in detail.
7 D. Ron, M. Zannini, M. Lewis, R. B. Wickner, L. T. Hunt, G. Graziani, S. R. Tronick, S. A. Aaronson, and A. Eva, New Biol. 3, 372 (1991). s A. Bender and J. R. Pringle, Proc. Natl. Acad. Sci. U.S.A. 86, 9976 (1989). 9 M. J. Hart, A. Eva, T. Evans, S. A. Aaronson, and R. A. Cerione, Nature 354, 311 (1991). 10j. M. Adams, H. Houston, J. Allen, T. Lints, and R. Harvey, Oncogene 7, 611 (1992). 11A. Musacchio, T. Gibson, P. Rice, J. Thompson, and M. Saraste, Trends Biochem. Sci. 18, 343 (1993). 12C. Shou, C. L. Farnsworth, B. G. Neel, and L. A. Feig, Nature 358, 351 (1992). 13N. Heisterkamp, V. Kaartinen, S. van Soest, G. M. Bokoch, and J. Groffen, J. Biol. Chem. 268, 16903 (1993). 14D. Toksoz and D. A. Williams, Oncogene 9, 621 (1994). 15A. M.-L. Chan, E. S. McGovern, G. Catalano, T. P. Fleming, and T. Miki, Oncogene 9, 1057 (1994). 16A. M.-L. Chan, S. Takai, K. Yamada, E. S. McGovern, and T. Miki, submitted for publication. t7 G. G. M. Habets, E. H. M. Seholtes, D., Zuydgeest, R. A. van der Kammen, J. C. Stam, A. Berns, and J. G. Collard, Cell 77, 537 (1994). 18y. Horii, J. F. Beeler, K. Sakaguchi, M. Tachibana, and T. Miki, EMBO J. 13, 4776 (1994). 19X. R. Bustelo, J. A. Ledbetter, and M. Barbacid, Nature 356, 68 (1992). 2o B. Margolis, P. Hu, S. Katzav, W. Li, J. M. Oliver, A. Ullrich, A. Weiss, and J. Schlessinger, Nature 356, 71 (1992). 21 y. Maru and O. N. Witte, Cell 67, 459 (1991). 22 D. Diekman, S. Brill, M. D. Garrett, N. Totty, J. Hsuan, C. Monfries, C. Hall, L. Lira, and A. Hall, Nature 351, 400 (1991).
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GUANINE NUCLEOTIDEEXCHANGE
Baculovirus-infectedSf9 cell extract
[1 l]
GlutathioneSepharose 4B
1
Dialyze
1
Pre-clearby GST beads 1 Incubatewith fusion protein beads
~
[
GST Small GTPase
Collectand wash beads SDS-PAGE
1 ' Blotting I
Detectwith anti-FLAGmAb
0
Ect2
FLAG epitope
" ~ Anti-FLAG mAb 1
FIG. 1. Principle of the method to detect specific association of small GTPases and Ect2. A brief procedure is shown on the left and the principle is schematically shown on the right.
Principles of M e t h o d Figure 1 shows principles of the method to detect association of small GTPases with their regulators. Small GTPases are expressed as fusion proteins with glutathione S-transferase (GST) and purified by affinity chromatography on glutathione-Sepharose. 23 This procedure can be performed using commercially available systems and also as described in [1] in this volume. DH-containing proteins are expressed as F L A G fusion proteins in a baculovirus system. 24 F L A G is composed of eight hydrophilic amino acid residues, 25 and oligonucleotides encoding this sequence can be inserted into expression vectors. The F L A G portion of the expressed fusion proteins is usually detected by commercially available anti-FLAG monoclonal antibodies ( a F L A G MAb). Extracts of baculovirus-infected insect cells are first "precleared" using G S T beads to reduce background and then incubated with G S T - G T P a s e fusion proteins. The complexes formed are precipitated by glutathione-Sepharose and separated by polyacrylamide gel electrophoresis. The protein bands are blotted onto nitrocellulose membranes and probed with a F L A G MAb. 23D. B. Smith and K. S. Johnson, Gene 67, 31 (1988). 24M. D. Summers and G. E. Smith, Texas Exp. Station Bulletin N.1555 (1987). 25K. S. Prickett, D. C. Amberg, and T. P. Hopp, Biotechniques 7, 580 (1989).
[11]
BINDINGOF Ect2 AND Dbl TO RhO-LIKEGTPases
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Expression of Rho Family GTPases in E s c h e r i c h i a coli Because coding sequences of small GTPases are relatively short, we amplified the sequences, cloned them in our prokaryotic vector containing the GST coding sequence, and then verified the sequences. 4 GST vectors 23 are also commercially available. GST fusion proteins can be purified from lysates of E. coll. In our hands, various Rho family proteins show different solubilities, although their structures are very similar. Figure 2 shows purified preparations of six Rho family proteins. Cdc42 is the most soluble, whereas RhoC and RhoG are mostly insoluble. Preparations of insoluble proteins usually contain a soluble, nonfused GST protein which may arise from premature translation termination (Fig. 2; lower bands). Since the free GST protein is very soluble, it often contaminates the preparations, even though present in very low concentrations. In order to accurately compare the ability of GST fusion proteins to bind GTPases, the concentration of the former should be carefully determined, taking into account any free GST protein. Standardization can also be achieved by determining the GTP-binding capacity of each preparation and adjusting the concentration accordingly. Expression of DH Domain-Containing Protein in Insect Cells Since biochemical analyses of Dbl were carried out using baculovirusexpressed protein, 9 we used this system to express other DH-containing proteins. These proteins may require post-translational modification, such as phosphorylation that mainly occurs in eukaryotic cells, for their activities. For example, R a s - G R F contains a DH domain as well as a Cdc25 homology
# o_o
Mr(K) 97.4 69463021.5 14.3 -
FIG.2. Preparationsof the Rho-relatedsmallGTPases expressed as GST fusionproteins in E. coli and purified with glutathione-Sepharoseaffinitychromatography.Lower bands represents premature translationterminationproducts (see text).
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GUANINENUCLEOTIDEEXCHANGE
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domain. It has been reported that the DH domain of Ras-GRF expressed in E. coli does not possess exchange activity on several Rho family proteins, whereas the Cdc25 homology domain expressed in a same manner efficiently catalyzes guanine nucleotide exchange on Ras proteins. I2 When specific antibodies are available, epitope tagging is obviously not necessary. However, detection with otFLAG MAb is usually more specific. Furthermore, the addition of short stretches of amino acids at either N or C termini does not affect the binding capability of DH-containing proteins. Other types of epitope tagging may be used. Therefore, DNAs for epitopetagged DH domain-containing proteins are preferably cloned in a baculoviral expression vector. The expression vector is cotransfected with linearized baculoviral DNA into insect cells to produce recombinant virus. A large culture of insect cells is infected with the recombinant virus and incubated for 60-64 hr. Expression of FLAG-tagged DH domain-containing proteins is monitored by immunoblotting of cell extracts.
Detection of Interaction of DH Domain-Containing Proteins with Rho Family Proteins Glutathione-Sepharose-immobilized GST-Rho fusion proteins are used to precipitate DH-containing proteins. The beads are first washed and then the proteins bound to the beads are analyzed by polyacrylamide gel electrophoresis following by immunoblotting. This procedure is described below in detail. A highly sensitive small-scale immunoblotting method using precast minigels and chemiluminescent detection is also described. Reagents HNTG: 20 mM HEPES, pH 7.0, 150 mM NaC1, 0.1% Triton X-100, 10% (v/v) glycerol HNTG-PI: 1 ml HNTG + 20/zl phenylmethylsulfonyl fluoride (10 mM in methanol) + 10/xl each of leupeptin (10 mg/ml), aprotinin (10 mg/ml), and pepstatin (1 mg/ml methanol) Precast minigels, minielectrophoresis apparatus and blotting module (e.g., NOVEX) 2× sample buffer: 4% SDS, 200 mM dithiothreitol, 120 mM Tris-HCl, pH 6.8, 0.002% bromophenol blue Transfer buffer: 12 mM Tris, 96 mM glycine, 20% (v/v) methanol, pH 8.3 PBS-T: phosphate-buffered saline containing 0.05% Tween 20 PBS-TM: 3% (w/v) Nonfat dry milk in PBS-T
[111
BINDINGOF Ect2 AND Dbl TO RhO-LIKEGTPases
95
Procedure 1. Transfer 0.5 ml of glutathione-Sepharose CL-4B (GSH beads; Pharmacia) to a microcentrifuge tube and wash three times with HNTG. The beads can be collected by low-speed centrifugation and supernatants aspirated by using capillary pipette tips. Resuspend the beads in 0.5 ml of HNTG by gentle inversion. 2. Prepare GST beads and GST-X (fusion protein) beads as follows: Into a tube containing 200/xl of GSH beads, add 10/xg of GST protein or the same number of moles of GST-X protein. Mix the suspensions by continuously inverting the tubes at 4° for 1 hr. Wash with 0.5 ml of HNTG three times. Resuspend the beads in 200/xl of HNTG. 3. Preclear lysates as follows: In a microcentrifuge tube, mix 250/zl of HNTG-PI and 25/xl of lysate, and 50/xl of GST beads. Invert at 4° for 2 hr. Centrifuge at 14,000 rpm for 5 min and transfer the supernatant into a microcentrifuge tube. 4. Centrifuge the supernatant again and carefully transfer 100-/xl aliquots to two microcentrifuge tubes labeled as " G " and "X". Add 50/xl of GST beads and GST-X beads to tubes labeled " G " and "X", respectively. Invert the tubes at 4° for 2-4 hr. 5. Centrifuge at 2000 rpm for 2 min at 4° and remove the supernatants by aspiration. Resuspend the beads in 0.5 ml of HNTG-PI. Repeat the washing step three times. 6. Carefully remove all the supernatant and resuspend the beads in 50/xl of 2× sample buffer. In a tube labeled "L", mix 10/xl of precleared lysate and 10/xl of 2× sample buffer. 7. Heat the samples "L", "G", and "X" at 100° for 3 rain in a heating block. Load 20/xl of the protein samples on 10-well gels. For molecular weight standards, load 2 /xl of Rainbow markers (Amersham). Run at 50 V (for 6 hr for 8-16% gradient minigels). 8. Soak a sheet of nitrocellulose paper in transfer buffer for 20 min. Wash the gel in transfer buffer for 10 rain. Transfer the proteins to a nitrocellulose paper in a miniblotting module at 30 V for 3 hr in an ice bath. 9. Mark the protein side of the membrane with a marking pen. Incubate the blot in PBS-TM at room temperature for 2 hr on a rocking shaker. 10. In a shallow tray with a lid, place a sheet of Parafilm with its cover sheet (cover sheet side up) and attach the film inside the tray by scraping both ends. Peel off the cover sheet and place 0.5 ml of PBS-TM containing a primary antibody on the Parafilm. Drain excess PBS-TM from the blot using a sheet of filter paper but do not dry the blot. Place the blot on the surface of the antibody solution (protein side down). Incubate at room temperature for 1 hr.
96
GUANINE NUCLEOTIDE EXCHANGE
[l
1]
11. Briefly wash the blot with PBS-TM. Wash with PBS-TM for 5 min on a rocking shaker three times. 12. Incubate the blot in 0.5 ml of PBS-TM to which was added 1/xl of horseradish peroxidase-conjugated anti-rabbit or mouse IgG (Amersham) for 1 hr as in step 10. 13. Wash the blot briefly with PBS-TM and then for 15 min, twice. Wash the blot three times with PBS-T for 5 min. 14. In order to orient the blot following chemiluminscent detection, place pieces of fluorescent tape on a used X-ray film. Wrap the film in Saran wrap, or any plastic film, and place it in a X-ray cassette. 15. In a dark room, drain the excess buffer from the washed blot and place it in a new container. Add 10 ml of the ECL detection reagent (Amersham) on the blot. Wait for 1 min. 16. Drain excess solution. Place the blot, protein side up, on the marked X-ray film in the cassette. Cover the blot with a sheet of Saran wrap and gently smooth out air pockets. 17. Switch off the lights and place a sheet of X-ray film on top of the blot. Close the cassette and expose for 30 sec. Develop the film. Examples of an association assay are shown in Fig. 3. To obtain reliable data, the presence of equal amount of small GTPases in each lane should be confirmed. This can be carried out by reprobing the blot with antibodies to the small GTPase used or with anti-GST antibodies. Because horseradish peroxidase is not active after overnight incubation, blots can be reprobed following blocking with PBS-TM.
Beads I
I
~1- Ect2
~1- Dbl FIG. 3. Detection of specific association of Ect2 or Dbl with Rho family proteins. Baculovirus-expressed Ect2 and Dbl were precipitated with Rho family GTPase beads as indicated and detected with anti-FLAG and anti-Dbl antibodies, respectively.
[11]
BINDINGOF Ect2 AND Dbl TO RhO-LIKEGTPases
97
Nucleotide I
I
Beads Racl
Cdc42
~'
~
41--Dbl
FIG. 4. Effect of the presence of guanine nucleotides for the association of Dbl with Racl or Cdc42. Nucleotide was added to the reaction mixture to a concentration of 0.5 mM and a binding assay was performed. ( - ) , no nucleotide; others, a mixture of CTP, ATP, and UTP.
Dissociation of DH Domain-Containing Protein/GTPase Complex by Addition of Guanine Nucleotides We have previously shown that the Dbl-Cdc42 complex was dissociated when GTP or GDP was added to the reaction mixture, whereas such a dissociation was not observed in the case of a Dbl-Racl complex. Dbl has been reported to catalyze guanine nucleotide exchange on Cdc42, but has very little or no activity on Racl.9 Therefore, guanine nucleotide-dependent dissociation of the Dbl-Cdc42 complex might reflect exchange activity because exchange factors should be released from GTPases after the exchange reaction. Thus, examination of the guanine nucleotide sensitivity of binding provides straightforward assays for the exchange activity. This can be performed by adding guanine nucleotides to a final concentration of 0.5 mM in the reaction mixtures. Examples of such assays are shown in Fig. 4.
Applications A growing number of DH domain-containing proteins have been discovered. These proteins are encoded by oncogenes or other growth regulatory genes. Since these molecules are implicated as regulators of Rho family GTPases, it will be important to determine whether these molecules can associate with members of the Rho or other subfamilies of GTPases.
98
GUANINENUCLEOTIDEEXCHANGE
1121
Once the interaction is demonstrated, it will become possible to study the regulatory effect on the GTPase and the biological consequences in vivo. Acknowledgment The author thanks Dr. Steven R. Tronick for support and critical reading of this manuscript.
[12] S o l u b i l i z a t i o n o f C d c 4 2 H s f r o m M e m b r a n e s by Rho-GDP Dissociation Inhibitor By DAVID A. LEONARD and RICHARD A. CER1ONE Introduction The discovery of proteins that can prevent the dissociation of GDP from low molecular weight GTPases added a third class of GTP binding/ GTPase regulatory factors to the two that had been previously known. GDP dissociation inhibitors (GDIs) have been discovered for the Rho subfamily ( R h o - G D I ) and the Rab subfamily (Rab-GDI), but a GDI has not been identified for any member of the Ras or Arf subfamilies thus far. Interestingly, the R a b - G D I and the R h o - G D I , unlike many exchange factors and GTPase-activating proteins (GAPs), show little discrimination within their respective target GTPase subfamilies, although there is no crossover to other subfamilies. Despite the lack of any apparent homology between R a b - G D I ( - 5 5 kDa) and R h o - G D I ( - 2 7 kDa), they both require that the target GTPase be modified at the carboxyl terminus with a geranylgeranyl moiety for a functional interaction, m Inhibition of GDP dissociation is not the only activity exhibited by the members of this class of regulatory proteins. It was known from early studies of the interaction of R h o - G D I with the RhoA protein that the GDI interfered with ability of RhoA to associate with membranes. 3 Later work demonstrated that the same GDI could also prevent the Cdc42Hs protein from associating with lipid vesicles, and also was able to solubilize Cdc42Hs from its native location in biological membranes. 1 Up to 50% of endogenous Cdc42Hs from A431 human adenocarcinoma or human placental membranes could be sequestered to the soluble phase simply by a D. Leonard, M. J. Hart, J. V. Platko, A. Eva, W. Henzel, T. Evans, and R. A. Cerione, J. Biol. Chem. 267, 22860 (1992). 2 S. Araki, K. Kaibuehi, T. Sasaki, Y. Hata, and Y. Takai, Mol. Cell. Biol. 11, 1438 (1991). 3 M. Isomura, A. Kikuehi, N. Ohga, and Y. Takai, Oncogene 6, 119 (1991).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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the addition of purified R h o - G D I from bovine brain. This extraction activity seemed to be independent of whether GTP or GDP was bound to the GTPase. While little is known about the in vivo function of GDIs, some evidence supports the notion that this solubilization activity is an important biological phenomenon. Specifically, it has been shown that a large percentage of certain members of the Rab and Rho subfamilies are located in the cytoplasm, despite the demonstration that they are isoprenylated. It has been further shown that the cytoplasmic populations of these GTPases are complexed to their respective GDIs. 4 It has been suggested, therefore, that the GDIs serve to regulate the localization of the GTP-binding proteins, in some cases sequestering them in the cytoplasm and in other cases releasing them to the membranes where they encounter, perhaps, another regulatory factor or an effector target. Some of the investigations described earlier have required the development of a suitable in vitro assay to measure the ability of R h o - G D I to solubilize the Cdc42Hs GTP-binding protein. The procedures used in this chapter allow the purification of the necessary components and the reproduction of the assay protocol.
Assay for Solubilization of Cdc42Hs from Biological Membranes The assay as it was originally described 1 made use of R h o - G D I purified from bovine brain. The GDI is an abundant protein in this source and is easily purified by several simple column chromatography steps. However, we have shown that a glutathione S-transferase (GST)/Rho-GDI fusion protein ( G S T - R h o - G D I ) is fully capable of substituting for the native brain GDI in this assay. Procedures for isolating both the brain and the recombinant protein are given here. Purification o f Bovine Brain R h o - G D I
A single bovine brain is prepared by removal of the cerebellum and any blood clots, and is then cut into small pieces. These pieces are homogenized in a Waring blender (2 × for 10 see each) in -300 ml ice-cold 20 mM Tris-HCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), pH 8.0, 0.25 mM phenylmethylsulfonyl fluoride (PMSF), 5 txg/ml aprotinin, 5 /zg/ml leupeptin, and 2 tzg/ml pepstatin A. This lysate is then clarified by centrifugation in a Beckman L8-70M ultracentrifuge for 30 min at 30,000g and 4°. The R h o - G D I is purified from the cytosolic fraction by ammonium sulfate 4R. Regazzi,A. Kikuchi,Y. Takai, and C. B. Wollheim,J. BioL Chem. 267, 17512 (1992).
100
GUANINE NUCLEOTIDE EXCHANGE
[12]
precipitation. The supernatant is brought to 40% saturation with ammonium sulfate, and after a 30-rain incubation at 4°, the sample is spun at ll,300g for 20 min at 4° in a Beckman JA-20 rotor. The supernatant of this spin contains the Rho-GDI, which is then precipitated by bringing the sample to 80% saturation of ammonium sulfate. The previous incubation and spin are repeated, but the pellet is now resuspended in - 1 0 ml of 20 mM Tris-HC1, 1 mM EDTA, 1 mM DTT, pH 8.0 (buffer A). This fraction is then dialyzed against a 1.5-liter total of the same buffer (three changes for a total of at least 20 hr). The dialyzed sample is then applied to a DEAE-Sephacel column (2.5 × 10 cm), previously equilibrated with buffer A. After washing this column with the equilibration buffer until no protein is detected in the eluent, a 300-ml gradient (0-300 mM NaC1 in buffer A) is used to elute the bound protein. Fractions are assayed for GDI activity (see below) and those which test positive are pooled together. The center of the R h o - G D I peak is typically at --80 mM NaC1. In order to prepare for the next column step, the pooled fractions are again dialyzed against buffer A as described earlier. The dialyzed sample is then applied to a Pharmacia Mono Q column (HR 5/5) equilibrated with buffer A. This column is washed with the same buffer until a stable baseline is achieved, and then a 20-ml gradient (0-300 mM NaC1 in buffer A) is used to elute the bound protein. Once again, fractions that contain GDI activity are pooled. In this case, -150 mM NaC1 is necessary to elute Rho-GDI. The final purification step is cation-exchange chromatography on the Pharmacia Mono S column (HR 5/5). Because of the nature of the interaction of R h o - G D I and this column matrix, it is necessary to dialyze the Mono Q pool against a low pH buffer (buffer B). This buffer contains 1.5 liters of 20 mM potassium phosphate, 1 mM EDTA, 1 mM DTT, pH 5.8 (three changes for a total of at least 20 hr). The dialyzed sample is applied to the Mono S column equilibrated with buffer B. The column is washed with the same buffer until a stable baseline is achieved, and then a gradient (25 ml, 0-300 mM NaC1 in buffer B) is used to elute the bound protein. At this stage, it is typically not necessary to test fractions with the standard GDI assay. Instead, aliquots of the Mono S fractions are analyzed by SDS-PAGE on a 10% acrylamide gel. The R h o - G D I should elute from the column at -100 ml NaC1 and will be visible on the gel as a single band at 27 kDa. (Note: the predicted molecular mass of R h o - G D I is 23,421 Da, 5 but it appears to migrate slower than predicted.) 5 y . Fukumoto, K. Kaibuchi, Y. Hori, H. Fujioka, S. Araki, T. Ueda, A. Kikuchi and Y. Takai, Oncogene 5, 1321 (1990).
[ 121
SOLUBILIZATIONOF Cdc42Hs
101
The R h o - G D I can now be used in both GDP dissociation inhibition assays and Cdc42Hs solubilization assays, even in the low pH buffer. However, it may be desirable to dialyze the pure protein against buffer A for long-term storage. The pure protein can be frozen at - 2 0 ° for at least 2 months, and it will still retain good activity in both assays.
Assay of Inhibition of GDP Dissociation from Cdc42Hs by Rho-GDI In order to purify R h o - G D I from bovine brain, an assay must be used to detect its activity in column fractions. The inhibition of GDP dissociation from Cdc42Hs represents a quick and easy assay for this purpose. In order for this assay to be effective, however, it is necessary to use geranylgeranylated Cdc42Hs, as the R h o - G D I requires this modification on the GTPbinding protein for functional coupling. The protocol for the purification of prenylated Cdc42Hs has been described. 6 Geranylgeranylated Cdc42Hs (5-50 ng) is first combined with 0.625 /zM GTP supplemented with [a-32p]GTP (90,000 cpm/pmol) in a buffer containing 20 mM Tris-HC1, pH 7.5, 100 txM DTT, 10 mM MgCI2, 50 mM NaC1, and 100/xM 5'-adenylylimido-diphosphate (AMP-PNP) (total volume 48 /xl). This binding reaction is allowed to incubate for 25 rain at room temperature, during which time the Cdc42Hs is saturated with nucleotide and also hydrolyzes all of the bound GTP to GDP. During this incubation, 24/zl of the fraction to be assayed is incubated with 3/zl of a reaction mixture containing 16 mM Tris-HCl, pH 7.5, 100/zM DTT, 40 mM NaC1, 2.5 mM EDTA, 200/zM AMP-PNP, 200/~M GDP, and 100/zg/ ml bovine serum albumin (final volume 27 ~1). After the binding reaction has proceeded for 25 min, a 3-/,1 aliquot is added to the GDI sample for a total of 30 tM. This combination is allowed to incubate for an additional 6 min at room temperature. During this incubation, the radiolabeled GDP will exchange completely with the "cold" GDP in the reaction mixture, but only if there is no R h o - G D I present. The samples are diluted with 500 /xl of ice-cold 20 mM Tris-HC1, pH 8.0, 10 mM MgC12, and 100 mM NaCI and are filtered on BA-85 nitrocellulose filters (Schleicher & Schuell). The filters are washed three times with the dilution buffer, and then are counted in Liquiscint (National Diagnostics). Filters with radioactivity higher than background indicate the presence of R h o - G D I in that sample (see Fig. 1).
Purification of Recombinant GST-Rho-GDI from Escherichia coli The R h o - G D I can be purified as a glutathione S-transferase fusion protein from E. coli. This procedure requires the pGEX-KG plasmid with 6 M. J. Hart, K. Shinjo, A. Hall, T. Evans, and R. A. Cerione, J. BioL Chem. 266, 20840 (1991).
102
GUANINENUCLEOTIDEEXCHANGE
[ 121
40-
q~ 30fO
rn ¢:
20-
Q.
~,
&
10-
l/ I
i
i
i
0
1
2
3
GST-GDI
(gM)
FIG. 1. Dose-dependent inhibition of G D P dissociation from Cdc42Hs by R h o - G D I . The indicated amounts of R h o - G D I were incubated with [a-32p]GDP-bound Cdc42Hs in the presence of E D T A as described in the text. The activity represents the amount of [a-32p]GDP still bound to Cdc42Hs after a 6-min incubation, as detected by filtration on nitrocellulose.
the R h o - G D I gene inserted into the polylinker region. A suitable strain of E. coli, such as JM101 transformed with this plasmid, can yield several milligrams of pure G S T - R h o - G D I per liter of LB media. A single colony of the proper bacteria is used to start a 10-ml overnight culture of LB media supplemented with 100/zg/ml of ampicillin. When this culture has reached saturation, it is added to 1 liter of LB media, 100 /zg/ml ampicillin in a 2-liter flask. This culture is grown with continuous shaking at 37 ° until the OD540 is approximately 0.5. The culture is then brought to 100/xM with isopropyl-/3-o-thiogalactopyranoside to induce the expression of the fusion protein and is allowed to continue shaking until the OD540 reaches at least 1.0. The cells are then harvested with a Beckman JA-10 rotor (15 min, 4400g, 4°). The cell pellet is then frozen in liquid nitrogen and can be stored at - 8 0 ° until needed. The frozen pellet is thawed in - 2 5 ml lysis buffer, which consists of 20 mM Tris-HCl, 5 mM EDTA, 1 mM DTT, pH 8.0, 1 mM sodium azide, 2 /zg/ml aprotinin, 2/xg/ml leupeptin, 2 /zg/ml benzamidine, and 200/zM PMSF. When the cell pellet is fully resuspended in this buffer, lysozyme (Sigma) is added to a final concentration of 1 mg/ml. The sample is incubated on ice until lysis is complete, usually 30 min, or when the lysate becomes very viscous with bacterial DNA. At this point, the sample is supplemented with MgCI2 to a final total concentration of 10 mM (free MgCIe 5 mM), and 250/zg DNase I is added. The enzymatic digestion of D N A is allowed to occur on ice and is usually finished within 30 min. The lysate is then clarified by centrifugation in a JA-20 rotor (12,000g, 20 min, 4°).
[12]
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103
The purification of G S T - R h o - G D I can be accomplished in a single step by affinity chromatography on a column of glutathione-agarose (Sigma). The lysate is applied to a 5-ml column equilibrated with buffer A (see earlier). The column is washed with this same buffer until there is no protein detected in the effluent. The fusion protein is released from the column with buffer A supplemented with 10 m M glutathione. Fractions of this protein analyzed on a 10% S D S - P A G E gel reveal a single band at 55 kDa. While the G S T - R h o - G D I purified in this manner is fully active, the GST domain can be removed by the following protocol. Pooled fractions of G S T - R h o - G D I from the glutathione-agarose column (up to 10 mg total fusion protein) are incubated on ice with 100/zg bovine thrombin. The cleavage of the GST protein and R h o - G D I is usually completed within 4 hr at 4 °, at which time the detergent C H A P S is added to a final concentration of 0.1%. The sample is then applied to a Pharmacia Mono Q column equilibrated with buffer A and 0.1% CHAPS. The bound protein is eluted with a 25-ml gradient (0-500 m M NaC1 in buffer A, 0.1% CHAPS). The GST protein elutes first at approximately 100 m M NaC1, and R h o - G D I follows at 225 m M NaCI. The G D I protein is now completely free of both the GST and thrombin proteins.
Purification of Biological Membranes That Contain Cdc42Hs The C d c 4 2 H s / R h o - G D I solubilization assay as originally described used plasma membranes prepared from the human epidermal adenocarcinoma cell line, A431. The method for the purification of these membranes is well described, 7,s and the abundance of Cdc42Hs in these preparations makes them an ideal choice for use in this assay. However, we have developed a source of Cdc42Hs-containing membranes that is easy to produce but still works as well as the A431 membranes in this assay. This source was dependent on the construction of a recombinant baculovirus designed to overexpress Cdc42Hs in Spodoptera frugiperda insect cells. The construction of this virus has been described elsewhere. 6 Ten 75-cm 2 flasks are seeded with S. frugiperda (Sf9) cells at a density of 5 × 105 cells/ml in Grace's insect cell medium (Gibco B R L ) supplemented with 10% fetal calf serum. The cells are incubated at 28 ° until they reach - 7 0 % confluence, at which time 100/zl of a recombinant Cdc42Hsbaculovirus stock is added. The incubation is continued until most of the cells are floating (1-2 days). The cells are harvested by rapping the flask gently, and then decanting into a 15-ml disposable conical centrifuge tube. 7 K. L. Carraway III, J. G. Koland, and R. A. Cerione, J. BioL Chem. 264, 8699 (1989). s D. Thorn, A. J. Powell, C. W. Lloyd, and D. A. Rees, Biochem. J. 168, 187 (1977).
104
GUANINENUCLEOTIDEEXCHANGE
Cdc42Hs~( ~Q~~~
[12]
qlllb ]
,p s, p s, ,p s, p s,P s, ys, GDI (pg):
1.0
0.G3
0.21
0.07
0.02
0
FIG. 2. Rho-GDI-stimulated solubilization of Cdc42Hs from the human placental membranes. Mono S-purified bovine brain R h o - G D I was used to solubilize Cdc42Hs from human placental membranes as described in the text. After treatment with the indicated amounts of R h o - G D I and centrifugation, the pellet and supernatant were loaded on a 10% SDSpolyacrylamide gel and then transferred to Immobilon. The polyclonal antibody used to detect Cdc42Hs was raised against a synthetic peptide identical to the last 22 amino acids of Cdc42Hs. The secondary antibody was 125I-labeled protein A. From Leonard et al. ] with permission.
They are then spun in a tabletop centrifuge (1000g, 10 min, 25°). The pellet is frozen in liquid nitrogen and stored at - 8 0 ° for later use. The cells are thawed in - 8 ml lysis buffer consisting of 20 mM Tris-HC1, 6 mM EDTA, 1 mM DTT, pH 8.0, 25/zg/ml aprotinin, 25/xg/ml leupeptin, and 200/zM PMSF and are then lysed with 10 strokes of a 7-ml glass/glass Dounce homogenizer. The nucleus and any unbroken cells are removed from the sample by centrifugation in an IEC tabletop centrifuge (1000 rpm, 12 min, 4°). The supernatant, containing the cytosol of the lysed cells as well as the cellular membranes, is spun in a microcentrifuge (14,000g, 10 rain, 4°) to pellet the cellular membranes. After removal of the supernatant, the membranes are washed twice in lysis buffer (see earlier), with centrifugation in the microcentrifuge in between each wash. The membranes are finally resuspended in 2.5 ml of 20 mM Tris-HC1, 1 mM EDTA, 1 mM DTT, pH 8.0, and are aliquoted for long-term storage at -80 °.
Assay for Solubilization of Cdc42Hs with Rho-GDI Cdc42Hs-containing membranes (human placental membranes, A431 plasma membranes, or recombinant insect cell membranes as described earlier) are thawed on ice. A reaction mixture containing 100 mM Tris-HC1, 250 mM NaC1, 25 MgC12, pH 8.0, is prepared, and 8 ~1 is added to 10/zl of the membranes and 22/zl of the R h o - G D I protein (either recombinant or from bovine brain). This mixture is incubated at room temperature for 25 rain in a Beckman 5 × 20-mm airfuge tube. When the incubation is finished, the mixture is centrifuged in a Beckman airfuge at 30 psi for 1 min. (Note: If an airfuge is not available, a microfuge can be substituted, although it is necessary to allow 10 min for centrifugation to separate the membranes from the soluble fraction.) The supernatant and the pellet are separated from each other and then boiled in SDS-PAGE buffer. In order or determine how much Cdc42Hs has been released into the soluble frac-
[ 13]
PURIFICATIONOF p190 Rho-GAP
105
tion, it is necessary to analyze these samples by immunoblotting, First, both the supernatant and pellet samples are run on a 10% SDS-polyacrylamide gel. The separated proteins are then transferred to Immobilon-P (Millipore), blocked with 2% milk, and then probed with an anti-Cdc24Hs antibody. As shown in Fig. 2, up to 50% of the Cdc42Hs can be solubilized by bovine R h o - G D I . It is not known why the remaining Cdc42Hs is insensitive, but it may be that these proteins are on lipid surfaces that are not exposed to the R h o - G D I . Because R h o - G D I can inhibit GDP dissociation from Rho, Rac, and Cdc42Hs proteins, 1'9 it is highly likely that this assay would work with a membrane source rich in any one of the Rho subfamily of GTP-binding proteins, provided a suitable antibody can be found to detect it by immunoblotting. It should also be noted that the D4 protein, a homolog of the R h o - G D I (-65% identity) t° can also solubilize Cdc42Hs from insect cell membranes, although at a much lower level of efficacy (D. Leonard, unpublished results, 1994). 9 T. Ueda, A. Kikuchi, N. Ohga, J. Yamamoto, and Y. Takai, J. Biol. Chem. 265, 9373 (1990). l0 J.-M. Lelias, C. N. Adra, G. M. Wulf, J.-C. Guillemot, M. Khagad, D. Caput, and B. Lim, Proc. Natl. Acad. Sci. U.S.A. 90, 1479 (1993).
[13] P u r i f i c a t i o n a n d G T P a s e - A c t i v a t i n g P r o t e i n A c t i v i t y of Baculovirus Expressed p 190
By JEFFREY
SETTLEMAN a n d ROSEMARY FOSTER
GTPase-activating proteins (GAPs) are important regulators of the nucleotide state of GTP-binding proteins and may additionally serve as effector targets of activated GTPases. ~ In mitogenically stimulated and tyrosine kinase-transformed cells the major Ras-specific GAP, p120 RasGAP, forms an abundant complex with the cellular protein p190. 2 p190 was purified by virtue of its association with p120 Ras-GAP, and corresponding cDNA clones have been isolated and sequenced. 3 Sequencing of the cDNAs revealed an open reading frame containing two distinct domains of sequence similarity to previously described proteins. At its amino terminus, p190 has a group of small sequence motifs found in all of the known guanine 1 D. R. Lowy and B. M. WiUumsen, Annu. Rev. Biochem. 62, 851 (1993). 2 M. Moran, P. Polakis, F. McCormick, T. Pawson, and C. Ellis, Mol. Cell BioL U , 1804 (1991). 3 j. Settleman, V. Narasimhan, L. C. Foster, and R. A. Weinberg, Cell 69, 539 (1992).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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GUANINENUCLEOTIDEEXCHANGE
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nucleotide-binding proteins, suggesting that p190 is likely to bind GTP and to exhibit GTPase activity. At the carboxy terminus of p190 is a region of homology to several proteins that exhibit GAP activity for the Ras-related Rho family GTPases, and p190 has been reported to function biochemically as a Rho-GAP. 4 The Rho GTPases, which are 30% identical to the Ras proteins, have been implicated in the regulation of cytoskeleton and cell morphology.5 Thus, the R a s - G A P : p190 complex establishes a direct physical connection between the Ras and Rho GTPase pathways, and raises the possibility that such a complex may serve as an important regulator for the coupling of Ras- and Rho-mediated signals. The expression and purification of the full-length p190 protein in the baculovirus-insect cell system have provided a useful reagent for addressing the biochemical properties of p190. Production of p 190 Baculovirus The complete coding sequence of p190 was subcloned into a baculovirus expression vector (pEV55). The plasmid DNA (2/zg) was then cotransfected into Sf9 insect cells at 50% confluence with linearized Baculogold DNA (25 lag, Pharmingen) as a calcium phosphate precipitate. After 4 days, the virus-containing medium was collected and used to reinfect Spodoptera frugiperda (Sf9) cells to plaque-purify recombinant virus, as described in the Baculogold handbook (Pharmingen). The plaque-purified virus was then repassaged on fresh monolayers of Sf9 cells twice to amplify the virus titer. Virus-containing medium was collected, filtered through a 0.45-/xm filter, and stored at 4°. Purification of p 190 from Baculovirus-lnfected Insect Cells High Five (Invitrogen) insect cells are plated at 80% confluence in 15cm tissue culture plates in Grace's insect medium containing 10% fetal calf serum, p190 baculovirus is added to each plate to achieve a multiplicity of infection of two plaque-forming units per cell. The virus is left in Parafilmsealed plates for 40 hr at 28° after which time most of the infected cells become detached from the flask and are seen floating in the medium. Detachment of the High Five cells is a good indicator that infection was successful. Sf9 insect cells can also be used for infection; however, the protein yield per flask is generally severalfold lower than that achieved with High Five cells. Additionally, the infected Sf9 cells remain attached 4j. Settleman,C. F. Albright, L. C. Foster, and R. A. Weinberg,Nature 359, 153 (1992). 5A. Hall, Science249, 635 (1990).
[ 131
PURIFICATIONOF p190 Rho-GAP
107
to a surface and therefore must be scraped from the flasks for collection. For virus plaquing in agar, Sf9 cells are generally used. Forty hours postinfection at 28 °, the cells are collected and pelleted by centrifugation at 3000g for 5 min. Pellets are then washed in cold phosphatebuffered saline (PBS) and spun again. The PBS is then decanted, and the pellets are quick-frozen in a dry ice/ethanol bath and placed at - 8 0 °. Pellets can be stored in this way for at least 1 year without a significant reduction in yield of purified protein. Frozen pellets are thawed in a solution (buffer A) containing 25 mM MES, pH 6.5, 0.1 mM EDTA, 1 mM EGTA, 2 mM MgC12, 0.1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 5/xg/ml leupeptin, and 5/xg/ml aprotinin (0.75 ml/15-cm plate). The suspension is briefly vortexed to ensure complete lysis and is then clarified by centrifugation at 100,000g for 15 min. A lysate from two 15-cm plates of cells typically yields 6-8 mg of soluble protein, of which about 400/xg is p190. The clarified lysate is passed through a 0.22-/zm syringe filter before loading it onto a column. Between 1.5 and 2 ml of lysate (about 8 mg) is loaded onto a Pharmacia 5 x 5 Mono S column. The column is washed with 3 column volumes of buffer A, and elution is performed with a linear gradient from 0 to 400 mM NaC1 in buffer A. The total gradient elution volume is 25 ml, and 50 fractions of 0.5 ml are collected. To detect p190 protein, 2/~1 of each fraction is spotted onto a nitrocellulose sheet that has been pregridded in pencil with 1-cm squares. The nitrocellulose is air-dried and then blocked for 30 min in 5% dry milk in TBST (25 mM Tris, pH 8.0, 150 mM NaC1, and 0.05% Tween 20) at room temperature. The filter is then washed three times (5 min each) with TBST and is incubated for 30 min in anti-pl90 antibody diluted in TBST. The filter is then washed five times (5 min each) with TBST, incubated in secondary antibody conjugated to H R P for 30 min, washed again five times, and developed with ECL (Amersham). Strong positive signals should appear on an exposure of only a few seconds in the peak fractions (Fig. 1). Alternatively, 5/xl of selected fractions can be analyzed by S D S - P A G E followed by Coomassie blue staining to visualize protein bands, p190 elutes around 250 mM NaC1 as two bands (190 and 170 kDa) that are somewhat resolved by Mono S chromatography. The lower band is a carboxy-terminal breakdown product that is devoid of R h o - G A P activity. Coomassie staining of the gel readily reveals the p190 protein. It is helpful to run samples in parallel consisting of total cell lysates from uninfected and infected cells to assess the efficiency of infection. A band at 190 kD that is specific to the infected cell lysate should be easily seen by staining, and in an efficient infection the expressed p190 should constitute about 5% of the total soluble protein in the lysate (Fig. 2, lanes A and B).
108
[ 13]
GUANINE NUCLEOTIDE EXCHANGE
1
p+i++i( +'
9
. 21
....
.......
.......
29
......... • ++
+, ++++++++++++++++i++++ :
+
39
}
4,
49
FIG. 1. Dot-blot immunoassay of p190. Two microliters of each of the odd-numbered Mono S column fractions of a p190 purification was spotted on nitrocellulose and immunoblotted to detect pl90-containing fractions.
The Mono S purification step results in substantial enrichment of p190 protein as most of the insect cell proteins do not bind to the Mono S column under the conditions used, whereas nearly all of the p190 is retained. In fact, by Coomassie stain of the peak p190 fractions after this first column step there are only a few other detectable protein bands (Fig. 2, lane C). Four or five tubes representing the peak Mono S fractions of full-length p190 are pooled and dialyzed against a solution identical to buffer A except A
B
C
D
a----pl90
FIG. 2. S D S - P A G E of p190 purification. Ten microliters each of (A) uninfected insect cell lysate, (B) pl90-infected insect cell lysate, (C) Mono S-pooled fractions, and (D) Mono Q-pooled fractions were analyzed by SDS-PAGE and Coomassie blue staining.
[ 13]
PURIFICATIONOF p190 Rho-GAP
109
that the MES is replaced by HEPES, pH 7.5, at an equivalent concentration (buffer B). The dialyzed sample is then applied to a Pharmacia 5 x 5 Mono Q column that has been equilibrated in buffer B. An elution gradient identical to the one used in the previous column step is then performed. A single major A2s0 absorbance peak is detected around 200 mM NaC1, which corresponds to the major pl90-containing fractions. Peak fractions are dialyzed against a solution containing 20 mM HEPES, pH 7.5, 2 mM MgC12, 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM DTT, and 20% glycerol. Following dialysis, the protein is aliquoted and frozen at -80 °. The p190 protein concentration in this final form is typically 100-500 tzg/ml. This preparation of p190 is typically free of other detectable protein bands by Coomassie staining following SDS-PAGE (Fig. 2, lane D). Comments Excellent purification of p190 can also be achieved by replacing the Mono Q column step with a gel-filtration step using a Superose 12 column (Pharmacia). In this case, the pooled fractions from the Mono S step are first concentrated to 200 ~1 using a Centricon-3 concentrator (Amicon) and then applied to the column. The protein is eluted isocratically in buffer B. As has been observed with p190 isolated from mammalian fibroblasts, 3 insect cell-produced p190 appears to partition between two major cellular 120
100
,-
:3 0 .Q a.
8O
6O
I0
o~
40 20
FIG. 3. p190 G A P activity. Results of a filter-binding assay in which each of the 14 indicated GTPases was tested as a substrate for p190 G A P activity. II, No p190; [], plus p190. Reproduced from Settleman e t al. 4
110
GUANINENUCLEOTIDEE X C H A N G E
[ 131
A 100q
80'
60
~g 40"
20" -o 0
o
io
A
30
40
plgO protein (ng) FIG. 4. Rho-GAP activity of p190 as determined in a filter-binding assay. (A) Protein concentration dependence (15 min, 30°). (B) Time dependence (20 ng, 30 °) II, RhoA, O, RhoA + p190. (C) Activity in total cell lysates; II, uninfected lysate; O, pl90-infected lysate.
subfractions. Approximately 70% of the p190 is in a freely soluble, presumably cytosolic fraction. The remaining 30% requires extraction from a nonidet p-40 (NP-40) "resistant" fraction with NaC1 at concentrations above 200 mM. Thus, the yield of p190 obtained from an infection can be increased significantly by extracting as described earlier, but with the addition of 250 mM NaC1. However, this material must be dialyzed to remove the NaC1 prior to column application. Dot-blot immunotitrations of the starting material and the purified protein indicate that recovery of antigenic activity following the two column purification is about 25%. Presumably, the other 75% consists of the 170-kDa breakdown product, other breakdown products, and peak tails that are not recovered, p190 appears to be an extremely stable protein both in vivo and following purification. The half-life of the baculovirusproduced protein, as determined in a pulse-chase experiment, is at least 20 hr (R. Foster and J. Settleman, unpublished observation, 1994), and the purified protein remains undegraded even after overnight incubation at 37°.
B lOOt
b
80
60'
40"
20'
;o
o
2'0
Time (rain)
C
8O
i:[
60'
40'
20 O 0
100
200
300
Cell lysate (ng) FIG. 4.
(continued)
400
112
GUANINENUCLEOTIDEEXCHANGE
[ 131
Measuring Rho-GAP Activity of p 190 To detect R h o - G A P activity of p190, purifed bacterially produced Rho family GTPases are prepared as substrates. For a set of 20 GAP assays, 2-5/~g of purified GTPase is loaded with [y-32p]GTP in an EDTA-facilitated exchange reaction. The reaction consists of the GTPase, 10/~Ci [Y3tp]GTP (3000 Ci/mmol, NEN), 20 mM HEPES, pH 7.5, 50 mM NaC1, 1 mg/ml bovine serum albumin, 1 mM DTT, and 1 mM EDTA in a 100-/~1 volume. After 10 min at 37°, the exchange reaction is stopped by the addition of MgC12 to a final concentration of 5 raM, and the tube is placed on ice. For GAP assays, various amounts of purified p190 are added to a 50/~1 reaction consisting of 5/~1 of [y-32p]GTP-loaded purified GTPase and 45/M of 20 mM HEPES, pH 7.5, 50 mM NaC1, 2 mM MgC12, 1 mM DTF, 0.01 mM GTP, 0.1 mM ATP. The samples are incubated for various amounts of time at 30 °. To stop the reaction, 500/.d of an ice-cold solution of 20 raM HEPES, pH 7.5, 5 mM MgC12, and 0.1 mM DTT (buffer C) is added to the reaction tube and the contents of the tube are then immediately passed over a 0.45-/xm nitrocellulose filter (Schleicher & Schuell, BA85) in a Millipore vacuum filtration apparatus. The filters are washed with 10 ml of buffer C and are then placed in scintillation vials and counted. The vast majority of free GTP or phosphate passes through the filter, whereas GTP that is bound to protein is retained. Thus, a reduction of filter-retained radioactivity is indicative of GTP hydrolysis. An alternative version of the R h o - G A P assay can be performed using thin-layer chromatography (TLC) instead of filter binding to assay the reaction products. For this type of assay, the substrate GTPase is loaded as described earlier except that [a-32p]GTP is used. The GTP-loaded protein is then purified away from free GTP by passage of the sample over a PD-10 desalting column (Pharmacia). Following application of the 100-/~1 sample to the column, free GTP is eluted by the addition of 2.5 ml of buffer C. An additional 1 ml of buffer C is then applied to the column to elute the GTP-bound protein, which is nearly free of unbound GTP. The sample is then concentrated 10-fold by ultrafiltration through a Centricon-3 concentrator. Rho GAP assays are performed as described earlier except that the reaction volume is reduced to 20/~1. Five microliters of each reaction is assayed by spotting on a TLC plate (PEI cellulose F, EM Sciences), and chromatography is performed in 0.75 M KPO4, pH 3.4, to resolve GTP and GDP. The baculovirus-produced p190 protein has been found to function biochemically as a specific GAP for members of the Rho GTPase family. 4 The members of this family that have been tested and found to function as p190 substrates include RhoA, RhoB, Racl, Rac2, and CDC42. p190
[ 13]
PURIFICATIONOF p190 Rho-GAP
113
does not exhibit GAP activity toward seven different Ras family members or two Rab family members that have been tested (Fig. 3). p190 is also reported to exhibit higher specific GAP activity toward Rho proteins relative to Rac and CDC42 proteins. 6 The R h o - G A P activity of p190 exhibits the features characteristic of all catalytic enzymes, including temperature, concentration, and time dependence. R h o - G A P activity of p190 in a 15-min reaction is linearly dose dependent in the 2-20 ng range of p190 protein concentration (at 30 °, 50 /zl), and is maximal above 50 ng (Fig. 4A). Using 20 ng of p190 per assay, R h o - G A P activity is somewhat linear in the first 5-10 min of the reaction (at 30°) (Fig. 4B). Intrinsic GTPase activity of RhoA in the absence of p190 is detectable, with about 25% of the initial radioactivity lost after a 20-min reaction. The rate of intrinsic GTPase activity at 37 ° is somewhat higher, and R h o - G A P activity is more difficult to measure at this temperature. The Rac GTPases have a significantly higher rate of intrinsic GTPase activity than the Rho proteins and are generally easier to assay at 25 °. Measuring p190 Rho-GAP Activity in Insect Cell Lysates The high level of expression of p190 obtained in infected insect cells (5% of soluble protein) permits a reliable assay of pl90-mediated R h o GAP activity from total cell lysates. In a 15-min R h o - G A P reaction at 30 °, the pl90-containing insect cell lysate promotes GTP hydrolysis by purified RhoA in a linearly dose-dependent manner in the range of 5-50 ng of total pl90-containing cell lysate (Fig. 4C). In this range of protein concentration, R h o - G A P activity from uninfected cell lysate is minimal. However, specific R h o - G A P activity can be detected with higher concentrations of uninfected cell lysate. Lysates for such assays are prepared in a solution containing 25 mM HEPES, pH 7.5, 150 mM NaC1, 0.1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, and protease inhibitors. The final protein concentration is 2-4 mg/ml. Comments In addition to its R h o - G A P activity, purified p190 is able to form a specific stable complex with R a s - G A P by brief mixing of the two proteins or mixing of insect cell lysates containing each of the proteins, at 4 °. The purified p190 is also bound to GDP and GTP (approximately 1 mol/mol protein) through an amino-terminal GTPase-like domain of the protein (R. Foster, K. Q. Hu, D. A. Shaywitz, and J. Settleman, Mol. Cell Biol. 14, 7173 (1994)). 3 6 A. J. Ridley, A. J. Self, F. Kasmi, H. F. Paterson, A. Hall, C. J. Marshall, and C. Ellis, E M B O J. 13, 5151 (1993).
114
GUANINENUCLEOTIDEEXCHANGE
[ 14]
[ 14] G T P a s e - A c t i v a t i n g P r o t e i n A c t i v i t y of n(al)-Chimaerin and Effect of Lipids B y SOHAIL A H M E D , R O B E R T KOZMA, CHRISTINE HALL,
and Louis
LIM
Introduction n(al)-Chimaerin cDNA encodes a 38-kDa GTPase-activating protein (GAP) for Racl and Cdc42Hs that possesses a protein kinase C (PKC)like cysteine-rich regulatory domain. 1-3 The cysteine-rich domain (with the motif HX12CX2CX13CX2CX4HX2CX7C,where X is any amino acid residue) of n-chimaerin coordinates 2 mol of Zn 2+ per mole protein 4 and binds phorbol esters. Zn 2+ is required for n-chimaerin and PKC to bind phorbol esters. 5 The cysteine-rich domain of n-chimaerin binds phorbol esters with characteristics similar to those observed with PKC 8-e (i.e., phospholipiddependent, high-affinity, Ca2+-independent and stereospecific)5'6 and allows phorbol ester/phospholipid regulation of its GAP activity.7 Thus, n-chimaerin is a novel functional target for phorbol esters and phospholipids. Other proteins with sequence identity to the cysteine-rich regulatory domain of n-chimaerin are the oncogene products, Raf and Vav, diacylglycerol kinase, and the Caenorhabditis elegans unc-13 gene product. 5 Similarly, a number of proteins have sequence identity to the C-terminal GAP domain of n-chimaerin, including Cdc42Hs-GAP, breakpoint cluster region gene product (Bcr), active Bcr-related gene product (Abr), and the Ras-GAP binding protein p190. The chimaerin family of proteins consists of a cysteine-rich domain coupled to a GAP domain with variable N-terminal sequences8-1° (Fig. 1). 1 D. Diekmann, S. Brill, M. D. Garret, N. Totty, J. Hsuan, C. Monfries, C. Hall, L. Lim, and A. Hall, Nature 351, 400 (1991). 2 C. Hall, C. Monfries, P. Smith, H.-H. Lim, R. Kozma, S. Ahmed, V. Vanniasingham, T. Leung, and L. Lira, J. Mol. Biol. 211, 11 (1990). 3 S. Ahmed, J. Lee, L.-P. Wen, Z. Zhao, J. Ho, A. Best, R. Kozma, and L. Lim, J. Biol. Chem. 269, 17642 (1994). 4 S. Ahmed, unpublished data (1993). 5 S. Ahmed, R. Kozma, J. Lee, C. Monfries, N. Harden, and L. Lim, Biochem. J. 280, 233 (1991). 6 S. Ahmed, R. Kozma, C. Monfries, C. Hall, H.-H. Lim, P. Smith, and L. Lim, Biochem. J. 272, 767 (1990). 7 S. Ahmed, J. Lee, R. Kozma, A. Best, C. Monfries, and L. Lim, J. Biol. Chem. 268, 10709 (1993). 8 T. Leung, B.-E. How, E. Manser, and L. Lim, J. Biol. Chem. 268, 3813 (1993). 9 C. Hall, W.-C. Sin, M. Teo, G. J. Michael, P. Smith J.-M. Dong, H.-H. Lim, E. Manser, N. K. Spurr, T. A. Jones, and L. Lim, Mol. Cell. Biol. 13, 4986 (1993).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
[14]
n(Oq)-CHIMAERIN GAP ACTIVITY
Protein n(Ctt)-chimaerin
~2-ehimaerin ~31-chi. . . . in ~2-eh,maor,° Hat.2 Rotund pl.7
kDa
Structure L~'I
115 Expression
cysteine-richdomaln
GTP. . . . rivalingdomain
38 (35*) Brain
cysteine-richdomain
GTPase activating domain
50 (45) Brain/Testes
[i:iiiiiiii~iii:] cysteineaichdomain
GTP. . . . ctivating domain
38 (30) Testes
¢ysleine-richdomain
GTP. . . . . tivalingdomain
54 (46) Brain
I cysteine-dchdomain
GTPase activating domain
>38 (-) Brain
[ SH2domain ~
I >2oo..,o H
~-///~.~JJ,~
cysteine-richdomain
GTP. . . . . rivaling domain
44 (-) ImaginalDiscs
Fin. 1. The chimaerin family of proteins. The chimaerin family members are shown schematically and consist of a cysteine-rich domain coupled to a GAP domain with highly variable N-terminal sequences, n(cq) and/31 isoforms are the products of two separate genes,2.s with alternate splicing of mRNAs giving rise to products possessing an additional SH2 domain (c~2 and/32 isoforms,9'1°respectively). There is preliminary evidence for the existence of a 3' isoform that is expressed in lymphocytes.11 The cDNA Hat-2 encodes the canary homolog of nchimaerin as it has 96% amino acid sequence identity to human n-chimaerin.12 Hat-2 mRNA is enriched in the forebrain in areas associated with learning. DNA isolated in the R o t u n d region of D r o s o p h i l a melanogaster detects transcripts of sizes 1.7 and 5.3 kb in Northern analysis. The DNA sequence of the pl.7 transcript encodes a protein with 20% amino acid sequence identity to human n-chimaerin23 pl.7 is expressed in the imaginal discs and in primary spermatocytes. Molecular weights in kilodaltons are those predicted from primary sequence whereas those in brackets represent observed values from tissue extracts. The asterisk indicates that the native protein has not been identified clearly. An open box indicates that the Hat-2 cDNA clone is not full-length. In c~2-and/32-chimaerins, part of the amino acid sequence flanking the cysteine-rich domain (shaded area) is replaced by a SH2 domain. The three shading patterns indicate the possible presence of different localization signals or motifs in the N-terminal region of the proteins. For a more detailed analysis of amino acid sequences see Hall et al. 9 and Leung et aL a° (SH2 domain), Ahmed et aL 5 (cysteine-rich domain), and Ahmed et al. 3 (GAP domain).
We believe that one function of these N-terminal sequences is to determine the cellular localization of the family members, c~2-,/31- and/32-chimaerins possess R a c l - G A P activity. 8-1° The chimaerin family are the only GAPs with a well-defined lipid binding domain, p120 R a s - G A P has sequence identity to the C2 domain of PKC, which is thought to confer Ca 2÷ and lipid dependence on enzyme activity, but the function of this domain in R a s - G A P has not been investigated. 10T. Leung, B.-E. How, E. Manser, and L. Lim, J. BioL Chem. 269, 12888 (1994). u T. Leung, E. Manser, and L. Lira, unpublished data (1993). 12 j. M. George and D. F. Clayton, MoL Brain Res. 12, 323 (1992). 13 M. Agnel, L. Roder, C. Vola, and R. Griffin-Shea, Mol. Cell. Biol. 12, 5111 (1992).
116
GUANINENUCLEOTIDEE X C H A N G E
[ 141
Identification of Chimaerin Proteins in Brain Since n-chimaerin was cloned as a brain-specific cDNA it is important to establish that a protein product is also synthesized, n-Chimaerin-specific antibodies detect a protein of 45 kDa in brain cytosol and a labile protein of 35 kDa in brain membrane fractions, both of which have R a c l - G A P activity. The 45-kDa protein has been purified 427-fold to approximately 1.6% purity (see Hall et al. 9 for details of purification procedure). Fractions containing partially purified p45 have a phosphatidylserine-stimulated R a c l - G A P activity. Peptide sequencing of the SDS gel-purified 45-kDa protein reveals that it is the o~2isoform. 9Thus the 35-kDa protein is probably n-chimaerin, the o~1isoform. The abundance of the 45-kDa protein in brain is less than 0.004%. Expression and Purification of n-Chimaerin Expression Systems
n-Chimaerin cDNA has been expressed in Escherichia coli in four different fusion expression systems:/3-galactosidase (pUR292), TrpE (pATH21), glutathione S-transferase (GST; pGEX-2T), and maltose-binding protein (MBP; p997). 3-7 Each system has its own advantages and disadvantages. The C-terminal GAP domain of n-chimaerin can be easily purified when expressed as a GST fusion protein and gives yields of about 5 mg/liter of culture (see Smith and Johnson TM for details of purification). The system of choice for biochemical analysis of full-length n-chimaerin, however, is the MBP fusion system, as it allows a combination of high-level expression and solubility (approximately 2-4 mg of protein has been purified per liter of culture). Expression of n-chimaerin in the other three systems gives predominantly insoluble protein. In general, there are no clear rules with regard to the solubility of proteins when expressed in E. coli. However, we have found that if proteins expressed as GST fusions are insoluble they will probably be soluble if expressed as MBP fusions and vice versa. Preparation of Protein-Induced Cell Extracts
To express protein from any of the systems just described, plasmid constructs are transformed into a Lon protease-deficient strain of E. coli (Y1090 t°~-) or XL-1 blue and single colonies grown overnight in 20 ml Luria broth (LB) with 50 ~g/ml ampicillin. Most reproducible expression 14D. B. Smith and K. S. Johnson, Gene 67, 31 (1988).
[14]
n(O~I)-CHIMAERINGAP ACTIVITY
117
of protein is obtained with freshly transformed E. coli. The next day this starter culture is diluted 50-fold into prewarmed (500 ml in 2 x 2.5-liter flasks) LB/ampicillin and cells are grown at 37° until an OD6s0 of 0.5 is reached. At this point isopropyl/3-D-thiogalactoside (IPTG) is added to 1 mM final concentration and cells are grown for a further 2-3 hr (reaching an OD650 of 1-2.0). Cells are then harvested, washed once and resuspended, after 100-fold concentration, in lysis buffer (20 mM Tris-HC1, pH 7.4/2 mM EDTA/10 mM EGTA/20/zg/ml leupeptin/0.5% Triton X-100). A cocktail of protease inhibitors can be added if the expressed protein is unstable. Cells are either used immediately or frozen at -20 °. When required, cell extracts are thawed rapidly and sonicated (6 x 15 sec with 1-min pauses). After sonication, cells are spun at 14,000g for 20 min, the supernatant is used for affinity purification, and the pellet can be used as a source of insoluble proteins to be refolded after urea denaturation (see below). Protein Purification
To purify MBP-n-chimaerin from the E. coli cell extracts the following procedure is used: sonicated cell extracts are fractionated by centrifugation and the supernatant is applied to prewashed amylose resin (1.5 ml) in a 10-ml syringe (New England Biolabs; binding capacity 3 mg MBP/ml bed volume) and washed with 10 column volumes of buffer (50 mM Tris-HC1, pH 8.0/0.15 M NaC1). MBP-n-chimaerin protein is eluted with column buffer containing 10 mM maltose, dialyzed against buffer (3 x 2 hr; 1 : 1000) without maltose, and concentrated to approximately 1 mg/ml using Centricon-10 units (Amicon). To cleave MBP-n-chimaerin, purified protein is incubated with factor Xa for 2 hr at room temperature (500:1, cleavage conditions determined empirically for each fusion protein). Cleaved protein is applied to amylose resin to remove MBP, leaving n-chimaerin in the flow through, n-Chimaerin purified in this system is >95% pure, and the protein is either used immediately or stored at - 7 0 ° with 15% glycerol in aliquots of 50-100/xl. The amylose resin can be regenerated by washing with 10 column volumes of 3 M NaC1 followed by 10 column volumes column buffer and stored in the same buffer containing 0.02% sodium azide at 4°. Purification of Rho family proteins, which are used as substrates for n-chimaerin in GAP assays, is described in [1], this volume). We have found that a polyglycine spacer is placed between GST and RhoA/Cdc42Hs dramatically improves cleavage by thrombin.
is K. Guan and J. E. Dixon,Anal Biochem. 192,262 (1991).
118
GUANINE NUCLEOTIDE EXCHANGE
[ 141
n-Chimaerin as Metalloprotein The presence of a cysteine-rich motif in n-chimaerin is indicative that it is a metalloprotein. To assess whether n-chimaerin is a metalloprotein, two different techniques have been used (i) The zinc-blot technique in which proteins transferred to nitrocellulose filters are probed with 65Zn2+ (see Ahmed et al. 5 for details). This technique, although qualitative, allows analysis of partially pure proteins. (ii) The second technique quantifies the amount of Zn 2+ coordinated by the purified recombinant protein using a Perkin-Elmer 2380 atomic absorption spectrophotometer (k214 nm). Zinc standard solutions are prepared in 6% (v/v) butanol and are compared with samples of purified protein (50-100/zM). Background Zn 2+ is estimated in the buffer used to prepare protein and has been found to be below 1 /~M. As long as proteins can be prepared to between 50 and 100 /~M concentrations there is no particular problem with background Zn 2+ levels. Data obtained from atomic absorption suggest a stoichiometry of 2 tool Zn 2+ per mole protein for n-chimaerin. 4 To ensure that recombinantly expressed metalloproteins are replete with Zn 2+, and therefore properly folded, Zn 2+ should be added to growth media (10-50/~M final concentration) prior to induction with IPTG. Phorbol ester-binding experiments (see below) have shown that supplementation of growth medium with Zn 2+ is essential to obtain maximal binding. 4
n-Chimaerin as Phospholipid-Dependent Phorbol Ester Receptor Phorbol Ester-Binding Assay
n-Chimaerin has sequence identity to the phospholipid-dependent phorbol ester binding regulatory domain of PKC. To determine the ability of n-chimaerin to bind phorbol esters, a binding assay with purified protein is performed. Phorbol esters (30 nM [3H]PDBu, 19 Ci/mmol Amersham or 20 Ci/mmol NEN) are incubated with protein samples in the absence/ presence of 100/~g/ml phosphatidylserine or phosphatidic acid (see below for preparation of lipids) for 30 min at room temperature followed by 30 min at 4 ° and the protein is separated from the surrounding buffer by rapid filtration on Whatman GF/C filters. Following filtration, samples are washed three times with 5 ml of ice-cold buffer [50 mM Tris-HC1, pH 7.5/0.15 M NaCI/1 mg/ml bovine serum albumin (BSA)] and radioactivity is counted in scintillant. In the absence of phospholipids, n-chimaerin does not bind phorbol esters significantly. 6 Background binding of [3H]PDBu to filters is between 4 and 25% of total protein bound radioactivity with PDBu concentrations varying from 3 to 500 nM.
[14]
n(Otl)-CHIMAER1N GAP ACTIVITY
119
Phorbol Ester Binding to Cell Extracts Phorbol ester binding to overexpressed but not purified protein present in E. coli cell extracts can be carried out as follows: a 10-ml IPTG-induced culture is centrifuged at 12,000g for 10 min and the cell pellet is resuspended in 100 t~l lysis buffer [20 m M Tris-HC1, p H 7.4/0.25 M sucrose/2 m M dithiothreitol (DTT)/2 m M phenylmethylsulfonyl fluoride/lysozyme 0.6 mg/ml] and sonicated for 1 rain. The lysed cell suspension (10/M) is then diluted 20-fold into 50 m M Tris-HC1, p H 7.4/100 t~g/ml phosphatidylserine and used in the binding assays as described earlier.
Refolding of Protein from Inclusion Bodies' To refold proteins present in inclusion bodies, cell pellets are isolated (as described above) and solubilized in 3.0 ml 8 M urea/50 mM TrisHC1, p H 7.5/1 m M E D T A / 1 m M D T T for 1 hr. A small glass rod should be used to disrupt/loosen the pellet before resuspension in 8 M urea. Refolding of protein is achieved by removing urea by dialysis in two steps at 4°: (i) dialysis against 500 ml of 4 M urea/1 m M Tris-HCl, p H 5.0/1 m M D T T for 1 hr, and (ii) dialysis against 2 liters of 50 m M Tris-HC1, p H 7.5/ 50 m M NaCI/2 m M M g C 1 j 2 0 0 / z M D T T / 1 0 0 / x M ZnC12 overnight with two changes of buffer. Protein obtained from cell pellets after washing with buffer containing 0.1% Triton X-100 is >90% pure and after refolding has G A P activity. An example of a phorbol ester-binding experiment using protein (approximately 5/xg) refolded in the presence or absence of Zn 2+ is shown in Table I. In attempting to refold the protein in the absence of TABLE I ZINC DEPENDENCEOF PHORBOLESTERBINDINGACTIVITYOF n-CH1MAERIN,PKC, AND
DIACYLGLYCEROL KINASE a
[3H]PDBu binding (cpm) Protein n-Chimaerin Protein kinase C Diacylgycerolkinase
- Zn
+ Zn
231 (_+58) 153 (+_77) NDb
5775 (_+1235) 7670 (_+722) 522 (_+43)
" Phorbol ester binding was measured with [3H]PDBu as described in the text. Inclusion bodies were made from E. coli cells followed by Triton X-100 washing in buffer containing 0.1% Triton X-100 and refolding in the presence or absence of Zn2+. b Not determined.
120
GUANINE NUCLEOTIDE EXCHANGE
[14]
Zn 2+ it is important to remove any background/contaminating Zn 2+. This can be achieved by washing glassware in acid and purifying buffers with chelexing resin prior to use. PKC and n-chimaerin bind phorbol esters to similar levels whereas diacylglycerol kinase does not bind phorbol esters (Table I). These data suggest that a Zn2+-dependent structure plays a role in the binding of phospholipids/phorbol esters by PKC and n-chimaerin. Furthermore, phorbol ester binding to the cysteine-rich domain must require specific sites of interaction since diacylglycerol kinase does not bind phorbol esters.
n-Chimaerin as GAP for Rac 1 and Cdc42Hs n-Chimaerin was first recognized as a GAP through work by Alan Hall's group on a putative R h o A - G A P (now thought to be a Cdc42Hs-GAP). Peptide sequencing of a gel-purified protein with R h o A - G A P activity revealed amino acid sequence identity with n-chimaerin and Bcr. Using recombinant proteins expressed in E. coli, Diekmann et al. 1 showed that n-chimaerin and Bcr are GAPs for Racl but not for RhoA. n-Chimaerin is a 37-fold more potent R a c l - G A P than a Cdc42Hs-GAP. 3 The GAP activity of n-ehimaerin can be measured by (i) loading p21s (Racl, RhoA, Cdc42Hs, and K-Ras) with [y-32p]GTP using their intrinsic G D P / G T P exchange activity at low Mg 2+, (ii) adding Mg 2+ to inhibit exchange, (iii) purifying the p21-[y-32p]GTP complex (an optional step), and (iv) estimating the amount of p21-[y-32p]GTP complex, by filtration and scintillation counting, over a 20-min time course, in the absence or presence of nchimaerin. The experimental details of these steps are described below:
1. GTP loading by [yJ2P]GTP/GDP exchange. Mix, at 4°, 15 /zl of buffer A, 2/xl of 50 mM EDTA, pH 8.0, and 6/xl of [y-32p]GTP (2/xCi/ /xl; 30 Ci/mmol) in a tube containing 2/~1 of Racl (1 mg/ml). There is sufficient GTP-loaded Racl to carry out eight experiments with four time points per experiment (0, 5, 10, and 15 rain or variation thereof). Total volume is 25/zl with Racl at 4 tzM. Incubate 3/xl of the above reaction at 30 ° for 10 min to allow GDP/[y-32P]GTP exchange and then replace on ice. 2. Inhibition of exchange. Add 1/xl of 0.1 M MgCI2 to the above reaction mix to inhibit exchange. This mixture should be kept on ice to reduce intrinsic GTPase activity and be used as soon as possible for a time course (within 1 hr of preparation). 3. Purification of complex. Unbound radioactivity can be removed using a 1-ml G-25 superfine Sephadex column. G-25 columns are equilibrated in the appropriate buffer and then spun dry by centrifugation (2 × 10,000g
[14]
n(O~I)-CHIMAERINGAP ACTIVITY
121
for 2 min). The sample is then applied to the column, the column is spun, and the eluate containing the p21-[T-32p]GTP complex is collected. 4. GTPase assay. The amount of p21-[T-32p]GTP complex can be estimated by immediate filtration on nitrocellulose filters. To measure GTPase activity, set up the following mix on ice: 4/xl p21-[y-32p]GTP complex (from step 2 or step 3) 1 /xl GTP (30 mM) 3 ~1 BSA (13 mg/ml) X/zl buffer B 2 2 - X ~1 protein extract or purified protein (GAP added last) 30/xl total volume Allow the reaction to proceed for 20 min at 15°. At different time points remove 5-/xl aliquots and mix with 1 ml cold buffer C, filter on nitrocellulose, and then assay the radioactivity in scintillant. The reaction is nonlinear above 50% hydrolysis. Each sample should give initial counts of between 20 and 40,000 cpm. At 15° the half-life of the Racl/Cdc42Hs-GTP complex is 15-20 rain. Alternatively, 5-/,1 aliquots of the reaction mix can be taken and directly spotted onto precut nitrocellulose filters, the filters washed (three times) in 10-20 ml of ice-cold buffer C in petri dishes, and the filters radioassayed.
Solutions Buffer A: 20 mM Tris, pH 7.5/25 mM NaCl/0,1 mM DTT Buffer B: 20 mM Tris, pH 7.5 Buffer C: 50 mM Tris, pH 7.5/50 mM NaC1/5 mM MgCI2 A typical GAP assay is shown in Fig. 2 with Racl as the substrate and time points 0, 5, 10, and 15 min. The data are plotted as a percentage of the p21-[y-32p]GTP cpm present at zero time. The R a c l - G A P activity of n-chimaerin is compared with a C-terminal protein lacking the cysteinerich domain.
Substrate Specificity of n-Chimaerin GAP Activity When the GAP activity of n-chimaerin is measured with Racl, RhoA, Cdc42Hs, or K-Ras as substrates as a function of n-chimaerin protein concentration, an activity constant (Kactivity) can be derived (Table II). Kactivity is defined as the amount of protein required to reduce the half-life of the p21-GTP complex by 50%. This type of analysis allows the substrate specificity of GAP proteins to be determined (Table II) and also allows a comparison between different GAPs to be made. Results from such experiments suggest that n-chimaerin interacts with Racl and probably Cdc42Hs in vivo, but not with RhoA or K-Ras.
122
[14]
GUANINE NUCLEOTIDE EXCHANGE
100
~
80
[~
40
20
~
o
0
5
10
15
Time (min) FI~. 2. G A P activity of full-length and the C-terminal of n-chimaerin. G A P activity was m e a s u r e d as described in the text using R a c l as a substrate. R a c l GTPase activity in the absence of any additions (11), in the presence of 0.1/xM n-chimaerin (O), or in the presence of 0.1/zM C-terminal of chimaerin ( G A P domain) ( 0 ) . A t 15 ° the half-life of R a c l - G T P is between 15 and 20 min, decreasing to 5 min at r o o m temperature. Values of 100% represent about 40,000 cpm.
T A B L E II SUBSTRATE SPECIFICITY OF G A P ACtiVITY OF n-CHIMAERIN C-TERMINAL a Substrate Racl Cdc42Hs RhoA K-Ras
gactivity
(/xM)
0.054 2.00 NA b NA b
G A P activity was m e a s u r e d with different substrates (Racl, Cdc42Hs, R h o A , or K-Ras) as a function of n-chimaerin C-terminal protein concentration. Similar results were obtained with full-length n-chimaerin protein, gactivity is an activity constant which represents the concentration of protein required to decrease the half-life of the p 2 1 - G T P complex by 50% (e.g., 20 to 10 min in the case of R a c l and Cdc42Hs). b No activity at concentrations up to 10/zM.
[14]
n(al)-CHIMAERIN GAP ACTIVITY
123
Lipid Modulation of n-Chimaerin GAP Activity
Classes of Lipids When assessing the ability of lipids to regulate enzyme activity it is appropriate to classify lipids into five different classes: (1) phospholipids present in cell membranes (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol); (2) phospholipids produced by cell stimulation (e.g., phosphatidic acid, lysophosphatidic acid, and phosphatidylinositol phospholipids); (3) diacylglycerols derived from phospholipase C or D activity; (4) phospholipase Az-derived products such as arachidonic acid, prostaglandins, and leukotrienes (also phosphatidic acid and lysoderivatives); and (5) sphingosine and derivatives. Lipids from each of these different classes can be used in an initial screen to determine which lipids should be analyzed in more detail.
Preparation of Lipids Lipids can be presented to proteins in two main ways: either as Triton X-100 (detergent) micelles or as liposomes. For lipid effects on n-chimaerin the latter method of presentation has been used. Liposomes are prepared as follows; chloroform solutions of lipids (1 mg/ml, stored at - 2 0 °) are dried under vacuum, rehydrated for 1-2 hr in 20 mM Tris-HC1, pH 7.5 (1 mg/ml final concentration), and sonicated until the solution is clear. Since the degree of sonication determines the size of the liposome, conditions for preparation should be uniform between experiments. Liposomes should be prepared immediately before use and not stored. Crude membrane fractions can be used as an undefined preparation of lipid and are made as follows: 108 COS7 cells/ml, or other mammalian cells, are homogenized in PBS, sonicated (6 × 5 sec with pauses of 15 sec) followed by low-speed centrifugation (3000g) to remove debris (whole cells and nuclei), and then membranes isolated by high-speed centrifugation (100,000g).
Liposome Sedimentation Assay To determine whether proteins can physically interact with lipids a sedimentation assay can be utilized. Protein (2/zg) in 50 mM Tris-HC1, pH7.5, 100 mM KCI, 2 mM MgCI2 is incubated with liposomes at 200/~g/ ml for 1 hr at 37° to allow interaction. The liposomes are then isolated by centrifugation at 90,000g for 20 min in a Beckman TL-100 ultracentrifuge. The pellet is taken up in a volume equivalent to the supernatant and both fractions are analyzed by SDS-polyacrylamide gels. If the protein physically interacts with a particular lipid it will partition into the pellet fraction.
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~=
~'~
[1 41
1
1
2
3
4
5
6
7
8
9
10 11 12
Lipids
FIG. 3. Lipid modulation of the GAP activity of n-chimaerin. GAP activity was measured as described in the text using Racl as a substrate, n-Chimaerin was present at 0.1 ~M (lanes 1-5) or at 0.4/zM (lanes 6-12) to examine activation or inhibition of GAP activity,respectively. Additions were as follows: (1) none, (2) phosphatidylcholine, (3) phosphatidylethanolamine, (4) phosphatidylserine, (5) phosphatidic acid, (6) none, (7) lysophosphatidic acid, (8) phosphatidylinositol, (9) phosphatidylinositol phosphate, (10) phosphatidylinositol bisphosphate, (11) arachidonic acid, and (12) arachidic acid. All lipids were present at 100/~g/ml. The GTPase activity of Racl is not affected by any of the just listed lipids.7
Lipid Effects on GAP activity Lipids are p r e p a r e d as described earlier and incubated with protein 5 - 1 0 rain prior to the addition of the p21-[y-32p]GTP complex. G A P assays are carried out as described above. It is important to mix the components of the reaction during the time course of the experiment to prevent liposomes from sedimenting. Figure 3 shows the effect of a range of lipids on n-chimaerin R a c l - G A P activity. Two different concentrations of nchimaerin are used to examine stimulatory and inhibitory lipid effects on G A P activity: 0.1 and 0 . 4 / z M (lanes 1-5 and 6-12, respectively). A p a r t from arachidonic acid, all these lipids require the presence of the cysteinerich domain to modulate n-chimaerin G A P activity. Lipids can also be added in combination to examine synergistic or competitive effects. For example, phorbol esters synergize with phospholipids in activating n-chimaerin (at limiting phospholipid concentrations, 5/~g/ml). 7 Conclusion The chimaerin family of proteins represents a new class of phorbol ester receptors which act as lipid responsive G A P s , with R a c l , and possibly
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Cdc42Hs, as in vivo substrates, Thus, PKC cannot be viewed as the only physiological target for phorbol esters. The use of phorbol esters as specific modulators of PKC must be reevaluated. The different chimaerins characterized so far are specifically expressed in the brain and/or testes, and the variable N-terminal sequences may allow particular intracellular localizations. The Rho family p21 proteins are implicated in signal transduction pathways leading to modulation of the cytoskeleton and cell morphology. Since n-chimaerin is a GAP, its cellular function could be to down-regulate the signaling pathways of Racl and Cdc42Hs. On the other hand, n-chimaerin could be an effector for these pathways and thus the role of its GAP activity might be to allow dissociation and recycling of p21s instead of down-regulating. The fact that n-chimaerin binds specifically to the GTPbound form of Racl supports its potential role as an effector.3 Work is currently underway, using microinjection and transfection of mammalian cells with n-chimaerin, to determine its cellular function.
Acknowledgment We thank the Glaxo-SingaporeResearch Fund for support.
[15] C h a r a c t e r i z a t i o n o f B r e a k p o i n t C l u s t e r R e g i o n Kinase and SH2-Binding Activities By DANIEL E. H. AFAR and OWEN N. WITTE Introduction The bcr (breakpoint cluster region) gene is the site of breakpoints exploited in the generation of the Philadelphia chromosome translocation (t9 : 22; q34 : q l l ) found in chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). 1 The molecular consequence of this translocation is the generation of the chimeric bcr-abl tyrosine kinase oncogene. The oncogenic potential of the ABL tyrosine kinase is activated by bcr sequences fused upstream of the second exon of c - a b & Sequence analysis of bcr shows that it encodes a protein of 160 kDa
1R. Kurzrock,J. Gutterman,and M. Talpaz, N. Engl. J. Med. 319, 990 (1988). 2A. J. Muller,J. C. Young,A. M. Pendergast,M. Pondel,N. R. Landau,D. R. Littman,and O. N. Witte, MoL CeIL Biol. 11, 1785 (1991). METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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[15/
EXON 1
SERINE KINASE
• """""--
I o""1 c
SH2 BINDING
FIG. 1. Schematic representation of B CR. The B CR protein contains a unique serine kinase domain encoded by the first exon, a central region homologous to the dbl oncogene and the yeast CDC24 gene, and a GTPase-activating domain at the C terminus. The serine kinase domain also contains two motifs termed the A and B boxes, which are capable of binding to SH2 domains in a phosphotyrosine-independent manner.
with multiple functional d o m a i n s (Fig. 1). T h e first exon of bcr encodes a unique serine kinase activity. 3 This region is also capable of interacting with the sr¢ h o m o l o g y (SH)2 d o m a i n of various signaling proteins in a p h o s p h o t y r o s i n e - i n d e p e n d e n t manner. 4'5 T h e central s e g m e n t of bcr displays h o m o l o g y to the dbl o n c o g e n e and the yeast C D C 2 4 gene, molecules with putative G T P / G D P exchange factor activities t o w a r d small G T P binding proteins. 6 T h e carboxyl terminus of B C R exhibits GTPase-activating activity t o w a r d p21Rac. 7 T h e s e findings suggest that B C R is a molecule that functions at the junction of multiple signaling pathways, involving small G proteins, kinase substrates, and SH2-containing molecules. D e l e t i o n mutagenesis of bcr-abl reveals that the bcr sequences necessary for A B L kinase activation reside in the first exon of bcr. 2 This region of the B C R protein e n c o m p a s s e s the serine kinase as well as the SH2-binding activities. This implies a potential role for one or both of these activities in the o n c o g e n i c activation of B C R - A B L . This chapter deals with practical m e t h o d s o f assaying B C R kinase and SH2-binding activities. Assaying BCR Kinase Activity T h e B C R serine kinase region shows no h o m o l o g y to the superfamily of protein kinases, which are characterized by a series of conserved domains e n c o d e d by five to seven separate exons, s T h e kinase d o m a i n of B C R is 3 y. Maru and O. N. Witte, Cell 67, 459 (1991). 4 A. M. Pendergast, A. J. Muller, M. H. Havlik, Y. Maru, and O. N. Witte, Cell66, 161 (1991). 5 A. J. Muller, A. M. Pendergast, M. H. Havlik, L. Puil, T. Pawson, and O. N. Witte, MoL Cell, Biol. 12, 5087 (1992). 6 D. Ron, M. Zannini, M. Lewis, R. B. Wickner, L. T. Hunt, G. Graziani, S. R. Tronick, S. A. Aaronson, and A. Eva, New Biol. 3, 372 (1991). 7 D. Diekmann, S. Brill, M. D. Garrett, N. Totty, J. Hsuan, C, Monfries, C. Hall, L. Lira, and A. Hall, Nature 351, 400 (1991). S. K. Hanks, A. M. Quinn, and T. Hunter, Science 241, 42 (1988).
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127
encoded by a single exon, which exhibits a unique ATP-binding site that involves the sulfhydryl groups of cysteine (Cys) residues. The first exon of BCR contains four Cys residues, one of which, Cys-332, is essential for kinase activity? BCR is capable of interacting with a variety of cellular proteins. Therefore, one may want to partially purify BCR from cell lysates prior to determination of kinase activity.
Purification of BCR The best available source of BCR is baculovirus-produced BCR in SF9
(Spodoptera frugiperda, fall armyworm ovary) insect cells, 3,4 although BCR hyperexpression in 293T cells by calcium phosphate transfection is also a good source. 9 BCR represents approximately 5% of total protein in baculovirus-infected SF9 cells. A single-step purification protocol using the weak cation-exchange resin Bio-Rex 70 may be utilized to obtain a 10-fold enrichment of BCR. Using baculovirus-infected SF9 cells, 5 × 107 cells (7 mg total protein) are Dounce homogenized in 10 ml of buffer 1:25 mM potassium phosphate, pH 7.0, 2 mM EDTA, 100 mM NaC1 with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 50 /zg/ml leupeptin, 100 units/ml aprotinin, and 20 mM benzamidine). The cell lysate is centrifuged at 3000 g for 10 min. The supernatant is retained and clarified at 100,000 g for 90 min. The high-speed supernatant is applied at a flow rate of 0.1 ml/ min to a Bio-Rex 70 column (1 ml) that has previously been equilibrated in buffer 1. The column is washed at 0.6 ml/min with 30 column volumes of buffer 1 containing 200 mM NaC1. The BCR protein is eluted at 0.1 ml/ min into 10 fractions of 5 column volumes of buffer 1 containing 900 mM NaC1. The amount of eluted protein may be monitored by OD280. The quality of the purification can be assessed by Coomassie blue or silver staining of the fractions resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The recovery of BCR protein can be analyzed by anti-BCR immunoblotting, immune complex kinase assays, or SDS-PAGE analysis of BCR isolated from [35S]Met-labeled cells. 4,5 Routinely, BCR elutes in fractions 2-5. At this point BCR may be stored for several weeks at - 7 0 ° in 50% glycerol. If necessary, further enrichment of BCR may be achieved using subsequent purification over Q Sepharose and Sephadex G-25 as described by Maru and Witte. 3
BCR Kinase Assay BCR kinase activity can be assessed by immune complex kinase assays. Antibodies directed against both the amino and carboxyl termini of BCR 9 D. E. H. Afar and O. N. Witte, unpublished results (1994).
128
GUANINE NUCLEOTIDE EXCHANGE
I15l
have been generated. 4,1°'u All of these antibodies are competent for immunoprecipitation and work well for in vitro kinase assays. An aliquot of partially purified BCR can be diluted into 1 ml of buffer 2:10 mM Tris, pH 7.5,150 mM NaCI, 1% Triton X-100, and protease inhibitors (see above, benzamidine may be excluded). When starting with tissue culture cells, cells may be lysed in ice-cold buffer 2 at up to 1 mg protein/ml of buffer 2. The cell lysate should be clarified at 100,000 g for 90 min. Antibodies directed against the amino or carboxyl termini of BCR are added for a 1-hr incubation on ice. Protein A-Sepharose (30/xl of packed beads) is added for an additional hour at 4 ° on a rocking platform. The immunoprecipitate is washed twice in buffer 2 and twice in kinase buffer (20 mM PIPES, pH 7.0, 10 mM MnC12). The kinase reaction is initiated by the addition of 10/zCi [y-32p]ATP at 30°. After 10 min the kinase reaction is terminated by the addition of 1 ml of ice-cold buffer 2 containing 5 mM EDTA. The samples are pelleted and eluted with SDS sample buffer for analysis by SDS-PAGE. BCR will also transphosphorylate exogenous added substrates such as histones and casein in vitro. To assess transphosphorylation activity, 5/xg of exogenous substrate is added to an immune complex kinase reaction. After 10 rain the reaction is terminated by the direct addition of SDS sample buffer. Assaying SH2-Binding Activity of BCR The SH2-binding activity of BCR resides in two regions of exon 1, which are termed the A box and the B box. 4 Both regions are rich in potential serine and threonine phosphorylation sites. The SH2-binding activity of BCR is dependent on phosphorylation. Although BCR/SH2 interactions are phosphotyrosine independent, tyrosine phosphorylation of BCR can greatly enhance the binding activity toward certain SH2-containing proteins. 12 SH2-binding reactions are usually performed using glutathione S-transferase (GST)-SH2 fusion proteins, which can be purified to homogeneity using glutathione(G)-Sepharose 4B beads (Pharmacia). 13 The G-Sepharose-GST-SH2 complex is used as a solid-state support to assess the binding activity to various proteins. We routinely use 35 ~g of purified GSTSH2 proteins to measure BCR-binding activity.5 As mentioned earlier, the 10M. S. Timmons and O. N. Witte, Oncogene 4, 559 (1989). 11 M. L. Campbell, W. Li, and R. B. Arlinghaus, Oncogene 5, 773 (1990). 12 y. Maru, K. L. Peters, D. E. H. Afar, M. Shibuya, O. N. Witte, and T. E. Smithgall, Mot Cell Biol. 15, 835 (1995). 13 D. B. Smith and K. S. Johnson, Gene 67, 31 (1988).
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BCR KINASEAND SH2-BINDING ACTIVITIES
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best source of BCR is baculovirus-produced BCR in SF9 cells. Since BCR prepared by crude cell lysates may interact with certain SH2 domains via a third binding partner, we recommend using partially purified BCR for the binding reactions. Binding reactions are carried out with 5/xg of partially purified BCR (see purification procedure above) added to 35/xg of GST-SH2 in buffer 3:20 m M HEPES, pH 7.0, 150 m M NaC1, 0.1% Triton X-100, 10% glycerol, 500/xM Na3VO4,20 m M NaF, and protease inhibitors (see above, benzamidine may be excluded). The binding reaction is carried out for 90 min at 4 ° on a rocking platform. The binding complexes are washed three times in buffer 3, and proteins are eluted with SDS sample buffer. The amount of GST-SH2 protein used in the assay can be monitored by Coomassie blue staining of samples resolved by S D S - P A G E . BCR-binding activity can be measured using in vitro kinase reactions of the bound material, evaluation by anti-BCR immunoblotting, or S D S - P A G E analysis of BCR isolated from [35S]Met-labeled cells. Summary BCR is an interesting signaling protein, whose cellular function is currently unknown. Its biochemical properties include serine kinase activity, SH2-binding activity, and a GTPase-activating activity. The SH2-binding activity is particularly interesting because it may link BCR to signaling pathways involving SH2-containing molecules. Since tyrosine phosphorylation of BCR has been detected in CML-derived cell lines 14 and since tyrosine-phosphorylated BCR shows increased affinity toward certain SH2 domains, it seems particularly important to further characterize this activity. This chapter described a simple purification scheme for partial purification of BCR, which can be used to assess in vitro kinase and SH2-binding activities. Acknowledgments This work was supported by Grant CA53867 from the NIH (O, N. W.) and by a fellowship from the Medical Research Councilof Canada (D. E. H. A.). O. N. W. is an Investigatorof the Howard Hughes Medical Institute.
14D. Lu, J. Liu, M. Campbell, J. Qiang Guo, N. Heisterkamp, J. Groffen, E. Canaani, and R. Arlinghaus,Blood 82, 1257 (1993).
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GUANINENUCLEOTIDEEXCHANGE
[16]
1161
Identification of GTPase-Activating Proteins by Nitrocellulose Overlay Assay
By E D W A R D
MANSER, THOMAS LEUNG,
and LOUIS LIM
Introduction The major GTPase-activating protein (GAP) for Ras, p120 Ras-GAP, was the first protein to be identified that could modulate the nucleotide state of p21 Ras. 1 Subsequently, GAPs were identified for every class of p21, for example, Rapl, 2 Rab3, 3 Cdc42, 4 and RhoA. 5 Interestingly, the sequence of a peptide from this purified R h o A - G A P showed similarity to the breakpoint cluster region (BCR) protein and neuronal (n)-chimaerin of a previously unknown function. These proteins were then shown to exhibit GAP activity. 6 At the outset, therefore, it appeared that a number of GAPs for Rho family p21s existed, although the relative contribution of these and other species in cell or tissue extracts was unresolved. In order to facilitate the study of chimaerin and other brain R a c l - G A P s , an overlay method was developed to visualize proteins exhibiting GAP activity after they are separated by SDS-polyacrylamide gel electrophoresis. 7 Figure 1 summarizes the steps involved for the method. The sensitivity of the technique lies in the ability to label p21s to high specific activity with [y-32p]GTP. Under the conditions given in the next section, a fraction of the added recombinant GST/p21 is labeled (as an upper limit, 1/xg of the 5/.~g added). If this is diluted in 2 ml of buffer, of which 200/xl is absorbed onto the filter containing the immobilized proteins, then the concentration of [y32p]GTP-labeled p21 is 25 ng/cm 2. Within the local area of a GAP protein band (estimated as -0.1 cm 2) there are perhaps 2.5 ng of labeled p21. This is probably lower than the amounts of the major R h o - G A P s present in 200 /xg of a typical extract, as estimated from signals produced by recombinant protein. 7 Although only a fraction of the GAP proteins may refold on the 1 M. Trahey and F. McCormick, Science 238, 542 (1987). R. Rubinfeld, S. Munimetsu, R. Clark, L. Conroy, K. Watt, W. J. Crosier, F. McCormick, and P. Polakis, Cell 65, 1033 (1991). 3 E. S. Burstein and I. G. Macara, Proc. Natl. Acad. ScL U.S.A. 89, 1154 (1992), 4 M. J. Hart, K. Shinjo, A. Hall, T. Evans, and R. A. Cerione, J. Biol. Chem. 266, 20840 (1991). 5 M. D. Garrett, G. N. Major, N. Totty, and A. Hall, Biochem. J. 276, 833 (1991). 6 D, Diekmann, S. Brill, M. D. Garrett, N. Totty, J. Hsuan, C. Monfries, C. Hall, L. Lira, and A. Hall, Nature 351, 400 (1991). 7 E. Manser, T. Leung, C. Monfries, M. Teo, C. Hall, and L. Lira, J. Biol. Chem. 267, 16025 (1992).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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nitrocellulose filter, this is sufficient to catalyze the complete hydrolysis of terminal phosphate from p21-bound [y-32p]GTP, releasing the labeled phosphate, thus generating the loss of signal that produces a white band against a dark background. The use of an in situ detection method has proved to have a number of advantages over more conventional G A P assays: (1) Many extracts (8 per minigel) can be analyzed and compared simultaneously. (2) Sample handling time is minimal (with less chance of protein degradation) since tissues extracts are immediately denatured and analyzed. (3) On the basis of their size, all major GAPs can be analyzed, without need for specific reagents (e.g., antibodies). (4) Potential G A P inhibitor proteins present in the extracts are dissociated during SDS-polyacrylamide electrophoresis. (5) Filters can be sequentially reprobed with different p21 "substrates," thus giving information about the p21 specificity of each G A P band. Since the m e t h o d is applicable to all the Rho p21s and GAPs we have tested, it should prove to be a useful tool in the analysis of those related p21s identified in lower organisms 8'9 for which mammalian homologs have not been identified.
Labeling of R e c o m b i n a n t p21 P r o t e i n s Rho p21 proteins are extremely well conserved, for example, human Cdc42 is 80% identical to that of S a c c h a r o m y c e s cerevisiae, and can partially complement yeast Cdc42 temperature-sensitive mutants, l°'u For this reason the origin of the mammalian p21 cDNAs to generate recombinant protein for the assays is of no consequence to the final results; even invertebrate homologs behave identically in in vitro G A P assays. 12 The cDNAs can be obtained by polymerase chain reaction (PCR) using published sequences (usually human) or by screening of a single library plate using a homologous probe (the m R N A s are relatively abundant). Purification and analysis of these proteins in Escherichia coli are described elsewhere in this volume. Ras-related G proteins have extremely high affinity for G T P (and G D P ) in the presence of millimolar magnesium. 13 In order to facilitate exchange with labeled GTP, the p21s are incubated in an E D T A buffer; when they 8y. Matsui and A. Toh-e, Gene 114, 43 (1992). 9j. Bush, K. Franek, and J. Cardelli, Gene 136, 61 (1993). i0 S. Munemitsu, M. A. Innis, R. Clark, F. McCormick,A. Ullrich, and P. Polakis, Mol. Cell, BioL 10, 5977 (1990). u K. Shinjo,J. G. Koland, M. J. Hart, V. Narasimhan, D. I. Johnson, T. Evans, and R. Cerione, Proc. Natl. Acad. Sci. U.S.A. 87, 9853 (1990). 12W. Chen, H.-H. Lim, and L. Lira, J. Biol. Chem. 268, 320 (1993). 13R. S. Goody, M. Frech, and A. Wittinghofer, T1BS 16, 327 (1991).
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are returned to buffer containing magnesium, the labeled nucleotide is locked into the guanine nucleotide-binding site.
Methods
Purified GST/p21 fusion protein (1 mg/ml) or cleaved p21 (0.5 mg/ml) are stored in aliquots at - 7 0 °. For each labeling use 5/xl of protein (100 pmol) in 50/zl of exchange buffer containing 1-10/zl of [T-32p]GTP (NEN -6000 Ci/mmol) corresponding to 1.5-15 pmol of GTP. The decay in specific activity of [y-32p]GTP can be compensated with time by adding more [y-32p]GTP since the molar amounts of label are much lower than that of the p21, even assuming that 50% of the recombinant protein may be denatured during purification and storage. Bovine serum albumin (BSA) or Triton X-100 is present in the buffers to stabilize the diluted p21 protein. Mix and leave at room temperature for 4 min. Put this exchange mix back on ice and use within 15 min. Exchange buffer: 50 mM NaC1, 25 mM morpholine-ethanesulfonic acid (MES)-NaOH, 2.5 mM EDTA, 0.05% Triton X-100. Make up 2× stock and keep at 4 °.
Identification of GAPs in Cell and Tissue Extracts In solution assays, all tissue extracts demonstrate varying levels of GAP activity against [-),-32p]GTP-labeled Rho proteins. Different proteins which contribute to this activity can be detected by the GAP nitrocellulose overlay protocol as outlined in Fig. 1. On the basis of the behavior of recombinant GAP domains and native purified GAP proteins, this assay appears to be able to detect individually all the major species that contribute to various R h o - G A P activities. The use of high-resolution SDS-polyacrylamide electrophoresis not only allows single step separation of GAP proteins, but also gives important information on the apparent molecular size of these proteins. When probed with different p21 substrates, conclusions can be drawn as to the type of GAP protein generating the observed signal (see next section). Of all the tissues tested, the brain appears to contain the strongest and most diverse R h o - G A P activities. In the case where GAPs have similar mobility in SDS gels, a simple fractionation of the extract by ion-exchange chromatography can sometimes resolve the different bands. 7 This is illustrated in Fig. 2 where detergent extracts of rat brain cortex, cerebellum, and rat testis were subjected to ion-exchange fractionation and probed for R a c - G A P activity. Here the c~2-chimaerin (present in cortex
[161
DETECTION OF G A P s SEPARATED BY S D S - P A G E
133
cell or tissue extracts SDS-PAGE
2dq2q2q_r2.Utdq2-
nitrocellulose filter
-- 2__
electro-blot
soak in ['d-32p]GTP-labeled p21
overlay with nitrocellulose
1% agarose plate remove replica filter se to X-ray film
m m
~
m
m m
FiG. 1. Schematic diagram of the G A P nitrocellulose overlay protocol.
and testis TM) and p50 R h o - G A P 15 are clearly separated. The smaller oqand/31-chimaerins 16'17 appear to be the only Rho family GAPs that are predominantly associated with an "insoluble," detergent extractable fraction. All of the other GAPs are released in hypotonic buffers; however, 14 C. Hall, W.-C. Sin, M. Teo, G. J. Michael, P. Smith, J.-M. Dong, H.-H. Lim, E. Manser, N. K. Spurr, T. A. Jones, and L. Lim, MoL Cell BioL 13, 4986 (1993). 15 C. A. Lancaster, P. M. Taylor-Harris, A. J. Self, S. Brill, H. E. van Erp, and A. Hall, J. BioL Chem 269, 1137 (1994). 16 C. Hall, C. Monfries, P. Smith, H.-H. Lim, R. Kozma, S. A h m e d , V. Vanniasingham, T. Leung, and L. Lim, J. MoL BioL 211, 11 (1990). 17 T. Leung, B.-E. How, E. Manser, and L. Lim, J. Biol. Chem. 268, 3813 (1993).
134
[161
GUANINE NUCLEOTIDE EXCHANGE Cortex
kDa 1
2
Cerebellum 3
1
2
3
Testis 1
2
GAP species 3
2O0 97
p190 BCR - - -
ABR ?
68 RhoGAP J 43
~2-
-
-
~2-
-
-
0~1-
29 Fro. 2. In situ R a c - G A P activity of fractionated detergent-extracted proteins and assignm e n t of the bands to known G A P s . Rat brain cortex and cerebellum and rat testes were subjected to the hypotonic extraction procedure (see methods), pellets were then homogenized in 25 m M M E S - N a O H , p H 6.5, 1% deoxycholate, and clarified. This extract was diluted to 10 m g / m l and loaded onto 1/2 volume S-Sepharose column. T h e flow-through fraction was collected (lane 1), the column was washed with 25 m M M E S - N a O H , p H 6.5, 0.5% deoxycholate, containing 50 m M NaC1, and fractions were collected at 250 m M NaC1 (lanes 2) and at 500 m M NaC1 (lanes 3) in the same buffer. A n equal volume of sample buffer was added to each fraction and 40/xl of each was r u n on 9% polyacrylamide gels and processed for G A P nitrocellulose assay. T h e identities of the various G A P activities are summarized: a l , c~2,/31 and/32 refer to the various chimaerin isoforms. 14.16 18
brain extracts, which contain a complexity of membrane compartments, trap a considerable amount of "soluble" material that is released by subsequent detergent extraction. In Fig. 2 the cerebellar/32-chimaerin protein (the SH2containing alternate-spliced form of p30 testis Rac-GApas), which is larger than cortical o~2-chimaerin, fails to be resolved from p50 Rho-GAP. In fact,/32-chimaerin is only detected in the 0.25 and 0.5 M NaC1 fractions by Western blotting (not shown). The reciprocal distribution of/31- and o~2chimaerins in the testis versus c~1- and/32-chimaerins in the cerebellum is intriguing. Identities for most of the brain GAPs can be inferred from published data (all the mammalian Rho-GAPs act on Racl in vitro), but the indistinct bands at a position corresponding to -170 kDa probably contain both BCR 19 and "p190" GAP. 2° The GTPase inhibitory proteins which produce the darker bands are discussed in [24] in this volume. Unidentified GAPs (on the basis of their molecular weight) are at lower levels and may represent novel or alternative-spliced variants of those already cloned. Although not reported as yet, novel GAPs can be detected in other soluble extracts, for example, from blood cells such as platelets. While signal intensity is only qualitative, the method has been used to follow the 18 T. Leung, B.-E. How, E. Manser, and L. Lim, J. Biol. Chem. 269, 12888 (1994). 19 M. S. T i m m o n s and O. N. Witte, Oncogene 4, 559 (1989). 20 j. Settleman, C. F. Albright, L. C. Foster, and R. A. Weinberg, Nature 359, 153 (1992).
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DETECTION OF GAPs SEPARATEDBY SDS-PAGE
135
purification of a2-chimaerin (estimated by overlay to represent ~20% of R a c - G A P activity in brain extracts) to a point where quantitative solution assays can be performed. 14 Methods Rat tissues or cells are freshly homogenized in 4 vol of hypotonic buffer using 20 strokes of a hand-held Dounce homogenizer. The cytosol is clarified by spinning at 30,000 rpm for 40 rain (Beckman Ti50 rotor), made 5% in glycerol or diluted with an equal volume of SDS sample buffer (containing a final concentration of 1% SDS), and stored at -70 °. Polyacrylamide gels are cast using the Bio-Rad Mini-PROTEAN system or equivalent using the gel mix given at the end of the chapter. This includes dithiothreitol (DTT), magnesium, and glycerol which, in addition to stabilizing the proteins, produces sharper bands that are less prone to diffusion during electrophoretic transfer to nitrocellulose. Since all GAPs have molecular mass >30 kDa, 9% gels or less are run in order to allow good protein resolution and efficient transfer. Use a semidry blotting apparatus (now available from many suppliers), which minimizes heating. Despite manufacturers claims, efficient transfer of high molecular weight proteins (>100,000) requires long transfer times, and the gel sandwich should be thoroughly soaked in buffer before putting the upper electrode in place. Use prestained protein markers (BRL) so that the efficiency of transfer can be seen. Transfer overnight at 4° using 20 mA per minigel (set maximum voltage at 10 V). The nitrocellulose filters are blocked in the renaturing buffer for at least 1 hr at 4° (up to 24 hr). An agarose plate (for each filter) is taken from storage and allowed to warm up to room temperature. Make up enough cold GAP buffer mix for the filters (2 ml for one or two filters) and add 0.5 mM cold GTP (5 tzl of 100 mM stock per ml buffer) and the [yYP]GTP-labeled p21 to the GAP buffer. Evenly soak each filter in this buffer, remove the excess by scraping against the side of the container, and lay them carefully onto the 1% agarose plates (all excess liquid must be absorbed by the agarose at this stage). Leave for 5 min at room temperature and then transfer the plate to the cold room and leave for 10 min. Wet a nitrocellulose filter of the same size with wash buffer, and blot with Whatman 3MM paper. Starting from one side, slowly lay this across the top of the original filter (bubbles must be avoided). Leave for 5 min to absorb the labeled p21, then remove the filter and wash (three changes of 50 ml, 1 min each). Blot the filters to remove excess liquid, and place the filter between Saran wrap. Place these in a precooled cassette and expose to film overnight at - 7 0 ° without an intensifying screen. The [y-32p]GTP-labeled p21 absorbed onto the filter provides an even dark background on the autoradiograph, the intensity of which should be adjusted by exposure time:
136
GUAr~NZNVCI.EOTmEZXCI~ANGE
[ 16]
GAP activity produces a local loss of this signal. Because these bands are sometimes difficult to visualize in the final reproduction, a clearer image can be obtained by making a contact print from the autoradiograph directly onto high-contrast photographic paper. In this case, GAP activity is represented as black bands as is clearly illustrated in Fig. 3. Hypotonic buffer: 10 mM Tris, pH 8.0, 5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 1/xg/ml of aprotinin and pepstatin. Polyacrylamide gel mix: The polyacrylamide separating gel of required percentage (using 29.2% acrylamide/0.8% bisacrylamide w/w from Millipore) is cast containing 125 mM Tris-HC1, pH 8.8 10% glycerol (v/v), 5 mM DTT, 0.5 mM MgC12, 0.1% SDS (w/v). Stacking gel (4%): 125 mM Tris-HC1, pH 6.8, 10% glycerol, 0.1% SDS. Blot transfer buffer: 50 mM Tris (base), 40 mM glycine, 10% methanol, 0.5 mM MgCI2, 0.02% Triton X-100 (Boehringer Mannheim), 0.02% SDS. Renaturing buffer: phosphate-buffered saline (PBS) containing 1% BSA (Sigma, protease-free), 5 mM DTT, 0.5 mM MgC12,0.1% Triton X-100. Store at 4 ° for up to 1 week. GAP buffer: 50 mM NaC1, 25 mM M E S - N a O H , pH 6.5, 2.5 mM DTT, 1.25 mM MgCI2, 0.05% Triton X-100. Make up as 2× stock and keep at 4 °. Agarose plates: 1% agarose is melted in PBS; when cooled to 60 °, add from stock solution to give final concentrations of 25 mM M E S RacGAP overlay kDa
b
h
k
li
lu
Reverse image m
s
t
Tissue
b
h
k
li
lu
m
s
t
200 97 68 43 29
FIG. 3. Reverse e n h a n c e d image of R a c - G A P activity detected in tissue extracts. Soluble tissue proteins were extracted and separated on a 9% polyacrylamide gel. Samples of 200/zg/ lane were from the following tissues: brain, b; heart, h; kidney, k; liver, li; lung, lu; muscle, m; spleen, s; and testis, t. The proteins were transferred to nitrocellulose and assayed for R a c - G A P activity as described in the m e t h o d section. A reverse image of the autoradiograph was obtained by making a contact print using Ilford grade 4 paper.
[16]
DETECTION
OF G A P s SEPARATED BY S D S - P A G E
137
~-Chim
DINIITGALKLYFRDLPIPLITYDAYPKFIESAKIMDPDEQLETL
..... HEALKL
~-Chim
DINIITGALKLYFRDLPIPIITYDTYTKFIEAAKISNADERLEAV
..... HEVLML
BCR
DVNAIAGTLKLYFRELPEPLFTDEFYPNFAEGIALSDPVAKESCM
..... LNLLLS
ABE
DINAIAGTLKLYFRELPEPLLTDRLYPAFMEGIALSDPAAKENCM
..... MHLLRS
BEM3
GVNTVSGLLKLYLRKLPHLLFGDEQFLSFKRWDENHNNPVQISL.GFKELIESGL
RHOGAP
ELHLPAVILKTFLRELPEPLLTFDLYPHWGFLNIDESQRVPATL
...... QVLQT
p190
TVNTVAGAMKSFFSELPDPLVPYSMQIDLVEAHKINDREQKLHAL
..... KEVLKK
BEM2
EVNAIAGCFKMYLRELPDSLFSHAMVNDFTDLAIKYKAHAMVNEEYKRMMNELLQK
GAP25
.........
KLYFRDLPQPLVPPLLLPHFRAALALSESEQCLSQI
..... Q E L I G S
p21
Sensitivity
Rac
Cdc
~-Chim
LPPAHCETLRYLMAHLKRVTLHEKENLMNAENLGIVFGPTLMRS
+++
+
~-Chim
LPPAHYETLRYLMIHLKKVTMNEKDIqLMNAENLGIVFGPTLMRP
+++
+
BCR
LPEANLLTFLFLLDHLKRVAEKEAVIWKMSLHNLATVFGPTLLRP
++
++
ABR
LPDPNLITFLFLLEHLKRVAEKEPINKMSLHNLATVFGPTLLRP
++
++
Rho
BEM3
VPHANLSLMYALFELLVRINENSKF~d~NLRI~'LCIVFSPTLNIP
?
++
RHOGAP
LPEENYQVLRFLTAFLVQISAHSDQNKMTNTNLAVVFGPNLLWA
++
+++
+ ++
p190
FPKENHEVFKYVISHLNKVSHNNKVI~LMTSENLSICFWPTLMRP
++
++
+++
BEM2
LPTCYYQTLKRIVFHLNKVHQHVVIVNKMDASNLAIVFSMSFINQ
?
GAP25
MPKPNHDTLRYLLEHLCRVIAHSDKNRMTPHNLALVFGPTL
(+)
FIG. 4. Alignment of conserved residues among identified Rho-GAPs. The region of the GAP domain containing well-conserved amino acid residues to which degenerate oligonucleotides were designed (underlined sequences) for PCR of novel GAPs including/3-chimaerin 17 and ABR 19 is shown. The relative ability of these various GAP domains to activate the intrinsic GTPase activity of Racl, Cdc42, and RhoA is shown; the original references describing these GAPs are given in the text. N a O H , p H 6.5, 5 m M D T T , 5 m M MgC12. Pour e n o u g h to cover a number of 12 × 12-cm 2 tissue culture plates. Wash buffer: Phosphate-buffered saline containing 25 m M M E S N a O H , p H 6.5, 5 m M MgC12, 0.05% Triton X-100. C l o n i n g o f N o v e l GAPs D e t e c t e d b y Overlay A considerable number of R h o - G A P s are n o w k n o w n (Fig. 4), but there are u n d o u b t e d l y others yet to be identified. The most interesting of these m a y be tissue- or cell-specific G A P s . A l t h o u g h the nitrocellulose overlay assay can be used to detect G A P activity, it has b e e n found to be technically difficult to extend this for expression screening because small bubbles trapped b e t w e e n the two nitrocellulose filters lead to a large number of false-positive signals. In contrast, screening for p21 GTPase inhibitory proteins has proved more successful. 21 Fortunately, sequence conservation 21 E. Manser, T. Leung, H. Salihuddin, L. Tan, and L. Lim, Nature 363, 364 (1993).
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GUANINE NUCLEOTIDEEXCHANGE
[ 16]
within the G A P domain allows novel G A P cDNAs to be obtained by PCR using degenerate oligonucleotides. The c D N A products from a particular tissue can be matched to known and unknown G A P sequences on the basis of the observed size and activity in G A P overlays. For example, the 30kDa G A P specific for Rac in the insoluble fraction of rat testis (see Fig. 2),/3-chimaerin, 17 was cloned by use of degenerate PCR oligonucleotide primers. The similarity of the predicted amino acid sequence of the new /3-chimaerin PCR product to o~-chimaerin suggested at the time of isolation that this corresponded to the p30 R a c - G A P . The conserved regions underlined in Fig. 4 proved the most successful in our hands, also allowing amplification and isolation of c D N A for a brain-enriched 100-kDa Rac/ Cdc42 GAP, 22 a small portion of which was previously isolated and termed the active BCR-related protein (ABR23). Figure 4 shows alignment of part of the G A P domain (boxes 2 and 3 according to Lancaster e t al. 15) of those proteins whose selectivity for Rho p21s has been determined. The amino acid residues, which are well conserved within the G A P domain, are marked in bold. After PCR of reverse-transcribed m R N A and identification of a product of the correct size(s), known sequences can be eliminated by digestion of the c D N A product with restriction enzymes which can cleave these within the intervening (less conserved) region. After agarose gel electrophoresis the remaining c D N A band contains potentially uncharacterized products. With the recent publication of two mammalian R h o A - G A P sequences, the p190 Ras-GAP-associated protein 2° and p50 R h o - G A P , a8'24 and the identification of yeast R h o - G A P s including Bem2 and Bem3, 25'26 it is possible to design primers with more certainty of cloning unknown GAPs. Such an example is given in Fig. 4 which shows the predicted partial sequence of an unidentified protein (GAP25) obtained from PCR of rat brain cDNA. The conservation of conserved residues (marked as bold) between the two primers confirms this as a bona fide product.
Conclusions The characterization of Rho p21 modulatory/interacting proteins is far from complete. Five classes of proteins have been identified: the guanine 22E.-C. Tan, T. Leung, E. Manser, and L. Lim, J. Biol. Chem. 268, 27291 (1993). 23N. Heisterkamp, C. Morris, and J. Groffen, Nucleic Acids Res. 17, 8821 (1989). 24E. T. Barfod, Y. Zheng, W.-J. Kuang, M. J. Hart, T. Evans, R. A. Cerione, and A. Ashkenazi, J. Biol. Chem. 268, 26059 (1993). 25y. Zheng, M. J. Hart, K. Shinjo, T. Evans, A. Bender, and R. Cerione, J. Biol. Chem. 268, 24629 (1993). 26y. Zheng, R. Cerione, and A. Bender, J. Biol. Chem. 269, 2369 (1994).
[16]
DETECTION OF GAPs SEPARATEDBY SDS-PAGE
139
nucleotide release factors, which are sequence related to Cdc24p and db127; the GAPs; guanine nucleotide dissociation inhibitors, which are also GTPase inhibitory28; GTP-p21-associated kinases such as ACK and PAK2a,29; and the neutrophil oxidase complex proteins. 3° At present GAPs appear to be the most diverse (and perhaps abundant) regulatory proteins, suggesting that they function as more than negative regulators of GTPbound p21s. While in vitro assays show that many of these GAPs are promiscuous in terms of their substrate p21s, they appear to be more selective in vivo, 31 perhaps as a consequence of interactions with other proteins. The observation that the testis/31-chimaerin is expressed at a time of gross morphological transformation of the developing spermatid provides indirect evidence for specific roles for R h o - G A P s . 15 In considering functional targets for new GAPs it must also be kept in mind that probably not all mammalian Rho family p21s have yet been identified. While in vitro assays can quantify the expected interaction between p21s and the proteins discussed earlier, studies of Ras signaling suggest the existence of signal transduction complexes. The challenge in studying the Rho p21s is to use the information now available from various experimental protocols and develop methods to identify proteins that may link extracellular signaling through Rho p21s to the cytoskeletal reorganization (as implied by p21 microinjection experiments). It could be that GAP proteins have an integral role in signal transduction in signaling complexes. The GAP overlay assay therefore promises to be useful not only in the general identification of GAPs, but also in the further characterization of their function. Acknowledgments We thank the Glaxo-Singapore Research Fund for support and Christine Hall for help with photography.
27 M. J. Hart, A. Eva, T. Evans, A. Aaronson, and R. Cerione, Nature 354, 311 (1991). 28 M. J. Hart, Y. Maru, D. Leonard, O. N. Witte, T. Evans, and R. A. Cerione, Science 258, 812 (1992). 29 E. Manser, T. Leung, H. Salihuddin, Z.-S. Zhao, and L. Lim, Nature 367, 40 (1994). 3o A. Abo, E. Pick, A. Hall, N. Totty, C. G. Teahan, and A. W. Segal, Nature 353, 668 (1991). 31 A. J. Ridley, A. J. Self, F. Kasmi, H. F. Paterson, A. Hall, C. J. Marshall, and C. Ellis, E M B O J. 12, 5151 (1993).
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GUANINE NUCL]EOTIDE EXCHANGE
[ 17] I d e n t i f i c a t i o n o f 3 B P - 1 i n c D N A E x p r e s s i o n by SH3 Domain Screening
[ 17]
Library
By PIERA CICCHETFI a n d D A V I D BALTIMORE Introduction We have developed a screening procedure to assay for protein-protein interactions using a biotinylated glutathione S-transferase (GST) fusion protein probe to screen cDNA expression libraries. This method has been developed to allow nonradioactive probing which has the additional advantages of both a very low background signal and a low incidence of falsepositives. Since neither the fusion protein probe nor the protein in the expression library is denatured, this assay allows for the structural components of the binding reaction to remain intact. We have found that this screening procedure allows for a practical and highly efficient method of identifying protein-protein interactions. For our purposes, we have screened a ~ gtll cDNA expression library derived from the mouse pre-B cell line 22D6, with a GST fusion protein probe containing the Src homology 3 (SH3) domain of the Abelson (Abl) nonreceptor tyrosine kinase. 1 The SH3 domain comprises approximately 55 amino acids and is mainly found in nonreceptor tyrosine kinases such as Src and Abl, although various signaling molecules, such as phospholipase C and GTPase-activating proteins (GAP), as well as cytoskeletal proteins such as myosin and spectrin also contain SH3 domains. 2 Until recently, this domain, based on sequence homology, had no known function. There were indications that the SH3 domain might have an inhibitory effect on transformation since deletions or mutations of this region in Src or Abl activated transformation by these kinases. 3'4 The high incidence of this modular domain in a large number and variety of proteins, along with its effect on transformation, made it seem likely that SH3 domains, independent of their protein context, might be capable of interacting with a protein or proteins that could potentially shed light on SH3 function. Using this screening procedure to detect proteins which bound to the Abl SH3 domain, we isolated five cDNA clones out of 7 million cDNA1 p. Cicchetti, B. J. Mayer, G. Thiel, and D. Baltimore, Science 257, 803 (1992). 2 A. Musacchio, T. Gibson, V.-P. Lehto, and M. Saraste, F E B S 307, 55 (1992). 3 p. Jackson and D. Baltimore, E M B O J. 8, 449 (1989). 4 C. Seidel-Dugan, B. E. Meyer, S. M. Thomas, and J, S. Brugge, Mol. Cell. Biol. 12, 1835 (1992).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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141
containing plaques screened. Of these five, three contained the identical cDNA clone, termed 3BP-1, while the other two were identical for a different clone, termed 3BP-2. On sequencing these clones, we found only a short stretch of approximately 40 amino acids where sequence similarity existed between these proteins. The most striking characteristic of this similarity was the high incidence of proline residues, and this proline-rich area was hypothesized to constitute the SH3-binding domain. In addition, we found that 3BP-1 shares sequence homology with the GAPs Bcr, n-chimaerin, and Rho-GAP.
Making Probes The GST-Abl SH3 fusion protein along with other GST-SH3 fusion proteins were made using the pGEX expression system (Pharmacia) and purified as described. 5The purified proteins were then biotinylated using the following protocol which was adapted from that used for the biotinylation of antibodies. 6 1. Exchange fusion proteins at a concentration of at least 1 mg/ml into 100 mM sodium borate buffer, pH 8.8. 2. Immediately before use, resuspend N-hydroxysuccinamide biotin ester (biotin amidocaproate N-hydroxysuccinamide ester, Sigma B2643) in dimethyl sulfoxide at a concentration of 10 mg/ml. 3. Add biotin solution to fusion protein at a ratio of 50/zg ester to 1 mg fusion protein. 4. Mix and incubate at room temperature for 4 hr. 5. Add 20/xl of 1 M NH4C1 per 250 tzg biotin ester used and continue incubation for 10 min at room temperature. 6. Exchange buffer into phosphate-buffered saline and, at the same time, remove unbound biotin from the fusion protein solution using PD10 columns (Pharmacia). 7. Aliquot and add 15% glycerol before storing the biotinylated probes at - 2 0 °, thawing on ice before each use. It is important to test whether the biotinylation procedure has worked optimally before the probe is used. In almost all cases if the biotinylation procedure is carried out carefully, the biotinylation level is proportional to the protein level so that probes can be standardized by relative protein 5 F. Ausubel et aL, eds., in "Current Protocols in Molecular Biology." John Wiley, New York, 1988. 6 E. Harlow and D. Lane, eds.,"Antibodies:ALaboratoryManual." Cold Spring Harbor, 1988.
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GUANINENUCLEOTIDEEXCHANGE
[ 17]
concentration. This is important for control probes and also allows for determining relative affinities of binding to a particular ligand for a variety of related probes. If too much biotinylation reagent is used or too little protein (concentration should be at least 1 mg/ml), overbiotinylation may result. This is sometimes observed as an upward mobility shift of the protein on SDS-PAGE. If this happens, the probe should be discarded and the biotinylation procedure repeated because overbiotinylation is likely to interfere with the binding properties of the fusion protein probe. The probes must therefore be checked for protein content and for the level of biotinylation. For this purpose, probes were run on SDS-PAGE and one set was stained with Coomassie blue while another set was transferred to nitrocellulose. The Coomassie-stained gel allowed approximation of protein content of the probes while the nitrocellulose transfer was incubated with streptavidin alkaline phosphatase (SA-AP) to assay the relative biotin content of the probes. The S A - A P development of the nitrocellulose transfer was performed as follows: 1. Wash twice with d.HeO then once with TBST (50 mM Tris, pH 8.0, 150 mM NaC1, and 0.1% Tween). 2. Block with TBST/G (TBST + 0.2% w/v gelatin) for 30 min at room temperature. 3. Add S A - A P (1000 units/ml Boehringer Mannheim No. 1089 161) into blocking buffer at a dilution of 1 : 5000 and incubate at room temperature for 45 min. 4. Wash three times in TBST and once in alkaline phosphatase (AP) buffer (100 mM Tris, pH 9.5, 100 mM NaC1, 5 mM MgC12). 5. Develop color reaction with nitroblue tetrazolium (NBT) and 5bromo-4-chloro-3-indolyl phosphate (BCIP) (Promega) as per manufacturer's protocol. Figure i shows an example of various GST-SH3 domain fusion proteins assayed for relative protein and biotinylation levels. A Coomassie-stained gel reveals the protein level of the probes whereas an identical gel, transferred to nitrocellulose and incubated with SA-AP, indicates the relative biotin content of the protein probes. The biotinylation of these proteins was consistent in that the level of biotin is essentially proportional to the level of protein for each probe. Screening cDNA Expression Library Phages are plated out and transferred to nitrocellulose as described 7 with the following exceptions: 7 R. Singh, G, Clerc, and J. H. Le Bowitz, Biotechniques 7, 258 (1989).
[ 171
SH3 DOMAINSCREENING
143
| m Q
P
o
11t
FIG. 1. Protein/biotin standardization of GST fusion protein probes: (Top) Coomassiestained GST fusion protein probes after SDS-PAGE. (Bottom) Identical samples transferred to nitrocellulose and assayed for biotin level.
1. The plaques are screened at a very high density (at least 2 × 105 plaque-forming units per 150-mm plate) in order to maximize the number of clones assayed. This has the disadvantage that since the plaques are in contact with each other and their morphology is disturbed, positive plaques appear simply as dark stained spots and are not easily identifiable as dark colored rings (as described later). We screened approximately 7 million plaques, isolating seven intense dark spots over background. Five of these seven remain positive after secondary and tertiary screening with control probes, and show up as the familiar dark rings at these lower density screenings. On sequencing the five positive plaques isolated, two contain the identical cDNA, clone I, whereas the other three are identical for a different cDNA, clone II. Although it is thus possible to screen at a very high density, screening at a lower density can be preferable, though more time-consuming. It is important, however, when first testing the system, to screen at a lower density in order to be confident that the screening proce-
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GUANINE NUCLEOTIDE EXCHANGE
[ 171
dure is working and that the color reaction is occurring specifically on plaques which can be identified as having the familiar ring morphology. 2. The incubation of filters at 37 ° can go for 3 hr instead of 6 hr and then small numbers of plates should be removed to room temperature to mark the position of the filter on the plate (filters and plates should be numbered with a pen and marked with a syringe needle containing ink in three places to allow for later alignment). Lift the filter off the plate and place into TBST (once the filters have been incubated with the phage, do not allow them to dry out). Steps 3 and 4 are used for secondary and tertiary screening only. 3. Place new isopropyl fl-D-thiogalactoside-soaked/dried filters (control set to be probed with a control biotinylated GST fusion probe) on plates for another 3 hr at 37 ° (use the same numbers and alignment marks as for original filters). 4. Remove second set of filters and place in TBST.
Probing of Nitrocellulose Filters 1. Block flters for 30 mill in TBST/G at room temperature. Steps 2-5 are necessary for eliminating a particularly high background signal obtained by simply adding S A - A P to the filters. These steps may not be necessary, and it should first be ascertained if there is a background problem and, if so, if it is due to binding of S A - A P nonspecifically. In our screening, a protein was present in each of the plaques that bound S A AP. We therefore blocked the filters first with avidin and then with biotin. As biotin has only one binding site for avidin, once bound, it cannot bind to the streptavidin-alkaline phosphatase used later in the protocol. This procedure eliminates the high background signal and results in a very light nonspecific staining of plaques. This light outline of plaques is very useful for orientation purposes. Figure 2 shows plaque-containing filters at various stages of this procedure and indicates the clarity of the positive signal obtained over background. 2. Add 1 nM avidin (Pierce) to 10 ml TBST/G blocking solution and incubate for 1 hr at room temperature. 3. Wash three times in TBST. 4. Incubate with 100 nM biotin (Sigma) in 10 ml TBST/G for 1 hr at room temperature. 5. Wash three times in TBST (cold). 6. Incubate filters with 1/xg biotinylated peptide per ml TBST/G for 2 hr to overnight at 4 ° (our probe was unstable at room temperature so all buffers and incubations with the probe were at 4°; if probe is stable at room
[171
SH3 DOMAIN SCREENING
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A
G
I
Fie. 2. Biotin/avidin blocking procedure for plaque-containing filters: (A) blocked in only TBST, then SA-AP added and color developed as described; (B) same as A except blocked in TBST/G; (C) same as B plus blocked with avidin and biotin (as described below), before the addition of SA-AP and color development; and (D) same as C except incubated with biotinylated fusion protein probe after biotin/avidin block, and then incubated with S A - A P and color developed as described.
t e m p e r a t u r e all i n c u b a t i o n s can b e d o n e at r o o m t e m p e r a t u r e a n d initial i n c u b a t i o n can b e for 1 - 2 hr). 7. W a s h f o u r t i m e s in T B S T (4°). 8. I n c u b a t e w i t h S A - A P at 1 : 5000 in T B S T / G for 1 h r at 4 °. 9. W a s h t h r e e t i m e s in T B S T (4 °) a n d o n c e in A P buffer. 10. C o l o r d e v e l o p w i t h N B T / B C I P ( P r o m e g a ) , as p e r m a n u f a c t u r e r ' s protocols.
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[ 17]
11. When color develops to satisfaction, wash filters in water quickly but thoroughly and immediately place filters onto paper towels or filter paper to dry. Isolating Positive Plaques and Preparing High Titer Phage Stocks 1. Positive plaques on probed filters have a strong color reaction over background in the shape of a plaque. Once aligned with the plaque containing LB/amp plate, locate the positive plaque(s) on the plate and, using the back end of a pasteur pipette, remove the plaque containing agar plug within the pipette. 2. Place each agar plug in an eppendorf tube containing 1 ml k diluent (10 mM Tris, pH 7.5, 10 mM MgC12; sterilized by autoclaving). Incubate for at least 1 hr at room temperature. 3. Replate the phage from the above stock (titer approximately 1 x 107) and repeat screening at lower phage concentrations (about 1 x 104) so that second or third screening allows for isolation of a single positive phage and that final screening shows only positive clones on plate. These screenings are done along with a control set of filters (steps 11 and 12 of filter preparation) for use with a control GST fusion protein as probe. Discard any positives also seen on these control filters. 4. Make a few agar plug phage stocks from each of the final screening positives. These can be saved at 4° and later used to make k lysates to isolate the kDNA. Isolation of Recombinant Phage Lysogens and Preparation of Lysogen Extracts This step can be useful for confirming that a positive clone is in fact a cDNA fused to the/3-galactosidase of the phage since it allows for high production of the recombinant fusion protein which can then be analyzed on SDS-PAGE. The isolation of lysogens and preparation of lysogen extracts were performed as described 7with the exception that the lysogen extract preparation was completed after a 15-min incubation with lysozyme. Lysogen Extract Blot 1. Take two 15-/zl samples each of induced and uninduced extracts from each lysogen extract prepared earlier and add 5/zl of sample buffer. 2. Boil samples and load two sets of induced and uninduced culture samples on a low percentage gel (7%) and run SDS-PAGE.
[ 171
SH3 DOMAINSCREENING Probes:
GST-Abl S H 3
GST-Sr¢ S H 3
+
+
147 GST
11697-
42-
Induction Clone
+ I
II
+ I
+ II
+ I
II
FIG. 3. Binding of SH3 domains to/3-galactosidase fusion proteins from selected k gtll clone lysates (clones I and II). (Left) Probed with the biotinylated Abl-SH3 domain fusion protein probe; (middle) probed with the biotinylated Src-SH3 domain fusion protein probe; and (right) probed with the control GST protein. Molecular size markers are indicated to the left in kilodaltons. Reproduced from Cicchetti e t al. 1 with permission.
3. Transfer gel to nitrocellulose, and divide transfer in half so that each piece has one set of induced and uninduced samples. 4. Wash twice with distilled H 2 0 and once in TBST. 5. Block in T B S T / G for 30 min at room temperature. 6. Add anti-/3-galactosidase antibody (Pierce) to half of the transfers and the biotinylated G S T fusion protein probe (1/xg/ml T B S T / G ) to the other half. Proceed as in steps 6-11 of Probing of Nitrocellulose Filters, with the exception that in step 8, use protein A/G-alkaline phosphatase (Pierce) instead of S A - A P for the antibody blot. 7. A band at approximately 114 kDa should be visible in the induced cultures with both the antibody to/3-galactosidase and with the biotinylated fusion protein probe. The fusion protein will be quite large since the /3-galactosidase portion of each fusion protein produced by the recombinant phage lysogen accounts for 114 kDa. Figure 3 is an example of a lysogen culture run on S D S - P A G E , transferred to nitrocellulose, and probed with multiple GST fusion protein probes. Conclusions The procedure described in this chapter is useful for identifying novel p r o t e i n - p r o t e i n interactions and is highly efficient, stringent, and reproduc-
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[ 17]
ible. This method has enabled us to identify the first SH3-binding proteins and to recognize a commonality between the two SH3-binding proteins cloned. The sequence alignment between 3BP-1 and 3BP-2 points to a proline-rich region of about 40 amino acids. Subsequent investigations determined that a 10 amino acid proline-rich sequence in 3BP-1 and 3BP-2 constitutes the core binding sequence for the Abl SH3 domain. 8 It has since become clear that many newly discovered SH3-binding proteins share this characteristic of proline-rich sequences within their SH3-binding domains.9,10
8 R. Ren, B. J. Mayer, P. Cicchetti, and D. Baltimore, Science 259, 1157 (1993). 9 M. Rozakis-Adcock, R. Fernley, J. Wade, T. Pawson, and D. Bowtell, Nature 363, 83 (1993). 10 I. Gout, R. Dhand, I. D. Hiles, M. J. Fry, G. Panayotou, P. Das, O. Truong, N. F. Totty, J. Hsuan, G. W. Booker, I. D. Campbell, and M. D. Waterfield, Cell 75, 25 (1993).
Section III Cell Expression and in Vitro Analysis
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[18] Serum Induction of RhoG Expression By
PHILIPPE FORT
and
SYLVIE VINCENT
Introduction T h e e u k a r y o t i c cell cycle involves three m a j o r phases during which gene expression is tightly regulated: 1-3 the transition f r o m a resting to a proliferative state (G0/Gl), the onset of D N A synthesis (GI/S), and the mitotic induction in cells that have replicated their c h r o m o s o m a l stock ( G : / M ) . In the absence of g r o w t h factors, the cell exits f r o m the n o r m a l cycle to the Go resting state. S u b s e q u e n t g r o w t h stimulation allows the cell p o p u l a t i o n to progress s y n c h r o n o u s l y t o w a r d the S phase. T h e mitogenic signal is t r a n s d u c e d t h r o u g h receptors and leads to the sequential biochemical activation of a wide variety of proteins, 4-6 followed by cascades of gene activations. 7,8 M o r e than 100 genes were f o u n d to be activated within 4 hr (early G 0 after g r o w t h stimulation. 9,1° Several R N A s encoding small G T P a s e s such as Ras, u Rat2,12 R h o B ] 3 and R h o G 14 have b e e n characterized as being growth regulated. H o w e v e r , the complexity and cell specificity of the m R N A p o p u l a t i o n that accumulates in late G1 r e m a i n far less documented. N u m e r o u s studies have investigated the regulatory m e c h a n i s m s that lead to changes in m R N A level, as these are readily a m e n a b l e to recombinant D N A technology. In particular, differential screening p r o c e d u r e s of t c. D. Scher, R. C. Shepard, H. N. Antoniades, and C. D. Stiles, Biochim. Biophys. Acta 560, 217 (1979). 2 A. B. Pardee, Science 246, 603 (1989). 3 R. Baserga, D. E. Waechter, K. J. Soprano, and N. Galanti, Ann. N.Y. Acad. Sci. 397, 110 (1982). 4 S. J. Leevers and C. J. Marshall, Trends Cell Biol. 2, 283 (1992). 5 C. M. Crews and R. L. Erikson, Cell 74, 215 (1993). 6 S. L. Pelech and J. S. Sanghera, TIBS 17, 233 (1992). 7 R. Bravo, Cancer Biol. 1, 37 (1990). H. R. Herschman, Annu. Rev. Biochem. 60, 281 (1991). 9 j. M. Almendral, D. Sommer, J. Perera, J. Burckardt, H. MacDonald-Bravo, and R. Bravo, Mol. CeIL Biol. 8, 2140 (1988). to L. F. Lau and D. Nathans, in "Molecular Aspects of Cellular Regulation" (P. Cohen and G. Foulkes, eds.), p. 257. Elsevier, Amsterdam, 1991. u K. Lu, R. A. Levine, and J. Campisi, Mol. Cell, Biol. 9, 3411 (1989). 12L. Reibel, O. Dorseuil, R. Stancou, J. Bertoglio, and G. Gacon, Biochem. Biophys. Res, Commun. 175, 451 (1991). 13D. Jahner and T. Hunter, Mol. Cell Biol. 11, 3682 (1991). 14S. Vincent, P. Jeanteur, and P. Fort, Mol. Cell, Biol. 12, 3138 (1992).
METHODS IN ENZYMOLOGY,VOL. 256
Copyright © 1995 by AcademicPress, Inc. All rights of reproduction in any form reserved.
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cDNA libraries have been specifically established for isolating RNAs which are unequally expressed in two or more cell populations. Using such methods, cDNAs coding for GTPases RhoB, 13 RhoG, 14 and Rad a5 have been isolated. This chapter describes the methodology for the characterization and regulation analysis of genes activated by serum or purified growth factors, using the small GTPase RhoG as a model.
Conditions for Cell Culture and Growth Stimulation Growth Stimulation of the Cell Culture To maximize the finding of growth-induced genes, the most widely used methods work with cells synchronized by serum starvation or densitydependent growth arrest. Conditions for both methods have to be adjusted depending on the cell type: Subconfluent fibroblastic murine NIH 3T3 cells are starved in 0.5% fetal calf serum (FCS) for 48 hr whereas hamster CCL39 cells are starved without serum for 24 hr. 16 Under these conditions, >95% of the cell population is found containing a G1 DNA content. At this stage, the culture can be treated with various agents such as serum, purified growth factors, inhibitors of DNA, RNA, or protein synthesis, or a combination of several of them. In practice, CCL39 cells are maintained in Dulbecco's modified Eagle's medium (DMEM) in 10% FCS. For serum starvation, cells are seeded at 105 m1-1 until the subconfluent state (approximately 3 days), rinsed with 10 ml of serum-free DMEM, and then incubated for at least 22 hr in serum-free DMEM. About 30 to 40% cell mortality is expected. Northern Analysis The biological system from which RNA has been extracted must be checked with suitable controls. Procedures for RNA isolation and Northern blotting have been described in detail elsewhere, a7,18 As far as growthstimulated genes are concerned, the most commonly used probes are c-los, 19'20 whose gene is induced by a wide variety of agents in most cell 15 C. Reynet and C. R. Kahn, Science 262, 1441 (1993). 16j. Pouyss6gur, C. Kahan, and K. Seuwen, in "Growth Factors: From Gene to Clinical Application" (W. Sara, ed.), p. 80. Raven Press, New York, 1990. 17 p. S. Thomas, Proc. Natl. Acad. Sci. U.S.A. 77, 5201 (1980). 18j. Sambrook, E. F. Fritsch, and T. Maniatis, in "Molecular Cloning: A Laboratory Manual" (C. Nolan, ed.), p. 18. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. 19 U. RUther, E. F. Wagner, and R. M/iller, E M B O J. 4, 1775 (1985). 2o p. Fort, J. Reeh, A. Vi6, M. Piechaezyk, A. Bonnieu, P. Jeanteur, and J.-M. Blanchard, Nucleic Acids Res. 15, 5657 (1987).
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types, GAPDH (glyceraldehyde-3-phosphate dehydrogenase),21'22 or $26 ribosomal p r o t e i n y highly expressed in all tissues or cell types. Although the GAPDH probe is widely used, its level of expression differs from one tissue to one another and increases two- to threefold in cells stimulated to grow. 23,24 All probes are available from our laboratory.
Assay for Growth Factor Activity Depending on the mitogenic strength and the amount of growth factor used for the stimulation, only part of the cell population will initiate a new round of division: the addition of o~-thrombin allows 30-40% of resting CCL39 cells to reinstate DNA synthesis.16 A better yield (-60%) can be achieved using a combination of growth factors. The mitogenic strength can be assayed by either [3H]thymidine or bromodeoxyuridine (BrdU) incorporation. [3H]TTP labeling is a quantitative method which gives an estimate of DNA synthesis rate on the whole cell population. This method has long been used in cellular biology25 and will not be described. BrdU labeling allows the detection by immunofluorescence of cells that have reached the S phase, giving the proportion of cells that have responded to the growth factors. The procedure described next has been used for NIH 3T3 and CCL39 fibroblastic cells.
Cell Labeling Grow the cell culture in 35-mm dishes up to subconfluence and starve for the appropriate time (see Growth Stimulation of the Cell Culture). Remove the medium and stimulate the cells for 18 hr with 1 ml of medium containing the appropriate concentration of growth factor and 50 mM BrdU (Boehringer-Mannheim).
Fixation 1. Remove the medium, rinse twice with 3 ml of phosphate-buffered saline (PBS), and add 1 ml of 3.7% formaldehyde in (PBS). Incubate for 5 min. 2. Rinse with 3 ml of PBS and then with 3 ml of sterile water. 3. Add 1 ml of acetone (at -20°), incubate for 1 min, and then repeat step 2. 21 p. Fort, L. Marty, M. Piechaczyk, S. E1 Sabrouty, C. Dani, P. Jeanteur, and J. M. Blanchard, Nucleic Acids Res. 13, 1431 (1985). 2: S. Vincent, P. Jeanteur, and P. Fort, Nucleic Acids Res. 10, 3054 (1990), 23 S. Vincent, L. Marty, and P. Fort, Nucleic Acids Res. 21, 1498 (1993). 24 L. M. Matrisian, G. Rautman, B. E. Magun, and R. Breathnach, Nucleic Acids Res. 13, 711 (1985). 25 L. P. Everhart, P. V. Hauschka, and D. M. Prescott, Methods Cell Biol. 7, 329 (1973).
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4. Add 1 ml of 4 N HCI, incubate for 10 min and rinse three times with PBS. Rinse with 3 ml of PBS containing 0.2% gelatin or 1% BSA.
Detection Add 0.5 ml of mouse monoclonal anti-BrdU antibody in PBS/gelatin, as recommended by the supplier (Boehringer-Mannheim). Incubate for 1 hr. Rinse three times with 3 ml of PBS. Add 0.5 ml of fluorescein isothiocyanate (FITC)-conjugated antimouse antibody, as recommended by the supplier. Incubate for 30 min. Rinse three times with 3 ml of PBS. Add a 100-/xl drop of Mowiol solution (see below), and place a coverslip. Cells are visualized with an epifluorescence microscope equipped with interference filters; the cell layer is observed under phase-contrast microscopy (Fig. 1B), whereas fluorescent cells (having replicated their DNA) are visualized using the appropriate interference filter (Fig. 1A).
Preparation of Mowiol 4.88 Solution Mix 6 g of glycerol (analytical grade) and 2.4 g of Mowiol 4.88 (Calbiochem) in a 50-ml Falcon centrifuge tube. Add 6 ml of sterile H20 and incubate for 2 hr at room temperature. Add 12 ml of 0.2 M Tris-HC1 (pH 8.5) and incubate at 50° for 10 rain with intermittent shaking. Centrifuge at 5000g for 5 min. Aliquot the supernatant in sterile tubes and store at - 2 0 °.
Working Concentrations of Purified Growth Factors Working concentrations of purified growth factors must be determined according to the cell type. For CCL39 fibroblast cells, the following concentrations were used: 14,26a-thrombin (bovine plasma, 2.5 U/txg), 0.1 to l NIH unit ml-1; epidermal growth factor (EGF), 10 ng ml 1; fibroblast growth factor (FGF, bovine pituitary), 20 to 35 ng ml-a; insulin (24 U/rag), 0.1 to 1 /zg mlq; 8Br-cAMP, 1 raM; and transforming growth factor/3, 0.1 to 1 ng ml -a. Procedures for Isolating Growth-Stimulated Genes
cDNA Cloning Whatever the methods used for isolating differentially expressed clones, a comprehensive cDNA library of the system of interest must be available. 26 J.-C. Chambard and J. Pouyssdgur, J. Cell. Physiol. 13& 101 (1988).
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FIG. 1. BrdU antibody staining of stimulated NIH 3T3 cells. Cells were starved in 0.5% FCS for 48 hr and then stimulated for 18 hr with 10% FCS in the presence of BrdU, as described in the text. Cells were visualized by fluorescence (A) or under phase-contrast microscopy (B).
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Commercially supplied libraries can be used, although the exact conditions at which poly(A) + R N A has been isolated are never well defined. Alternately, it is now quite simple to construct a library using commercial cloning kits. A few observations have to be pointed out concerning the cDNA cloning procedure. (i) For reverse transcription, R N A can be primed with either random oligonucleotides or oligo(dT). Although large RNAs are overrepresented in a random-primed library, it circumvents the problems due to premature termination or poorly adenylated RNAs. However, it generates nonoverlapping cDNA clones derived from the same mRNA, which can be a source of trouble during the screening procedure. (ii) Size selection eliminates cDNAs shorter than 500 bp that are more efficiently cloned. It should be kept in mind that a fraction of m R N A is made of small RNAs and may be lost during the size fractionation.
Differential Hybridization Various strategies for selecting cDNA clones that are differentially expressed in two or more cell systems have already been described. 27 Because of its simplicity, we used a classical differential screening to isolate genes induced in the late G1 phase. 1. An oriented cDNA library from resting fibroblastic CCL39 cells stimulated for 5 hr with 10% FCS was constructed in pT7T318U phagemid (Pharmacia LKB), using a primer/adapter method. 28 The library is plated at low density (5 to 10 colonies per cm 2) on 20 x 20-cm HybondN membranes (Amersham) layered on LB + 150/zg m1-1 ampicillin agar plates. Bacterial clones are grown until they reach a mere pinhead size, usually achieved upon overnight incubation at 20-22 °. 2. Strip the nylon membranes off the agar plate and place on Whatman paper, colonies facing up. Place a second membrane on the master filter, add a Whatman paper sheet and a rigid plate, and press moderately to ensure an even contact between both membranes. After plate and paper removal, use a needle to perforate both membranes (tightly bound) to generate landmarks. Several replicas can be obtained by repeating the procedure. However, our own experience has shown that differential hybridizations must be performed on the same replica. Layer replica and master filters back on fresh agar plates and allow to grow for 3 to 4 hr at 37 °. Transfer the master filter onto a fresh agar plate supplemented with 10% glycerol and store at - 2 0 °. 27 B. H. Cochran, P. Zumstein, J. Zullo, B. Rollins, M. Mercola, and C. D. Stiles, this series, Vol. 147, p. 64. 28 D. Caput, personal communication (1990).
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3. Replica filters are then submitted to a classical alkali lysis. TM Prehybridize for 12 hr at 42 ° in 50% formamide containing 0.75 M NaC1, 0.1% SDS, 5x SSC, 5× Denhardt's solution, 10 mM phosphate buffer (pH 7.5), and 100/~g m1-1 salmon sperm DNA. 4. For [a-32P]cDNA probe synthesis, mix 0.5 /~g poly(A) + RNA and 0.6/~g oligo(dT)~2 primer in 11 ~1 of H20. Heat at 65 ° for 5 min; cool down on ice; and sequentially add 4/~1 of 3.3 mM each of dATP, dGTP, and dTTP, 1 /~1 of freshly prepared 0.1 mM dCTP, 10 /M (100 /~Ci) of [a-32p]dCTP (3000 Ci/mmol, Amersham), 1 ~1 (30 units) of RNase inhibitor (RNasin, Promega), 7 /~1 of 5× MuLV (murine leukemia virus) buffer (supplied by BRL), and 200 units of MuLV reverse transcriptase (BRL). Incubate at 37° for 1 hr. Unincorporated nucleotides are removed through a spun dialysis P-0 column (Bio-Rad), equilibrated in 10 mM Tris-HC1 (pH 7.5), 1 mM EDTA. Hydrolyze RNA at 65 ° for 30 min in 0.15 N NaOH. Neutralize by adding 1/20 volume of 1 M Tris-HC1 (pH 7.5), 3 N HC1. A classical reaction produces 3 × 107 disintegrations per minute (dpm) of labeled probe. 5. Add the probe to the filters at 2 x 10 6 (dpm) ml -~. Hybridize for 48 hr at 42 °. Wash filters three times for 20 rain in 2× SSC, 0.1% SDS at room temperature, and then once for 10 min in 0.2x SSC, 0.1% SDS at 65 °.
Selection of Differentially Expressed cDNAs Analysis of differentially hybridized filters is now greatly facilitated by the use of automatic/3-scanners (e.g., Phosphorimager, Molecular Dynamics), which provide within minutes or hours a digitized image of the radioactivity present on a filter. These devices offer a wide dynamic range, combined with a high linearity. Alternatively, classical autoradiographic films can be digitized using a high-resolution scanner or camera. Numerical images are then processed for spot detection and quantification. We used the Millipore Bio-lmage 2D software package, originally dedicated to twodimensional protein gel analysis, which allows the automatic detection of thousands of spots and the determination of coordinates and optical density of each of them. Comparison between two or more images indicates matched and unmatched spot positions after having corrected spatial differences. Spots are then sorted out according to various criteria, e.g., spots present in one image and absent in the others or spots present in two images whose integrated intensity ratio is greater than a given value. We easily handled three different images (obtained with three distinct probes) containing more than 3000 spots, and isolated 88 clones which displayed more than twofold a greater signal with the "stimulated" probe
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CELL EXPRESSION
[ 18]
versus the "unstimulated" probe. Further analysis showed that 70% corresponded to genuine growth-stimulated RNAs. 29 cDNA Characterization Automatic Sequencing The best strategy for a rapid characterization of c D N A clones is undoubtedly the use of automatic sequencing, which allows a fast and accurate systematic sequence determination of over 500 nucleotides on both ends of a c D N A insert. If such facilities are available, the whole characterization step can be solved within 1 week. Sequence Comparison To sort out rapidly known sequences and to detect regions of similarity which could help in determining to which class the deduced proteins belong, D N A sequences are then compared to international nucleic acid and protein databases. We used two packages, F A S T A 3° and BLAST, 31 to search for similar sequences and found that seven serum-stimulated m R N A sequences correspond to vascular o~-actin, y-actin, a-tubulin, plasminogen activator inhibitor (type I), thrombospondin, a mitochondrial proton/phosphate symporter, and R h o G ) 4,29 R e g u l a t o r y Analysis of G r o w t h - R e g u l a t e d Clones Further analysis is required to check that isolated clones correspond to genuine growth-stimulated RNAs. Serum stimulation is a complex mixture of mitogenic and nonmitogenic agents, which might exert various positive or negative effects on gene regulation. It is therefore necessary to address which type of agent is responsible for the induction and which level of gene control is altered. Time Course R N A Accumulation in Absence of Protein Translation Growth-stimulated genes fall into three main classes: immediate/early genes (e.g., c-los) are activated within 1 to 3 hr after the stimulation, 7 whereas early/early genes (e.g., R h o G TM) and delayed/early genes (which require the expression of immediate/early genes to be induced, e.g., PAIl 29) are activated within 6 to 10 hr. 29 If cells are serum stimulated in the 29S. Vincent, L. Marty, L. Le Gallic, P. Jeanteur, and P. Fort, Oncogene 8, 1603 (1993). 30D. J. Lipman and Pearson, Proc. Natl. Acad. Sci. U.S.A. 85, 2444 (1988). 31S. F. Altschul, W. Gish, W. Miller, E. W. Myers, and D. J. Lipman, J. Mol. Biol. 215, 403 (1990).
[18]
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RhoO m U
GAPDH
E Q 1 3 6 8 10 Q 1 3 6 8 10 E FCS
FCS + CItX
FIG. 2. Time course of RhoG RNA expression and effect of cycloheximide. RNA was extracted from exponentially growing (E), resting (Q) CCL39 cells, or cells stimulated for the indicated times with 10% FCS, alone (FCS), or in combination with 10/zg m1-1cycloheximide (FCS + CHX), and probed for RhoG and GAPDH sequences.
presence of 10/xg m1-1 cycloheximide (an inhibitor of protein translation, Sigma), delayed/early genes are no longer induced, whereas most immediate/early and early/early R N A s are overexpressed. This latter feature results from an increased gene transcriptional activity and f r o m a reduction in m R N A turnover. Serum induction of R h o G in the absence or presence of cycloheximide is shown in Fig. 2. Change in m R N A Turnover in Response to Growth Factors
A change in the m R N A steady state is directly proportional to a change in m R N A half-life. Moreover, changes in R N A turnover and transcription activity may equally modulate the steady state level of a given m R N A . The most c o m m o n procedure for half-life determination consists of treating cells with a R N A polymerase II inhibitor (usually actinomycin D, at 1 to 5/xg ml-~). Cells are b r o k e n at various periods of time after actinomycin D treatment, and R N A is analyzed by Northern blotting. The decay of R N A is obtained through Eq. (1): C = C0(2-"T,,~),
(1)
where C and Co represent the amount of R N A at time t of sampling and at t nought, respectively, and "rl/2 represents R N A half-life. Although suitable for numerous studies, actinomycin D may interfere with metabolic pathways and provide misleading results. Alternative methods are gene transcription inactivation by U V irradiation of the cell culture 32 or kinetics of cytoplasmic accumulation monitored by continuous labeling of cellular 32C. Dani, M. Piechaczyk, Y. Audigier, S. E1 Sabrouty, G. Cathala, L. Marty, P. Fort, J. M. Blanchard, and P. Jeanteur, Eur. J. Biochem. 145, 299 (1984).
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RNA. 33 In this latter case, cells are continuously labeled with 3H-labeled uridine, and poly(A) + R N A is then extracted at different times during the labeling period. Each R N A sample is then hybridized exhaustively to membrane-bound D N A fragments. Radioactive signals are in proportion with mRNA turnover through Eq. (2) C
1
2- 7~-~
(2)
C~
where C and C~ represent radioactivity at time of sampling and at equilibrium, respectively, tD the cell doubling time, and "rl/2 the half-life of RNA. Changes in m R N A turnover in exponentially growing, resting, or growthstimulated cells can be detected by either method. Run-On Assays on Isolated Nuclei This method estimates R N A polymerase density at different times on a given gene. Nuclei are isolated from a cell culture and are incubated in the presence of a-3Zp-labeled ribonucleotides to elongate nascent primary R N A transcripts. R N A is then extracted and hybridized to gene fragments bound to membranes. Under the hypothesis that (i) R N A polymerase density directly reflects the rate of R N A synthesis, (ii) no transcriptional initiation takes place during the in vitro elongation, and (iii) RNA synthesis rate is not time and sequence dependent, this method gives correct estimates of the transcriptional activity. The original protocol 34 has been adapted as follows. 35 Isolation of Purified Nuclei from Cell Culture 1. Nuclei are prepared from 5 x 107 cells. Rinse Petri dishes twice with cold PBS. Add 2 ml of cold sterile 10 mM Tris-HC1 (pH 7.4), 10 mM NaC1, 3 mM MgCI2, and 0.5% Nonidet P-40 (NP-40) (solution A). Scrape cells on ice with a rubber policeman, transfer the suspension to a Dounce homogenizer, and break the cells on ice. The extent of cell breakage is monitored by mixing 10/zl of cell suspension to 10/xl of 0.5% trypan blue on a coverslip and observation under optical microscopy. Intact cells are hardly stained, whereas nuclei appear bright blue. 2. Layer the cell lysate on top of a 5-ml cushion of solution B [30% sucrose (w/v) in solution A] and pellet nuclei by centrifugation (2000g, 5 min at 4°). Resuspend the pellet carefully and thoroughly in 300/zl of cold 33 j. R. Greenberg, Nature (London) 24tl, 102 (1972). 34 M. E. Greenberg and E. B. Ziff, Nature (London) 311, 433 (1984). 35 A. Bonnieu, J. Rech, P. Jeanteur, and P. Fort, Oncogene 4, 881 (1989).
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50 mM Tris-HC1 (pH 8.3), 5 mM MgC12, and 40% glycerol. The suspension should not become viscous, which would signal the presence of lysed nuclei, unsuitable for the next steps. Dispatch in 100-/.d aliquots in 1.5-ml microfuge tubes with pierced caps. Freeze immediately in liquid nitrogen. Transcription on Isolated Nuclei
1. Thaw 100/zl of frozen nuclei on ice and add 100/zl of 2x transcription buffer [40 mM Tris-HCl (pH 8.0), 10 mM MgC12,20 mM MnCI2, 300 mM KC1, 2 mM each of ATP, GTP, and CTP, and 10 mM dithiothreitol]. Add 100/zCi [o~-32p]UTP (400 Ci/mmol) and incubate at 26° for 30 rain with intermittent shaking to avoid nuclei sedimentation. 2. Add 10/~g of DNase (RNase-free, BRL) to the labeled nuclei and incubate for 20 min at 37 °. Adjust the volume to 4 ml with 10 mM TrisHC1 (pH 8.0), 1 mM EDTA, 0.1% SDS (TES) buffer, and add 100/~g yeast tRNA and phenol extract at 65°. 3. Adjust the aqueous phase in 2 M ammonium acetate and precipitate with 2 vol of ethanol. 4. Repeat step 3 twice to get rid of unincorporated labeled nucleotides. Rinse the pellet with 4 ml of 70% ethanol and resuspend in 50/~1 of sterile pure water. Take a 2-/zl sample and count the amount of radioactivity. Immobilization o f D N A Probes on Membranes
Several types of nucleic acids can be prepared. Single-stranded ribo- or deoxyribonucleic acid probes can be synthesized from recombinant multifunctional plasmids or M13 vectors. Such single-stranded probes are required when sense and antisense nuclear transcriptional activities have to be assayed. Otherwise, double-stranded polymerase chain reaction insert DNA or recombinant plasmid DNA are suitable. For each transcription assay, 5-10 /~g of recombinant of DNA or 0.5-1 /~g of purified insert is required. 1. For n transcription assays, boil the required amount of each doublestranded DNA for 10 min in n x 50 /~1 of 0.1 N NaOH. Neutralize by adding n x 100/.d of cold sodium acetate (pH 5.5). Single-stranded probes are diluted in n x 150/~1 of sterile 2x SSC. 2. Spot nucleic acids (150 /~1) under light vacuum onto a HybondN (Amersham) membrane using a slot-blot apparatus (Schleicher & Schuell). When using riboprobes, autoclave membrane and solutions, and soak the apparatus for 30 min in 0.1 N NaOH. Rinse thoroughly in sterile water. The membrane must be wetted in 2× SSC before spotting. Rinse each slot with 1 ml 2xSSC after spotting. Air dry the membrane and bake at 80° for 1 hr.
162
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Hybridization and Washing Conditions 1. Cut the membrane filters into narrow strips so that each contains a set of independent probes and prehybridize for 12 hr at 42° in hybridization buffer (see step 3 under Differential Hybridization). 2. Dilute the labeled RNA from isolated nuclei in hybridization buffer at a final concentration of 2-5 x 106 dpm m1-1. Pour off the prehybridization solution and add the probe. Do not exceed 0.2 ml per cm 2 of membrane. Hybridize for 48 hr at 42°. The use of a rotating hybridization oven is recommended to ensure a constant redistribution of the probe. 3. Wash filters three times for 15 min at 22° in 2x SSC, 0.1% SDS, and then once for 30 min at 50° in 0.2x SSC, 0.1% SDS. Rinse in 2x SSC, and then incubate filters for 30 min at 22° in 2x SSC with 100/xg m1-1 boiled RNase A. Rinse in 2x SSC, 0.1% SDS, and expose to autoradiographic films with intensifying screens.
Acknowledgments This work was supported by institutional grants from CNRS and the University of Montpellier I and II, and by contracts from INSERM, the Association pour la Recherche contre le Cancer, and the Fondation pour la Recherche M6dicale.
[19] M i c r o i n j e c t i o n o f E p i t o p e - T a g g e d R h o F a m i l y c D N A s and Analysis by Immunolabeling
By
H U G H PATERSON, PETER ADAMSON, and DAVID ROBERTSON
Introduction Analysis of the intracellular distribution of small GTP-binding proteins in both normal and mutant forms can convey useful information regarding the function of these molecules. In particular, immunolabeling of these proteins in situ can give a more precise indication of their location within the cell than is obtainable by cell fractionation techniques. The conventional approach has been to raise antibodies against each protein or against synthetic peptides derived from the amino acid sequence. However, producing antibodies against proteins of the Ras superfamily is complicated by the high sequence homology which typifies this group of molecules. Mammalian RhoA,B, and C proteins share approximately 80% of the amino acid seMETHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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quence 1'2 such that most antibodies raised against one protein show crossreactivity with other members of the group, and consequently yield limited information. Antibodies raised against peptides corresponding to the unique amino acid sequences found primarily in the C-terminal region of the proteins l'2 have the potential for high specificity but require separate immunizations for each member of the family. Such antibodies may also fail to bind native or cross-linked proteins. Furthermore, even after obtaining specific antibodies, low levels of expression of the endogenous protein may render immunolabeling so weak as to be barely distinguishable from background. This problem is exacerbated with many members of the Ras superfamily of proteins whose ubiquitous expression means that negative control cells are not available for comparison. In order to circumvent these problems, alternative methods for immunolocalization of proteins have been developed. A D N A sequence representing a short antigenic epitope, known to be the binding site for a preexisting high affinity antibody, is engineered into the coding sequence of a mammalian expression vector containing the gene of interest. Cells into which this vector is introduced synthesize exogenous protein carrying an antigenic tag which enables it to be recognized by the antibody. This technique known as "epitope tagging" has the advantage that a single, well-characterized antibody can be used to detect a potentially infinite variety of tagged proteins. Additionally, since endogenous protein remains undetected in this system, changes in intracellular distribution accompanying mutations introduced at key sites in the tagged protein can be observed without interference from the endogenous, wild-type signal. Levels of tagged protein can be varied according to the promoter employed in the expression vector together with the amount of plasmid DNA introduced into the cells. Although almost any method of delivery may be used to introduce the D N A constructs into the target cells, we have found microinjection to be particularly well suited for this purpose, not least because of its high efficiency in a wide variety of adherent cell types. Microinjected cells are not significantly disturbed by the procedure and can easily be relocated for analysis. Cells in neighboring areas of the same culture dish can be microinjected with different plasmid constructs, and the tagged proteins which are subsequently expressed can be immunolabeled and analyzed together by immunofluorescence. This chapter describes how microinjection of expression vectors coding for epitope-tagged proteins can be used to analyze the intracellular localization of Rho family proteins. P. Chardin, P. Madaule, and A. Tavitian, Nucleic Acid Res. 16, 2717 (1988). 2 p. Yeramian, P. Chardin, P. Madaule, and A. Tavitian, Nucleic Acids Res. 15, 189 (1987).
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Methods Choice o f Epitope Tag
A number of different epitope tags have been described in the literature, and can be successfully employed for immunolocalization. Most widely used are peptides derived from influenza hemagglutinin, 3vesicular stomatitis virus (VSV) G protein, 4 and human c-myc, 5,6 although in principle any peptide corresponding to the epitope recognized by an existing antibody is a suitable candidate. The most important attributes of the antibody are that it should not show affinity for endogenous cellular proteins and that it should bind with high affinity to the epitope tag even after the rigors of cell fixation. We have used exclusively the Myc epitope tag, a peptide sequence derived from the human c-myc protein which is recognized by the mouse monoclonal antibody 9E10. 7 Although this epitope is present within the endogenous c-myc protein of human and other mammalian cells, it remains concealed from the 9 E l 0 antibody. 7 We have not detected interference from endogenous c-myc in any of our experiments employing this antibody. Construction o f Epitope- Tagged Plasmids
Plasmids are designed to express the Myc epitope sequence, as recognized by the 9 E l 0 antibody, at the amino terminus of Rho family proteins. A previously described eukaryotic expression vector 5 containing the Rho c D N A ( p E X V 3 - R h o A ) was linearized at the natural AccIII site at codon 3 and ligated to a double-stranded oligonucleotide encoding the peptide sequence M E Q K L I S E E D L to create the epitope tag at the amino-terminal end. An analogous construct, p E X V 3 - m y c - R h o B , was made by first engineering a PvuII site into a partial RhoB c D N A at codon 60 by oligonucleotide site-directed mutagenesis and then ligating this sequence to the natural PvuII site in p E X V 3 - m y c - R h o A . The plasmid generated in this way differs from the authentic RhoB at two amino acids which were corrected by transfer to M13 mp19, and then oligonucleotide site-directed mutagenesis was used to create p E X V - m y c - R h o B , p E X V - m y c - R h o C was constructed in an analogous way to p E X V - m y c - R h o B , p E X V - m y c - R a c l was epitope tagged at the 5' end using the polymerase chain reaction and subcloned into pEXV3. 8 3 y. T. Chen, C. Holcomb, and H. P. Moore, Proc. Natl. Acad. Sci. U.S.A. 90, 6508 (1993). 4M. Algrain, O. Turunen, A. Vaheri, D. Louvard, and M. Arpin, J. Cell Biol. 120, 129 (1993). 5 p. Adamson, H. F. Paterson, and A. Hall, J. Cell. Biol. 119, 617 (1992). 6 M. Meichsner,T. Doll, D. Reddy, B. Weisshaar, and A. Matus, Neuroscience54, 873 (1993). 7 G. I. Evan, G. K. Lewis, G. Ramsay, and J. M. Bishop, Mol. Cell. Biol. 5, 3610 (1985). 8 A. J. Ridley, H. F. Paterson, C. L. Johnston, D. Deikmann, and A. Hall, Cell 70, 401 (1992).
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It is important that this family of proteins is epitope tagged in such a way that the additional sequence does not prevent the normal function and posttranslational processing of the protein. Therefore these proteins cannot be tagged at the C terminus which undergoes a series of essential posttranslational modifications.9 However, cDNAs encoding other proteins have been successfully epitope tagged at the carboxy terminus. The methionine residue at the end of the epitope sequence is not necessary for antigenicity but is essential as an initiation codon when used as an N-terminal tag. In the case of C-terminal tagging the methionine residue is not essential, but conversely it is most important that the stop codon is removed and replaced at the end of the epitope tag sequence.
Choice of Cells Microinjection as a means of delivering plasmid DNA into the cell nucleus has the advantage of allowing almost any adherent cell type to be used. Obtaining adequate levels of expression of the injected DNA vectors is rarely a problem, and where this is experienced it commonly reflects shortcomings in the construction or preparation of the plasmids. In general we have found that immortalized rodent fibroblast cell lines, such as NIH 3T3, Swiss 3T3 and Rat 2, with their flat uniform morphology are well suited for the analysis of predominantly cytoplasmic proteins. Plasma membrane associated protein, however, is most easily visualized in confluent epithelial cells which present a tall, vertical stack of membrane at their basolateral margins. We have routinely used the MDCK (Madin-Darby canine kidney) epithelial cell line for analyzing the plasma membrane association of tagged normal and mutated small GTPases.
Preparation of Cell Cultures for Microinjection For immunofluorescence studies the cells to be microinjected are cultured either on 60-mm-diameter plastic tissue culture petri dishes (Nunc, Denmark) or on round 13-mm glass coverslips (No. i !2, Chance Propper, England). When plastic culture dishes are used, the area of cells to be injected is selected at low magnification on an inverted microscope immediately prior to use, and a fine cross is cut into the upper (cell culture) surface of the dish with a curved sterile scalpel blade. This produces precisely marked quadrants which allow up to four neighboring areas of cells to be injected with different plasmid constructs for side by side comparison. Marking the underside of the dish is not recommended since such lines are indistinct at higher magnifications both during microinjection and subse9p. Adamson,C. J. Marshall,A. Hall, and P. A. Tilbrook,J. Biol. Chem.267, 20033 (1992).
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quent immunofluorescence imaging. Glass coverslips are similarly engraved with a cross on the top surface using a diamond-tipped marker prior to sterilization by flaming in methanol. The coverslips are then placed in the bottom of 4- or 24-well multidishes (Nunc, Denmark), and cells are seeded in 0.5 ml of culture medium. Immediately prior to microinjection, coverslips are transferred to a 60-mm petri dish in 5 ml of culture medium. Each coverslip is then gently pressed down to exclude bubbles of air trapped beneath, which can otherwise allow it to drift across the bottom of the dish during the microinjection session. For immunoelectron microscopy experiments, cells are seeded onto 10mm square sheets of Thermanox plastic (Nunc), previously sterilized by immersion for 10 min in methanol, and placed in the bottom of 60-mm petri dishes in 5 ml of culture medium. Prior to microinjection, four marker lines are cut with a sterile scalpel blade at right angles to each other into the top surface of the Thermanox sheet to delineate a rectangle enclosing an area of cells approximately 1 mm square for microinjection. Culture medium both for routine passaging and for all experimental procedures is Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Swiss 3T3, Rat-2 and MDCK) or 10% donor calf serum (NIH 3T3). Microinjection
Detailed accounts of the basic techniques involved in microinjecting mammalian somatic cells may be found in Graessmann and Graessmann I° and Ansorqe. H For most experiments we have used a Zeiss-motorized microinjection workstation equipped with a temperature and CO2 controlled incubator jacket, which allows lengthy experiments involving several plasmids to be performed without subjecting the cells to undue stress. Problems may be experienced with this equipment when microinjecting cells on Thermanox plastic coverslips for immunoelectron microscopy. In this case the objective lens of the inverted microscope is unable to resolve a sharply focused image of the cells through the combined layers of the petri dish and plastic coverslip (glass coverslips are not affected in this way). Consequently, for immunoelectron microscopy experiments we have employed a conventional upright microscope focusing on the cells through the culture medium from above, in conjunction with a Leitz mechanical micromanipulator. The volume of culture medium in the petri dish must be reduced to less than 2 ml before attempting microinjection with this upright system to avoid immersing the microscope objectives. 10M. Graessmannand A. Graessmann,this series, Vol. 101, p. 482. 11W. Ansorqe, Exp. Cell Res. 140, 31 (1982).
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Microinjection needles are loaded with plasmid D N A solution at between 1 and 500/zg/ml. Precise D N A concentration must be determined empirically according to cell type, plasmid construction, and levels of expression required, but we routinely use 50/zg/ml with the EXV3-Rho D N A constructs described here. Dulbecco's phosphate-buffered saline is the standard diluent for stock plasmid solutions. Working strength plasmid solutions are freshly prepared for each experiment, spun at 14,000 rpm for 15 rain, maintained on ice, and discarded after use. Cells (100-200) contained within a marked area are injected in the nucleus with a volume of D N A just sufficient to produce a momentary swelling. This volume has been estimated at approximately 1-2 × 10 -11 ml per cell. 1° Dishes containing the injected cells are then returned to the incubator to allow time for expression of the injected genes.
Preparation of Microinjected Cells for Immunofluorescence Although myc-tagged Rho proteins can be detected in cells as little as 3 hr following plasmid microinjection, we have found that overnight expression times of up to 15 hr are generally more informative since they produce a wide range of levels of expression for analysis. Longer expression times with activated Rho proteins result in increasingly severe distortion of cell morphology. 12In order to eliminate the possibility of artificial changes in localization resulting from overexpression, it is important to confirm that the fluorescent pattern remains constant over the full range of staining intensities being examined. Artifacts of this nature may arise as a result of saturating the capacities of the enzymes involved in the post-translational processing of Ras family proteins. Fixation and immunofluorescent labeling of microinjected cells are performed according to the following protocol. All stages are performed at room temperature with gentle agitation on an orbital shaker. 1. Rinse briefly in Dulbecco's phosphate-buffered saline (PBS). 2. Fix for 15 min in 3% (v/v) formaldehyde diluted in PBS. 3. Rinse five times, and wash for 30 rain in PBS. 4. Quench residual formaldehyde in 50 m M ammonium chloride or 100 m M glycine in PBS for 10 min. 5. Permeablize in PBS containing 0.2% Triton X-100 for 10 min. 6. Rinse in PBS, and shake in PBS containing 10% fetal bovine serum for 30 min as a blocking step. 12H. F. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell Biol. 111, 1001 (1990).
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7. Incubate at room temperature in 9El0 antibody at 5 tzg/ml in PBS (we use ascites fluid diluted 1 : 400) for 1 hr. 8. Rinse five times, and wash for 30 min in PBS. 9. Incubate for 1 hr in donkey anti-mouse antibody conjugated fluorescein isothiocyanate (Jackson Immunoresearch) diluted 1 : 200 in PBS. 10. Rinse five times, and wash for 30 rain in PBS. 11. Mount in moviol mountant 13 containing 0.1% p-phenylenediamine as an antiquenching agent. Coverslips containing injected cells are gently wiped dry on the underside with a paper tissue and inverted onto a drop of mountant on a glass microscope slide. Injected areas of cells on plastic petri dishes are mounted in situ under a glass coverslip, and the sides of the dish are cut away with a pair of scissors to facilitate handling on the microscope stage. If double labeling is performed, in conjunction with antibodies from a different species directed against another cellular protein, then both primary and secondary antibody incubations are carried out concurrently with those for 9E10. Where double labeling for actin is desired then 0.1/~g/ml tetramethylrhodamine isothiocyanate (TRITC) phalloidin (Sigma) is included in the secondary antibody incubation.
Analysis by Immunofluorescence Mounted coverslips or petri dishes may be examined on a conventional fluorescence microscope, but superior resolution of cellular fine structure can be obtained with a confocal imaging system. Confocal optics also help to eliminate the flare associated with mounting fluorescent specimens on plastic dishes. For our studies we have used a Bio-Rad MRC 600 confocal system equipped with a krypton-argon laser, in conjunction with a Nikon Optiphot fluorescence microscope and plan-apo objectives. An additional advantage of this system is the ability to overlay ("merge") the images obtained from different fluorophores in the same specimen when double labeling has been used. This enables the intracellular localization of myc-tagged proteins to be closely compared with that of other cellular proteins, or superimposed on the phase-contrast image of the cell. Figure 1 shows the intracellular distribution of myc-tagged Rho proteins in Swiss 3T3 cells immunolabeled with the 9E10 antibody 15 hr following plasmid microinjection. It is evident that whereas RhoA and RhoC exhibit a floccular but relatively even distribution throughout the cytoplasm, RhoB shows a highly punctate cytoplasmic pattern of expression. By triple fluorescent labeling5 and immunoelectron microscopy (see below), this pattern 13 G. V. Heimer and C. E. Taylor, J. Clin. Pathol. 27, 254 (1974).
[19]
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PROTEINS
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0 0
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o
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o
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CELLEXPRESSION
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of RhoB expression has been shown to be associated with the membrane of vesicles of the endocytic pathway. Traces of all three tagged Rho proteins can sometimes be seen adjacent to the plasma membrane in some specimens at this time point; however, as longer expression times give rise to increasingly distorted cell morphology, a progressively greater proportion of the protein becomes localized at or near the plasma membrane. 5 Figure 2 shows the fluorescent intracellular localization of myc-tagged Racl protein in Rat2 cells 15 hr following microinjection. Expression of the racl gene coding for the activating Val-12 mutation induces ruffling of the plasma membrane, and when subconfluent a dramatic stimulation of pinocytosis occurs in these cells, s The Rac protein associates with the plasma membrane and is particularly evident both on the membrane of the internalized pinocytotic vesicles and on cell surface ruffles.
Preparation of Microinjected Cells for Immunoelectron Microscopy After a suitable incubation period following microinjection (6-20 hr), the cells are fixed for 1 hr at room temperature in 2% paraformaldehyde
FIG. 2. Rat2 cells expressing myc-tagged Racl protein 15 hr following microinjection of plasmid imaged by immunofluorescence confocal microscopy. (a) Racl protein labeled with 9El0 antibody. (b) Phase-contrast image of same cell. The protein localizes to the pinocytotic vesicles and membrane ruffles induced by rac expression. Bar: 10/xm.
[19]
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plus 0.05% glutaraldehyde in PBS ("2.05" fixative), rinsed in PBS, and the coverslip carefully trimmed to a size that would fit inside a plastic B E E M embedding capsule. Samples are then dehydrated by the progressive lowering of temperature technique (PLT) and are embedded in Lowicryl HM20 resin at - 5 0 ° as described in detail elsewhere. 14 The UV-polymerized blocks are trimmed to release the piece of T h e r m a n o x which peels away leaving the cells embedded in the resin. A replica of the marked rectangles is clearly visible in the resin allowing the group of microinjected cells to be identified. The blocks are then carefully trimmed to include only the marked rectangle, mounted in the microtome, and sectioned en face. Care is taken in aligning the block to the knife edge (in both lateral and horizontal planes) to maximize the number of sections that can be collected once sectioning has begun. Ultrathin (0.1/xm) sections are collected on uncoated nickel grids and are immunolabeled on both sides by immersion incubations using the protocol described below. 1. Incubate in blocking buffer (BB) consisting of PBS + 0.8% (w/v) bovine serum albumin (BSA) + 0.1% (w/v) gelatin + 5% (v/v) fetal calf serum (FCS) for 30 min. 2. Incubate in antibody 9E10 diluted 1 : 200 in BB overnight in a moist chamber at room temperature. 3. Wash 3 × 5 min in washing buffer (BB-FCS:WB). 4. Incubate in goat anti-mouse immunoglobulin G (IgG) conjugated to 5 nm colloidal gold diluted 1 : 50 in BB for 90 min. 5. Wash 3 x 5 min in WB. 6. Wash 3 × 2 min in U H Q water (18 ml~). 7. Silver enhance in Amersham IntenSE for 6 min. 8. Rinse 3× in U H Q and blot dry. Control incubations are undertaken in the absence of a primary antibody and also using a mouse antiactin antibody in place of the 9E10. Grids are then contrasted with uranyl acetate and lead citrate using the standard program in a Leica Ultrostainer and examined in a Philips CM10 at 80 kV.
Analysis by Immunoelectron Microscopy Figure 3a is a transmission electron micrograph showing immunogold localization of myc-tagged R a c l protein in Rat2 cells 15 hr after microinjection. The Rat2 cells used in this experiment were subconfluent, and in this condition racl expression induces large pinocytotic vesicles within the cells. ~4D. Robertson, P. Monaghan, C. Clarke, and A. J. Atherton, J. Microsc. 168, 85 (1992).
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m~
m
FlG. 3. (a) Subconfluent Rat2 cells immunogold labeled for expression of myc-tagged Racl protein. The label is associated with the membrane of the large pinocytotic vesicles. Original magnification: x26,000. Bar: 0.5/xm. (b) MDCK cell immunogold labeled for expression of myc-tagged RhoB. Two multivesicular bodies (mvb) in the micrograph show that the membrane of the mvb is labeled and also the membrane of the vesicles within the mvb.
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The micrograph (Fig. 3) shows a portion of the cytoplasm of a Rat2 cell in which several large pinocytotic vesicles are visible. The immunogold label is clearly associated with the periphery of these vesicles. Figure 3b is part of a MDCK cell at a much higher magnification than Fig. 3a (× 145,000 compared to × 26,000) and shows immunogold localization of myc-tagged RhoB to multivesicular bodies (mvb) within the MDCK cell. The labeling shows that RhoB is on the membrane of the multivesicular bodies and also on the vesicle membrane within the mvb. Concluding Remarks The combined techniques of microinjeetion and eptope tagging have proved particularly useful in enabling the intracellular distributions of closely related small GTP-binding proteins such as the Rho group to be analyzed. The principal advantages may be summarized as follows: (1) Delivery of plasmids by microin)ection allows high levels of expression in almost any adherent cell type. (2) The short time course required for these experiments enables activated forms of the proteins to be studied despite their potentially lethal effects on the cell. (3) Expression of exogenous myc-tagged genes allows detection of closely related Ras family proteins without problems of cross-reactivity and without the necessity of raising and characterizing separate antibodies. (4) Analysis of intracellular localization of tagged protein can be performed where levels of endogenous protein are too low to be reliably detected by conventional antibodies. (5) Changes in intracellular localization induced by specific mutations in microinjected plasmids can be observed without interference from endogenous protein. We have used m y c epitope tagging to investigate the intracellular distribution of a substantial number of Ras superfamily proteins, including HaRas, N-Ras, and Ki-Ras, RhoA, RhoB, and RhoC, G25K, Racl, R-Ras, and Rab6 together with a variety of unrelated proteins involved in signal transduction such as Ras-GAP, Raf-1, and ERK-2. In all cases where conventional antibodies have also been available we have been able to confirm that the activity and distribution of the myc-tagged proteins are identical to those of the endogenous product. Microinjeetion of plasmids coding for epitope-tagged gene products therefore constitutes a widely applicable system for analyzing the function of proteins in vivo.
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[20] Purification a n d A s s a y of R e c o m b i n a n t C3 T r a n s f e r a s e B y SIMON T. DILLON a n d LARRY A . F E r n
Introduction E x o e n z y m e C3 is a b a c t e r i a l l y p r o d u c e d p r o t e i n t h a t A D P - r i b o s y l a t e s a n d i n a c t i v a t e s t h e R h o A , B, a n d C p r o t e i n s f r o m t h e R h o s u b f a m i l y o f R a s - l i k e G T P a s e s . m C3 b i n d s to N A D + a n d c a t a l y z e s t h e a d d i t i o n of an A D P - r i b o s e o n t o an a s p a r a g i n e r e s i d u e at p o s i t i o n 41 o f R h o . 3 This activity o f C3 has b e e n e x p l o i t e d to r e v e a l t h a t R h o p r o t e i n s f u n c t i o n to m o d u l a t e t h e p o l y m e r i z a t i o n o f actin in cells. 4 I n p a r t i c u l a r , w h e n R h o is i n a c t i v a t e d in a cell b y C 3 - i n d u c e d A D P - r i b o s y l a t i o n , actin f i l a m e n t s d e p o l y m e r i z e . 5'6 Thus, m a n y r e s e a r c h e r s h a v e b e g u n to use e x o e n z y m e C3 to s t u d y R h o function. C3 was o r i g i n a l l y p u r i f i e d as a 24- to 2 6 - k D a p r o t e i n f r o m t h e c u l t u r e s u p e r n a t a n t s o f c e r t a i n strains o f C l o s t r i d i u m b o t u l i n u m . 7,8 N o n a t u r a l cellb i n d i n g c o m p o n e n t t h a t w o u l d efficiently a l l o w C3 to e n t e r t a r g e t cells has b e e n identified; t h e r e f o r e , it is classified as an e x o e n z y m e r a t h e r t h a n a toxin. O t h e r p a t h o g e n i c strains o f b a c t e r i a h a v e also b e e n f o u n d to e x p r e s s a C3-1ike activity. 9-12 C l o s t r i d i a l C3 efficiently m o d i f i e s o n l y R h o A , B, a n d C. T h e o t h e r R h o f a m i l y p r o t e i n s such as R a c l a n d C D C 4 2 a r e v e r y p o o r
1 K. Aktories, U. Braun, B. Habermann, and S. Rosener, in "ADP-Ribosylating Toxins and G Proteins" (J. Moss and M. Vaughan, eds.), p. 97. American Society for Microbiology, Washington, D.C., 1990. 2 A. Hall, Science 249, 635 (1990). 3 S. Narumiya, S. Akhiro, and M. Fujiwara, J. Biol. Chem. 263, 17255 (1988). 4 A. Hall, MoL Biol. Cell 3, 475 (1992). 5 p. Chardin, P. Boquet, P. Madaule, M. R. Popoff, E. J. Rubin, and D. M. Gill, EMBO J. 8, 1087 (1989). 6 H. F. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell Biol. 111, 1001 (1990). 7 K. Aktories, U. Weller, and G. S. Chhatwal, FEBS Lett. 212, 109 (1987). 8 E. J. Rubin, D. M. Gill, P. Boquet, and M. R. Popoff, Mol. Cell. BioL 8, 418 (1988). 9 y. F. Belyi, I. S. Tartakovskii, Y. V. Vertiev, and S. V. Prosorovskii, Biomed. Sci. 2,169 (1991). 10I. Just, G. Schallehn, and K. Aktories, Biochem. Biophys. Res. Commun. 183, 931 (1992). 11I. Just, C. Mohr, G. Schallehn, L. Menard, J. D. Didsbury, J. Vandekerckhove, J. van Damme, and K. Aktories, J. Biol. Chem. 267, 10274 (1992). 12 M. Sugai, K. Hashimoto, A. Kikuchi, S. Inoue, H. Okumura, K. Matsumoto, Y. Goto, H. Ohgai, K. Morishi, B. Syuto, K. Yoshikawa, H. Suginaka, and Y. Takai, J. Biol. Chem. 267, 1 (1992).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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PURIFICATIONOF C3 TRANSFERASE
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substrates in vitro u'13 and are not modified in platelet extracts. TM Therefore, a C3 assay including [32p]NAD provides a very sensitive marker to quantitate R h o in an experimental sample. Both recombinant C3 and the forms purified from its naturally producing organism have been used on a wide variety of mammalian cell types to study R h o expression and cellular function. It has also been shown to modify R h o from X e n o p u s laevis 8 and Saccharomyces cerevisiae. 15 When it is added in micrograms per milliliter amounts to the external media, C3 enters some tissue culture cells 5's'16 and human platelets. 17 A more certain route of cell entry has been by microinjection. 6 Construction of a fusion protein between C3 and the cell-binding component of diphtheria toxin has also been used to deliver C3 to the cytosol of intact cells (see [34] in this volume). 1~ This chapter describes a relatively simple method for the purification of milligram amounts of recombinant C3 for use in cell studies as well as in in vitro assays to detect Rho proteins.
E x p r e s s i o n of a G l u t a t h i o n e S - T r a n s f e r a s e - C 3 F u s i o n in E s c h e r i c h i a coli
To express high levels of the C3 protein in E. coli, the p G E X vector expression system (Pharmacia) was chosen. These vectors generate a fusion with glutathione S-transferase(GST) and allow a one-step purification of the fusion protein. Construction o f Expression Vector
The exoenzyme C3 gene is cloned from C. b o t u l i n u m type D strain 1873.19 A 2.0-kb H i n d I I I fragment of D phage D N A is cloned into the H i n d I I I site located in the polylinker cloning region of pUC19 to create 13L. Menard, E. Tomhave, P. J. Casey, R. J. Uhing, R. Snyderman, and J. R. Didsbury, Eur. J. Biochem. 206, 537 (1992). 14y. Nemoto, T. Namba, T. Teru-uchi, F. Ushikubi, N. Morii, and S. Narumiya,J. Biol. Chem. 267, 20916 (1992). 15M. McCaffrey,J. S. Johnson, B. Goud, A. M. Myers, J. Rossier, M. R. Popoff, P. Madaule, and P. Boquet, J. Cell Biol. 115, 309 (1991). 16T. Tominaga,K. Sugie,M. Hirata, N. Morii, J. Fukata, A. Uchida, H. Imura, and S. Narumiya, J. Cell BioL 120, 1529 (1993). 17N. M0rii, T. Teru-uchi, T. Tominaga, N. Kumagai, S. Kozaki, F. Ushikubi, and S. Narumiya, J. BioL Chem. 267, 20921 (1992). 18p. Aullo, M. Giry, S. Olsnes, M. R. Popoff,C. Kocks, and P. Boquet, EMBOJ. 12, 921 (1993). t9 M. Popoff, P. Boquet, D. M. Gill, and M. W. Eklund, Nucleic Acids Res. 18, 1291 (1990).
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p M R P l l , z° From this vector a 859-bp SacI-SpeI fragment containing the C3 gene is then ligated into the SacI-XbaI sites of the trpE fusion expression vector pRX-121 to generate pBC11. To create a GST-C3 fusion vector, the plasmid pBC11 is first digested with ClaI, blunt ended using Klenow, and is then digested with BamHI. This generates a 0.9-kb fragment containing the C3 gene that is cloned into the BamHI and SmaI sites of pGEX2T(Pharmacia) to give a plasmid referred to as pGEX2T-C3.
Culture of E. coli Expressing GST-C3 (1 Liter) The protocol is adapted from a published procedure for the general expression of proteins from p G E X vectors 22 and yields approximately 1 mg of C3. The pGEX2T-C3 D N A is transformed into any standard E. coli strain since the lacIq repressor allele is cloned into the p G E X DNA. RR1 is the strain used here. A single colony is picked from a fresh transformation on plates containing 50/xg/ml ampicillin and is innoculated into 100 ml of LB medium containing 10/zg/ml ampicillin. This is grown overnight at 37 ° on a shaker table. The stationary phase bacteria is added to 900 ml of LB containing 10/xg/ml ampicillin and is grown at 37 ° on a shaker for 1 hr. Isopropyl/3-D-thiogalactopyranoside (USB) is then added to a final concentration of 100/~g/ml and the cells are grown at 37 ° with shaking for an additional 5-9 hr. In general, with p G E X vector fusions we find it important to not allow the bacterial culture to grow overnight since once the cultures reach stationary phase there is proteolysis of the fusion protein. The cells are then pelleted at 5000g for 10 min and the medium is drained off. The cell pellet is stored at - 8 0 ° unless proceeding directly to the lysis. The GST-C3 fusion runs at 50 kDa on S D S - P A G E and it represents 2-3% of total cellular protein in E. coli (Fig. 1A).
Purification of G S T - C 3 from Bacterial Cell Lysate
Preparation of Cell Lysate To maximize lysis, the bacteria are first resuspended in 40 ml of an icecold phosphate-buffered saline(PBS) solution containing I mg/ml lysozyme, 1% Triton X-100, 25% sucrose (w/v), 1 m M EDTA, 5 m M dithiothreitol 20 M. R. Popoff, D. Hauser, P. Boquet, M. W. Eklund, and D. M. Gill, Infect. lmmun. 59, 3673 (1991). 2a D. L. Rimm and T. D. Pollard, Gene 75, 323 (1989). 22 D. B. Smith and L. M. Corcoran, in "Current Protocols" (F. M. Ausubel, R. Brent, R. E. Kingston, D. M. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds.), unit 16. John Wiley and Sons, New York, 1990.
[20]
PURIFICATION
A
C3 TRANSFERASE
OF
B
C
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i
Fie. 1. SDS-PAGE analysis of GST-C3 and purified C3. (A) Relative level of expression of GST-C3 fusion protein in the E. coli strain RRI (12.5% SDS-PAGE). Lane 1, GST-C3; lane 2, GST; and lane 3, control bacteria with no pGEX plasmid. (B) Purity of GST-C3 precipitated on glutathione-agarose beads (12.5% SDS-PAGE). Lane 1, GST-C3; lane 2, GST; and lane 3, control bacteria. (C) Purity of recombinant C3 (15% SDS-PAGE). Lane 1, 1 /~g of purified recombinant C3. The numbers to the left of each gel represent the Mr (×10 -3) of the protein standards.
(DTT), 1% aprotinin, and 1 m M phenylmethylsulfonyl fluoride (PMSF) on ice. This solution is then stirred gently(magnetic stirrer) for 30 rain at 4°. The solution should become viscous. To complete cell lysis and begin the breakdown of the DNA, the sample is sonicated on ice using a probe sonicator for 2-3 min at the maximum setting. There should be a significant decrease in the viscosity of the solution. Deoxyribonuclease I (DNase I, Boehringer Mannheim) is then added to a final concentration of 0.1 mg/ ml, and the solution is stirred for an additional 20 min at 4°. The insoluble material is removed by centrifuging the bacterial lysate at 10,000g for 10 min. It is possible to stop here and store the soluble fraction at - 8 0 °. Precipitation o f Fusion Protein on Glutathione Beads
One milliliter of a 50% slurry of preswelled and washed glutathioneagarose beads (Sigma or Pharmacia) is then added to the soluble bacterial lysate and is incubated overnight at 4 ° with constant mixing. The beads are then centrifuged away from the lysate at 500g for 5 min. Care should be
178
CELL EXPRESSION
[20]
taken to not spin the beads at too high a centrifugal force as they will be crushed. The beads are then washed three times with 20 bead volumes of PBS containing 1% Triton, 1% aprotinin, and 1 mM PMSF. The beads can be stored in this solution at 4° for 1-2 days before proceeding to the thrombin cleavage step. A small aliquot of the bead solution (1-2 tzl) is then run on SDS-PAGE (10 or 12.5%) to estimate the amount of fusion protein that is present on the beads. The GST-C3 fusion on glutathione beads is not significantly contaminated by other bacterial proteins (Fig. 1B).
Cleavage with Thrombin The pGEX2T vector is designed with a thrombin cleavage site just before the multiple cloning site for inserting the gene. Therefore, incubation of the fusion protein on the beads with this protease results in the release of the fused protein from the beads. The protocol for the cleavage is adapted from a published procedure. 22 All manipulations are performed either on ice or at 4°. The glutathione beads containing GST-C3 are washed three times with 20 vol of 1% Triton X-100 in PBS, three times with 20 vol of a 50 mM Tris (pH 7.5), 150 mM NaC1 solution, and three times in 20 vol of 50 mM Tris (pH 7.5), 150 mM NaC1, 2.5 mM CaC12. The beads are then resuspended in a roughly equal volume of the CaC12 buffer (0.5-1.0 ml). To cleave the protein from the beads, highly purified bovine a-thrombin (Haemotologic Technologies, Essex Junction, VT) is added to the bead solution. Thirty NIH units of thrombin activity is added per mg of fusion protein. The cleavage is carried out for 12-24 hr at 4° with constant mixing. The solution is centrifuged at 500-1000g for 5 min, and the supernatant is retained. The beads are then washed three more times with an equal volume of the 50 mM Tris(pH 7.5), 150 mM NaC1 buffer and these washes are pooled together with the original supernatant from the cleavage reaction. Typically 90-95% of the total fusion protein is cleaved. Once this level of cleavage is attained, adding more thrombin does not generate more cleaved protein.
Ion-Exchange Chromotography Clostridial exoenzyme C3 is a basic protein 1a'19 and is therefore suitable for purification by cation-exchange chromotography. The buffer containing the C3/thrombin mixture is replaced with 50 mM Tris (pH 7.5), 30 mM NaC1, 1 mM DTT by either passing the C3 solution from the thrombin cleavage reaction over a small desalting column (Bio-Rad) or by dialysis of the sample. The cleavage mixture is then applied to a Mono S HR 5/5 column (0.5 × 5 cm, Pharmacia). It is eluted with a 30-ml linear gradient
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PURIFICATIONOF C3 TRANSFERASE
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of 0-500 mM NaC1 in 50 mM Tris (pH 7.5), 1 mM DTT. The fractions containing the major protein peaks are pooled and the DTT is removed by desalting or by dialysis. The protein is then concentrated to a 1-mg/ml solution using a Centricon-10 (Amicon, Beverly, MA). Purified recombinant C3 runs at 24 kDa on 15% SDS-PAGE and is >95% pure (Fig. 1C). The majority of the thrombin is removed; however, there are still trace amounts of thrombin activity that are present with the purified C3. 23
Use of C3 as Probe for Rho Proteins
Preparation of Cell Lysates Whole cell extracts from tissues are made by homogenization in 4 vol of the following medium: 50 mM HEPES (pH 7.3), 130 mM NaC1, 1 mM PMSF, 1 mM DTT, 5/xg/ml leupeptin, 0.02% azide. All steps here should be performed on ice. The tissue is cut up into small pieces and is then homogenized by 20 strokes in a Dounce homogenizer (tight fitting). The homogenates are then centrifuged at 500g for 5 min at 4° to remove unbroken cells and nuclei. All cell extracts are either used immediately in the C3 assay or snap frozen in a dry ice/EtOH bath and stored at -80 ° until use. Cells grown in tissue culture are gently removed with a rubber policeman, washed in PBS, and collected by centrifuging at 1000g for 10 min. Three volumes of the following buffer is added to the cell pellets: 20 mM Tris (pH 7.5), 3 mM MgCI2, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1% aprotinin, 10/xg/ml leupeptin, and 1% Triton X-100. The mixture is then vortexed to resuspend and lyse the cells. If the cell extract is to be fractionated into cytosol and particulate then the cells are resuspended in the above buffer minus the Triton X-100. The cells are lysed with 20 strokes of a Dounce homogenizer. Triton X-100 is added to the cell fractions to a final of 1% after all of the fractionations are completed.
Assay and Activity of Purified Recombinant C3 on Rho Proteins To assay for the presence of Rho proteins in a broken cell extract, the following components are added together on ice to the stated final concentration: 25-200/zg of cell extract protein, 20 mM HEPES (pH 8.0), 2 mM MgCI2, 10 mM thymidine, 0.1% sodium deoxycholate, 200/zM GTP, 10 tzM NAD +, [32p]NAD+ (either ICN or NEN/Dupont; 0.5-2.5/xCi/tube, 10,000-50,000 cpm/pmol), and 40/xg/ml purified recombinant C3 in a final volume of 20-30/~1. This mixture is then incubated for 1 hr at 30°. Laemmli 23S. E. Rittenhouse,personal communication(1993).
180
CELL EXPRESSION
1
2
3
4
5
[20]
6
66_ 43_ 36~ 29 ._j 24--
,11 Rho-ADPR
20
14 ~
|
~
FIG. 2. Activity of purified recombinant C3 on Rho in NIH 3T3 cell extracts. The broken cell extract was made and the C3 assay was performed as described in the text. Lanes 1-3, control reactions with all of the assay reagents added except for C3. Lanes 4-6, C3 added to the reaction mixtures. Lanes 1 and 4 contain 50/xg of a whole cell extract. Lanes 2 and 5 contain the cytosolic fraction of a 100,000g, 1-hr centrifugation of 50/~g whole cell extract. Lanes 3 and 6 contain the pellet from the same centrifugation. The numbers to the left represent the Mr ( x l 0 -3) of the protein standards.
sample buffer (2x) 24 is added, and the samples are boiled for 5 rain and resolved on SDS-PAGE (12.5 or 15%). Afterward, the gel is stained with Coomassie blue and destained. Unincorporated [32P]NAD will increase the background signal in the gel so it is useful to soak the gel in several changes of tap water before drying the gel. The gel is then dried and autoradiographed. A typical C3 assay performed with NIH 3T3 broken cell extracts, cytosol, and particulate fractions is shown in Fig. 2. In these cells, R h o - A D P R runs as a single band at 22 kDa on one-dimensional SDS-PAGE (12.5%). In other tissues and cell lines, R h o - A D P R can resolve into several bands in this region. This may be due to the presence of more than one family member, partial proteolysis of the Rho, or differences in some endogenous covalent modification such as occurs at the COOH-terminal C A A X box. Depending on the tissue or cell line examined, other labeled bands appear (see the band below R h o - A D P R in Fig. 2); however, these are not specific 24 U. K. Laemmli, Nature (London) 227, 680 (1970).
[20]
PURIFICATION OF C3 TRANSFERASE
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for C3 since they are also present when C3 is omitted in control reactions. Recombinant C3 is active on both cytosolic and particulate Rho (Fig. 2). The recombinant C3 described here has an extra 26 amino acids on the NH2 terminus compared to the enzyme purified from clostridia: 19 amino acids from the pUC19 polylinker, and the last 7 amino acids from the signal sequence of C3 in C. botuIinum. The relative activities of the recombinant C3 presented here versus those purified from C. botulinum have not been assessed. However, using a very similar C3 clone it was found that the recombinant form had 50% of the activity of the wild-type form of the protein. 2° In human platelets, maximal modification of Rho by the purified recombinant C3 presented here was achieved with a final concentration of 40 ixg/ml. 25
Known Inhibitors of C3 Reaction in Broken Cell Extracts In their cytosolic form, Rho family proteins are found complexed to a 28-kDa protein called Rho-GDI. 26'27 It has been shown directly that R h o GDI is able to inhibit the ability of C3 to modify Rho proteins. 28 This is the only known inhibitory protein for cytosolic Rho, but there may well be others. Detergents, certain phospholipids, and dilution are known to stimulate the ability of C3 to modify cytosolic Rho. 27'29 This phenomenon appears to be due to the disruption of the interaction between R h o - G D I and Rho. 27 There are no known inhibitory proteins of the C3 reaction for membrane-bound Rho.
Identification and Resolution of RhoA, B, and C on Two-Dimensional Isoelectric Focusing (IEF) S D S - P A G E On one-dimensional SDS-PAGE the resolution of the three Rho proteins after the C3 assay can be difficult since they all run about the same size. Thus, two-dimensional IEF SDS-PAGE 3° was used to identify the different Rho proteins and their relative levels. The RhoA spot was positively identified using a NIH 3T3 cell line expressing a point mutant of RhoA that makes the protein more basic. With this cell line an additional 25 j. Zhang, W. G. King, S. Dillon, A. Hall, L. Feig, and S. E. Rittenhouse, J. Biol. Chem. 268, 22251 (1993). 26 R. Regazzi, A. Kikuchi, Y. Takai, and C. B. Wolheim, J. BioL Chem. 267, 17512 (1992). 27 N. Bourmeyster, M.-J. Stasia, J. Garin, J. Gagnon, P. Boquet, and P. V. Vignais, Biochemistry 31, 12863 (1992). 28 A. Kikuchi, S. Kuroda, T. Sasaki, K. Kotani, K. Hirata, M. Katayama, and Y. Takai, J. Biol. Chem. 267, 14611 (1992). 29 K. C. Williamson, L. A. Smith, J. Moss, and M. Vaughan, J. BioL Chem. 265, 20807 (1990). 3o j. I. Garrels, J. Biol. Chem. 254, 7961 (1979).
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pH 3-10 IEF
acidic
basic
liver
t
I
"tl
spleen
brain
A RhoB
RhoA
RhoC
F~6. 3. Two-dimensional IEF S D S - P A G E of various murine tissue extracts after the C3 assay. Fifty micrograms of tissue was used in each C3 assay. The IEF gel used pH 3-10 ampholines (Pharmacia LKB). The second dimension gel was 12.5% SDS-PAGE. Thick arrows represent internal marker proteins that are included with each sample to allow comparisons between different gels and experiments.
spot the same size as R h o A but shifted slightly to the right (basic end) was observed. 31 The calculated size of RhoB is slightly larger than R h o A and its calculated pI is more acidic (to the left). 32'33 It is with this evidence that we have identified the RhoB as the spot to the left, resolving with a slightly larger apparent molecular weight (see Figs. 3 and 4). RhoC should resolve at about the same size as R h o A and, because it is slightly more basic than RhoA, it should appear to the right side of RhoA. A relatively weak spot is observed that resolves to the basic side of R h o A and this is designated as RhoC (see Fig. 4). Using immunoblots in combination with twodimensional gel electrophoresis, a spot to the basic side and a slightly lower apparent molecular weight of R h o A was identified as RhoC. 34 The relative amounts of RhoA, B, and C in various mouse tissues fall 31 S. T. Dillon, unpublished observation (1990). 32 p. Yeramian, P. Chardin, P. Madaule, and A. Tavitian, Nucleic Acids Res. 15, 1869 (1987). 33 p. Chardin, P. Madaule, and A. Tavitian, Nucleic Acids Res. 16, 2717 (1988). 34 p. Lang, F. Gesbert, J.-M. Thiberge, F. Troalen, H. Dutartre, P. Chavrier, and J. Bertoglio, Biochem. Biophys. Res. Commun. 196, 1522 (1993).
[20]
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pH 5-8 IEF acidic
basic
homogenate
cytosolic
particulate
"
RhoB
Z2
(?)
RhoC
'
Fro. 4, Membrane versus cytosol distribution of RhoA, B, and C in murine brain extracts by two-dimensional IEF S D S - P A G E . The IEF gel was run with a 2 : 1 ratio of pH 5-8 to p H 3-10 ampholines. The second dimension gel was 12.5% S D S - P A G E .
into three categories (Fig. 3). Tissues such as liver contain mostly RhoA with little RhoC and no detectable RhoB by the C3 assay. In this IEF system, using a broad range of ampholines (pH 3-10), the RhoC spot does not resolve well from RhoA but is observed as a streaking to the basic side of RhoA. The second type of tissue distribution observed is illustrated by spleen in which RhoB is detectable but is present in lower amounts than RhoA and there is little or no detectable RhoC. Finally, the mouse brain is unique in that it has high and roughly equal amounts of both RhoA and RhoB, with RhoC at a low level (Figs. 3 and 4).
Dealing with Problem of High NADase in Certain Tissues Some mouse tissues were found to possess high amounts of NADase activity which resulted in the degradation of [32p]NAD. NADase activity in certain tissue extracts has been shown to cause problems in assays with other ADP-ribosyltransferases.35To overcome this problem in cholera toxin assays on membrane Gs, three known inhibitors of NAD glycohydolase activity were included.36 In both the brain and spleen extract assay (Figs. 3 35 D. M. Gill and M. J. Woolkalis, this series, Vol. 195, p. 267. 36 D. M. Gill and J. Coburn, Biochim. Biophys. Acta 954, 65 (1987).
184
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[21 ]
and 4), isonicotinic acid hydrazide (INH, isoniazid, Sigma), 3-acetylpyridine adenine dinucleotide (3-APAD, Sigma), and DTT were added to a final concentration of 15, 1, and 5 mM, respectively. Membrane versus Cytosol Distribution of RhoA, B, and C in Murine Brain Extracts
Two-dimensional IEF SDS-PAGE was used to investigate the relative distribution of RhoA, B, and C proteins in mouse brain extracts (Fig. 4). RhoB is primarily found in the particulate fraction (100,000g pellet) whereas RhoA is found in equal amounts between the cytosol (100,000g supernatant) and particulate fractions. The spot to the basic side of RhoA, designated as RhoC, appears to be present more in the particulate fraction. It is unclear which of the Rho proteins is resolving to the acidic side of RhoA, perhaps a modified form of RhoA. Conclusions Exoenzyme C3 has been used by a growing number of researchers interested in the regulation of the actin cytoskeleton. It is hoped that the procedures described in this chapter will help those investigators who wish to use C3 to specifically inhibit RhoA, B, and C in cells. The pGEX2T-C3 clone described here will be deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852.
[21] I n V i t r o A D P - R i b o s y l a t i o n o f R h o b y B a c t e r i a l ADP-Ribosyltransferases B y KLAUS AKTORIES a n d INGO JUST
Introduction A growing family of intracellularly acting bacterial protein toxins is characterized by ADP-ribosyltransferase activity (for review see Refs. 1-6). These toxins split NAD into ADP-ribose and nicotinamide and trans1 I. Pastan and D. FitzGerald, J. Biol. Chem. 264, 15157 (1989). 2 K. Aktories and I. Just, in "GTPases in Biology I" (B. F. Dickey and L. Birnbaumer, eds.), p. 87. Springer-Verlag, Berlin/Heidelberg, 1993. 3 j. Moss and M. Vaughan, eds., "ADP-Ribosylating Toxins and G Proteins," American Society for Microbiology, Washington, D.C., 1990. 4 K. Aktories, ed., Curt. Top. Microbiol. Immunol, 175 (1992).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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fer the A D P - r i b o s e moiety onto eukaryotic target proteins. T h e covalent modification f o r m e d is highly selective and causes gross changes in the physiological functions of the target proteins. The toxins are, therefore, of importance not only as bacterial virulence factors, but also as tools in cell biology to study structure and functions of their eukaryotic target proteins. Well-known examples of this family of toxins are diphtheria toxin, Pseud o m o n a s aeruginosa exotoxin A, cholera toxin, and pertussis toxin (for reviews see Refs. 1-6). For unknown reasons, nucleotide-binding proteins (mostly GTPases) are c o m m o n substrates for these toxins. Diphtheria toxin and P s e u d o m o n a s exotoxin A modify elongation factor 2 (EF2), a G T P a s e that is involved in protein synthesis; and cholera and pertussis toxins A D P ribosylate heterotrimeric GTP-binding proteins (G proteins) that are key regulators of t r a n s m e m b r a n e signal transduction. A n o t h e r group of bacterial ADP-ribosylating toxins including Clostridium b o t u l i n u m C2 toxin] Clostridium perfringens iota toxins, s and related toxins modifies actin, an ATPase. Several bacterial ADP-ribosyltransferases have been described which modify small G T P a s e s of the R h o family. The best studied m e m b e r of this group is C. b o t u l i n u m ADP-ribosyltransferase C3. C l o s t r i d i u m b o t u l i n u m A D P - R i b o s y l t r a n s f e r a s e C3 C. b o t u l i n u m C3 transferase is produced by various strains of C. bofulihUm type C and D. 9-12 The transferase modifies selectively the low molecu-
lar mass GTP-binding proteins R h o A , B, and C, which appear to be involved in the organization and regulation of the actin cytoskeleton) 3-15 A p p a r ently, a large heterogeneity exists a m o n g C3 ADP-ribosyltransferases. Whereas the c D N A f r o m C. b o t u l i n u m strains D1873 and C468 encodes a 5 j. E. Aloug and J. H. Freer, eds., "Sourcebook of Bacterial Protein Toxins," Academic Press, London, 1991. 6 B. D. Spangler, Microbiol. Rev. 56, 622 (1992). 7 K. Aktories, M. Barmann, I. Ohishi, S. Tsuyama, K. H. Jakobs, and E. Habermann, Nature 322, 390 (1986). 8 B. Schering, M. B~irmann, G. S. Chhatwal, U. Geipel, and K. Aktories, Eur. J. Biochem. 171, 225 (1988). 9 K. Aktories, U. Weller, and G. S. Chhatwal, FEBS Letr 212, 109 (1987). 10K. Aktories, S. R6sener, U. Blaschke, and G. S. Chhatwal, Eur. J. Biochem. 172,445 (1988). n E. J. Rubin, D. M. Gill, P. Boquet, and M. R. Popoff, Mol. Cell Biol. 8, 418 (1988). 12K. Moriishi, B. Syuto, N. Yokosawa, K. Oguma, and M. Saito, J. Bacteriol. 173, 6025 (1991). 13p. Chardin, P. Boquet, P. Madaule, M. R. Popoff, E. J. Rubin, and D. M. Gill, EMBO Z 8, 1087 (1989). 14H. F. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell BioL 111, 1001 (1990). 15A. J. Ridley and A. Hall, Cell 70, 389 (1992).
186
CELLEXPRESSION
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211 amino acid protein (without a signal peptide) with a molecular mass of 23,546 Da, 16 Nemoto et aL 17 reported a cDNA for C3 (strain C003-9) encoding a protein of 204 amino acids (Mr 23,119) with about 60% identity at the amino acid level. C3 transferases are heat stable (1 min, 95°), are resistant to short-term trypsin treatment, and are very basic proteins (pI -10), a property that has been utilized for purification. 9'1° As with other bacterial toxins, C3 catalyzes a mono(ADP-ribosyl)ation and, therefore, phosphodiesterase treatment of [32p]ADP-ribosylated Rho protein releases [32p]5'-AMp1° but not [32p]phosphoribosyl-AMP, a cleavage product of poly(ADP-ribose). 11 Accordingly, ADP-ribosylation is neither blocked by thymidine, a well-known inhibitor of poly(ADP-ribose) polymerase, nor isonicotinic acid hydrazide, an inhibitor of NAD glycohydrolases. The reaction is specific for N A D and is not observed with ADP-ribose, which can serve as a cosubstrate for nonenzymatic ADPribosylation. The Km value for NAD is about 0.4/zM ___ 0.04 (_SD), and the specific enzyme activity is 6.4 _+ 0.6 ( _ S D ) nmol/min/mg. 18 As known for other bacterial transferases, C3 exhibits N A D glycohydrolase activity. 1° However, this activity is very low and its physiological function is questioned.
ADP-Ribosylation of Rho Proteins at Asparagine-41 Three mammalian Rho proteins are now known (RhoA, B, and C) to serve as substrates for C3-catalyzed ADP-ribosylation. Other members of the Rho protein family (Racl and 2, RhoG, CDC42 (G25K), and TCI0) are essentially not substrates for ADP-ribosylation. Is It has been shown that C3 modifies Rac proteins in the presence of SDS (0.01%) maximally by about 10% TM (and see below); however, the physiological meaning of this finding is unclear. Heterotrimeric G proteins, tubulin, or actin are not substrates for C3-catalyzed ADP-ribosylation. Protein chemistry and site-directed mutagenesis have shown that C3 ADP-ribosylates R h o A at asparagine-41.19 Most likely, all C3-1ike transferases modify Rho at the identical amino acid residue. Asparagine is unique as an acceptor for ADP-ribosylation by C3-1ike exoenzymes; cholera toxin and C. b o t u l i n u m C2 toxin ADP-ribosylate arginine residues in heter-
16M. R. Popoff,P. Boquet, D. M. Gill, and M. W. Eklund, Nucleic Acids Res. 18, 1291 (1990). 17y. Nemoto, T. Namba, S. Kozaki, and S. Narumiya,J. Biol. Chem. 266, 19312 (1991). 18I. Just, C. Mohr, G. Schallehn, L. Menard, J. R. Didsbury, J. Vandekerckhove, J. van Damme, and K. Aktories, J. Biol. Chem. 267, 10274 (1992). 19A. Sekine, M. Fujiwara, and S. Narumiya,J. Biol. Chem. 264, 8602 (1989).
[21]
ADP-RIBOSYLATIONOF Rho
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otrimeric GTP-binding proteins 2° and actin, 21 respectively, whereas pertussis toxin modifies G proteins selectively at cysteine residues. 22 The ADPribose-asparagine bond, which is formed by C3-1ike transferases, is highly stable toward hydroxylamine (0.5 M, 2 hr) and mercury ions (2 raM, 1 hr), whereas arginine- and cysteine-specific ADP-ribosylation is sensitive toward these agents, respectively. 23 It appears that ADP-ribosylation of Rho at asparagine-41 renders the GTP-binding protein biologically inactive. 14 This was concluded from the finding that activated Rho protein [(Val-14) Rho], microinjected after in vitro ADP-ribosylation, loses its ability to induce formation of stress fibers. 14 Because asparagine-41 is located in the so-called effector region of Rasrelated GTP-binding proteins, it has been suggested that ADP-ribosylation disturbs the interaction with a putative effector. However, because the exact signal transduction cascade involving Rho has not been fully elucidated, precise molecular consequences of ADP-ribosylation of Rho are not known (see other chapters of this volume). O t h e r C3-1ike E x o e n z y m e s In addition to C3, various other Rho ADP-ribosylating transferases have been described. Clostridium l i m o s u m produces a 25-kDa protein that is about 70% identical with C3 (strain C468). TM Specific enzyme activity and Km for N A D of the C. l i m o s u m transferase-catalyzed reaction are similar to C3. The C. l i m o s u m exoenzyme modifies RhoA, B, and C but not Rac, CDC42, or R h o G proteins (even in the presence of SDS) at the same asparagine as C3. In contrast to C3, the C. l i m o s u m exoenzyme is autoADP-ribosylated in the presence of SDS (0.01%). Certain strains of Staphylococcus aureus produce an exoenzyme called E D I N (epidermal differentiation inhibitor), 24'25 which belongs to the family of C3-1ike transferases. The mature protein (EDIN) of 212 amino acids shares about 35% identity (amino acid level) with C3 and appears to modify the identical eukaryotic substrates RhoA, B, and C. 24 Finally, a 28-kDa exoenzyme from Bacillus 20c. Van Dop, G. Yamanaka, F. Steinberg, R. D. Sekura, C. R. Manclark, L. Stryer, and H. R. Bourne, J. Biol. Chem. 259, 23 (1984). 2! j. Vandekerckhove, B. Sehering, M. B~irmann, and K. Aktories, J. Biol. Chem. 263, 696 (1988). 22R. E. West, J. Moss, M. Vaughan, T. Liu, and T.-Y. Liu, J. Biol. Chem. 260, 14428 (1985). 23K. Aktories, I. Just, and W. Rosenthal, Biochem. Biophys. Res. Commun. 156, 361 (1988). 24S. Inoue, M. Sugai, Y. Murooka, S.-Y. Paik, Y.-M. Hong, H. Ohgai, and H. Suginaka, Biochem. Biophys. Res. Commun. 174, 459 (1991). 25M. Sugai, K. Hashimoto, A. Kikuchi, S. Inoue, H. Okumura, K. Matsumota, Y. Goto, H. Ohgai, K. Moriishi, B. Syuto, K. Yoshikawa, H. Suginaka, and Y. Takai, J. Biol. Chem. 267, 2600 (1992).
188
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cereus has been described that ADP-ribosylates Rho proteins. 26 Again this
enzyme exhibits kinetic properties very similar to C3 but it appears to be rather distantly related to the other C3-1ike clostridial transferases. The polyclonal antibody against C3 does not cross-react with the B. cereus transferase. 27 All these ADP-ribosyltransferases are very basic proteins (pI > 9) and appear to modify selectively Rho proteins at the identical amino acid acceptorJ 8'27,28Accordingly, analysis of the active site structure of C3 has identified Glu-174 as part of the catalytic center of the transferase which appears to be conserved in all other C3-1ike transferases, including B. cereus exoenzyme and EDIN. 27"29 ADP-Ribosylation Assay Because C3 needs no activation to elicit transferase activity, no other factors in addition to Rho proteins and NAD are essential for in vitro ADPribosylation by C3. To identify the C3-modified Rho proteins, [adenylate32p]NAD is usually used for ADP-ribosylation reactions. Rho proteins are very abundant GTP-binding proteins and are present in all tissues and cell lines studied so far. Therefore, ADP-ribosylation by C3 occurs with all cell types studied. C3 labels Rho proteins in cell lysates or in the cytosolic and membrane fractions of lysates. For unknown reasons the total amount of [32p]ADP-ribosylated Rho proteins of the cell lysate is not entirely the sum of the labeled Rho proteins in the cytosol and in the membrane fractions. Similarly, C3-1ike transferases ADP-ribosylate purified endogenous Rho proteins or recombinant Rho proteins. Even Rho-glutathiontransferase fusion proteins are substrates for ADP-ribosylation by C3. The typical assay conditions are as follows. I°'17 About 20 to 150 ~g of cellular protein is incubated in an ADP-ribosylation buffer containing 50 mM triethanolamine hydrochloride (pH 7.5), 2 mM MgCI2, i mM EDTA, i mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.2-i /zM [32p]NAD (about 0.2 to 0.5 ~Ci), and 0.15-i ~g/ml C3 or C3-1ike transferase at 37 ° for 5-30 rain. Assay Protocol
10/El buffer A 5/El C3 (10/zg/ml) 26 I. Just, G. Schallehn, and K. Aktories, Biochem. Biophys. Res. Commun. 183, 931 (1992). 27 I. Just, J. Seizer, M. Jung, J. van Darnme, J. Vandekerckhove, and K. Aktories, Biochemistry 34, 334 (1995). 28 K. Aktories, C. Mohr, and G. Koch, Curr. Top. Microbiol. Immunol. 1757 115 (1992). 29 M. Jung, I. Just, J. van Damme, J. Vandekerckhove, and K. Aktories, J. Biol. Chem. 268, 23215 (1993).
[21]
ADP-RIBOSYLATIONOF Rho
189
5/xl [32p]NAD (10/xM, about 0.3 tzCi) 10/xl additions to be tested (e.g., nucleotides) 20/zl cell lysate (20-150/zg of cellular protein or 0.5-1/xg of purified Rho protein) 50/xl total volume Buffer A: 250 mM triethanolamine hydrochloride (pH 7.5), 10 mM MgC12, 5 mM EDTA, 5 mM DTF, and 1 mM PMSF. The various assay components are premixed at 0° (ice bath). The ADPribosylation reaction is started with the addition of the cell lysate and is continued for 15-30 min at 37 °. The reaction is stopped by the addition of 10 /xl of 5× Laemmli sample buffer3° and is heated for 10 min at 95°. Thereafter, proteins are subjected to SDS-polyacrylamide gel electrophoresis. Alternatively, the reaction is stopped by the addition of 900/xl trichloroacetic acid (20%, w/v). The samples are kept on ice for 30 min. Thereafter, the pellet is collected by centrifugation (15 min, 14,000g, 4°C), washed three times with 1 ml ether/ethanol (1 : 1, v/v), and resuspended in 1 × Laemmli sample buffer and used for SDS-polyacrylamide gel electrophoresis. For gel electrophoresis, 12% gels can be used and run in a Mini-PROTEAN II (Bio-Rad) system. Gels are stained with Coomassie blue, destained, and subjected to autoradiography (Kodak X-Omat AR) for 12 to 72 hr or are analyzed by phosphorimaging for 2 to 12 hr. Dithiothreitol and phenylmethylsulfonyl fluoride are not essential for ADP-ribosylation by C3-1ike transferases, especially not with highly purified Rho preparations. If the amount of poly(ADP-ribosyl)ation is very high in cell lysates, it can be blocked by the addition of 10 mM thymidine. Otherwise, NAD is consumed for poly(ADP-ribose) formation. Figure 1 shows the gel and the autoradiogram of C3-catalyzed ADPribosylation of NRK (normal rat kidney) cell lysate. On SDS-polyacrylamide gels, ADP-ribosylation causes only small changes in the migration behavior (small increase in Mr) of modified Rho proteins. By using nondenaturing gels, ADP-ribosylation of Rho by C3 can be detected by a significant increase in migration compared to nonmodified protein (Fig. 2); ADPribosylated Rho is detected by the anti-Rho antibody below nonmodified Rho. The assay conditions for native gels are as follows. Nondenaturing gel electrophoresis of Rho is performed with modification according to Safer31 with the buffer containing 25 mM Tris-base, 194 mM glycine, 0.1% Triton X-100, 100/xM GDP, and 7.5% (w/v) acrylamide (acrylamide/bisacrylamide, 37.5/liter) in slab gels. 3o U. K. Laemmli, Nature 227, 680 (1970). 31 D. Safer, A n a l Biochem. 178, 32 (1989).
190
CELL EXPRESSION 10 20 40 130
10 20 40 130
[2 1 ] p.g protein
66 36 24
14
SDS gel
autoradiogram
FIG. 1. ADP-ribosylation of Rho protein in cell lysate of NRK cells. Cell lysates of NRK cells (lane 1, 10/zg; lane 2, 20/zg; lane 3, 40/zg; and lane 4, 130/~g of protein) were incubated with C3 (0.15/~g) and 0.5/zM [32p]NAD in an incubation buffer as described in the text for 30 rain at 37°. Labeled proteins were analyzed by SDS-polyacrylamide gel electrophoresis and subsequent autoradiogram (Kodak X-Omat AR, 24 hr).
R u n n i n g buffer: 25 m M T r i s - b a s e plus 194 m M glycine L o a d i n g buffer: 10 m M T r i s - H C 1 , p H 8.0, 1 0 0 / z M MgC12, 1 0 0 / z M G D P , 0.5 m M D T F , a n d 50% (w/v) g l y c e r o l plus b r o m p h e n o l blue. T e n m i c r o l i t e r s o f t h e s a m p l e is m i x e d with 2 / z l l o a d i n g b u f f e r a n d is
start -
R h o ~."q
ADP-rib. Rho
FtG. 2. ADP-ribosylation of Rho protein in rat brain cytosol. Rat brain cytosol (6 mg/ml of protein) was ADP-ribosylated with C3 (0.3/zg/ml) and unlabeled NAD in the incubation buffer as described in the text for 15 min at 37°. Proteins (10/~g) were analyzed by nondenaturing gel electrophoresis as described in the text. After immunoblotting according to Towbin et aL (Proc. Natl. Acad. Sci. U.S.A. 76, 4350, 1979) the Rho protein was probed with an antiRho antibody (1:1000, Santa Cruz Biotechnology) using the ECL system according the manufacturer's protocol (Amersham).
[21]
ADP-RIBOSYLATIONOF Rho
191
centrifuged for 5 min at 14,000g. Maximally, 8/.d is loaded per slot. The gel is prerun for 1 hr at 140 V followed by separation run for 45 min at 140 V. For quantitative determination of the amount of [32p]ADP-ribose incorporated into Rho proteins, a filter assay is used. The ADP-ribosylation reaction is stopped by the addition of 400 tzl solution containing sodium dodecyl sulfate (2%, w/v) and bovine serum albumin (1 mg/ml), and the proteins are precipitated with 500/xl trichloroacetic acid (30%, w/v). After incubation for 30 rain on ice the proteins were collected on nitrocellulose filters [BA85, 0.45 /~m, Schleicher & Schuell, Dassel (Germany)]. The filters are washed with 20 ml of 6% trichloroacetic acid and are placed in scintillation fluid for counting of retained radioactivity. The filter blank, obtained in the absence of toxin, is usually 0.2-0.5% of added [32p]NAD and is subtracted from retained radioactivity. Influences of Temperature, Mg2+ Ions, and Guanine Nucleotides ADP-ribosylation of Rho proteins also occurs at 00.32 Although the rate of C3-catalyzed ADP-ribosylation is considerably decreased at 0°, phosphodiesterase activities, which degrade NAD, or proteolytic cleavage of Rho proteins may be reduced at this temperature, occasionally leading to increased labeling by C3. Rho proteins are very unstable in the absence of guanine nucleotides,l°'u For example, in the presence of EDTA at a concentration surmounting free magnesium ions, Rho is rapidly denatured and is no longer a substrate for ADP-ribosylation. Therefore, it is important to stabilize the GTP-binding protein during preparation and storation with free Mg2+ ions (2-10 raM). Monovalent cations (e.g., Na +) decrease ADP-ribosylation at concentrations >50 raM. ADP-ribosylation of Rho is apparently influenced by guanine nucleotides. Purified endogenous Rho, recombinant Rho proteins, and the membranous Rho protein are better substrates for ADP-ribosylation when bound to GDP rather than GTP; the addition of GDP (300/~M) to the assay mixture increases ADP-ribosylation, whereas GTP or GTP[S] (300/xM) decreases modification of Rho. 32 In contrast, ADP-ribosylation of cytosolic Rho proteins appears to be increased with GTP or GTP[S]. 18 These differences may be due to different amounts of complexation of Rho with regulating factors like GDI (guanine nucleotide dissociation inhibitor) found in the cytosolic fraction. In the GDI complex, Rho proteins are apparently poor substrates for ADP-ribosylation.33 Therefore, phospha32B. Habermann,C, Mohr,I. Just, and K. Aktories,Biochim. Biophys. Acta 1077,253 (1991). 33A. Kikuchi,S. Kuroda,T. Sasaki,K, Kotani,K. Hirata, M. Katayama,and Y. Takai,J. Biol. Chem. 267, 14611 (1992).
192
CELLEXPRESSION
[2 11
tidylinositides or sodium dodecyl sulfate (see also below) or GTP[S] that dissociate the G D I - R h o complex increase C3-catalyzed ADP-ribosylation of Rho in the cytosolic fraction. 34'35 Influence of Lipids and Detergents C3-catalyzed ADP-ribosylation is influenced by various lipids and detergents. Sodium cholate (0.2%), deoxycholate, dimyristoylphosphatidylcholine (3 mM), and SDS (0.01%) increase C3-catalyzed ADP-ribosylation. In contrast, CHAPS, Lubrol-PX, and SDS (>0.03%) impair ADPribosylation. 18'35'36 The stimulatory effect of SDS depends on the type of Rho protein. Whereas the ADP-ribosylation of human platelet cytosolic and recombinant RhoA is increased with the detergent, membrane Rho, recombinant RhoB, and Rho from bovine brain cytosol are almost not affected by low concentrations of SDS.18 ADP-ribosylation of recombinant RhoA is increased four- to fivefold at 0.01% SDS. Most likely, the effect of the detergent occurs on the Rho protein or on the ternary complex. In line with this, the detergent has no stimulatory effect on the NAD glycohydrolase activity of C3 but decreases the Km value for the ADPribosylation of recombinant Rho from about 10 to 0.5/zM. is Also, amphiphilic agents like mastoparan, mellitin, and compound 48/80, which affect heterotrimeric G proteins, influence ADP-ribosylation of Rho by C3-1ike exoenzymes. These agents inhibit ADP-ribosylation, an effect that is accompanied by an increase in the steady-state GTPase activity of Rho. 37 De(ADP-Ribosylation) for Testing Acceptor Amino Acid of C3-1ike Transferases In intact cells and under in vitro assay conditions, ADP-ribosylation of Rho proteins is practically irreversible. However, similar to other bacterial mono(ADP-ribosyl)transferase reactions, ADP-ribosylation by C3-1ike transferases is reversed in the absence of NAD and at high concentrations of nicotinamide. 32 Under these conditions, C3 releases ADP-ribose that is previously incorporated into Rho and forms NAD. The reverse reaction 34 N. Bourmeyster, M.-J. Stasia, J. Garin, J. Gagnon, P. Boquet, and P. V. Vignais, Biochemistry 31, 12863 (1992). 35 I. Just, C. Mohr, B. Habermann, G. Koch, and K. Aktories, Biochem. Pharmacol. 45, 1409 (1993). 36 T. Maehama, K. Takahashi, Y. Ohoka, T. Ohtsuka, M. Ui, and T. Katada, J. BioL Chem. 266, 10062 (1991). 37 G. Koch, B. Habermann, C. Mohr, 1. Just, and K. Aktories, Eur. J. Pharmacol. MoL Pharmacol. 226, 87 (1992).
[2 11
ADP-RIBOSYLATION OF Rho
193
[de(ADP-ribosylation)] can be used to test whether other C3-1ike transferases modify Rho at the identical acceptor amino acid (Asp-41) as C3.18 For this purpose, membranous Rho is [32p]ADP-ribosylated by the C3-1ike transferase (e.g., C. l i m o s u m exoenzyme, B. c e r e u s exoenzyme). Thereafter, the m e m b r a n e s are washed and the release of the previously incorporated radioactive label is induced by the C3 toxin in the presence of high concentrations of nicotinamide (30 mM). In contrast to the ADP-ribosylation reaction which shows a p H o p t i m u m at 7.5, the rate of the de(ADP-ribosylation) is maximal at p H 5.5. 32
D e t e c t i o n of C 3 - C a t a l y z e d A D P - R i b o s y l a t i o n in I n t a c t Cells In intact cells, studies of the C3-catalyzed ADP-ribosylation are hamp e r e d by the fact that the exoenzyme contains no translocation c o m p o n e n t like other bacterial ADP-ribosyltransferases. 38 Therefore, high concentrations (10 to 100 /xg/ml) of the transferases and rather long incubation times (12-48 hr) are necessary. 39 To improve uptake, electroporation, 4°'41 permeabilization with detergent, 42 or osmotic shock 16 can be applied. A further approach is the usage of chimeric C3, i.e., fusion protein of the C3 exoenzyme with the transport c o m p o n e n t of diphtheria toxin. This chimeric C3 enters the cell via receptor-mediated endocytosis, allowing the application of low concentrations and short-term incubation of the toxin. 43 The successful introduction of C3 into ceils results in rounding up of cells (Fig. 3). Depending on the cell type and the m e t h o d used, less than 60 to 80% of the cells get round. In contrast, microinjection of C3 into cells causes complete rounding of all cells treated. In intact cells, ADP-ribosylation of R h o by C3 is m o r e difficult to detect because N A D is not m e m b r a n e permeable. One approach in identifying ADP-ribosylation in intact cells is the prelabeling of ceils with ortho[ 32 P]phosphate. 44 A n o t h e r possibility is the differential ADP-ribosylation of the GTP-binding protein in cell lysate. The rationale of the assay is that R h o proteins which are modified in intact cells are no longer substrate 38M. R. Popoff, D. Hauser, P. Boquet, M. W. Eklund, and D. M. Gill, Infect. Immun. 59, 3673 (1991). 39W. Wiegers, I. Just, H. Miiller, A. Hellwig, P. Traub, and K. Aktories, Eur. J. Cell BioL 54, 237 (1991). 40M.-J. Stasia, A. Jouan, N. Bourmeyster, P. Boquet, and P. V. Vignais, Biochem. Biophys. Res. Commun. 180, 615 (1991). 41G. Koch, J. Norgauer, and K. Aktories, Biochem. J. 299, 775 (1994). 42V. Adam-Vizi, S. ROsener, K. Aktories, and D. E. Knight, FEBS Letr 238, 277 (1988). 43po Aullo, M. Giry, S. Olsnes, M. R. Popoff, C. Kocks, and P. Boquet, EMBOJ. 12, 921 (1993). 44K. H. Reuner, P. Presek, C. B. Boschek, and K. Aktories, Eur. J. Cell BioL 43, 134 (1987).
194
CELL EXPRESSION 100-
[21 ] -100
//'e //
I
80
g
60-
60
E ~
40-
40 ~
80I
:'
g n,"
20
20-
n 121
<, D.
O'
0'.3
1'o
,
0
30
C3 (l.tg/ml) FIG. 3. C3-induced rounding up of rat hepatoma FAO cells and differential in vitro ADPribosylation of Rho protein. FAO cells were grown on tissue culture plates as described39 for 2 days with the indicated concentrations of C3. The number of rounded cells (given in percentage of total cells) was determined from randomly selected fields of the dish. Cells were washed several times with PBS and lysed as described in the text. Thereafter, ADPribosylation of proteins was performed with C3 (0.15 /xg/ml) in the presence of 0.1 /xM [32p]NAD (0.5/xCi) in an incubation buffer as described in the text. The labeled proteins were autoradiographed (Kodak X-Omat AR, 48 hr) and analyzed by densitometry using a GS 300 scanning densitometer. The relative incorporation of [32p]ADP-ribose is given as a percentage of untreated control cells. Data are from Wiegers et aL 39
for ADP-ribosylation in cell lysate. 13'39,4°'45 For example, cells (e.g., rat hepatoma FAO cells) are treated with C3 for 24 hr at a concentration of 30/zg/ml. Thereafter, cells are washed three to five times with phosphatebuffered saline to remove excess C3 (note that C3 binds rather tightly to membranes). After washing, the cells are harvested and lysed in ice-cold buffer containing 10 mM triethanolamine hydrochloride (pH 7.5), 2 mM MgC12, and 1 mM PMSF. Thereafter, the cell lysate is ADP-ribosylated in the presence of C3 and [32p]NAD under essentially the same assay conditions as described earlier. A decrease in [3Zp]ADP-ribosylation indicates modification of Rho in intact cells (Fig. 3). To exclude that ADPribosylation of Rho occurred during the lysis process, C3-treated cells are 45 K.-D. Hinsch, B. Habermann, I. Just, E. Hinsch, S. Pfisterer, W.-B. Schill, and K. Aktories, F E B S Lett. 334, 32 (1993).
[21 ]
ADP-RIBOSYLATIONov Rho
195
lysed with buffer containing 0.5/zM [32p]NAD (2/zCi). No labeling of Rho should be detected on the SDS-polyacrylamide gel. F u r t h e r Application of C3-Catalyzed in Vitro ADP-Ribosylation a n d Concluding Remarks C3-1ike ADP-ribosyltransferases are valuable tools because they modify Rho proteins with high selectivity and allow the detection of the GTPbinding proteins. The exoenzymes cause biological inactivation of Rho and are excellent pharmacological devices for functional studies of Rho proteins (see other chapters in this volume). Moreover, because C3-catalyzed ADPribosylation depends on the structural integrity of Rho, this modification is useful to test structural changes of Rho. This application was particularly successful in studies on the mode of action of the Clostridium difficile toxins A and B. 46 C. difficile toxins A and B, which are the causative agents of the antibiotic-induced diarrhea, act on the actin cytoskeleton. 47 However, the exact eukaryotic target of the toxins has not been elucidated. The finding that treatment of tissue culture cells (e.g., CHO, NIH 3T3 cells) with C. difficile toxins causes inhibition of subsequent in vitro ADPribosylation of Rho by C3 led to the hypothesis that Rho proteins are the pathophysiological targets of the toxins. 46 One pitfall in working with C3 has to be mentioned. Because Rhoassociated factors may affect ADP-ribosylation, it is difficult to extrapolate the total amount of Rho protein in cell lysates, cytosol, or membrane fractions from the amount of C3-catalyzed [32p]ADP-ribosylation. The usage of an anti-Rho antibody appears to be more reliable and appropriate. With this precaution in mind, C3 and other C3-1ike transferases are as useful for studying the low molecular mass Rho proteins as cholera and pertussis toxins have been for elucidating the role of heterotrimeric G proteins.
46I. Just, G. Fritz, K. Aktories, M. Giry, M. R. Popoff, P. Boquet, S. Hegenbarth, and C. Von Eichel-Streiber,J. Biol. Chem. 269, 10706 (1994). 47M. C. Shoshan, C. Fiorentini, and M. Thelestarn,J. Cell Biochem. 52, 107 (1993).
196
CELLEXPRESSION
[22]
[22] P r e p a r a t i o n o f N a t i v e a n d R e c o m b i n a n t Clostridium botulinum C3 ADP-Ribosyltransferase and Identification of Rho Proteins by ADP-Ribosylation By NARITO M O R I I a n d SHUH NARUMIYA Clostridium botulinum C3 ADP-ribosyltransferase (C. botulinum C3 exoenzyme) is the enzyme that catalyzes the transfer of an ADP-ribose moiety of NAD to the asparagine residue located at the 41st position from the amino terminus of the mammalian rho gene product (Rho proteins, Rho p21s). 1 The reaction catalyzed is depicted below: Rho p21 + NAD ~ ADP-ribose-Asn 41 Rho p21 + nicotinamide + H +. Mammalian rho genes consist of three members, rhoA, rhoB, and rhoC. 2-4 All of these gene products are ADP-ribosylation substrates for the C3 exoenzyme. 5-7 Although other members of the Rho subfamily of small GTPases such as Rac and CDC42Hs contain an asparagine at the analogous position, they are not subject to ADP-ribosylation unless they have been denatured with detergents. 8 Since A s n 41 is in the putative effector domain of Rho p21, it has been suggested that this ADP-ribosylation interferes with the signal transduction of Rho p21 in the cell. 1 Indeed, treatment of cells with the C3 exoenzyme induces the ADP-ribosylation of Rho p21 and inhibits Rho p21-mediated actin cytoskeletal organization and cell a d h e s i o n . 7'9-14 In these experiments, the ADP-ribosylated state of Rho p21 1 A. Sekine, M. Fujiwara, and S. Narumiya, J. Biol. Chem. 264, 8602 (1989). 2 p. Madaule and R. Axell, Cell 41, 31 (1985). 3 p. Yeramian, P. Chardin, P. Madaule, and A. Tavitian, Nucleic Acids Res. 15, 1869 (1987). 4 p. Chardin, P. Madaule, and A. Tavitian, Nucleic Acids Res. 16, 2717 (1988). 5 S. Narumiya, A. Sekine, and M. Fujiwara, J. Biol. Chem. 263, 17255 (1988). 6 M. Hoshijima, J. Kondo, A. Kikuchi, K. Yamarnoto, and Y. Takai, Mol. Brain Res. 7, 9 (1990). 7 p. Chardin, P. Boquet, P. Madaule, M. R. Popoff, E. J. Rubin, and D. M. Gill, E M B O J. 8, 1087 (1989). 8 I. Just, C. Mhr, G. Schallehn, L. Menard, J. R. Didsbury, J. Vandekerckhove, J. van Damme, and K. Aktories, J. Biol. Chem. 267, 10274 (1992). 9 T. Nishiki, S. Narumiya, N. Morii, M. Yamamoto, M. Fujiwara, Y. Kamata, G. Sakaguchi, and S. Kozaki, Biochem. Biophys. Res. Commun. 167, 265 (1990). 70H. F. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell Biol. 111, 1001 (1990). 11 A. J. Ridley and A. Hall, Cell 70, 389 (1992). 12 N. Morii, T. Teruuchi, T. Tominaga, N. Kumagai, S. Kozaki, F. Ushikubi, and S. Narumiya, J. Biol. Chem. 267, 20921 (1992).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
[22]
C3 TRANSFERASEAND Rho PROTEINS
197
is the dominant negative form because microinjected ADP-ribosylated Rho p21 interferes with actions of normal Rho p21.1°'15 Therefore, the C3 exoenzyme can be used to identify and quantify Rho p21 in various cells and tissues, and also to examine the role of Rho p21 in various cellular responses. This chapter describes the in vitro ADP-ribosylation reaction, the purification of the C3 exoenzyme, and application of the C3 exoenzyme in biological systems. ADP-Ribosylation Reaction 16,17 The ADP-ribosyltransferase activity of the C3 exoenzyme and the amount of Rho p21 in the cells are assayed by an in vitro ADP-ribosylation reaction using [a-32P]NAD.
Sample Preparation Cells and tissues are suspended in an appropriate volume of homogenization buffer consisting of 20 mM Tris-HC1, pH 7.5, 0.25 M sucrose, 5 mM MgC12, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, and 5 /xM leupeptin and are homogenized on ice by three sonication bursts of 5-sec duration each or by three strokes in a Potter-Elvehjem homogenizer. The homogenate is centrifuged at 700g for 10 min, and the supernatant (100-200/xg protein) is used for the ADP-ribosylation reaction.
ADP-Ribosylation Reaction Mixture The reaction mixture contains 100 mM Tris-HCl, pH 8.0, 10 mM thymidine, 10 mM nicotinamide, 10 mM DTT, 5 mM MgCI2, 50/xM [32p]NAD (900 cpm/pmol), purified C3 exoenzyme or crude enzyme preparation, and 13T. Tominaga, K. Sugie, M. Hirata, N. Morii, J. Fukata, A. Uchida, H. Imura, and S. Narumiya, J. Cell Biol. 120, 1529 (1993). 14 I. Mabuchi, Y. Hamaguchi, H. Fujimoto, N. Morii, M. Mishima, and S. Narumiya, Zygotes 1, 325 (1993). 15 T. Nishiyama, T. Sasaki, K. Takaishi, M. Kato, H. Yaku, K. Araki, Y. Matsumura, and Y. Takai, Mol. Cell Biol. 14, 2447 (1994). 16N. Morii, A. Sekine, Y. Ohashi, K. Nakao, H. Imura, M. Fujiwara, and S. Narumiya, J. Biol. Chem. 263, 12420 (1988). ~7N. Morii, Y. Ohashi, Y. Nemoto, M. Fujiwara, Y. Ohnishi, T. Nishiki, Y. Kamata, S. Kozaki, S. Narumiya, and G. Sakaguchi, J. Biochem. (Tokyo) 107, 769 (1990). 18 y. Nemoto, T. Namba, S. Kozaki, and S. Narumiya, J. Biol. Chem. 266, 19312 (1991). 19 N. Kumagai, N. Morii, K. Fujisawa, Y. Nemoto, and S. Narumiya, J. BioL Chem. 268, 24535 (1993).
198
CELLEXVRESSION
[221
recombinant RhoA p21 or crude homogenate in a total volume of 100 t~l. The reaction is performed at 30° and is terminated by the addition of 400 ~1 of 0.02% sodium deoxycholate and 200/A of 24% trichloroacetic acid. The mixture is chilled on ice for 20 min and is then centrifuged at 10,000g for 15 rain at 4 °. The pellet is dissolved in Laemmli sample buffer and is applied to a 12.5% SDS-polyacrylamide gel for electrophoresis. After staining with Coomassie brilliant blue R-250, the gel is dried and subjected to autoradiography. The radioactive bands at Mr 21-23K are excised and the radioactivity is determined by liquid scintillation counting. As shown in Fig. 1, ADPribosylated Rho p21 shows a slower migration rate on SDS-polyacrylamide gel electrophoresis than the unmodified form. For two-dimensional polyacrylamide gel electrophoresis analysis, aliquots of the reaction mixture are taken before the addition of sodium deoxycholate and trichloroacetic acid, and are subjected to nonequilibrium pH gradient gel electrophoresis) 4 Comments on Enzyme Assay For the measurement of enzyme activity, we use either 140 ~g protein of bovine adrenal cytosol or 5 pmol of recombinant RhoA p21 as a substrate. Under these conditions, a linear relationship is observed between the amount of C3 exoenzyme and [3zP]ADP-ribosylation, up to 2.5 ng of the purified enzyme after 1 hr incubation (Fig. 2). The reaction reaches a plateau after 1 hr with more than 5 ng of the enzyme, indicating that most of the substrate is ADP-ribosylated under these conditions. To determine the amount of Rho p21, 50 ng of purified C3 exoenzyme and 100/xg of
2
3
67 ~n~ 43 30
=(=(ADP- ri bosyl ated rho p21 unmodified rho pZ1
20
14
iiiii!!i~ii:!!
FIG. 1. ADP-ribosylation-induced decrease in the mobility of Rho p21 on SDS-polyacrylamide gel electrophoresis. Native (lane 1), ADP-ribosylated (lane 2), and [32p]ADPribosylated Rho p21 (lane 3) (1 pmol of each protein) were electrophoresed and transferred to a PVDF membrane. Lanes 1 and 2 show the results of Western blotting with an anti-Rho p21 antibody, and lane 3 shows the results of [32p]ADP-ribosylation autoradiography.
[22]
C3 TRANSFERASEAND Rho PROTEINS I
I
199
I
4
g
r~ 1
I
I
I
2.5
5
7.5
Amount of C3 exoenzyme (ng)
FIG. 2. Dose-dependent ADP-ribosylation as a function of the amount of C3 exoenzyme. Bovine adrenal cytosol (140/zg) was incubated for the ADP-ribosylation reaction with the indicated amounts of C3 exoenzyme for 1 hr at 30°. Incorporation of the radioactivity was determined as described in the text.
cell h o m o g e n a t e protein are used and the reaction is allowed to proceed for 2 hr as per the standard assay. The amount of the ADP-ribosylation substrate varies with the type of tissues and cells. In rats, it is most abundant in the brain, which contains 25 to 40 p m o l per mg of h o m o g e n a t e protein; in contrast, it is barely detectable in skeletal muscle. 16 Therefore, it is necessary to increase the specific radioactivity of [32p]NAD in order to study samples containing a low level of substrate. For optimal ADP-ribosylation, it is essential to keep R h o p21 in the intact guanine nucleotide-binding conformation. The presence of free magnesium ion is required for the reaction, and decreasing the concentration of Mg 2+ to submicromolar levels greatly attenuates the reaction. The addition of guanine nucleotides such as G D P and GTPTS is also helpful, especially in conditions such as the a m m o n i u m sulfate fractionation during which R h o p21 tends to release the bound nucleotide. 2° Detergents such as Triton and T w e e n inhibit this reaction. When the use of such detergents is unavoidable for sample preparation, they should be removed before the reaction is begun by the use of, for example, Extractigel (Pierce). In addition, some other cellular components have been reported to affect 20S. Narumiya, N. Morii, K. Ohno, Y. Ohashi, and M. Fujiwara, Biochem. Biophys. Res. Commun. 150, 1122 (1988),
200
CELLEXPRESSION
[22]
the ADP-ribosylation r e a c t i o n . 21-23 For these reasons, a preliminary study is recommended to determine the optimal incubation time, the quantity of sample, and the amount of enzyme used in the assay. The three substrate proteins, RhoA, RhoB, and RhoC p21s, have almost the same molecular weights; thus, they cannot be separated by SDSpolyacrylamide gel electrophoresis. Although their isoelectric points, calculated from their deduced amino acid sequences, are different (5.76, 4.69, and 6.20 for RhoA, RhoB, and RhoC p21s, respectively), the identification of each protein by the ADP-ribosylation reaction is difficult. This is because of isoprenylation and carboxyl methylation of these proteins and charge changes by the ADP-ribosylation. The comigration of a sample with a standard protein on two-dimensional gel electrophoresis helps with such identification. The purification of native RhoA and RhoB p21s has been reported previously. 6'16 In addition, RhoA p21 has been identified as the major, if not only, ADP-ribosylation substrate in human platelets. 24Accordingly, the ADP-ribosylation product of the human platelet cytosol is a convenient substitute for ADP-ribosylated, purified RhoA p21) 4 Purification of the C3 Exoenzyme
Purification of the C3 Exoenzyme from Culture Filtrate of C. botulinum 17 C. botulinum, type C, strain 003-9 is cultured in a medium composed of 5% trypticase peptone (Becton-Dickinson), 0.5% Bacto-peptone (Difco), 1% yeast extract (Oriental Yeast, Japan), 1% glucose, 1% ammonium sulfate, 0.5% CaCO3, and 0.1% L-cysteine hydrochloride (pH 7.6) at 37 ° for 2 days in an anaerobic jar. The whole culture is then filtered through gauze, and the culture supernatant is obtained by centrifugation at 3800g for 15 min. Ammonium sulfate is added to 1 liter of the culture supernatant to 60% saturation, and the mixture is stirred for 30 rain at 4 ° and then centrifuged at 10,000g for 30 min. The precipitate is dissolved in 25 ml of 50 mM TrisHC1, pH 7.5, and dialyzed against the same buffer. The dialyzate is applied to a CM-Sephadex C-50 column (0.9 cm, i.d., x 10 era) equilibrated with this buffer, and the enzyme is eluted by a linear gradient between 0 and 0.3 M NaCI, in a total volume of 200 ml. The enzyme fraction is concentrated 21 T. Ohtsuka, K.-I. Nagata, T. Liri, Y. Nozawa, K. Ueno, M. Ui, and T. Katada, J. BioL Chem. 264, 15000 (1989). 22 N. Bourmeyster, M. J. Stasia, J. Garin, J. Gagnon, P. Boquet, and P. V. Vignais, Biochemistry 31, 12863 (1992). 23 G. Fritz and K. Aktries, Biochem. J. 300, 133 (1994). 24 y. Nemoto, T. Namba, T. Teruuchi, F. Ushikubi, N. Morii, and S. Narumiya, J. Biol. Chem. 267, 20916 (1992).
[22]
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201
by the use of a YM10 membrane (Amicon, Danvers, MA) and applied to a Sephadex G-75 column (1.6 era, i.d., x 94 cm) equilibrated with 50 mM Tris-HC1, pH 7.5, containing 0.2 M NaC1. The enzyme is eluted with the same buffer, and the fractions containing activity are pooled (the purified enzyme). About 9 mg of purified C3 exoenzyme can be obtained by this procedure.
Purification of Recombinant C3 Exoenzyme from Escherichia coli The C3 exoenzyme gene isolated from C. botulinum, type C, 003-9 TM was modified by PCR (polymerase chain reaction)-mediated site-directed mutagenesis. This mutant lacks the signal peptide sequence and instead codes for Met-Ala to Ser a of the mature form. The modified gene was then cloned into a pET-3a vector (pET-3a/C3). 19 E. coli BL21(DE3)pLysE is transformed with pET-3a/C3 and is cultured in 1 liter of M9 medium containing 1 mM MgSO4,0.1 mM CaC12,0.5% casamino acids, 0.4% glucose, 1/100 vol of LB medium, 100 mg/liter ampicillin, and 25 mg/ml chloramphenicol. When the absorbance of the cell culture at 600 nm reaches 0.5, 1 ml of 1 M isopropylthiogalactoside (IPTG) is added and the culture continues for another 3 hr. The cells are harvested by centrifugation at 3000g for 5 min. After one cycle of freeze-thawing, the cell pellets are sonicated in 50 ml of 50 mM Tris-HC1, pH 7.5, 2 mM EDTA, and 1 mM PMSF. The homogenate is incubated with 3 mg lysozyme for 20 min at 37°. DNase I (Sigma) and MgCI2 are then added to a final concentration of 10/xg/ml and 5 mM, respectively, and the lysate is centrifuged at 10,000g for 30 min. The supernatant is diluted with an equal volume of 20 mM HEPES-NaOH, pH 7.5, and applied to a CM-Sepharose column (2.5 cm, i.d., × 10 cm) equilibrated with the same buffer. Elution is performed by a linear gradient between 0 and 0.5 M NaC1 in the same buffer, and the enzyme typically elutes at 0.15 to 0.2 M NaC1. The enzyme fraction is concentrated by the use of Centriprep 10 (Amicon) and is applied to gel filtration on a TSK-gel 3000SW column (Toso, Japan). Elution is performed with 20 mM H E P E S - N a O H containing 150 mM NaCI. This procedure yields about 3 mg of the purified enzyme.
Properties of Enzymes The botulinum C3 exoenzyme consists of 244 amino acids with a calculated molecular weight of 27,362. It is a cationic protein with a calculated pI of 9.79.18 The first 40 amino acids serve as a signal peptide and are cleaved to yield an active enzyme of 23 kDa. The recombinant C3 enzyme contains no signal peptide, but instead has the dipeptide Met-Ala attached to the first Ser of the mature form. There is no difference in enzymatic
202
CELLEXPRESSION
[221
activity between the native and the recombinant enzymes. Both purified enzymes have a Kr, of 0.125 tzM for NAD and a catalytic activity of 1.3 pmol of ADP-ribose transferred/ng/hr under the conditions specified earlier. The activity is stable for at least 1 month at 4°. Applications in Biological Systems The C3 exoenzyme has been used in many biological systems to ADPribosylate Rho p21 and to inhibit Rho p21-mediated cell functions. These studies have revealed that Rho p21 is involved in the regulation of cell adhesion, actin cytoskeleton organization, cell motility, smooth muscle contraction, cytokinesis during cell division, and cell proliferation and differentiation (Table I). The C3 exoenzyme can be introduced into the cells by simple incubation with the cells in culture medium, by permeabilization, or by microinjection. Since the enzyme is not an A - B toxin and has no receptor on the cell surface, its incorporation appears to be mediated by nonspecific endocytosisY This accounts for the requirement of micrograms per milliliter concentrations of the enzyme and for the long incubation period (usually over 24 hr) required to elicit its effects as compared to the nanograms per milliliter concentrations required for cholera and pertussis toxins. It was also found that the sensitivity to C3 exoenzyme treatment differs among cell lines (Table I). Thus, both sensitivity and incubation time should be tested in a preliminary study. Incubation of cells with 1, 3, 10, 30, and 100 tzg/ml of the C3 exoenzyme for 24 to 48 hr is recommended. Once the Rho p21 in the cells has been ADP-ribosylated in situ with endogenous NAD, it can no longer serve as a substrate. Thus, the amount of ADP-ribosylated Rho p21 can be estimated by the reduction in substrate activity in cell homogenates subjected to the in vitro reaction with [32p]NAD. The C3 exoenzyme is a highly cationic protein and tends to stick to the cell surface; therefore, ADP-ribosylation of Rho p21 can occur during the preparation of homogenates from C3 exoenzyme-treated cells, which leads to misinterpretation of the effects of C3 exoenzyme. To avoid this, the cells are washed extensively with ice-cold phosphate-buffered saline to remove traces of the C3 exoenzyme from the sample. It is also recommended that the homogenization be performed in a reaction mixture containing [32p]NAD and that the homogenate be subjected to incubation immediately. Two examples of application of the C3 exoenzyme in cell culture are described below. 25 E. J. Rubin, M. D. Gill, P. Boquet, and M. R. Popoff, Mol. Cell. Biol. 8, 418 (1988).
[221
C3 TRANSFERASE AND R h o PROTEINS
203
TABLE I EFFECTS OF THE C3 EXOENZYMEIN VARIOUSCELL LINES
Cell type
Amount of C3 exoenzyme (/~g/ml)
Added directly to culture medium Swiss 3T3 1-30 5 NIH 3T3 Vero LLC-MK2 HeLa FL PC-12
GOTO N1E-115 NG108-15
15" 5 3.6 7 >100 >100 45 1-100 15 1 30 30
JY lymphoblastoid 3-30 cell Platelets 6.25-50 Electric or chemical permeabilization HL-60 0.01-9.6 Lymphocyte 0.4-4 Mesenteric arterial smooth muscle Microinjection Swiss 3T3 b
Xenopus egg b Sea urchin eggc
0.25
3-30 80-160 40 2.6 0.1-20
Effects
Refs.
Cell rounding, growth inhibition Inhibition of agonist-evoked tyrosine phosphorylation Cell rounding Disappearance of actin fiber Cell rounding Cell rounding Not observed Not observed Neurite-like process formation Neurite-like process formation Generation of short neurites Neurite-like process formation Inhibition of agonist-induced neurite retraction Inhibition of agonist-induced neurite retraction Inhibition of LFA-l-dependent cell adhesion Inhibition of aggregation
26 19
Inhibition of cell motility Inhibition of lymphocyte-mediated cytotoxity Inhibition of GTP-dependent Ca sensitization
29 30
Inhibition of stress fiber formation Inhibition of focal adhesion formation Inhibition of cell motility Inhibition of cell division Inhibition of contractile ring formation
10 11 32 33 14
a The C3 exoenzyme was delivered into the cells by an osmotic shock. b The concentration of the C3 exoenzyme in the injection solution is shown. c The calculated intracellular concentration is shown.
25 7 27 27 27 27 27 9 25 27 28 28 13 12
31
204
CELLEXPRESSION
[221
B
A kD 97.4 - - ~ 66.2 " - ~ 45.0--,"
31.0 - ' ~
21.5 14.4
C
I
1
kD 45
2 3 4
5
• olOOq
o
D O
~
c
.9
50
0 0-
u
-//
I
i
i
r
1
3
10
30
Ca Exoenzyme Concentration (pg/ml)
FIG. 3. Effects of C3 exoenzyme treatment on cell morphology and proliferation of Swiss 3T3 cells. Cells were incubated with 10/zg/ml (A and B) or with the indicated amounts (C) of the C3 exoenzyme for 3 days. (A) Autoradiogram of the ADP-ribosylation reaction of homogenates from the control (left lane) and C3-treated cells (right lane). (B) Morphology of the control (top) and C3-treated (bottom) cells. (C) Concentration-dependent inhibition of cell growth by the C3 exoenzyme. After incubation for 3 days with the indicated amounts of C3 exoenzyme, the number of cells was counted by the trypan blue dye exclusion method. No difference in cell viability existed between the control and C3-treated cells. Cell proliferation is expressed as a percentage of control cells cultured without the enzyme. The inset shows an autoradiogram of the ADP-ribosylation reaction of the cell homogenates. Lanes 1-5 are cells treated with 0, 1, 3, 10, and 30/zg/ml of enzyme, respectively. Reproduced from Yamamoto eta/. 26 by copyright permission of the publisher.
[22]
C3 TRANSFERASEAND R h o PROTEINS
205
A C3 (0g/ml)
0 1 10 3 0 1 0 0
31KDa'-P-
21KDa'-~
B
', 2~)
FIG. 4. Induction of neurite-like processes by the C3 exoenzyme in PC-12 cells. PC-12 cells were cultured with 0 to 100/xg/ml of C3 exoenzyme for 4 days. (A) Autoradiogram of the ADP-ribosylation reaction of homogenates from control cells and cells treated with the indicated amounts of C3 exoenzyme. (B) Morphology of control cell (left-hand side) and cell treated with 100/xg/ml of C3 exoenzyme (right-hand side).
Treatment o f Swiss 3T3 Cells with the C3 Exoenzyme 26 Swiss 3T3 cells were seeded at a density of 105/well in 6-well culture plates and were cultured in D u l b e c c o ' s modified E a g l e ' s m e d i u m ( D M E M ) s u p p l e m e n t e d with 10% fetal calf s e r u m for 24 hr. T h e cells were then t r e a t e d with 10 ~ g / m l of the C3 e x o e n z y m e a d d e d to the culture m e d i u m for 3 days. Figure 3 shows the [32p]ADP-ribosylation reaction of lysates f r o m control and C3 e x o e n z y m e - t r e a t e d cells (A) and their respective m o r p h o l o g i e s (B). A s shown, radiolabeling of R h o p21 in lysates f r o m the C3 e x o e n z y m e - t r e a t e d cells is significantly suppressed. T h e treated cells show m a r k e d cell rounding, with b e a d e d dendritic processes typical of the 26M. Yamamoto, N. Marui, T. Sakai, N. Morii, S. Kozaki, K. Ikai, S. lmamura, and S. Narumiya, Oncogene 8, 1449 (1993). 27y. Kamata, T. Nishiki, K. Matsumur, T. Hiroi, and S. Kozaki, Microbiol. ImmunoL 38, 421 (1994).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, inc. All rights of reproduction in any form reserved.
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morphological changes induced by Rho p21 inactivation in fibroblast cells. As shown in Fig. 3C, treatment of the cells with different concentrations of the C3 exoenzyme resulted in the dose-dependent ADP-ribosylation of Rho p21 in the cells: [32p]ADP-ribosylation in cells treated with 1, 3, 10, and 30/xg/ml was 44, 25, 13, and 5% of that in control cells, respectively. Because of this ADP-ribosylation, the growth of cells was also suppressed to 95, 71, 65, and 16% of controls, respectively. Thus, the biological effects of C3 exoenzyme do not appear to be linearly correlated with the extent of in situ ADP-ribosylation. Instead, it is proportional to the logarithm of the remaining amount of intact Rho p21, which is suggestive of a simple mass action relationship between the amount of Rho p21 and its action. Treatment o f PC-12 Rat Pheochromocytoma Cells with the C3 Exoenzyme 9
PC-12 cells were plated at a density of 105 per dish on poly(L-lysine)coated 35-ram plastic dishes and were grown in DMEM containing 10% horse serum and 5% fetal calf serum. After 2 days, the cells were washed and recultured in fresh medium containing the C3 exoenzyme for 4 days. [32p]ADP-ribosylation of lysates from control cells and cells treated with 1 to 100/xg/ml C3 exoenzyme and their respective morphologies are shown in Fig. 4. This cell line was relatively resistant to the C3 exoenzyme treatment, and a reduction in [32p]ADP-ribosylation can only be detected by treatment with more than 10 tzg/ml of the enzyme. Contrary to the morphology observed in Swiss 3T3 cells, enzyme treatment induced a fiat cytoplasm shape and the sprouting of neurite-like processes in PC-12 cells. This is consistent with the report by Jalink et al. 28 that Rho p21 plays a role in neurite retraction in neuroblastoma-derived cells.
28 K. Jalink, E. J. van Corven, T. Hengeveld, N. Morii, S. Narumiya, and W. H. Moolenaar, J. Cell Biol. 126, 801 (1994). 29 M.-J. Stasia, A. Jouan, N. Bourmeyster, P. Boquet, and P. V. Vignais, Biochem. Biophys. Res. Commun. 180, 615 (1991). 30 p. Lang, L. Guizani, I. Vitte-Mony, R. Stancou, O. Doreuil, G. Gacon, and J. Bertoglio, J. Biol. Chem. 267, 11677 (1992). 31 K. Hirata, A. Kikuchi, T. Sakai, S. Kuroba, K. Kaibuchi, Y. Matuura, H. Seki, K. Saida, and Y. Takai, J. Biol. Chem. 267, 8719 (1992). 32 K. Takaishi, A. Kikuchi, S. Kuroba, K. Kotani, T. Sasaki, and Y. Takai, Mol. Cell. Biol. 13, 72 (1993). 33 K. Kishi, T. Sasaki, S. Kuroba, T. Itoh, and Y. Takai, J. Cell Biol. 120, 1187 (1993).
[23]
I n V i t r o BINDINGASSAY
207
[23] In Vitro Binding Assay for Interactions of Rho and Rac with GTPase-Activating Proteins and Effectors By DAGMAR DIEKMANN and ALAN HALL Introduction M e m b e r s of the Rho subfamily of proteins are approximately 30% identical to Ras in amino acid sequence and, like Ras, they regulate a variety of signal transduction pathways in m a m m a l i a n cells. 1 In fibroblasts, for example, R h o controls the assembly of focal adhesions and the reorganization of actin into stress fibers in response to extracellular ligands such as lysophosphatidic acid and bombesin. 2 Rac, on the other hand, regulates the polymerization of actin at the plasma m e m b r a n e to form ruffles and lamellipodia in response to growth factors such as platelet-derived growth factor, epidermal growth factor, and insulin. 3 An additional function for Rac has been described in phagocytic cells, where it activates the N A D P H oxidase. 4-6 In order to have a clearer understanding of the way in which Rho-like proteins function, it is crucial to identify their downstream effector molecules. We describe here a simple and quick assay to test the ability of candidate target proteins to interact with small GTP-binding proteins of the R h o family. The in v i t r o assay uses glutathione S-transferase (GST) fusion proteins coupled to Sepharose beads to capture recombinant Rhorelated G T P a s e s that have b e e n loaded with radiolabeled G D P or GTP. This chapter describes the basic principle of the m e t h o d using the GTPaseactivating protein R h o - G A P as a model system. This protein has been shown previously to interact with and stimulate the G T P a s e activity of Rho, Rac, and CDC42. 7 The influence of p a r a m e t e r s such as protein concentrations, incubation times and temperature, and ionic strength are assessed. In addition, we describe the use of the assay to identify the target protein for Rac in the N A D P H oxidase complex. 1A. Hall, Science 249, 635 (1990). : A. J. Ridley and A. Hall, Cell 70, 389 (1992). 3 A. J. Ridley, H. F. Paterson, C. L. Johnston, D. Diekmann, and A. Hall, Cell 70, 401 (1992). 4 A. Abo, E. Pick, A. Hall, H. Totty, C. G. Teahan, and A. W. Segal, Nature 353, 668 (1991). 5 U. G. Knaus, P. G. Heyworth, T. Evans, J. T. Curnutte, and G. M. Bokoch, Science 254, 1512 (1991). 6 T. Mizuno, K. Kaibuchi, S. Ando, T. Musha, K. Hiraoka, K. Takaishi, M. Asada, H. Nunoi, I. Matsuda, and Y. Takai, J. Biol. Chem. 267, 10215 (1992). 7 C. A. Lancaster, P. M. Taylor-Harris, A. J. Self, S. Brill, H. E. van Erp, and A. Hall, J. Biol. Chem. 269, 1137 (1994).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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CZLLZXPRZSSION
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Materials [3H]GTP (10 Ci/mmol, code TRK 314) and [3H]GDP (10 Ci/mmol, code TRK 335) from Amersham [a-32P]GTP (3000 El/retool, code NEG-006H) and [y-32p]GTP (6000 Ci/mmol, code NEG-004Z) from NEN DuPont Glutathione-Sepharose beads (Pharmacia, code 17-0756-01) Reduced glutathione (Sigma, G-4251) 1 M dithiothreitol (DTT) 10 mM GTP Membrane filters (Schleicher & Schuell, NC 45) Nucleotide loading buffer: 0.5 mg/ml bovine serum albumin (BSA), 50 mM Tris, pH 7.5, 5 mM EDTA Wash buffer: 50 mM Tris, pH 7.5, 100 mM NaC1, 5 mM MgCle, 0.1% Triton X-100 Binding Assay
Purification of Proteins Rho and Rac, cloned into the pGEX-2T vector (Pharmacia), are expressed as GST fusion proteins in Escherichia coli. After purification on glutathione-Sepharose beads the native GTP-binding protein is released from GST by thrombin cleavage as described elsewhere in this volume (see [1]). Protein concentrations are determined using [3H]GTP in a nitrocellulose filter binding assay (see [1]). The target protein of interest, also expressed in E. coli as a GST fusion protein, is eluted from the glutathione-Sepharose beads with reduced glutathione (5 mM). The protein is dialyzed against 10 mM Tris, pH 7.5, 2 mM MgC12, 10 mM NaCI, and 0.1 mM DT-I" and is concentrated to at least 0.5 mg/ml. The final concentration and purity of the protein are checked after electrophoresis on a SDS-polyacrylamide gel. We find that most proteins can then be snap frozen and stored in liquid nitrogen without deterioration. For the experiments described here we have used a pGEX2T vector expressing the catalytic domain of Rho-GAP, p29 Rho-GAP. 7
Binding Assay Recombinant Rho or Rac (-0.4 t~g) is loaded with radioactive guanine nucleotide in a 200-~1 reaction volume (50 mM Tris, pH 7.5, 5 mM EDTA, 0.5 mg/ml BSA) containing 40 ~Ci of either [ o t - 3 2 p ] - o r [T-32p]GTP (3000 or 6000 Ci/mmol, respectively) for 10 min at 30°. The nucleotide exchange
[231
In Vitro BINDINGASSAY
209
reaction is stopped on ice after the addition of MgCI2 (to 10 mM). DTT (to 0.1 mM) and GTP (to 10 ~M) are added to reduce background signals. A 50-/xl sample is filtered through nitrocellulose to d~termine input counts, while additional 50-txl aliquots are incubated with (i) no addition, (ii) GST (10 tzg), or (iii) G S T - R h o - G A P (10 tzg) for 5 min on ice. GlutathioneSepharose beads (15 t~l) (1 : 1 suspension) are added and the samples are swirled for 30 min at 4 °. The beads are then washed three times with 1 ml cold wash buffer, resuspended in 400 /xl wash buffer, and collected by filtration on nitrocellulose filters. Radioactivity bound to the filters is determined by scintillation counting.
C h a r a c t e r i z a t i o n of Binding Assay Using Rho-GAP a. Use o f Activated Mutants of Rho/Rac
To test for an interaction between Rho or Rac and R h o - G A P we have made use of mutant proteins, containing a glutamine to leucine substitution at amino acid 63 in Rho (L63Rho) or at the equivalent position, amino acid 61, in Rac (L61Rac). This particular mutation was originally described in Ras (L61Ras), where it inhibits GAP-stimulated GTPase activity and behaves as an oncogenic mutation. In addition, however, this leucine substitution increases the affinity of Ras for R a s - G A P and NF1 by 10 to 50fold.* Using competition assays we have subsequently found that L63Rho binds to R h o - G A P with an affinity constant of approximately 10 nM, about 100 times tighter than wild-type Rho. 9 In addition L63Rho and L61Rac are insensitive to G A P activity and therefore, unlike wild-type Rho and Rac, will remain in the GTP-bound state during the course of the binding assay. We have also tested other activated versions of Rho and Rac. V14Rho, for example, behaves like the L61Rho mutant and is resistant to R h o GAP-stimulated GTP hydrolysis. However, we were not able to detect significant binding of V14Rho to G S T - R h o - G A P using this binding assay. We have previously estimated the binding affinity of V14Rho to R h o G A P in the low micromolar range and this appears to be insufficient to detect binding using this assay. A similar result was obtained using V12Rac. We conclude that the L61/L63 substitutions are a useful way to detect interactions between Rho-like proteins and their GAPs. Whether they also increase the affinity of Rho-like GTPases for effector proteins is not yet clear. 8 G. Bollag and F. McCormick,Nature 351, 576 (1991). 9 A. J. Self and A. Hall, ([8] this volume).
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CELLEXPRESSION
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b. R h o / R a c GTP-Binding Protein Concentration
Figure 1 shows the effect of increasing the concentration of L63Rho in the binding assay using a fixed amount of G S T - R h o - G A P (2/zg). It can be seen that the signal increases linearly, whereas the background is little affected. We routinely use 100 ng of GTP-binding protein per binding assay to test for an interaction with a candidate effector protein. c. G S T - R h o - G A P
Concentration
The effect of increasing the amount of G S T - R h o - G A P using a fixed amount of L63Rho (100 ng) is shown in Fig. 2. Saturation is reached at about 20/xg of protein under the conditions of the assay, but this is likely to depend on the affinity of the fusion protein used. d. Incubation Times
[y-32p]GTP-preloaded L63Rho was incubated with GST or G S T - R h o GAP for 5, 30, or 60 min on ice and then swirled with glutathione-Sepharose beads at 4° for half an hour. There was no difference in binding to GSTRho-GAP, although the background binding to GST seemed to increase with a longer incubation period. We now routinely incubate the two proteins for 5 min before adding beads. The mixture is then swirled for a further 30 min. 40
30 ¸
20-
/
/
/
/
/
/
/
/
j0~..~ I " 1 0
10
I
i
I
I
FIG. 1. Effect of increasing amounts of L63Rho with a fixed amount of ([[]) GST (2/zg) or (O) G S T - R h o - G A P (2/~g). Results are given in counts per minute. Input counts were on average 28,000 cpm/ng of L63Rho.
In Vitro BINDINGASSAY
[23]
211
40
30/
8
!
t
20-
10-
i
/
0
GST fusion protein (~g) FIG. 2. Effect of increasing amounts of ([~) GST or (O) G S T - R h o - G A P using a fixed amount of L63 Rho (100 ng). Results are given in percentage of input counts.
e. Incubation Temperature Somewhat surprisingly, the interaction between L63Rho or L61Rac and G S T - R h o - G A P seemed to be little affected by incubation temperature. Essentially the same results were obtained when incubation and washing were performed at room temperature and, in some cases, the binding even seemed better. We recommend that new proteins be tested under different conditions since the effect of changing the temperature on the relative on and off rates cannot be predicted.
f. Number of Bead Washes The number of bead washes after binding is critical since the interaction between the proteins is relatively weak. Washing has to be performed carefully to ensure reproducibility of the results: 1 ml of wash buffer is added, the beads are immediately collected by brief centrifugation in a microfuge, and the supernatant is removed with care. Three washes are sufficient to optimize the difference between the signal and background. By five washes most of the signal is lost (see Fig. 3).
g. Ionic Strength Effect In vitro GAP assays have shown that the stimulation of GTP hydrolysis of wild-type Rho by R h o - G A P is sensitive to the ionic strength and is inhibited by about 50% at high salt (150 mM) compared to low salt
212
CELL EXPRESSION
[23]
60 508
40-
\
302010I
I
1
2
I
3
zl
5
numberof washes Fio. 3. Effect of the number of bead washes after doing the binding assay. L63Rho (50 ng) was incubated with 5/zg ([]) GST or (O) GST-Rho-GAP and washed for one to five times with 1 ml of wash buffer. Results are given in percentage of input counts. (10 mM). 7 On the other hand, the R a c / R h o - G A P interaction appears to be independent of ionic strength. 7,1° However, using the binding assay we find no difference between the binding of L63Rho and L61Rac to R h o G A P at 150 m M NaCI c o m p a r e d to 10 m M NaC1.
h. G T P Dependence Small GTP-binding proteins are active only in the G T P - b o u n d form and it is expected that any interaction with an effector protein will be G T P dependent. Since we were unable to obtain commercial [3Zp]GDP, we resorted to the less sensitive 3H-labeled nucleotides to test this. [3H]GTP is available at 10 C i / m m o l and we find that the signal to background ratio using [3H]GTP-bound R h o or Rac is not as high as with the 32p-labeled nucleotides. The concentration of Rho or Rac used in the binding assay was thus increased about three- to fivefold. Figure 4 shows the result using 300 ng L63Rho loaded with 30/xCi of either [3H]GDP or [3H]GDP and incubated with 40/~g G S T - R h o - G A P . It can be seen that the G T P form of L63Rho has a higher affinity for R h o - G A P than the G D P - b o u n d form. D e t e c t i o n of Effector P r o t e i n for Rac U s i n g in Vitro B i n d i n g A s s a y A similar in vitro binding assay to that described here has been used previously to detect an interaction between Ras and c-Raf. 11 Ras in the 10A. J. Self, H. F. Paterson, and A. Hall, Oncogene 8, 655 (1993). 11p. H. Warne, P. R. Viciana, and J. Downward, Nature 364, 352 (1994).
In Vitro BINDING ASSAY
[231
213
15-
108
5.
0~
rho GTP
rho.GDP
FIG. 4. Guanine nucleotide dependence of L63Rho binding to GST-Rho-GAP. [3H]GTPor [3H]GDP-loaded L63Rho (0.3 t~g)was incubated with 40/xg of either ([]) GST or (11) GSTRho-GAP, and bound protein was recovered using glutathione-Sepharose beads. Results are shown in percentage of input counts. Input counts were on average 300,000 cpm for RhoGTP and 220,000 cpm for Rho-GDP. Results shown are the means of three experiments.
G T P - b o u n d state binds to the a m i n o - t e r m i n a l region of c-Raf, expressed as a G S T fusion protein, and the interaction is abolished by the addition of a synthetic peptide c o r r e s p o n d i n g to the Ras effector domain. This in vitro assay has confirmed that c - R a f is a real effector target protein for the Ras G T P a s e . W e have used the binding assay to detect an effector protein for R a c in the N A D P H oxidase complex, a2 R a c has b e e n identified as an essential c o m p o n e n t required for the activation of the s u p e r o x i d e - p r o d u c i n g N A D P H oxidase e n z y m e in phagocytic cells. 4-6 T h e N A D P H oxidase is a m u l t i m o l e c u l a r e n z y m e c o m p l e x consisting of two integral m e m b r a n e c y t o c h r o m e b subunits and two cytosolic proteins, p47 ph°x and p67Ph°x. B O n activation of phagocytic cells, p47 ph°x and p67 ph°x translocate f r o m the cytosol to f o r m a c o m p l e x with the m e m b r a n e c o m p o n e n t s J 4 Superoxide p r o d u c t i o n can be reconstituted in vitro using purified c y t o c h r o m e b, r e c o m binant p47 ph°x and p67 ph°x, and R a c in the G T P form, 15'a6 and it is clear, therefore, that one of the four proteins must be the R a c target protein. T o see if R a c could interact with one of the cytosolic proteins, 100 ng [a32p]GTP-loaded R a c is incubated with 20/xg G S T - p 4 7 ph°x or G S T - p 6 7 ph°x 12D. Diekmann, A. Abo, C. L. Johnston, A. W. Segal, and A. Hall, Science 265, 531 (1994). 13A. W. Segal and A. Abo, Trends Biochem. Sci. 18, 43 (1993). 14S. Dusi, V. Della-Bianca, M. Grzeskowiak, and F. Rossi, Biochem. J. 290, 173 (1993). 15A. Abo, A. Boyhan, I. West, A. J. Thrasher, and A. W. Segal, J. Biol. Chem. 267,16767 (1992). 16D. Rotrosen, C. L. Yeung, T. Leto, H. Malech, and C. Kwong, Science 256, 1459 (1992).
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CELL EXPRESSION
123]
fusion proteins as described earlier. Figure 5a shows that wild-type Rac (G12Rac) as well as L61Rac both bind to GST-p67 ph°x, whereas L63Rho, which cannot activate the N A D P H oxidase system, does not. None of the proteins bind to G S T - p 4 7 ph°x. In order to confirm that the interaction is functionally significant, we carried out two further experiments. In Fig. 5b we show that G S T - p 6 7 ph°x interacts only with the G T P - b o u n d form and not with the G D P form of Rac. This is consistent with the observation that a
25 20-
•~
15-
b
8[
~
6-
g
•~"
~ 10-
4-
~6 2-
5-
rac G12
rac L61 C
rac.GTP
rho L63
rac.GDP
15"1"
10o o
5-
0 ~
rac
A35
K40
A38
FIG. 5. Binding of Rac to GST-p67 ph°x. (a) [c~-32p]GTP-loaded wild-type Rac (G12Rac), L61Rac, or L63Rho (100 ng each) was incubated with 20/zg of (D) GST, ( I ) GST-p67 ph°x, or (D) GST-p47 ph°x and binding was determined using the bead assay described earlier. (b) GTP Dependence of the Rac/p67 ph°x interaction. [3H]GTP- or GDP-loaded wild-type Rac (300 ng) was incubated with 40/xg ([]) GST or (11) GST-p67 ph°x. (c) Rac effector mutants do not bind to p67ph°x. Wild-type Rac or mutants with an amino acid substitution in the effector region (Thr to Ala 35, Asp to Ala 38, Tyr to Lys 40) (100 ng each) were loaded with [a-3ep]GTP and incubated with 30 /zg (11) GST-p67ph°x; (R) GST. Results are shown as percentage of input counts.
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Rac AND Cdc42-ASSOCIATEDKINASES
215
only the GTP form of Rac will activate superoxide production in the cellfree assay. 4 Furthermore, we have used altered Rac proteins having amino acid substitutions in the effector domain (amino acids 30-40), which had been shown previously to be inactive in the cell-free assay. Figure 5c shows that none of these mutants was able to bind to GST-p67 ph°x. On the basis of these results we propose that p67ph°x is the target protein for Rac in the N A D P H oxidase complex) 2 Acknowledgments This work was supported by the Cancer Research Campaign, UK (A. H.) and by an Medical Research Council student fellowship (D. D.).
[24] P u r i f i c a t i o n
By
and Assay of Kinases That Interact with Rac / Cdc42
E D W A R D MANSER, THOMAS LEUNG,
and L o u i s LIM
Introduction The pioneering work on Ras-mediated control of multiple signaling pathways has yielded significant new information and has demonstrated the importance of both biochemical and genetic methods to determine the underlying interactions of p21-mediated signaling. Similarly, Rho family signal transduction cascades, leading to coordinated cytoskeletal reorganization (and perhaps nuclear events), promise to be just as fascinating. Many genetically defined gene products have been found in Saccharomyces cerevisiae to interact with Cdc42 and Rhol, 2, 3, and 4 proteins; 1-3 of these the GTPase-activating protein (GAP) 4 and guanine nucleotide releasing factor (GRF) 5 homologs have been identified in mammalian cells. The best studied of these p21s is Cdc42Sc, first identified as a protein required for budding 6 and shown to affect morphology in Schizosaccharomyces pombe. 7 1 A. Bender and J. R. Pringle, Mol. Cell, Biol. 11, 1295 (1991). 2 j. Chant, K. Corrado, J. R. Pringle, and I. Herskowitz, Cell 65, 1213 (1991). 3 y. Matsui and E. A. Toh, Mol Cell. Biol. 12, 5690 (1992). 4 y. Zheng, M. J. Hart, K. Shinjo, T. Evans, A. Bender, and R. Cerione, J. Biol. Chem. 268, 24629 (1993). 5 y. Zheng, R. Cerione, and A. Bender, J. Biol. Chem. 269, 2369 (1994). 6 A. E. M. Adams, D. I. Johnson, R. M. Longnecker, B. F. Sloat, and J. R. Pringle, J. Cell Biol. U l , 131 (1990). 7 p. j. Miller and D. I. Johnson, Mol. Cell. Biol. 14, 1075 (1994).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
216
CELLEXVRESSION
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The Caenorhabditis elegans homolog has been cloned, 8 while two alternatespliced products of human Cdc42 are known. 9'1°To dissect the different Rho family signal transduction cascades, characterization of the p21-interacting proteins is required. As for the Ras pathway, genetic information has allowed the identification of a family of guanine nucleotide exchange/ release factors (GRFs) by virtue of their homology to Cdc24p 5 genetically upstream of Cdc42Sc. Conversely, we have shown that a brain p21 (Cdc42 and Rac)-activated kinase 11 (now designated oL-PAK) is highly related to the mating cascade kinase Ste20p, 12 which interacts with Cdc42.13 This chapter describes a method to detect a new family of Rho p21interacting proteins, and the use of the assay for expression screening and in the purification of an abundant Cdc42 and Racl "target," the brain serine/threonine kinase p65-PAK. H It is perhaps not a coincidence that all proteins of this class that interact with Rho p21s that have been studied and cloned in our laboratory are protein kinases, given that an important target of Ras are the Raf kinases. ~4 These methods allow the cloning of those proteins detected in [y-32p]p21 overlays of tissue extracts, and others such as the activated Cdc42-associated tyrosine kinase (ACK). ~5 The GAP overlay technique not only detects tissue levels of GTPaseactivating proteins (GAPs) after SDS electrophoresis, but also GTPase inhibitory proteins. 16 For these proteins to exert such an effect on the intrinsic GTPase activity of p21 they must remain bound (i.e., they cannot act in a catalytic manner, as do GAPs). After autoradiography of the nitrocellulose overlay filter, which holds the image of the nucleotide state of the test p21 within the original filter, some of these proteins can be observed as signals darker than the background (cf. [16] in this volume). However, if the [y-32p]GTP-labeled p21 binds very tightly to these proteins, a white signal, similar to that produced by GAPs, is observed 15 because this test p21 is locally sequestered. s W. Chert, H.-H. Lim, and L. Lira, J. Biol. Chem. 268, 13280 (1993). 9 S. Munemitsu, M. A. Innis, R. Clark, F. McCormick, A. Ullrich, and P. Polakis, Mol. Cell. Biol. 10, 5977 (1990). 10 K. Shinjo, J. G. Koland, M. J. Hart, V. Narasimhan, D. I. Johnson, T. Evans, and R. Cerione, Proc. Natl. Acad. Sci. U.S.A. 87, 9853 (1990). 11 E. Manser, T. Leung, H. Salihuddin, Z.-S. Zhao, and L. Lim, Nature 367, 40 (1994). 12 E. Leberer, D. Dignard, D. Harcus, D. Y. Thomas, and M. Whiteway, EMBO J. 11, 4815 (1992). 13 Z.-S. Zhao, T. Leung, E. Manser, and L. Lim, Unpublished data (1994). 14 p. H. Warne, P. R. Viciana, and J. Downward, Nature 364, 352 (1993). 15 E. Manser, T. Leung, H. Salihuddin, L. Tan, and L. Lim, Nature 363, 364 (1993). 16 E. Manser, T. Leung, C. Monfries, M. Teo, C. Hall, and L. Lira, J. Biol. Chem. 2~7, 16025 (1992).
[24]
Rac AND Cdc42-ASSOCIATEDKINASES
217
GTPase inhibitors can be detected by a more conventional protein overlay technique with [T-32p]GTP-p21 (illustrated in Fig. 1). A comparison of [y-32p]GTP-Cdc42 binding to rat brain and spleen extracts fractionated on 9% gels at 4 ° versus room temperature is shown: the weaker signals for those processed at a higher temperature is due both to the increased intrinsic [T-32P]GTP hydrolysis and to a more rapid dissociation of the p21 from proteins bound to the nitrocellulose during washing. Wild-type rather than GTPase-deficient p21s are the preferred probes to detect low levels of these p21-binding proteins in tissue extracts because GTPase inhibition by the binding proteins enhances the signal (relative to background). Even so, the higher molecular weight binding proteins are only clearly seen after separation on 7.5% gels. After purification the predicted GTPase inhibitors have been shown to exhibit such an activity when tested in solution assays. 11'15 This is not detectable in unfractionated tissue or cell extracts because of the counteracting effect of the many p21 modulatory proteins, of which GAPs are the dominant activity.
Cdc42 overlay 4°C kDa
B
S
24°C B
S
200 97 68 43 29
FIG. 1. Temperature dependence of [y-3Zp]GTP-Cdc42 binding to brain and spleen proteins. Proteins (200 ~g/lane) from total rat brain (B) and spleen (S) extracts were separated on a 9% polyacrylamide gel and transferred to nitrocellulose. GST/Cdc42 was labeled as described (see [16] in this volume) and used to probe identical flters which were processed at 4 and 24° (binding and washing steps). Autoradiography was performed for 4 hr using hyperfilm (Amersham). The 36-kDa band found only in spleen is a Cdc42-specific binding protein. The brain 45-kDa Cdc42 and Rac binding band may be a breakdown product of the p62-p68 PAK-related proteins.
218
CELLEXPRESSION
[24]
Detection of SDS-Polyacrylamide-Fractionated GTP-p21-Binding Proteins Since the protocol for GTP-p21 overlay was developed along with the GAP nitrocellulose overlay method, the reader should refer to [16] for details of electrophoresis, p21 labeling, and the various buffers. For reasons outlined in the introduction, care must be taken to keep all solutions cold and processing times must be kept to the values recommended otherwise the signals will be weak. Human recombinant Cdc42, Racl, and RhoA proteins can be obtained with good purity and high yield as GST fusion proteins; in our hands RhoG expresses rather poorly. When labeled with [y-3ap]GTP the p21s can bind to a number of protein bands on nitrocellulose blots of tissue or cell soluble extracts separated by SDS-PAGE. In contrast, no signal is detected when the p21 is labeled with [a-32p]GDP. Deletion analysis of a number of cDNAs coding for these p21-binding proteins have shown that the p21-binding domains are less than 50 amino acid residues and are able to refold after SDS-polyacrylamide electrophoresis. Methods
Because no additional bands can be detected in the high-speed pellet of tissues extracted with hypotonic buffer, only the soluble proteins need to be analyzed. Extracts are prepared at pH 8.0, and 100-200 k~g of total protein per lane is run on 9% polyacrylamide gels as described for the GAP nitrocellulose overlay assay. Proteins can be transferred to either nitrocellulose or polyvinylidene difluoride (PVDF) membranes. The advantage of the latter is that the blot can be stained with Coomassie to determine the efficiency of transfer and protein loading prior to "blocking" and p21 binding (for PVDF membranes SDS should be omitted from the transfer buffer). Problems associated with the inefficient transfer of high molecular mass proteins (>100 kDa) are remedied by running 7.5% gels. Mark the corners of the gel on the filter. Immerse the filter in 0.1% (w/v) Coomassie blue dissolved in 40% methanol/10% acetic acid (v/v) for 3 rain and destain in the same buffer. For convenience, a record of the blot can be made on a photocopy machine. Completely destain the proteins by washing in methanol for 5 rain and place in phosphate-buffered saline (PBS) containing 0.1% Triton X-100. Since a denature/renature step can improve binding, treat the filters for 5 min in 6 M guanidinium hydrochloride dissolved in buffer Q (see section on PAK purification) at 4°, then dilute this with an equal volume of buffer Q, and agitate for a further 5 min; the process is repeated five times total, then the filter is placed in GAP renature buffer. The test p21s can be used as GST fusion proteins or as the cleaved product using the appropriate protease; both are labeled and bind to the
[24]
Rac AND Cdc42-ASSOCIATEDKINASES
219
target sequences with equal efficiency. The [y-32p]GTP is exchanged into the GST/p21 fusion protein which is then diluted into 2 ml of GAP buffer containing 0.5 mM GTP (sufficient for two filters). Completely soak the filters in the radioactive solution in a small plate kept on ice. Remove excess solution by scraping filters against the side of the plate and lay the filter carefully onto a 1% agarose plate at room temperature. Leave for 5 min, then move this to the cold room. After 10 min wash the filters in three 50-ml changes of GAP wash buffer (1 min each). Blot with Whatman 3MM paper, cover with saran wrap, and arrange in a precooled X-ray cassette. Check the level of radioactivity using a hand-held monitor; it should register 50-200 cpm. A high-resolution film such as Hyperfilm (Amersham) is placed in the cassette and immediately exposed at - 7 0 °. Develop the film after 4 hr and then reexpose as appropriate. Expression Screening with [y-a2P]GTP-Labeled p21 s Because the Rho p21-binding regions are small independent domains, almost any construct expressing such a sequence is able to associate with labeled p21. This is ideal for the screening of expression libraries, although the labile nature of the labeled p21 and its apparent lower affinity of binding compared to other signal transduction interactions (e.g., that of SH3 domains to proline-rich sequences 17) mean that filters must be processed rapidly after binding. Protocols involving the use of secondary antibody or streptavidin-biotin are not appropriate in this case. We have successfully isolated clones from both Agtll (Clontech) and AZAP (Stratagene) libraries; the former giving expression products as fl-galactosidase fusion proteins while the latter contain only a small polylinker-derived leader peptide. The number of clones that need to be screened for a given target is probably related more to the quality of the library than to the abundance of the message; it is therefore advisable to use a random primed library which has been prechecked by D N A screening and polymerase chain reaction analysis of clone size for a medium abundance tissue-specific gene. Technically, direct expression screening with a labeled protein is more difficult than using the recently popular yeast two-hybrid system because positive signals are difficult to see on the primary plate. Figure 2 shows a tertiary screen of the ACK using [~/-3ap]GTP-labeled Cdc42. Although overlays of SDS-fractionated tissues u with [y-32p]GTP-RhoA give very weak signals, positives are strong in expression screens. This suggests that the [T-3ap]GTP-p21 overlay might be applied to clone target proteins that are not detected after SDS-PAGE. Once positive cDNAs are isolated, 17p. Cicchetti, B. J. Mayer, G. Thiel, and D. Baltimore, Science 257, 803 0992).
220
CELLEXPRESSION
[24]
Cdc42 overlay
ACK ~,ZAP FIG. 2. Detection of recombinant Cdc42-binding protein produced by bacteriophageinfected E. coil Bacteriophage containing part of the ACK cDNA 15 were plated as described in the method section. After induction overnight at room temperature the filter was blocked and subjected to [y-32p]OTP-Cdc42 overlay.
they should be grouped according to restriction pattern, and full-length cDNAs should be isolated by conventional D N A screening methods. The p21-binding domain can be mapped by N- and C-terminal deletions of the cDNA cloned into an appropriate expression vector, with analysis of purified protein products or total induced Escherichia coli lysates by the [y32p]GTP-p21 overlay. 15
Methods
Bacteriophage are plated on the appropriate bacterial strain early in the morning according to standard (or suppliers) protocols at ~40,000 plaque-forming units per 25 × 25-cm plates. When the plaques reach a visible size, leave for a further hour, then overlay with damp nitrocellulose membranes (20 × 20 cm) wetted with 10 mM isopropyl fl-D-thiogalactoside (IPTG) and blotted with Whatman 3MM paper. The filter is left overnight at room temperature. The next day mark filters and block in renature buffer for at least 1 hr. The filters are probed with [y-32p]GTP-labeled p21 according to the same scheme as described in the previous section. In this case twice the amount of labeled p21 is used, diluted into 10 ml in GAP buffer. False-positive signals due to nonspecific binding of the probe to
[241
Rac AND Cdc42-ASSOCIATEDKINASES
221
particulate matter in the labeled p21 are eliminated by passing it through a 0.45-~m filter prior to use. Regions from the primary plate corresponding to putative positive phage plaques are excised and replated for titering and secondary screening. Once positive clones are purified, the p21 specificity and size of the expression product can be established by inducing a confluent layer of phage in top agarose as described and harvesting 0.5 ml of this top agarose in an Eppendorf tube for S D S - P A G E and p21-binding analysis. Add an equal volume of 2 × SDS sample buffer, vortex, and incubate for 1 hr at room temperature: spin for 5 min at full speed and run 50 t~l on a 7.5% (for Agt11-derived extracts) or 12% acrylamide gel (for AZAP), then transfer and probe the blotted proteins for [y-32p]p21 binding. For AZAP libraries the cDNA can be excised in vivo as plasmid, then 1 ml of bacteria harboring the plasmid grown to an OD of 0.6 (600 nm) is induced with 0.5 mM IPTG for 2 hr at 37°. Pellet cells and suspend in 100/~1 of PBS/ 0.1% Triton X-100/1 mg/ml lysozyme and leave for 10 min. Sonicate the extract and add an equal volume of 2 × SDS sample buffer. Each lane requires 20/~1 of the total E. coli extract.
Purification of GTP-Cdc42-Associated Kinase p65-PAK Strong signals generated by [y-32p]GTP-Cdc42 overlays 15 (particularly in brain) suggest that GTP-Cdc42 might be used as an affinity ligand to purify the proteins. Most of the Cdc42-binding bands are also detected (although more weakly) by Racl. Preliminary experiments with GST/ Cdc42Hs columns loaded with different nucleotides showed that GTP and GTPyS can sequester a small proportion of the abundant p62-p68 Cdc42binding proteins from crude rat brain extracts after a single passage through ion-exchange media, n These proteins are coeluted with the GST/Cdc42. The low yield under these conditions could be due to the dilute concentration of Cdc42-binding proteins, interference by other proteins, and possibly effects of nucleotide exchange proteins and GAPs on the Cdc42 column. A purification scheme was therefore established to prepare an extract enriched in the p62-p68 Cdc42-binding proteins that was depleted of GAPs. The nitrocellulose overlay assay was used to assess the amount and type of GAPs present at each step. To minimize protein degradation, the fractionation times have been kept to a minimum by applying step gradients rather than linear salt (or pH) gradients. The enriched extract is then passed over the GTP-loaded GST/Cdc42 column and binding proteins are eluted at pH 8.5. The method used to prepare the brain p65-PAK is shown schematically in Fig. 3.
222
CELL EXPRESSION
[24]
high speed supernatant 40g rat brains, in 200 ml Q buffer
dilutewithequal
s
J
Zn-chelating Sepharose
\ (10 ml) pH 7.5-6.0 \ fractionI I I
W immobilized GTP-Cdc42 ~
(1 ml)
wash pH 6.0 collect PAK at pH 8.5
FIG. 3. Outline of the p65-PAK purification procedure.
Methods Weigh out 40 g of whole rat brains (from - 2 5 animals; these can be stored beforehand at -70°), mince the material with a pair of scissors, add 200 ml volumes of buffer Q containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM dithiothreitol (DTT), and apply 20 strokes in a hand-held Dounce homogenizer on ice. Spin the material in a high-speed centrifuge at 30,000 rpm at 4 ° for 40 min and decant off the supernatant. Apply this to a 80-ml column of S-Sepharose (Pharmacia-LKB) either by gravity or with a pump (all columns run at 4 ° unless indicated otherwise). For gravity feed it is best to pour columns with a height : diameter ratio of --3:1 to allow efficient flow; the flow will stop automatically when the solutions reach the top of the column. The p62-p68-binding proteins have
[241
Rac AND Cdc42-ASSOCIATEDKINASES
223
similar retention on S-Sepharose to hemoglobin, which is a convenient marker. Wash the column with 80 ml of buffer Q (no salt), then add 50 ml of Q + 0.25 M NaC1 (void volume), and collect the binding proteinenriched fraction by adding 80 ml of Q + 0.25 M NaC1 followed by 50 ml of Q + 0.5 M NaC1; all the hemoglobin should be visible in this fraction. Collect a remaining fraction by adding 50 ml of Q + 0.5 M NaC1. Dilute the 0.25 M NaC1 S-Sepharose fraction with an equal volume of ice-cold deionized water to reduce the salt concentration. Immediately load this onto a 30-ml Q-Sepharose column. The hemoglobin in this case is in the flow-through fraction. Wash with 24 ml of buffer Q and then collect 24-ml fractions with each change of the buffer containing 0.1, 0.2, 0.3, 0.4, and 0.5 M NaC1. Glycerol is added to each fraction to 5%; 20/zl of every fraction during the preparation is taken for analysis and the remainder is quick frozen and stored at - 7 0 °. Figure 4 shows the expected elution pattern of the rat brain Cdc42-binding proteins detected by the method described in the second section. Pool those frozen fractions from the Q-Sepharose column containing the binding proteins (usually the 0.2, 0.3, and 0.4 M fractions). Add 1/20 vol of 1 M Tris, pH 8, to increase the pH to --7.5 (check on the pH meter). A 10-ml chelating Sepharose column is saturated with zinc using 2 column vol of Q + 100 mM ZnC12 and washing with buffer Z + 25 mM Tris, pH 7.5. Load the pooled fraction (adjusted to pH 7.5), and wash with 2 column vol of Z + 25 mM Tris, pH 7.5. Fill the column with 7 ml of
kDa
FT ~
c;m FT o~
o~" c5~ o~' cSm NaCI (M)
200 97 68 43 29 S-sepharose
Q-sepharose
FIG. 4. Analysis of Cdc42-binding protein fractionation during p65-PAK purification. Aliquots of 20 ~1 taken from the fractions collected during S- and Q-Sepharose chromatography were run on a 9% SDS-polyacrylamide gel, transferred to nitrocellulose, and analyzed for [T-32p]GTP-Cdc42 binding. FT, flow-through fraction; other fractions refer to the step gradient of salt used to elute the proteins.
224
CELLEXPRESSION
[24]
buffer Z + 25 mM M E S - N a O H , pH 6.0, and then collect a 14-ml fraction at this pH. This material is ready to be directly loaded onto the Cdc42 affinity column. In order to charge a 1-ml glutathione-Sepharose column with >5 rag/ ml GST/Cdc42Hs fusion protein, an extract prepared from 400 ml of E. coli cells harboring the plasmid construct is required. These cells are induced at 0.60D600nm with 0.5 mM IPTG and are incubated for 6 hr at room temperature. The bacteria are pelleted and resuspended in 20 ml of cold GST buffer containing I mg/ml lysozyme, 0.5 mM PMSF, and 5 mM DTF. After 10 min the cells are sonicated until the viscosity returns to normal, and insoluble material is removed by centrifugation at 30,000 rpm for 40 min. Extracts are made 5% in glycerol and quick frozen prior to use. The extract is loaded onto the l-ml glutathione-Sepharose column, then washed with 10 ml of GST buffer. Either GTP or GTPyS can be exchanged into the Cdc42 by adding 1 column vol of exchange buffer (at room temperature) containing 0.5 mM nucleotide and leaving the column for 5 min. Return the column to the cold and wash with 1 ml of buffer Z + 25 mM MES, pH 6.0, containing 0.5 mM GTP or GTPyS. Load 7 ml of the chelating Sepharose pH 6 fraction. Two wash fractions, each of i ml with pH 6 buffer, are collected, followed by two separate fractions with buffer Z + 25 mM Tris-HC1, pH 8.5. The flow through is reloaded after the column is reequilibrated at pH 6.0 and taken through the same cycle. Then the GST/Cdc42 column is recharged with GTP or GTPyS and the other half of the preparation is treated in the same manner. Aliquots (20 tzl) of each of the eight pH 8.5 fractions are immediately run on duplicate 9% acrylamide SDS gels, one of which is Coomassie or silver stained to determine the purity of the samples. Run a standard containing 200 ng of bovine serum albumin (BSA) in the last lane. The second of the pH 8.5 fractions should contain predominantly a protein of 65 kDa, whereas the first fraction is more heterogeneous. The second gel is transferred to PVDF, stained to locate the purified bands, and then processed for [y-32p]GTP-Cdc42 binding. Signals should correspond with the major stained band(s): breakdown products of the kinase can also be detected by this method. The yield is 2-5/zg of kinase for each 1-ml fraction using an estimate of the protein concentration from the BSA standard: the pooled material with 5% glycerol can be concentrated prior to storage using Centricon membranes (Amicon, Danvers, MA). Buffer Q: 25 mM M E S - N a O H , pH 6.5, 0.5 mM MgCI2, 0.05 mM ZnC12, 0.05% Triton X-100 Buffer Z: 100 mM NaC1, 0.5 mM MgC12, 0.05% Triton X-100 GST buffer: phosphate-buffered saline containing 50 mM Tris-HCl, pH 8.0, 0.5 mM MgC12, 0.1% Triton X-100
[241
Rac AND Cdc42-ASSOCIATEDKINASES
225
Activation of Purified p65-PAK b y Cdc42 and Rac 1 The brain-enriched p65 Cdc42-binding protein was tested for kinase activity prior to its cloning because of its possible relationship to the Cdc42associated tyrosine kinase pl20-ACK, which was obtained by expression screening. The purified p65 exhibits variable levels of autophosphorylation and kinase activity toward exogenous substrates such as myelin basic protein and histones. It is a threonine/serine but not tyrosine kinase. 11The variability in basal activity is probably related to the degree of dephosphorylation of the protein during its purification. Our data indicate that the phosphorylated form of the enzyme has a lower affinity for activated Cdc42 and Racl. Since like many kinases its activation results from phosphorylation, the most tightly binding (affinity enriched) form should have the lowest basal activity. PAK is at present a unique kinase being directly activated by the p21 (either Cdc42 or Racl) in vitro. This activation can be assayed by including a test substrate in the reaction or by testing the activity of the kinase after allowing p21-mediated autophosphorylation.
Methods
Purified p65-PAK ( - 1 / x g ) is dialyzed against kinase buffer for 2 hr at 4°. In order to preload the recombinant GST/Cdc42 and GST/Racl with the nucleotide mix, take 10 txl of the 1-mg/ml stock and add 10 /~1 2× exchange buffer and 2/zl of 10 mM GTP~/S or GDP; also make up controls using 1 mg/ml GST protein with GTP~/S or GDP as a control. Leave these at room temperature for 5 min and return to ice. Make up six tubes containing 150 ng of purified kinase per reaction in a final volume of 40 t~l of kinase buffer. Add 6 txl of control, Cdc42, or Racl mix, each "exchanged" either with GDP or GTP7S. Then add 2 txl of 5 mg/ml myelin basic protein (bovine MBP, Sigma) and 2/zl of labeled ATP. This contains 1 mM cold ATP mixed with an equal volume of high specific activity [~/-32p]ATP (=10/xCi per reaction of >3000 Ci/mmol). The final concentration of ATP in the kinase reaction mixture is therefore 20/.~M. Incubate for 10 rain at 30°, then return to ice and add an equal volume of 2 x SDS sample buffer. Run half of each sample on a 12% polyacrylamide gel, stain the gel then dry, and expose to film for an appropriate time. Both autophosphorylation and MBP phosphorylation should be stimulated 10-100 times in the presence of the activated p21. The degree of observed activation is dependent on the "basal" activity of the kinase. If the p65 band is visible after drying, it may not align with the position of the strongest labeled autophosphorylated band. This is because under these activation conditions a
226
CELL
EXPRESSION
124]
minority of the kinase is fully phosphorylated, and this band runs above the position of the unphosphorylated starting material. Kinase buffer: 50 m M H E P E S , p H 7.3, 5 m M MgC12, 5 m M MnC12, 1 m M D T T , 0.05% Triton X-100.
Conclusions The use of predicted dominant positive and negative mutants has become a standard method of probing the pathway controlled by the growing n u m b e r of p21 that have been cloned. With this method, the early effects of R h o A and Racl activation have been shown to involve reorganization of actin cytoskeleton, 18'19which in nonmuscle cells is highly dynamic. Interestingly, long-term morphological transformation of monocytes correlates with a substantial increase in membrane-bound Cdc42. 2° While the studies of Ras signaling strongly suggest that the cellular end point of each Rho protein is cell-type dependent, it seems likely that different cells (and indeed organisms) will share many of the components up- and downstream of each p21. Both the similarity of the interaction of R h o - p 2 1 s with their associated kinases to that of Raf/Ras and the observation that these proteins selectively bind activated p21s suggest their roles in transducing p21 signals. Most cells (in culture) have at least four targets of this type for Cdc42 and Racl: the ubiquitous 62-kDa binding protein, 65- to 68-kDa PAK-related proteins, the A C K tyrosine kinase, and the 170-kDa family of binding proteins. How each of these is coupled to activated Cdc42 or Racl remains to be resolved. While the overlay detection method can identify p21 "targets," it has not yet been generally applied to other p21s. It would be of interest to determine if Rabphillin 3A can be detected since this could be a prototype for a family of activated Rab-associated proteins. 21 It seems pertinent to consider what the candidate "target" proteins for Rho p21s are in mammalian cells. Studies of p120 R a s - G A P 22 suggest that the first group to consider are the R h o - G A P s . Most of those exhibiting significant activity in various tissues appear to have been cloned (see [16] on G A P nitrocellulose overlay assay), but no evidence is yet published for an effector function. In overall structure, G A P proteins appear to be poorly 18A. J. Ridley and A. Hall, Cell 70, 389 (1992). 19A. J. Ridley, H. F. Paterson, C. L. Johnston, D. Diekmann, and A. Hall, Cell 70, 401 (1992). 20M. Aepfelbacher, F. Vauti, P. C. Weber, and J. A. Glomset, Proc. Natl. Acad. Sci. U.S.A. 91, 4263, (1994). 21H. Shiritaki, K. Kaibuchi, T. Sakoda, S. Kishida, T. Yamaguchi, K. Wada, M. Miyazaki, and Y. Takai, Mol. Cell, Biol. 13, 2061 (1993). 2~G. A. Martin, A. Yatani, R. Clark, L. Conroy, P. Polakis, A. M. Brown, and F. McCormick, Science 255, 192 (1992).
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Rac AND Cdc42-ASSOCXATEDKINASES
227
conserved between lower and higher organisms. The second group are those identified by the p21-binding method described here, of which P A K is directly activated by interaction with Cdc42 or Rac. The third class are Rac-activated neutrophil oxidase complex proteins, 23 none of which exhibit homology to the first two classes. While it is an important observation that the serine/threonine kinase p65-PAK can be activated by both Cdc42 and Racl in vitro, it is yet to be established which of these are in vivo activators. This might be resolved in an organism such as yeast which does not appear to contain Rac. The homology of P A K with the yeast kinase Ste20p may have implications for its activation and function. First, S T E 2 0 is believed to lie close to S T E 4 / 18 (encoding heterotrimeric G protein 13 and y subunits) in the mating signaling cascade: ~2the brain is particularly rich in serpentine receptors (e.g., neuropeptide receptors) that activate heterotrimeric G proteins, perhaps relating to the high level of P A K in this organ. Second, S T E 2 0 lies upstream of the well-studied S T E l l / 7 and F U S 3 kinase cascade, whose activation regulates mating-specific genes. Third, independent of this cascade, S T E 2 0 could be also involved in the cytoskeletal reorganization that leads to the formation of the mating projection toward the gradient of pheromone 24 since overexpression of a dominant truncated Ste20p mutant 25 in cells deleted for S T E 7 / l l remains lethal. The overlay method suggests that there is a family of PAK-like kinases of similar molecular weights. Comparison of the sequence of these related mammalian proteins, when they become available, should provide clues as to the important features of this new class of kinase. It should be fascinating to discover the means by which the interaction of G T P - C d c 4 2 and G T P Rac modulates their activity. Acknowledgment We thank the Glaxo-SingaporeResearch Fund for support.
23A. Abo, E. Pick, A. Hall, N. Totty, C. G. Teahan, and A. Segal, Nature 353, 668 (1991). 24C. L. Jackson and L. H. Hartwell, Cell 63, 1039 (1990). 25S. W. Ramer and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 90, 452 (1993).
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[25] Yeast Two-Hybrid S y s t e m to Detect Protein-Protein Interactions with Rho GTPases B y PONTUS ASPENSTRt3M and MICHAEL F. OLSON
Introduction The yeast two-hybrid system I has emerged as a powerful method to examine protein-protein interactions. Its greatest advantage lies in its potential for detecting novel targets for a protein of interest by library-screening procedures. 2 In addition, the two-hybrid system can be used to monitor interactions between known proteins. Several versions of this system have been reported, I-3 but in each case two plasmids encoding the proteins of interest are expressed simultaneously in Saccharomyces cerevisiae. In the system originally described by Field and Song, I one plasmid encodes the DNA-binding domain of the GAL4 transcription factor (GAL4DB), consisting of amino acids 1-147 fused to the N terminus of a protein of interest. The other plasmid encodes the GAIA activation domain (GAL4AD), consisting of amino acids 768-881 fused to the N terminus of the second protein of interest. The two plasmids are introduced into a yeast strain that has been engineered such that two reporter genes, HIS3 and lacZ, are under the control of the G A L l upstream activation sequence (UAS). The separately expressed domains of the GAL4 protein are unable to activate transcription of the reporter genes unless the two proteins of interest have the capacity to interact (see Fig. i). The lacZ expression is monitored by measuring the fl-galactosidase enzymatic activity. HIS3 selection is slightly less straightforward since a very low level of the HIS3 gene product, the enzyme imidazoleglycerol-phosphate dehydratase (IGPD), is sufficient to give rise to HIS prototrophy, even in the absence of exogenous histidine. In order to restore histidine auxotrophy the IGPD inhibitor, 3-aminotriazole (3AT), must be included in the culture medium. In addition, the fusion proteins must be able to enter the nucleus in order to form a functional G A L 4 transcription factor. For this reason it may be necessary to remove sequence motifs that direct the protein of interest to other compartments of the cell. We have used the yeast two-hybrid system to monitor the interaction S. Field and O. Song, Nature (London) 340, 245 (1989). z C.-T. Chien, P. L. Bartel, R. Sternglanz, and S. Fields, Proc. Natl. Acad. Sci. U.S.A. 88, 9578 (1991). 3 A. B. Vojtek, S. M. Hollenberg, and J. A. Cooper, Cell 74, 205 (1993).
METHODSIN ENZYMOLOGY.VOL. 256
Copyright© 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.
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R h o IN YEAST TWO-HYBRID SYSTEM
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Pmtein%"
A
AL4AD
GAL4 Inactive
Protein'X' AIADB I
IlUlIIMHlUUl
GALl UAS
HIS3 LacZ
} Reportergenes silent
B GAlA Active tein'Y' rein'X'
[
IIIIInlllMIIII
GALl UAS
D
j
p, HIS3 ]Reportergenestranscribed LacZ $
FIG. 1. Outline of the two-hybrid system. (A) The protein "X" fused to the GAL4 DNAbinding domain binds to the GALl UAS upstream of the reporter genes HIS3 and (lacZ) but it is unable to activate their transcription. (B) If the protein "Y" fused to the GAL4AD has the capacity to interact with "X", a functional GAL4 transcription factor is restored and the transcription of the reporter genes can be initiated.
between small GTPases and their putative target proteins. The procedure described below, designed to investigate the interaction between RhoA and R h o - G A P (GTPase-activating protein), can be used as a general protocol for the yeast two-hybrid system. Construction of Plasmids Standard protocols for DNA manipulations were followed.4 The polymerase chain reaction (PCR) was used to provide wild-type RhoA and the activated mutant L63RhoA cDNAs with NcoI and BarnHI restriction sites at their 5' and 3' ends, respectively. In addition, the cysteine at position 190 residing in the C-terminal CLVL sequence was altered to a serine. The integrity of this C A A X - b o x motif is essential for correct post-translational isoprenylation,5 which in turn is necessary for localizing RhoA to its proper 4 j. Sambrook, E. F. Fritsch, and T. Maniatis, in "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory Press, Plainview, NY, 1989. 5 p. Adamson, C. J. Marshall, A. Hall, and P. A. Tillbrook, J. Biol. Chem. 267, 20033 (1992).
230 A
CELL EXPRESSION NdeI.NcoI.SfiI.SrnaI.BamHI.SalI
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B SalI.NcoI.ClaI.EcoRI.PstI.SmaI.BgllI.SpeI.NotI.SaclI
C Ndel.NcoI.SmaI.BamHI.EcoRI.XhoI.BgllI
Fxa. 2. The plasmids used in the two-hybrid system: (A) pAS, (B) pYTH6, and (C) pACTII, Expression of the fusion proteins is under control of the constitutive yeast alcohol dehydrogenase promoter (ADH-P), and correct termination of the transcript is ensured by the A D H termination sequence (ADH-T). The plasmids also contain the/3-1actamase gene (Ap) for propagation in E. coli and either the TRP1 or the LEU2 gene which function as selectable markers in yeast. Ori, ColE1 origin of replication; fl+, fl origin of replication; 2/.t, yeast 2/zm origin of replication; HA, hemagglutinin epitope.
site in the cell. 6 By altering the C A A X box, we expected that the G A L 4 D B RhoA fusion proteins would enter the nucleus more efficiently and, in addition, would interfere less with endogenous signal transduction mechanisms. The NcoI/BamHI fragments of RhoA and L63RhoA were inserted into the GAL4DB-encoding plasmids pAS and pYTH6 (Figs. 2A and 2B) 6 p. Adamson, H. F. Paterson, and A. Hall, J. Cell BioL 119, 617 (1992).
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Rho IN YEASTTWO-HYBRIDSYSTEM
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that had been linearized with NcoI/BamHI and NcoI/BgIII, respectively. An EcoRI fragment of the R h o - G A P gene containing the C-terminal amino acids 230-439 was inserted into the EcoRI site of p A C T I I (Fig. 2C), thereby fusing it to G A L 4 A D . D N A sequencing was used to verify that the R h o - G A P and R h o A cDNAs were inserted in frame. The subcloned R h o A and L 6 3 R h o A cDNAs were fully resequenced to confirm that no errors had been introduced by PCR. The pAS and p A C T I I vectors (a generous gift from S. J. Elledge, Baylor College, Houston, TX) 7 are 2-/xm-derived, high-copy number plasmids which are maintained and replicated extrachromosomally in yeast whereas the p Y T H 6 plasmid (a generous gift from Julia White, Glaxo, UK) is integrated and stably maintained in a single copy in the yeast genome. The p A S R h o A construct could be used to create a RhoA-expressing yeast strain and subsequently transformed with the p A C T I I construct. Alternatively, the two plasmids could be simultaneously introduced into yeast cells. The p Y T H 6 R h o A construct was integrated first to create a stable yeast strain which was subsequently transformed with the p A C T I I plasmids.
Strains
Saccharomyces cerevisiae Y190 [MA Ta, gal4-542, gal80-538, his3, trpl901, ade2-101, ura3-52, leu2-3,112, URA3::GALI-lacZ, LYS2:: GAL1-HIS3cyh r] (a generous gift from S. J. Elledge, Baylor College, Houston, TX).
Escherichia coli: D H 5 a or XL-1 Blue. The plasmids used in the yeast two-hybrid system are larger than 7 kb and we have found them to be unstable in E. coli. For that reason only recAl-deficient bacteria are used for plasmid propagation.
Media The procedure to make up yeast media and plates is an adaptation of the protocol by Rose et al. s 20% Glucose (w/v). This solution should be autoclaved separate. Glucose tends to caramelize when it is autoclaved included in the medium. 7T. Durfee, K. Becherer, P.-L. Chen, S.-H. Yeh, Y. Yang, A. E. Kilburn, W.-H. Lee, and S. J. Elledge, Genes Dev 7, 555 (1993). s M. D. Rose, F. Winston, and P. Hieter, in "Laboratory Course Manual for Methods in Yeast Genetics." Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990.
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YEPD (Rich medium) 20 g peptone (Difco, No. 0118-01-8) 10 g yeast extract (Difco, No. 0127-01-7) Add water to 900 ml and autoclave. Add 100 ml of sterile 20% glucose. SC (synthetic complete) medium 6.7 g yeast nitrogen base without amino acids (Difco, No. 0919-15-3) 2 g of drop-out mix Add one or more of the following supplements as required by the selection conditions: 0.1 g tryptophan, 0.2 g leucine, 0.1 g histidine, 0.1 g uracil. Add water to 900 ml and autoclave. Add 100 ml of sterile 20% glucose. Drop-out mix is a mixture of amino acids and other supplements with those components that are to be used as selectable markers omitted. For all applications described in this chapter we use a drop-out mix lacking uracil, histidine, tryptophan, and leucine. The drop-out mix is made up from the following L-amino acids (Sigma, Kit No. LAA-21): 1 g each of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. In addition, the following non-amino acid components are included: 1 g of inositol, 0.25 g of adenine, and 0.1 g of paminobenzoic acid. The mixture is swirled in a 100-ml bottle on a rotating mixer for at least 15 min in order to break up any lumps of powder. This drop-out mix is stored at room temperature with the bottle wrapped in aluminum foil since some of the constituents are light sensitive. Plates Autoclave the agar, medium, and 20% glucose solution separately. We have found that autoclaving these together results in loose and mushy plates. Agar solution 20 g Bacto-agar (Difco, No. 0140-01). Add water to 450 ml and autoclave. YEPD plates Make up the constituents of the YEPD as above in 450 ml in a l-liter bottle and autoclave. To pour the plates add the 450 ml of agar solution and 100 ml of sterile 20% glucose to the YEPD medium; use approximately 25 ml of this solution per plate. SC plates Make up the appropriate SC medium in 450 ml of water in a l-liter bottle. Autoclave. Mix the melted agar solution and 100 ml of sterile 20% glucose with the SC medium in the l-liter bottle and pour the plates.
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R h o IN YEAST TWO-HYBRID SYSTEM
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3-AT plates Prepare SC-Trp-Leu-His plate solution as described earlier. Then add 25 ml of a 1 M, sterile-filtered stock of 3-amino-l,2,4-triazole (Sigma, No. A-8056) per liter of autoclaved medium immediately before pouring the plates.
Solutions Carrier D N A Herring testes D N A 10 mg/ml (Clonetech, No. K1606-A). Alternatively, carrier D N A can be made from salmon testes D N A by the method of Gietz e t al. 9 Dissolve 1 g of D N A (Sigma, No. D-1626) in 100 ml of TE, pH 8.0. Leave overnight at 4 ° on mild magnetic stirring since it takes some time to get the D N A into solution. The next day further dissolve the D N A by drawing it up and down in a 10-ml pipette. Fragment the D N A by sonication with three bursts of 30 sec with the sonicator set at moderate power. Extract the solution with 1 vol of phenol/CHC13 and separate the phases by centrifugation. The D N A in the aqueous phase is precipitated with 2.5 vol of ice-cold ethanol. Collect the D N A by centrifugation for 30 min at 4,200 rpm, partially air dry the pellet, and dissolve in TE, pH 8, to a final concentration of 10 mg/ml. Finally, the carrier DNA is denatured by boiling for 5 rain. The denatured carrier should not be reboiled since this reduces the efficiency of transformation. The D N A solution is highly viscous and is more easily pipetted when slightly heated. 0.1 M lithium acetate/TE, pH 7.5 (LiAcTE) Make a 1:10 dilution from an autoclaved 10× stock solution of 1 M lithium acetate, 100 mM Tris-HCl, pH 7.5, 5 mM EDTA. Autoclave. 44% polyethylene glycol (PEG) 3,350 (w/v). Autoclave. 40% PEG in 0.1 M LiAcTE. Make fresh with each use by mixing 9 parts of 44% PEG with 1 part of the 10× LiAcTE stock solution just prior to use. TE, pH 7.5 10 mM Tris-HC1, pH 7.5, 0.5 mM EDTA. Autoclave. Z buffer 60 mM Na2HPO4 • 7 H:O, 40 mM NaH2PO4 • H20, 10 mM KCI, 1 mM MgSO4" H20. Add water to 1 liter. Ensure that the pH is 7.0. Add 2-mercaptoethanol, 270/zl/100 ml Z buffer, freshly prior to use. 9 D. Gietz, A. St. Jean, R. A. Woods, and R. H. Schiestl, Nucleic Acids. Res. 20, 1425 (1992).
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X-Gal solution 20 mg/ml X-Gal (5 bromo-4-chloroindolyl-/3-D-galactoside, GIBCO/ BRL, No. 5520UC). Dissolve in N,N-dimethylformamide. Store at - 2 0 ° in a glass container covered with foil. Z buffer/X-Gal solution Add 270/xl 2-mercaptoethanol and 1.67 ml X-Gal solution to 100 ml of Z buffer. Make up fresh solution prior to use. ONPG solution. 4 mg/ml of o-nitrophenyl-/3-o-galactopyranoside (ONPG, Sigma No. N-1127). Make up fresh in water. 0.1% (w/v) sodium dodecyl sulfate (SDS) 1 M Na2CO3 Yeast Transformation This protocol for high efficiency transformation in yeast is essentially an adaptation of the method by Gietz et aL9
A. Transformation of Y190 with pAS and pYTH6 Constructs The pYTH6 plasmid cannot replicate autonomously and therefore must be integrated into the yeast genome. To facilitate integration, the plasmid is linearized with XbaI at a unique site in the TRP1 gene prior to its introduction into yeast. 1. Inoculate one colony of Y190 in 5 ml YEPD and incubate in a shaker at 30° overnight. Dilute the overnight culture into 50 ml of YEPD in a 250-ml conical flask (use enough of the overnight culture to give an initial A600 of about 0.2). Incubate in a shaker at 30° until the A600 reaches about 0.7-1.0 (it usually takes 4-5 hr) and harvest the cells by centrifugation at 2000 rpm for 3 rain at room temperature. 2. Resuspend the cells in 20 ml of TE, pH 7.5, and repellet at 2000 rpm for 3 min at room temperature. 3. Resuspend the cells in 1 ml of 0.1 M LiAcTE and transfer the suspension to a 1.5-ml microfuge tube. Centrifuge at 13,000 rpm in a microfuge for 20 sec, repeat once, and finally resuspend cells in 0.5 ml of 0.1 M LiAcTE. 4. Add 20/xl carrier DNA to a microfuge tube followed by 0.5-1/xg of pAS or linearized pYTH6 containing RhoA or L63RhoA cDNA inserts. 5. Add 100/zl of the Y190 suspension and 700/.d of 40% PEG in 0.1 M LiAcTE. Mix carefully without vortexing and incubate the tubes at 30° for 30 min. 6. Heat-shock the transformation mix at 42° for 20 min during which time the tubes are inverted occasionally.
[25]
Rho ~NYEASTTWO-hYBRIDSYSTEM
235
7. Pellet the cells at full speed in a microcentrifuge for 20 sec. 8. Carefully remove the supernatant. Resuspend the cells in 100/xl TE, pH 7.5, and spread on SC-Trp plates. Incubate at 30°. 9. Monitor the plates for colonies. Colonies are usually visible after 3-4 days on SC-Trp plates. Pick several transformants and streak out on fresh SC-Trp plates. 10. Yeast strains expressing RhoA or L63RhoA can be kept on SCTrp plates at 4° for periods up to 1 month. In addition, Y190:pYTH6 RhoA and Y190:pYTH6 L63RhoA can be stored frozen as glycerol stocks for prolonged periods, whereas Y190:pAS strains are unsuitable for long-term storage since this could lead to an accumulation of yeast cells carrying reorganized pAS plasmids. For that reason we transform Y190 with pAS RhoA and pAS L63RhoA on a regular basis.
B. Transformation of Y190:pAS RhoA and Y190:pYTH6 RhoA Strains Protocol A is followed with the following modifications to the numbered steps: 1. SC-Trp is used instead of YEPD for growth and the amount of medium to be inoculated is adjusted for the number of transformations to be performed. We use approximately 10 ml of SC-Trp per transformation. 4. pACTII constructs (0.5-1 /xg) (e.g., pACTII Rho-GAP) are used to transform the RhoA-expressing cells. In addition, we also transform cells with the empty pACTII vector as a negative control. 8. The transformation mixtures are spread on 3-AT plates. 9. The growth on 3-AT plates is dependent on the strength of the interaction between the fusion proteins encoded by pAS/pYTH6 and pACTII. Colonies usually appear also on plates where no interaction between the pairs of fusion proteins occurs. However, there is a noticeable difference in growth rates between transformants that express genuinely interacting fusion proteins, where colonies appear after 2-4 days, and transformants expressing noninteracting fusion proteins, where colonies do not appear until after approximately a week. These latter colonies represent background growth and are not positive when analyzed for/3-galactosidase activity. The colonies on the 3-AT plates are analyzed for/3-galactosidase activity by the filter transfer and liquid culture assays (see below).
C. Simultaneous Transformation of Y190 with pAS and pA CTII Constructs Protocol A is followed with the following modifications to the numbered steps: 4. Add 20 /zl of carrier DNA along with 1-2 /xg each of pAS and pACTII constructs.
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CZLLEXPRESSION
[251
8. Spread cells on 3-AT plates. When transformants appear (see protocol B:8-9) they are analyzed for/3-galactosidase activity. Assays to Monitor Production of/3-Galactosidase The strength of the interaction between the two sets of fusion proteins can be determined by monitoring the/3-galactosidase produced from the lacZ reporter gene. This is done in two ways: (A) the yeast colonies on the plates are replica plated to filter papers, lysed, and stained with X-Gal in order to detect ]3-galactosidase activity;I° and (B) as a complement, the liquid culture assay is used to quantify the/3-galactosidase enzymatic activity by measuring the generation of the yellow compound o-nitrophenyl (ONP) from the colorless substrate o-nitrophenyl-/3-D-galactoside (ONPG). 11 A. Filter Transfer Assay 1. Add one filter disk (Whatman No. 1, 85 mm when using 90-mm petri dishes) to a clean petri dish, one for each transformation to be assayed. Add 2 ml of Z buffer/X-Gal solution and let it soak into the filter completely. 2. Replica plate the yeast cells onto another Whatman filter disc, then carefully remove the filter and drop it into a Styrofoam box containing liquid nitrogen. After 5-10 sec remove the frozen filter from the liquid nitrogen and allow it to thaw. Carefully overlay the filter onto the presoaked filter with the lysed cells facing upwards. Ensure that no air bubbles are trapped between the filters. 3. Incubate the petri dishes at 30° until a blue color develops. The time for this to occur varies, but the interaction between L63RhoA and R h o GAP is sufficiently strong for blue staining to develop within an hour. We usually incubate for 5 hr to allow weak interactions to be detected. If the petri dishes have to be incubated for longer periods, wrap them in Saran wrap to prevent the solution from drying out and to reduce the exposure of 2-mercaptoethanol to the laboratory environment. B. [3-Galactosidase Assay, Liquid Cultures 1. Grow cells overnight in 2 ml of selective medium (SC-Trp-Leu) at 30°. The following day, dilute the culture 5- to 10-fold in 5 ml of fresh medium and incubate further until the A600is approximately 1.0 (roughly 2-3 hr). Record the Z600 for 1-ml samples taken from each culture. 10L. Breeden and K. Nasmyth,Cold Spring Harbor Syrup. Quant. Biol. 50, 643 (1985). aaj. H. Miller, ed., in "Experimentsin MolecularGenetics."Cold Spring LaboratoryPress, Cold SpringHarbor, NY, 1972.
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2. Transfer three aliquots of 1 ml to 12 × 75-mm polypropylene tubes (Falcon, No. 2053; these tubes were chosen since they fit into the blue adapters for a Beckman JA M6 centrifuge) and pellet the cells by centrifugation. Discard the supernatants and add i ml of Z buffer to each tube. Prepare triplicate 1-ml aliquots of Z buffer without cells in order to determine the spontaneous rate of ONPG hydrolysis. Add 50 txl of CHC13 and 50/xl of 0.1% SDS to the tubes and vortex vigorously for 10 sec to resuspend and lyse the cells. Prewarm the samples to 30° for 5 rain and add 0.2 ml of ONPG solution to each tube. Mix the solutions by a quick vortexing and incubate the reactions at 30°. The time of incubation depends on the speed with which color develops but should take from 15 min to 6 hr. If color develops rapidly then the reaction should be stopped as depletion of the ONPG substrate may reduce the linearity of the assay. 3. Stop the reaction by the addition of 0.5 ml of i M Na2CO3 followed by a quick vortexing to mix. Centrifugate the samples to spin down the cell debris, and remove 1 ml of each sample to a disposable cuvette (take care not to disturb the cell debris that is found at the interface between the aqueous and the CHC13 layers). Determine the A420for each of the samples. 4. /3-Galactosidase activity is calculated using the following equation: Activity (in U) = 1000[(A420 -
Ablank)/(tWA600],
(1)
where A420 is absorbance at 420 nm of the sample; Ablank, mean A420 for triplicate blanks; t, time (min) of incubation; V, volume (ml) of initial cell aliquot; and A60o, cell density of the culture. Calculate the values for the mean/3-galactosidase activity for each culture from the triplicate determinations.
Results and Discussion Three sets of experiments are described to illustrate the limitations of the two-hybrid system and to standardize the assay procedure. (i) A comparison of the interaction of wild-type RhoA and L63RhoA to R h o GAP. In addition, the effect of using RhoA inserted in either pAS or pYTH6 is examined. (ii) Parameters such as cell density and the time of incubation are examined in the/3-galactosidase liquid culture assay. (iii) The affinities of L61Racl, L61G25K, and L63RhoA for R h o - G A P are compared. (i) Yeast strains harboring the pAS or pYTH6 plasmids carrying either wild-type RhoA or L63RhoA were transformed with pACTII Rho-GAP. The/3-galactosidase activity was thereafter monitored by the filter transfer and liquid culture assays following the protocol described earlier. The
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TABLE I INTERACTIONBETWEENGAL4DB FUSIONPROTEINSANDGAL4AD : Rho-GAP tNTHE Two-HYBRIDSYSTEM fl-Galactosidase activity (U)" GAL4DB fusion protein
Membrane transfer assayb
pAS L63RhoA pAS RhoA pAS pYTH6 L63RhoA pYTH6 RhoA pYTH6 pYTH6 L61Racl pYTH6 L61G25K
Blue White White Blue White White Blue Blue
Liquid culture assayC 6.7 (SD = 0.1 (SD = 0.2 (SD = 1.7 (SD = 0.1 (SD = 0.1 (SD = 2.4 (SD = 2.4 (SD =
1.6) 0.1) 0.02) 0.6) 0.04) 0.01) 0.7) 0.4)
"The assays for measuring/3-galactosidase activity were performed as described in the text./3-Galactosidase activity units (U) were calculated using Eq. (1). b The filter transfer assays were performed as described in the text. The filters were incubated for 5 hr to develop the color fully. cThe liquid culture measurements were performed in triplicates on three independent transformants. results are summarized in Table I. No interaction could be detected between R h o - G A P and wild-type R h o A using the two-hybrid system, whereas the R h o - G a p : L 6 3 R h o A interaction is readily detected (within 1 hr using the filter transfer assay). Independent measurements of binding affinities 12have found that the interaction between wild-type R h o A and R h o - G A P is in the order of 1 /xM, whereas that between L 6 3 R h o A and R h o - G A P is 10 nM. It appears that the yeast two-hybrid system is unable to detect p r o t e i n - p r o t e i n interactions in the micromolar range. We have found that the /3-galactosidase signal is stronger when L 6 3 R h o A is inserted into the pAS plasmid compared to p Y T H 6 (6.7 U for pAS L 6 3 R h o A compared to 1.7 U for p Y T H 6 L63RhoA). This difference is likely to reflect the difference in copies of the G A L 4 D B - L 6 3 R h o A fusion proteins in the yeast strains; pAS is a high copy-number, autonomously replicating, plasmid whereas p Y T H 6 integrates into the yeast genome as a single copy. Despite the lower values obtained with the p Y T H 6 L 6 3 R h o A , we have found distinct advantages in using this integrated plasmid. The background growth on 3-AT plates is much reduced using integrated sequences and, in addition, pAS L 6 3 R h o A gives rise to occasional blue colonies when combined with the empty p A C T I I vector. We now routinely use yeast strains containing integrated p Y T H 6 constructs. 12A. J. Self and A. Hall, [8] in this volume.
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(ii). In order to standardize the/3-galactosidase assay, the yeast strains containing either pYTH6 L63RhoA and an empty pACTII vector or pYTH6 L63RhoA and pACTII Rho-GAP were examined for ONPG hydrolysis with increasing cell densities (Fig. 3A) and at varying time points A 0.20 -
0.15 t"q
"~ 0.10< 0.05 -
0.00 Cell Density (Abs600) 3.0-
B
L~ 2.0-
8 1.0,A
o.o
,*"*'-"~-.¢-~,, time (min)
FIG. 3. Assays for measuring/3-galactosidase activity were performed as described in the text. (A) The mean ONPG hydrolysis (A420) is shown for increasing cell densities (A6oo) of three independent transformants harboring pYTH6 L63RhoA and pACTII R h o - G A P (D) or pYTH6 L63RhoA and empty pACT ( 0 ). (B) The mean fl-galactosidase activity at varying times is shown for three independent transformants harboring pYTH6 L63RhoA and pACTII R h o - G A P ([]) or pYTH6 L63RhoA and empty pACTII (0)./3-Galactosidase activity was calculated using Eq. (1).
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(Fig. 3B). The results indicate that ONPG hydrolysis in yeast cells with pYTH6 L63RhoA and pACTII R h o - G A P is linear for cell densities ranging from A600 of 0.05 to 1.25 (Fig. 3A). At the same cell densities, no significant ONPG hydrolysis was detected in yeast cells cotransformed with pYTH6 L63RhoA and empty pACTII. Incubation time was, however, important (Fig. 3B); with 15 min incubation the/3-galactosidase activity was 2.8 U but this dropped to 1.9 U after 3 hr. The most likely explanation for this effect is that the amount of active/3-galactosidase protein diminishes during the time of incubation. In conclusion, although we use a standard initial cell density of A600 of 1.0, this is not critical since ONPG hydrolysis is linear over a broad range of initial cell densities. The time of incubation does alter the observed rates of/3-galactosidase activity, and this should be standardized. We routinely use 1-hr incubations. (iii) We have compared the affinity of R h o - G A P for two additional members of the Rho family, Racl and G25K, to that of RhoA. In this case L61Racl and L61G25K were transformed into Y190 using the pYTH6 vector, pACTII R h o - G A P was subsequently introduced into these strains. L61Racl and L61G25K both interact with R h o - G A P with similar strength to L63RhoA. This is consistent with the observation made by Lancaster et aL 13 that the affinities of R h o - G A P for wild-type RhoA, Racl, and G25K were similar.
Conclusions For the two-hybrid system to be successful it is vital that the fusion proteins enter the yeast nucleus, otherwise they are unable to function as transcription activators. For a correct interpretation of a negative result it is essential to establish that the proteins of interest are expressed. This can be done by analyzing yeast cell extracts by Western blotting using antibodies against the protein of interest. Alternatively, the hemagglutinin epitope (HA) present on the pAS vector can be used as a tag to detect protein production using an anti-HA antibody. The yeast two-hybrid system has been used successfully to detect interactions between small GTPases and their target proteins. In particular, a ras effector, c-raf, was identified in this way.3 This chapter described the use of RhoA, Racl, and G25K in the two-hybrid system and showed that each is capable of interacting with Rho-GAP. Our experiments suggest that the strength of interaction must be in the nanomolar range for detection. We are currently using the strains described in this chapter to screen cDNA 13 A. C. Lancaster, P. M. Taylor-Harris, A. J. Self, S. Brill, H. E. van Erp, and A. Hall, J. Biol. Chem. 269, 1137 (1994).
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libraries fused to the GAL4AD to look for novel proteins interacting with the Rho family of GTPases. Acknowledgments This work was supported by grants from the Commission of European Communities, Human Capital and Mobility Programme to P. A. and Alan Hall, and from the Cancer Research Campaign to M. F. O. and Alan Hall. P. A. was supported by a Wellcome-Swedish Travelling Research Fellowship. M. F. O. was supported by a fellowship from the Natural Sciences and Engineering Research Council of Canada. We are very grateful to Robin Brown, Glaxo, UK, for advise during the course of this work. We thank Alan Hall for critically reading this manuscript.
[26]
Assay for Rho-Dependent Phosphoinositide 3-Kinase Activity in Platelet Cytosol
By SUSAN ERIKA
RITTENHOUSE
Introduction In addition to being important in their own right as cells crucial to blood coagulation, platelets have proved to be a useful model system for the study of stimulus-induced cytoskeletal reorganization and phosphoinositide turnover. 1'2 An important physiological function of platelets is aggregation. Such aggregation is dependent on the formation of the active conformation of the integrin Odlib~3, whose major ligand is fibrinogen, which is present in the blood and is also released from activated platelets. Fibrinogen-bound integrin is linked to the cytoskeleton in a complex that also contains talin and vinculin, as well as polymerized actin, the classic components of socalled "focal adhesions". 3 A major goal in studies of these phenomena is the elucidation of the mechanism(s) by which integrin becomes "activated" and the cytoskeleton becomes reorganized. Functional Rho appears to be required for focal adhesion formation in fibroblasts, 4 and inactivation of Rho by ADP-ribosylation inhibits platelet aggregation. 5 IS. E. Rittenhouse, in "The Platelet: Advances in Molecular and Cell Biology" (E. G. Lapetina, ed.). JAI Press, Greenwich, CT, in press. 2 S. E. Rittenhouse, Sem. Hematol. (in press). 3 j. E. B. Fox, Thromb. Haemost. 70, 884 (1993). 4 A. J. Ridley and A. Hall, Cell 70, 389 (1992). 5 N. Morii, T. Teru-uchi, T. Tominaga, N. Kumagai, S. Kozaki, F. Ushikubi, and S. Narumiya, J. Biol. Chem. 267, 20921 (1992).
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Phosphoinositide metabolism in platelets exposed to a variety of physiological agonists is dependent on GTP-binding proteins. Intact platelets incubated with the physiological agonist, thrombin, or permeabilized platelets exposed to the nonhydrolyzable G T P analog, GTPyS, undergo an activation of phosphoinositide 3-kinase (PI 3-K6), which generates phosphatidylinositol 3,4,5-trisphosphate [Ptdlns(3,4,5)P3] by phosphorylation of Ptdlns(4,5)P2 at the 3-OH position of the inositol ring] Activated PI 3-K associates rapidly with the cytoskeleton of thrombin-stimulated platelets, 8 as does Rho. 9 Since it has been suggested that the PI 3-K product(s) may be causally involved in the modifications of the cytoskeleton that accompany mitogenic and chemotactic stimulation l°'u and platelet aggregation, 8 we have studied whether Rho and PI 3-K are functionally linked. 9 We have found it possible to stimulate PI 3-K activity in platelet cytosolic fractions with GTPyS. The activation is inhibited by prior incubation of cytosol with ADP-ribosylating enzymes for Rho, i.e., C3 transferase 9 or EDIN, 12 and is overcome by exogenous recombinant Rho 9 or R h o - G S T (glutathione S-transferase) fusion protein. 12Similar effects are achieved using permeabilized, stimulated platelets. 12 Thus, a significant part of PI 3-K activation in platelets appears to be dependent on active Rho. P r e p a r a t i o n of Platelet Cytosol One unit (approximately 450 ml} of fresh blood is collected in N I H citric acid/citrate/dextrose (Baxter, Fenwal USP bag} anticoagulant. Platelet-rich plasma (PRP} is obtained by centrifugation at 2900 rpm for 4 min at 25 ° (GH-3.8 horizontal rotor, Beckman GS-6 centrifuge} and transfer of the supernatant plasma (avoiding erythrocytes and '¢buffy coat" interface} to a satellite bag (Baxter, Penwal). Plastic or siliconized glassware is used for all platelet manipulations, which are performed at room temperature. Prostaglandin E1 (0.5/xM) and acetylsalicylic acid (1 raM) are then added to minimize platelet activation during washing. The former transiently elevates 6 G. L. Kucera and S. E. Rittenhouse, J. Biol. Chem. 265, 5345 (1990). 7 A. N. Carter, R. Huang, A. Sorisky, C. P. Downes, and S. E. Rittenhouse, Biochem. J. (in press). 8j. Zhang, M. J. Fry, M. D. Waterfield,S. Jaken, L. Liao, J. E. B. Fox, and S. E. Rittenhouse, J. Biol. Chem. 267, 4686 (1992). 9j. Zhang, W. G. King, S. Dillon, A. Hall, L. Feig, and S. E. Rittenhouse, J. Biol. Chem. 268, 22251 (1993). 10C. P. Downes and A. N. Carter, Cell. Signal. 3, 501 (1991). 1i M. Eberle, A. E. Traynor-Kaplan, L. A. Sklar, and T. Norgauer, Z Biol. Chem. 265, 16725 (1990). 12j. Zhang, J. Zhang, J. L. Benovic, M. Sugai, R. Wetzker, I. Gout, and S. E. Rinenhouse, J. Biol. Chem. 270, 6589 (1995).
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cyclic AMP, which inhibits platelet activation, and the latter inhibits thromboxane A2 (a platelet agonist) formation. After 20 min, PRP is recentrifuged in 30-ml portions at 3000 rpm (823gay) for 45 sec (SS-34 rotor, Sorvall RC5B) to decrease any erythrocyte and leukocyte contamination to <0.1%. PRP is spun at 4500 rpm (1872gay) for 90 sec and platelet-poor plasma is removed. The pellets (any traces of erythrocytes are avoided) are resuspended gently in 5 ml of a modified Tyrode's buffer containing 132 mM NaC1, 12.2 mM trisodium citrate, 5.6 mM dextrose, 2.0 mM HEPES, 10 mM Trizma, 2.8 mM KC1, 8.9 mM NaHCO3, 0.86 mM MgC12, 13 mM creatine phosphate, 0.36 mg/ml creatine phosphokinase (to remove platelet-activating ADP), pH 6.5, and recentrifuged as above. Pellets are resuspended gently in the above buffer, without creatine phosphate/creatine phosphokinase, pH 7.3, and recentrifuged. The washed platelets are then suspended to 101° platelets/ml in hypotonic buffer containing 10 mM HEPES, 1 mM EGTA, 1 mM sodium pyrophosphate, 2 mM sodium vanadate, 0.2 mM leupeptin, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 5/zg/ml antipain, and 50 ~M dithiothreitol (DTT), pH 7.0. Platelets are disrupted by microprobe sonication, 4 × 10s (the first sonication at room temperature, since cold temperatures promote platelet shape change; the remainder on ice) at intensity setting 3 (Heat Systems Ultrasonics Sonicator W-375). Supernatants are obtained after centrifugation of platelet lysates at 100,000g for 90 min at 4 ° and are stored as aliquots at - 8 0 °. Quantitation of Rho by ADP-Ribosylation Platelet supernatants (3 mg/ml) are incubated in covered Eppendorf tubes at 37 ° in a mixture containing 0-40/xg/ml recombinant C3 transferase 13 (UBI), 200/xM fl-NAD +, 20/xCi/ml [a-32p]~-NAD+, 10 mM thymidine, 12 mM HEPES, 5 mM DTT, 5 mM MgC12, pH 8.0, for 5-30 rain. Incubations are terminated with 0.5 vol of 3× concentrated Laemmli 14 sample buffer (reducing) and boiled for 5 min. After samples have cooled to room temperature, they are spun in a microfuge (13,000g for 10 sec) to bring down condensate and are vortexed. Aliquots are applied to 12.5% SDS-polyacrylamide reducing gels, as are stained standards, and electrophoresed. After vacuum drying of the gel on paper, [3ap]ADP-ribosylated Rho (which migrates slightly more slowly than the approximately 21-kDa free Rho, as detected by Western blotting) is located by autoradiography, cut out, and counted in a scintillation counter. Aliquots of the original mixture applied to the gels are also counted. Based on the specific activity 13 S. T. Dillon and L. A. Feig, [20] this volume. 14 U. K. Laemmli, Nature 227, 680 (1970).
244
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[261
of [ot-3zP]fl-NAD+ in the mixture, the amount of ADP-ribosylated Rho can be calculated. We have found that, either at 5 or at 30 min, the amount of Rho that is ADP-ribosylated by 24 txg/ml C3 transferase is approximately 90 pmol/mg protein. This is higher than the published amount, 9 which is attributable to the fact that the current procedure utilizes a higher concentration of/3-NAD + than used previously, rendering the variable contribution of platelet supernatant NAD + relatively insignificant. Therefore, the estimated specific activity of [32p]NAD+ is more accurate. Assay of PI 3-Kinase Platelet supernatants are incubated for 5 min in the above mixture, lacking [o~-32P]/3-NAD+, in the presence and absence of fl-NAD +. Incubation mixtures are then added to a P1 3-kinase assay mix, such that the final composition is 1 mg/ml supernatant protein, 80 mM KCI, 2.7 mM MgCI2, 16.7 mM NaC1, 0.7 mM NaH2PO4,0.7 mM EDTA, 10 mM HEPES, 138/~M CaC12, 417 /~M ATP, 100 ~Ci/ml [y-3Zp]ATP (prepared as described15), 8.3/~M DTT, 167/~M sodium pyrophosphate, 33.3/~M leupeptin, 1/xg/ml antipain, 16.6 /xM PMSF, and 100 ~g/ml substrate, in the presence or absence of 5/~M GTPyS or 200 pmol/ml recombinant Rho or R h o - G S T fusion protein, 16 pH 7.1, and incubated for 5 min at 37°. It is important that recombinant protein be washed free of any detergent such as Triton X-100, which is highly inhibitory for PI 3-K activity. 8 Lipid substrates consist of PtdIns, PtdIns4P, or PtdIns(4,5)P2, in an equimolar mix with PtdSer. Prior to assay, chloroform solutions of these lipids are dried down under N2 flow and lipids are suspended via sonication in 5 mM HEPES, pH 7.3. Assays (120/~1) are terminated with 400/~1 of cold CHC13/MeOH/1 N HC1 (1 : 2.5 : 0.25, v/v), followed by 50/~1 cold 1 N HCI and 100/~1 cold CHCI3. The CHC13 phase (lower) is removed and dried under N2 flow or speed vac concentration. The dried lipids are deacylated as described 17by incubation in 3.0 ml 25% methylamine/methanol/n-butanol(58 : 62 : 15, v/v) at 53 ° for 50 min and dried. The resulting glycerophosphoinositol phosphates are dissolved in 1 ml H20 and are washed twice (discarding upper phase) with 1.2 ml n-butanol/petroleum ether/ethyl formate (20 : 4 : 1) to remove fatty acids. The aqueous phase is removed and dried. Glycerophosphoinositol (GroPIns) phosphates are dissolved in H20 for injection onto a 25-cm Whatman Partisphere SAX column in a HPLC system pumping H20 (A) vs 1.25 M ammonium phosphate, pH 3.1, titrated with H3PO4 (B). Fractions are collected and counted, or an in-line scintillation counter is employed. 15 T. F. Walseth and R. A. Johnson, Biochim. Biophys. Acta 562, 11 (1979). 16 A. Self and A. Hall, [1] this volume. 17 M. W h i t m a n , C. P. Downes, M. Keeler, T. Keller, and L. Cantley, Nature 332, 644 (1988).
[26]
Rho AND PI 3-KINASEACTIVITY
245
TABLE I H P L C GRADIENT SCHEMEa
Time (rain)
%A
%B
0 10 60 120 125 130 131 145
100 100 80 40 0 0 100 100
0 0 20 60 100 100 0 0
a See text for description.
For pumping at 1 ml/min the ratios given in Table I are used. Under these conditions, retention times for the various glycerophosphoinositolcontaining labeled products (these will vary with the column) are shown in Table II. Other possible contributors [such as ATP, Ins(1,4,5)P3, Ins(1,4)P2, and Pi] should be ruled out with appropriate standards. Standards for the 3-phosphorylated phosphoinositides can be generated using purified PI 3-K, a tyrosine phosphate-directed immunoprecipitate derived from the activated platelet-derived growth factor receptor from fibroblasts or an immunoprecipitate of platelet PI 3-K, obtained using an antibody directed to the 85-kDa subunit of this enzyme. 12 We have observed that, under conditions of maximum ADP-ribosylation, a maximum inhibition of PI 3-K of 75-80% can be achieved. 9 Failure to inhibit completely may be due to incomplete (albeit the maximum achievable) ADP-ribosylation of functional Rho or to the presence of another GTPyS-stimulatable PI 3-K not dependent on Rho. Such an activity might, for example, be triggered by newly generated/37 subunits from a solubilized heterotrimeric GTP-binding protein(s), a phenomenon described recently T A B L E II RETENTION TIMES FOR GLYCEROPHOSPHOINOS1TOL PHOSPHATES
Product
Retention (min)
GroPIns(3)P GroPIns(3,4)P2 GroPIns(3,4,5)P3 GroPlns(4)P GroPIns(4,5)P2
30 55-57 82 34 59-61
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CELLEXPRESSION
[27]
for neutrophils. TM Inhibition is prevented when recombinant Rho is added simultaneously with GTP'yS. Thus, Rho can "rescue" PI 3-K activity, and does not have to be post-translationally modified to do so since the recombinant Rho used is expressed in E. coil The platelet supernatant described here is a complex system, and although it might be tempting to assume a direct interaction between PI 3-K and Rho, based on the Rho-GAP-like domain present in the 85-kDa subunit of the PI 3-K heterodimer, 19 such interaction is by no means proven by the results described. 18 L. Stephens, A. Smrcka, F. T. Cooke, T. R. Jackson, P. C. Sternweis, and P. T. Hawkins, Cell 77, 83 (1994). 19M. Otsu, I. Hiles, I. Gout, M. J. Fry, F. Ruiz-Larrea, G. Panayoutou, A. Thompson, R. Dhand, J. Hsuan, N. Totty, A. D. Smith, S. J. Morgan, S. A. Courtneidge, P. Parker, and M. D. Waterfield, Cell 65, 91 (1991).
[27] N e u t r o p h i l P h o s p h o l i p a s e D: I n h i b i t i o n b y R h o - G D P Dissociation Inhibitor and Stimulation by Small GTPase GDP Dissociation Stimulator B y EDWARD P. BOWMAN, DAVID J. UHLINGER, a n d J. DAVID LAMBETH
Introduction Neutrophils contain a phospholipase D that is activated by receptorcoupled agonists such as f-Met-Leu-Phe and protein kinase C activators such as phorbol esters. 1 Phospholipase D catalyzes the hydrolysis of phospholipids into phosphatidic acid, and has been implicated in signal transduction in the neutrophil as well as in a variety of other cells. 2 In broken cell preparations, neutrophil phospholipase D can also be activated by guanosine 5'-O-(3-thiotriphosphate) (GTPTS) and by phorbol esters. 3'4 Activity can be reconstituted by combining cytosol with plasma membrane, and protein factors in both fractions are implicated as phospholipase Drelated components. 3,4 Until recently, it was not clear whether the GTP1 D. E. Agwu, L. C. McPhail, M. C. Chabot, L. W. Daniel, R. L. Wykle, and C. E. McCall, J. Biol. Chem. 264, 1405 (1989). 2 j. D. Lambeth, in "Protein Kinase C" (J. F. Kuo, ed.), p. 121. Oxford University Press, New York, 1994. 3 S. C. Olson, E. P. Bowman, and J. D. Lambeth, J. Biol. Chem. 266, 17236 (1991). 4 j. C. Anthes, P. Wang, M. I. Siegel, R. W. Egan, and M. M. Billah, Biochem. Biophys. Res. Commun. 175, 236 (1991).
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binding/regulatory species was a classical heterotrimeric G protein or whether it was a member of the Ras family of small GTPases. Studies 5 using the inhibitory and stimulatory guanine nucleotide exchange factors R h o - G D I (GDP dissociation inhibitor) and smgGDS (small GTPase GDP dissociation stimulator) have implicated a small molecular weight GTPbinding protein, probably in the Rho subfamily, as the GTP-binding component that regulates phospholipase D activity. Herein, we document the methodologies used to assay phospholipase D and to prepare and use R h o GDI and smgGDS to investigate the identity of the GTP-binding species. Isolation of H u m a n Granulocytes Neutrophils are isolated based on a procedure by Pember et al. 6 Blood (450 ml) is obtained by venapuncture in a 600-ml transfer pack container (Baxter Healthcare Corp., Deerfield, IL) containing 10 ml of 0.1 M EDTA, pH 7.4/0.9% NaCI. Blood is diluted with 180 ml Hespan (Du Pont Pharmaceuticals, Wilmington, DE) and is allowed to settle in plastic cylinders for 1 hr at room temperature. At no time during the preparation are cells allowed to come into contact with glass. The plasma layer containing granulocytes is isolated and centrifuged in 50-ml polypropylene centrifuge tubes (Corning Incorp., Corning, NY) at 850g for 15 rain at 4 °. The cell pellet is resuspended in 20 ml of buffer A (8.0 mM NaePO4/1.5 mM KH2PO4/ 136 mM NaCI/2.6 mM KC1/0.5 mM MgCI2/0.6 mM CaCI2, pH 7.4), diluted with 60 ml deionized water for 70 sec, and brought to isoosmolarity by the addition of 20 ml of 4% (w/v) NaC1. This procedure lyses any residual red blood cells. Cells are centrifuged (850g for 10 min at 4 °) and resuspended in 20 ml of buffer A containing 5.5 mM glucose. Lymphocyte separation medium (10 ml, Organon Teknika, Durham, NC) is layered under the cell suspension and centrifuged (850g for 30 min at 4°). Cells at the interface are discarded, and the cell pellet is resuspended in buffer B (25 mM HEPES, pH 7.4, 125 mM NaC1, 0.7 mM MgC12, 0.5 mM EDTA) containing 11 mM glucose and 1 mg/ml crystallized bovine plasma albumin (Intergen Company, Purchase, NY) to a final concentration of 2 × 107 cells/ml. Labeling of Cells The transphosphatidylation assay for phospholipase D activity described below requires prior labeling of the phosphatidylcholine pool and utilizes 5E. P. Bowman, D. J. Uhlinger, and J. D. Lambeth, J. Biol. Chem. 268, 21509 (1993). 6S. O. Pember, R. Shapira, and J. M. Kinkade, Jr., Arch. Biochem. Biophys. 221, 391 (1983). 7F. H. Chilton, J. T. O'Flaherty, J. M. Ellis, C. L. Swendsen,and R. L. Wykle,J. Biol. Chem. 258, 7268 (1983).
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a variant of the methodologies of Chilton et al. 7 and Billah et al. 8 1 - O Alkyl-2-1ysophosphatidylcholine is added to the cell preparation and is acylated by the cells to form labeled phosphatidylcholine. In our hands, approximately 70% of the added lipid is taken up by the cells, and essentially all of this is acylated, as judged by thin-layer chromatography (TLC). 1-O[3H]octadecyllyso-platelet-activating factor [Amersham Corp., Arlington Heights, IL; 80-180 mCi/mmol in toluene/ethanol (1 : 1) solution] is dried in a glass tube under a stream of nitrogen. The dried lipid is resuspended by adding I ml of buffer B containing bovine plasma albumin and vortexing the tube for 20 sec. The radiolabeled lipid-containing solution is added to neutrophils (2-3 × 109 cells in 30 ml of buffer B), and the lipid resuspension procedure is repeated six more times. The cells are diluted to a final concentration of 2 × 107 cells/ml which gives a final specific activity of 1.5/zCi/ ml. Neutrophils are then incubated at 37 ° for 90 min in a slowly shaking water bath (50-ml tubes, each containing 30-40 ml of cells, were laid parallel to the direction of shaking). DNase I (50 U/ml, final concentration) (Type II from bovine pancreas, 2700 U/mg solid, Sigma Chemical Co., St. Louis, MO) is included to decrease the clumping of cells during the labeling period.
Isolation of Membrane and Cytosolic Fractions Following labeling, cells are centrifuged (850g for 5 min at 4°), resuspended in 10 ml of buffer B, and incubated with 4.6 mM final diisopropyl fluorophosphate (DFP, 5.8 M stock concentration. Sigma Chemical Co.) for 20 min at 0 °. The latter is added under a well-functioning fume hood. Because of the potential danger of this agent, DFP is used only when other personnel are present, and a syringe of atropine (0.4 mg/ml, Kendall McGaw Lab., Irvine, CA) is kept handy at all times in case of an accident. Residual DFP is inactivated by washing the syringe sequentially with 1 M NaOH, deionized water, and finally acetone. Pretreatment of neutrophils with the cell-permeant DFP is essential to limit proteolysis on breakage of cells and granules. 9 Following DFP treatment, ceils are immediately centrifuged (850g for 5 rain at 4 °) and resuspended in 10 ml buffer C (25 mM triethanolamine, pH 7.4, 100 mM KC1, 5 mM MgC12,3 mM NaCI) containing protease inhibitors [2/xM leupeptin (Sigma Chemical Co.), 2/xM pepstatin A (Sigma Chemical Co.), 0.5 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Chemical Co.), and 1/xg/ml aprotinin (Sigma Chemical Co.)]. Cells are broken by nitrogen cavitation using a Parr cell disruption 8M. M. Billah, S. Eckel, T. J. Mullmann, R. W. Egan, and M. I. Siegel,J. Biol. Chem. 264, 17069 (1989). 9p. C. Amrein and T. P. Stossel, Blood 56, 442 (1980).
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249
bomb (Parr Instrument Co., Moline, IL). Cells are pressurized at 500 psi for 20 min at 4° with agitation of the suspension every 10 min. After 20 min, the cells are broken by slowly releasing the chamber's contents into a 50-ml centrifuge tube. The lysate is centrifuged at 850g for 5 min at 4° to remove unbroken cells and nuclei, and the supernatant is layered over discontinuous sucrose gradients (5 ml per gradient) composed of 3 ml each of 50% (w/v) and 30% (w/v) sucrose dissolved in buffer C containing protease inhibitors, according to Markert et aL a° Gradients are centrifuged in a Beckman L8-80M ultracentrifuge at 4° for 90 min (209,000g) using a Beckman SW-41 Ti rotor. The "cytosolic fraction" is isolated from the upper layer of the gradient and is centrifuged for 90 rain (347,000g) at 4° in a Beckman 70.1 Ti rotor. The protein content of this fraction is typically in the range of 1 to 2 mg/ml, and the typical preparation yields 14 ml. Membranes at the 30%/50% interface are collected, diluted with buffer C, and centrifuged for 90 min at 4° (346,000g) in a Beckman TLA-100.3 rotor. The membrane pellet is resuspended in 1.5 ml of buffer C using a glass homogenizer, and the final protein concentration is approximately 2 rag/ ml. The membrane fraction has a specific activity of approximately 150,000 dpm of tritium/25 tzg membrane protein. Cytosol and plasma membrane fractions are used immediately or are kept on ice overnight and used on the day after isolation. No change in GTPyS-stimulated phospholipase D activity is observed over this period. Plasma membrane activity can be stored for longer times (1-2 weeks) by diluting the membrane fraction 1 : 1 (v/v) with 30% sucrose in buffer C before freezing on dry ice and storage at - 8 0 °. Cytosolic activity is stable for long periods of time (>4 months) by freezing on dry ice and storage at - 8 0 °. Phospholipase D Assay A variety of assays are available for measuring phospholipase D activity, which include formation of labeled phosphatidic acid, release of a labeled choline headgroup, and transphosphatidylation. Phosphatidic acid is subject to further metabolism by phosphatidic acid phosphohydrolase and by phospholipases A, and can also be formed by other metabolic routes. Hence, at least in crude systems, it is an unreliable quantitative marker for phospholipase D activity. Headgroup release can be used, but requires separation of choline from choline phosphate to differentiate phospholipase D from phospholipase C activities. We have utilized primarily transphosphatidylation as a more quantitative indicator of phospholipase D activity. In the presence of primary alcohols, phospholipase D catalyzes the transfer of the phosphatil0 M. Markert, G. A. Glass, and B. M. Babior, Proc. Natl. Acad. Sci. U.S.A. 82, 3144 (1985).
250
CELLEXPRESSION
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date group from phosphatidylcholine to ethanol to generate phosphatidylethanol, la Transphosphatidylation, instead of hydrolysis, by the neutrophil phospholipase D occurs nearly exclusively in the presence of 1.6% (v/v) ethanol, resulting in the production of phosphatidylethanol. The latter has a distinct migration by thin-layer chromatography and is therefore readily separated from the parent lipid and other potential metabolites. The assay is based on the procedure of Olson et al. 3 with minor modifications. Plasma membrane (100/zg/ml) and cytosol (200/zg/ml) in 0.25 ml of buffer C containing 1/xM CaC12 and 1.6% ethanol are incubated at 37° in a shaking water bath for 20-25 min. Either GTPyS (10/xM) or GTP (100/xM) is added. Incubations are terminated, typically after 20 min, by transfer of the reaction mixture to 1.5 ml chloroform/methanol (1 : 2, v/v), with mixing by vortexing for 5 sec. Phases are separated by the addition of 0.5 ml CHC13 and 0.5 ml 2% acetic acid. 12 Samples are vortexed and then centrifuged (850g for 2 min) to separate the layers. The organic lower layer is removed, dried using a SpeedVac concentrator (Savant Instruments Inc., Farmingdale, NY), and dissolved in 30/zl chloroform/methanol (95:5, v/v). Samples are spotted with capillary tubes onto silica gel 60 thin-layer chromatography plates (EM Science, Gibbstown, N J), and plates are developed using chloroform/methanol/concentrated acetic acid (90 : 10 : 10, v/v/v). Radioactivity is quantitated using a System 200 Imaging Scanner (Bioscan Inc., Washington, D.C.) equipped with two-dimensional image analysis software (Version 2.49). A typical two dimensional scan of a TLC plate is shown in Fig. 1. The phosphatidylethanol spot (PEth, Rf 0.62) is well separated from other labeled lipids and comigrates with an authentic phosphatidylethanol standard (Avanti Polar Lipids, Inc., Alabaster, AL). Notice that the GTPyS addition (Fig. 1, right lane) stimulates phospholipase D activity and increases the amount of label in the phosphatidylethanol spot compared to unstimulated activity (Fig. 1, left lane). Activity is expressed as the percentage conversion of label to phosphatidylethanol, or [(counts in PEth spot - counts in background spot)/E(counts in each spot - counts in background spot)] × 100%. Expression and Purification of Rho-GDI The cDNA for R h o - G D I is obtained by polymerase chain reaction (PCR) amplification from a human B-cell cDNA library using the N-terminal amplimer 5 ' - C G T G G A T C C A T G G C T G A G C A G G A G C C C and the C11 M. Liscovitch, Biochem. Soc. Trans. 19, 402 (1991). lz E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).
[27]
PHOSPHOLIPASE D, Rho GDI, AND smgGDS
=-
251
DG
tpdl ~'- " I 1 ~
=~=
I I GTP~
-
PEth
PA
PC +
FIc. 1. Typical thin-layer chromatography plate scan from System 200 Imaging Scanner. Plasma membranes (25/zg), cytosol (50/zg), 1/zM CaC12, and 1.6% ethanol were incubated in the absence (left lane) or presence of 10/zM GTPyS (right lane). Reactions were terminated, extracted, and spotted on TLC plates as described. The plates were eluted with chloroform/ methanol/concentrated acetic acid (90 : 10 : 10, v/v/v) and quantitated as described. Nonlabeled standards were spotted and chromatographed to identify the labeled spots (PC, phosphatidylcholine; PA, phosphatidic acid; PEth, phosphatidylethanol; DG, diacylglycerol).
terminal amplimer 5'-GATGAATTCTCAGTCCTTCCACTCCTTCTTG. PCR is carried out according to standard protocols 13 and permits the introduction of a 5'-BamHI restriction site and a 3'-EcoRI restriction site. Following restriction digestion, the DNA is directionally inserted into the corresponding restriction sites of the pGEX-2T vector (Pharmacia LKB Biotechnology Inc. Uppsala, Sweden), as detailed elsewhere. 13 The vector allows in-frame placement of the R h o - G D I coding sequence downstream of a region encoding a portion of glutathione S-transferase, with the do13 "Current Protocols in Molecular Biology" (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Seidman, J. G., and Struhl, K., eds.), John Wiley and Sons, New York 1994.
252
CELLEXPRESSION
[27]
mains separated by a thrombin site. The pGEX-2T vector containing R h o GDI is available from the authors on request. The recombinant plasmid is used to transfect competent DH5-a or BL21 E. coli cells, and cells are plated on LB plates containing 50 /zg/ml ampicillin. 13 Ampicillin-resistant colonies are picked and grown overnight in 3 ml of LB 13 containing 100/xg/ml ampicillin (LB-Amp). Fifty milliliters of LB-Amp is inoculated with 1 ml of the freshly saturated culture and grown overnight. The freshly saturated 50-ml culture is used to innoculate 1 liter of LB-Amp. One-liter cultures are grown at 37 ° to an A600 of 0.4, induced with 0.1 mM isopropylthiogalactopyranoside (IPTG) for 6 hr, and harvested by centrifugation in a Sorvall H600A rotor (4700g for 20 min at 4°). Cells are resuspended in 8% sucrose, buffer D (137 mM NaC1, 2.7 mM KC1, 4.3 mM NazPO4, 1.4 mM KH2PO4 pH 7.3), 1 mM EDTA, 1% Triton X-100, 0.5 mM PMSF, and 2 mM dithiothreitol (30 ml/liter culture). Cells (30 ml) are sonicated (1 min/0 °) on a power setting of 7 using a Sonicator Ultrasonic Processor XL (Heat Systems-Ultrasonics, Inc., Farmingdale, NY). The lysate is centrifuged at 27,000g for 15 min at 4 ° in a Beckman JA20 rotor. The resulting supernatant is transferred to a 50-ml polypropylene centrifuge tube (Corning Inc., Corning, NY) and is mixed with hydrated glutathione-agarose beads (0.5 ml hydrated beads/liter culture, 10/xmol glutathione/ml gel. Sigma Chemical Co.) preequilibrated with buffer D containing 1% Triton X-100 and 2 mM dithiothreitol. The fusion protein is allowed to bind to the support overnight with mixing on a rotating wheel at 4 °. Beads with bound R h o - G D I are pelleted by centrifugation (850g for 2 min at 22 °) and the solution is discarded. The beads are washed by transferring them to a 15-ml polystyrene centrifuge tube (Corning Inc.) and diluting them with a 10-fold excess (v/v) of buffer D/1% Triton X100/2 mM dithiothreitol. The beads are mixed on a rotating wheel (5 min at 22°), pelleted by centrifugation (850g for 2 rain), and the supernatant containing unbound protein is discarded. The procedure is repeated once more. After the second wash, beads are suspended in a 10-fold excess (v/ v) of thrombin cleavage buffer (TCB buffer: 50 mM Tris, pH 7.5, 150 mM NaC1, 2.5 mM CaC12, 2 mM dithiothreitol). Beads are mixed on a rotating wheel (5 min at 22°), pelleted by centrifugation (850g for 2 min), and the supernatant is discarded. The wash procedure is repeated six more times using TC buffer to remove all traces of detergent. The beads are suspended with 1.5 ml TC buffer, transferred to a 2.0-ml polypropylene microfuge tube, and incubated with thrombin (25 NIH units/0.5-ml beads, from human plasma, 1160 NIH units/mg protein, Sigma Chemical Co.) for 90 min at 22 °. Beads are pelleted by centrifugation (15,000g for 10 sec at 22°), and the supernatant is treated with benzamidine-Sepharose 6B beads (10 min at 22 °, 30/zl slurry of beads/liter culture; Pharmacia LKB Biotechnology
[271
PHOSPHOLIPASE D, Rho GDI, AND smgGDS
253
Inc., Uppsala, Sweden) to inactivate and remove thrombin. Beads with bound thrombin are removed by centrifugation (15,000g for 10 sec at 22°). The supernatant is concentrated (Centricon 10 miniconcentrators, W. R. Grace & Co., Beverly, MA) and diluted 10-fold with buffer C followed by reconcentrating twice to decrease the calcium concentration. Concentrated R h o - G D I is >90% pure by Coomassie-stained SDS-polyacrylamide gel electrophoresis and is stored frozen at -80 °. Approximately 0.5 mg of R h o - G D I is obtained per liter of E. coli culture. Expression and Purification of smgGDS The cDNA for smgGDS is obtained by PCR amplification from a human brain cDNA library using the N-terminal amplimer 5'-AAACTCG A G C A T G G A T A A T C T C A G T G A T and the C-terminal amplimer 5'-GGGAAGCTTTTCAGCTTTCCACAGTAA. PCR is carried out according to standard protocols 13 and permits the introduction of a 5'-XhoI restriction site and a 3'-HindIII restriction site. Following restriction digestion, the 1.7-kb cDNA is directionally inserted into the corresponding restriction sites of the pTrcHis B vector (Invitrogen Corp., San Diego, CA), as detailed elsewhereJ 3 The vector allows in-frame placement of the smgGDS coding sequence downstream of a region encoding six histidines. The pTrcHis B vector containing smgGDS is available upon request. DH5a E. coli ceils are transfected with the recombinant plasmid, arnpicillin-resistant colonies are selected, and cultures are grown as described earlier for R h o - G D I expression. Cells are induced with IPTG, harvested, and resuspended as described earlier except that 2 mM 2-mercaptoethanol is substituted for dithiothreitol and EDTA is omitted. Suspended cells are sonicated, and the lysate is centrifuged as described earlier. The supernatant is mixed with Probond resin (0.25 ml resin/liter culture, Invitrogen Corp.) which had been preequilibrated with buffer D containing 1% Triton X-100 and 2 mM 2-mercaptoethanol and allowed to bind for 1 hr at 4° with mixing on a rotating wheel. Unbound protein is removed and beads are washed twice with a 10fold excess of buffer D containing 1% Triton X-100 and 2 mM 2-mercaptoethanol, as described earlier. The beads are subsequently washed, as described earlier, seven times with a 10-fold excess of buffer D containing 2 mM 2-mercaptoethanol to remove detergent. The beads are resuspended, transferred to 2.0-ml microfuge tubes, and eluted with 1.5 rnl buffer C containing 300 mM imidazole (30 rain/4 °) with mixing on a rotating wheel. Using Centricon 10 miniconcentrators (Amicon, Danvers, MA), the supernatant is concentrated and diluted 10-fold with buffer C twice to decrease the imidazole concentration. Concentrated smgGDS is >95% pure by SDS-
254
CELLEXPRESSION
[271
P A G E and is stored at - 8 0 °. Approximately 18 mg of smgGDS is obtained per liter of E. coli culture.
Use of Rho-GDI and smgGDS to Investigate GTP-Binding Protein-Regulated Functions When neutrophil cytosol and plasma membranes are incubated at 37 ° for 25 rain in the absence of guanine nucleotides, low but detectable phospholipase D activity is apparent by the formation of phosphatidylethanol (filled circle, left side of Fig. 2). GTP~S causes a significant increase in PEth formation, ranging between 10 and 20% of the total counts (e.g., open circle, left side of Fig. 2). R h o - G D I inhibits GTP~S-stimulated phospholipase D activity with an IC50 of 1/~M (open circles). There is no requirement to preincubate membranes and cytosol with R h o - G D I before GTP~/S addition, as the degree of inhibition does not change with more prolonged preincubations.
IF--
12"
~,
+GTP~S
I.z
0 (3 =,J <
8
0
6
g
-.._
.c
4,
-GTP,fS ~
t
I
5
i
J
20
[RHO GDI] p,M FIG. 2. Inhibition of GTPyS-stimulated phospholipase D activity by R h o - G D I . Assay conditions were as described in the text, except that cell fractions were preincubated on ice for 15 min with the indicated concentrations of R h o - G D I . Either 10/xM GTPyS (open circles) or no nucleotide (filled circles) was added and incubations were continued for 25 min at 37 ° in the presence of 1.6% ethanol. Reactions were terminated and phosphatidylethanol (PEth) was quantified as described. Data points represent the mean + / - range of duplicate incubations. The experiment shown is representative of five using subcellular fractions from different donors.
[27]
PHOSPHOLIPASE D, Rho GDI, AND smgGDS
255
GTP is a relatively poor agonist for phospholipase D activation compared with GTP3,S (compare GTPyS stimulation in Fig. 2 with GTP stimulation in Fig. 3). This is likely due to the competing GTPase activity of the GTP-binding protein which converts bound GTP to GDP. However, the addition of increasing concentrations of smgGDS to a GTP-stimulated phospholipase D assay augments activity (Fig. 3). The stimulation requires GTP, as no effect of smgGDS is seen in the absence of GTP. Maximal stimulation occurs at approximately 200 nM smgGDS. Higher concentrations of smgGDS are inhibitory for unknown reasons. General Utility and Interpretation of Effects of Regulatory Factors The phospholipase D system provides a paradigm for the potential utility of regulatory proteins that modulate guanine nucleotide binding in defining a role for small GTP-binding proteins in biochemical processes. In principle, these as well as other such proteins can be used to study a variety of processes for which GTP or GTPTS regulation has been demonstrated. With 50 or more small GTP-binding proteins in the Ras superfamily,
7
Jl--
+GTP
z
8. j
5
o u. O
4
uJ a.
3 I
z 0.0
,
L
,
0.2
, 0.4
IP2
[smg GDS] I~M FIG. 3. Stimulation of GTP-activated phospholipase D activity by s m g G D S . Assay conditions were as described in the text. G T P (100/zM) was used to stimulate phospholipase D activity, and the indicated concentrations of s m g G D S were included at time zero. Reactions were terminated and phosphatidylethanol (PEth) was quantified as described. Data points represent the m e a n + / - range of duplicate incubations. T h e experiment is representative of three.
256
CELL EXPRESSION
[9,8]
as well as a growing family of heterotrimeric G proteins, the use of these regulatory proteins provides a potential short-cut toward narrowing the possibilities. With this in mind, it is important to discuss the limits of interpretation of these types of data. The most definitive conclusion that can be made from this approach is that the GTP-binding/regulatory species is a member of the Ras superfamily. Insofar as we know, neither smgGDS nor R h o GDI interact with any of the heterotrimeric GTP-binding proteins. Effects of both regulatory factors also strengthen the interpretation that this is a small GTP-binding protein, smgGDS has a fairly broad specificity within the Ras superfamily, exerting effects on Ki-Ras, RaplA, RaplB, RhoA, RhoB, Racl, and Rac2, but not on Ha-Ras and Rab3A. 14,15Hence, its main value is in demonstrating or confirming that a GTP effect is due to a small GTPase, but it is of little value in further narrowing the possibilities. R h o GDI, however, appears to be more specific for the Rho subfamily of small GTPases. It exerts effects on RhoA, RhoB, Racl, Rac2, and CDC42Hs, but not on Ki-Ras, RaplB, or Rab3AJ 4'16 However, it should be cautioned that effects of R h o - G D I have been tested on only about 10% of "representative" GTPases. Hence, a theoretical possibility exists that R h o - G D I will ultimately be found to interact with members of the Ras superfamily outside of the Rho subfamily. It therefore seems prudent to advise caution for the time being in interpreting specificity based on R h o - G D I effects. Acknowledgment This work was supported by N I H G r a n t CA46508.
14 y . Takai, K. Kaibuchi, A. Kikuchi, and M. Kawata, Int. Rev. Cytol. 133, 187 (1992). i5 T. Mizuno, K. Kaibuchi, S. A n d o , et al., J. BioL Chem. 267~ 10215 (1992). 16 K. Takaishi, A. Kikuchi, S. Kuroda, K. Kotani, T. Sasaki, and Y. Takai, Mol. Cell. Biol. 13, 72 (1993).
[28] M e a s u r e m e n t o f R a c T r a n s l o c a t i o n f r o m C y t o s o l t o Membranes in Activated Neutrophils By MARK T. OUINN
and
GARY M. BOKOCH
Introduction Activation of the superoxide (02-) generating NADPH oxidase of human neutrophils involves the assembly of several neutrophil components, METHODSIN ENZYMOLOGY,VOL. 256
Copyright© 1995by AcademicPress. Inc. All rightsof reproductionin any formreserved.
[28]
MEASUREMENTOF Rac TRANSLOCATION
257
some located on the plasma membrane and others in the cytosol (reviewed in Clark1), and previous studies showed that two of these cytosolic N A D P H oxidase components, p47-phox and p67-phox, were translocated to the plasma m e m b r a n e on phagocyte activation where they became associated with the active N A D P H oxidase complex. 2 A third cytosolic protein, the small GTP-binding protein Rac, has been shown to be absolutely required for N A D P H oxidase activation in a cell-free reconstitution assay system, 3'4 indicating that Rac is responsible for at least part of the G T P sensitivity of the N A D P H oxidase system. The absolute requirement for Rac in the activation of the N A D P H oxidase suggested the possibility that it might also be translocated from the cytosol to the plasma membrane during activation of the N A D P H oxidase. Therefore, to investigate whether changes in the subcellular distribution of Rac occur during assembly of the active oxidase, we developed procedures to analyze the subcellular distribution and kinetics of translocation of this cytosolic N A D P H oxidase protein in intact cells. Using these procedures, which are described here in detail, we found that Rac does translocate to the plasma membrane from the cytosol on neutrophil activation and that this translocation corresponds both temporally and quantitatively with p47-phox and p67-phox translocation and N A D P H oxidase activation. 5 Procedure Reagents and Buffers The general chemicals and reagents used were of the highest quality commercially available. Dulbecco's phosphate-buffered saline (DPBS) was purchased from Sigma Chemical Co. (St. Louis, MO). Nitrogen cavitation buffer consisted of 100 m M KC1, 10 m M NaC1, 3.5 m M MgC12, 1 m M ATP, 10 txg/ml chymostatin, 1 m M phenylmethylsulfonyl fluoride (PMSF), 10 m M H E P E S , p H 7.4. Membrane resuspension buffer consisted of 100 m M KC1, 10 m M NaC1, 1 m M E D T A , 10 ~g/ml chymostatin, 1 m M PMSF, 10 m M H E P E S , p H 7.4. Previously characterized antibodies used for Western blotting included I R. A. Clark, J. Infect. Dis. 161, 1140 (1990). 2 R. A. Clark, B. D. Volpp, K. G. Leidal, and W. M. Nauseef, J. Clin. Invest. 85, 714 (1990). 3 U. G. Knaus, P. G. Heyworth, T. Evans, J. T. Curnutte, and G. M. Bokoch, Science 254, 1512 (1991). 4 A. Abo, E. Pick, A. Hall, N. Totty, C. G. Teahan, and A. W. Segal, Nature 353, 668 (1991). 5M. T. Quinn, T. Evans, L. R. Loetterle, A. J. Jesaitis, and G. M. Bokoch, J. BioL Chem. 268, 20983 (1993).
258
CELL EXPRESSION
[28]
antibodies to gp91-phox peptide (residues 546-558), 6 p22-phox peptide (residues 162-174), 7 and p47-phox.8 Antibodies against Rac2 and GDP dissociation inhibitor proteins were prepared using purified recombinant Rac29 or human (Rho) GDI 1° as antigens. Antiserum to p67-phox was a kind gift of Dr. David J. Uhlinger (Emory University).
Preparation of Neutrophils Purified human neutrophils, isolated as previously described, H were treated with 3 mM diisopropyl fluorophosphate (DFP) for 15 min at 4 ° to inactivate serine proteases, washed with DPBS, and resuspended in DPBS at 108 cells/ml. DFP treatment is essential to ensure that the N A D P H oxidase proteins remain intact during subsequent preparation steps, and we have found that gp91-phox, p67-phox, and Rac are especially sensitive to proteolysis by neutrophil granule proteases.
Fractionation of Neutrophils For subcellular fractionation of neutrophils and analysis of the subcellular distribution of N A D P H oxidase components in resting and stimulated cells, the purified neutrophils (10 9 cells for each condition resuspended at 108 cells/ml in DBPS containing 0.1% glucose w/v, 0.1% bovine serum albumin (BSA) w/v, 250 U/ml catalase, and 50 U/ml superoxide dismutase (SOD) to protect the cell from oxidative damage) were warmed to 37 ° in a shaking water bath and stimulated for 6 min with 1/xg/ml phorbol myristate acetate (PMA). We chose 6 min because kinetic studies showed that essentially maximal translocation of Rac had occurred by this point. In addition, we used PMA because gradient fractions retain 02- generating activity in PMA-stimulated cells, whereas 02- generating activity shuts off in f-MetLeu-Phe (fMLP)-stimulated cells and cannot be analyzed in gradient fractions from these cells. Therefore, the conditions were optimal to analyze the subcellular distribution and translocation of N A D P H oxidase components as well as the relative subcellular distribution of N A D P H oxidase 6 M. T. Quinn, C. A. Parkos, L. Walker, S. H. Orkin, M. C. Dinauer, and A. J. Jesaitis, Nature 342, 198 (1989). 7 M. Y. Quinn, M. L. Mullen, and A. J. Jesaitis, J. BioL Chem. 267, 7303 (1992). 8 B. D. Volpp, W. M. Nauseef, J. E. Donelson, D. R. Moser, and R. A. Clark, Proc. Natl. Acad. Sci. U.S.A. 86, 7195 (1989). 9 U. G. Knaus, P. G. Heyworth, B. T. Kinsella, J. T. Curnutte, and G. M. Bokoch, J. Biol. Chem. 267, 23575 (1992). 10 T. H. Chuang, X. Xu, U. G. Knaus, M. J. Hart, and G. M. Bokoch, J. Biol. Chem. 26& 775 (1993). 11 M. T. Quinn, C. A. Parkos, and A. J. Jesaitis, Biochim. Biophys. Acta 987, 83 (1989).
[281
MEASUREMENT OF R a c TRANSLOCATION
259
activity. The reaction was stopped by the addition of ice-cold DPBS containing protease inhibitors (10 tzg/ml chymostatin and 1 mM PMSF). It was important to ensure that the cells are mixed well with stop buffer and kept on ice to prevent further activation of the cells. The cells were then washed twice with DPBS, resuspended in cavitation buffer, and disrupted by N2 cavitation at 4° (450 psi for 15 min with slow stirring). The cavitate was collected and centrifuged for 5 min at 1000g and 4° to form supernatant (1KS) and foam/pellet (1KP). The 1KP was resuspended in the smallest possible volume of cavitation buffer, rehomogenized with 10-15 strokes in a Dounce homogenizer, and again separated into 1KS and 1KP. The 1KS fractions were then pooled and fractionated by isopycnic sucrose density gradient sedimentation. Isopycnic sucrose density gradients, constructed by layering a 20-ml 2055% sucrose gradient on top of an 8.0-m160% sucrose cushion, were allowed to set overnight at 4°, and a 1.5-ml cushion of 15% sucrose was layered on top of the gradients immediately prior to application of 9.5-10 ml of the 1KS homogenate. The gradients were then sedimented at 163,000g for 45 min in a Beckman VAC-50 vertical rotor (Beckman Instruments, Inc., Palo Alto, CA), and 1.5-ml fractions were collected from the bottom of each gradient and analyzed for subcellular markers and O2- generating activity. For analysis of the kinetics of Rac translocation, the purified neutrophils were resuspended at 108 cells/ml in DBPS contairiing 0.1% glucose, 0.1% BSA, 250 U/ml catalase, and 50 U/ml superoxide dismutase; warmed to 37° in a shaking water bath; and stimulated for the indicated times with 1 tzg/ml PMA or 1 tzM fMLP with gentle agitation. We have also stimulated the cells in a single batch and removed aliquots at specific time points, and the results were identical for both methods. The reaction was stopped by the addition of ice-cold DPBS containing protease inhibitors (10 ~g/ml chymostatin and 1 mM PMSF) and the cells were cavitated as described earlier. The 1KS fractions were then combined and layered on top of discontinuous sucrose density gradients. For cavitates ---2.5 ml, we used gradients constructed of 2.5 ml of 20% sucrose layered on top of 3 ml of 38% sucrose in Beckman Ti 75 tubes. For cavitates >2.5 ml and -<4 ml, we used gradients constructed of 7 ml of 20% sucrose layered on top of 9 ml of 38% sucrose in Ti 60 tubes (all sucrose solutions are w/w made up in 10 mM HEPES, pH 7.4). The gradients were centrifuged at 144,000g (rav) for 45 min at 4°, and the plasma membrane was removed from the interface using an 18-gauge spinal needle (4 inches long) with a flat end (made by cutting off the tip). Usually, we aspirate --2-3 ml of sample containing plasma membrane. Previously, we confirmed using fractionation and analysis for plasma membrane (alkaline phosphatase) and azurophil granule (myeloperoxidase) markers that this method cleanly separates the
260
CELLEXPRESSION
[28]
plasma membrane from other neutrophil components. The membranes are then resuspended in 4 vol of membrane resuspension buffer and pelleted at 144,000g (ray) in a Beckman Ti60 rotor for 45 min at 4 °. The membrane pellets were sonicated into a small volume of membrane resuspension buffer using three 10-sec bursts with a Sonic Dismembrator 50 set at power level 5 (Fisher Scientific) and analyzed for protein content. Equal amounts of protein for each sample were then applied to 7-18% polyacrylamide gradient gels, which were then run and transferred to nitrocellulose for Western blotting. Western blots were developed using an alkaline phosphataseconjugated goat anti-rabbit IgG secondary antibody (Bio-Rad Laboratories, Richmond, CA). Western blots were quantitated by video densitometry using an image analysis system [Microcomputer Imaging Device (MCID) with Image Analysis software] (Imaging Research, Inc., Brock University, Ontario, Canada). The relative density of a sample (expressed in arbitrary units) represents the density of the sample relative to an arbitrary gray scale defined by the image analysis system to cover the total gray shade range of the image.
Biochemical Assays Neutrophil cytochrome b was quantitated by reduced-minus-oxidized difference spectroscopy on a Cary 3 dual-beam spectrophotometer, using a reduced-minus-oxidized Soret band (427 nm) extinction coefficient of 161 mM -1 cm-1.12 Alkaline phosphatase, myeloperoxidase, protein, and other markers were measured as described previously, n'13 Superoxide generating capacity in whole cells stimulated with either 1 ~g/ml PMA or 1 /xM fMLP was determined by incubating the cells (2 >( 10 6 cells/ml) in DPBS + 0.65 mg/ml cytochrome c -+ 500 U/ml SOD in stirred cuvettes at 25 ° and monitoring the reduction of cytochrome c at 550 nm with a Cary 3E dual beam UV-Vis spectrophotometer (Varian Analytical Instruments, Australia). SOD-inhibitable cytochrome c reduction was calculated using e = 18.5 mM -a cm -t. Superoxide generation in sucrose density gradient fractions was measured at 25 ° as described previously6 in 1.6-ml microcuvettes containing 650/zl detection buffer (0.65 mg/ml cytochrome c, 2 mM MgC12, 2 mM NAN3, 10 mM HEPES, pH 7.4, _+500 U/ml SOD) and 50/xl of membrane. 12 R. Lutter, M. L. J. van Schaik, R. Van Zwieten, R. Wever, D. Roos, and M. N. Hamers, J. Biol. Chem. 260, 2237 (1985). 13 A. J. Jesaitis, J. R. Naemura, R. G. Painter, L. A. Sklar, and C. G. Cochrane, J. Biol. Chem. 258, 1968 (1983).
[28]
MEASUREMENTOF Rac TRANSLOCATION
261
Results Subcellular Fractionation
The subcellular distribution of Rac, GDI, and other oxidase-associated proteins in resting or PMA-stimulated human neutrophils determined by isopycnic sucrose density gradient sedimentation is shown in Fig. 1. Distinct subcellular organelles were localized using specific markers that have been characterized previously [i.e., plasma membrane markers (alkaline phosphatase, RaplA, and cytochrome b; peak: 30-32% sucrose, fractions 1214), specific granule markers (cytochrome b and RaplA; peak: 41-43% sucrose, fractions 18-20), and an azurophil granule marker (myeloperoxidase; peak: 48-50% sucrose, fractions 22-24)]. 11,14 Figure 1C also shows the subcellular distribution of 02- generating activity in PMA-stimulated cells (peak activity: 34-36% sucrose, fractions 13-15). As was observed previously, H the peak 02 generating activity occurred in the heavy plasma membrane (enriched in membrane skeletal proteins) region of the gradient. No 02- production was observed in any of the fractions prepared from unstimulated cells. The fractions from these gradients were analyzed for the distribution of Rac by Western blotting with antibodies prepared against recombinant Rac. As Fig. 1A shows, Rac is localized completely to the cytosolic compartment of resting cells (fractions 1-10), but is translocated to the plasma membrane and 02- generating regions (fractions 12-15) after stimulation with PMA (Fig. 1B). Quantitation of the amount of translocated Rac using densitometric scans of the Western blots indicated that -10% of the total cytosolic Rac was translocated in these gradients. In addition, if the cells were stimulated with the chemotactic peptide fMLP, a similar redistribution of Rac to the plasma membrane was observed (not shown), indicating that Rac translocation is not stimulus specific and that translocation of this GTPbinding protein is a general phenomenon associated with oxidase activation for both receptor-mediated and nonreceptor-mediated stimuli. Because Rac is associated with GDI in human neutrophil cytosol,15 the subcellular distribution of GDI was also analyzed to determine if GDI could be translocating in association with Rac, or by itself, during neutrophil activation. The localization of GDI was assessed using both the GDIspecific antipeptide antibody and a more sensitive antiprotein antibody. Our detection level was down to <10 ng, which would have readily detected any GDI translocated at levels even approximating those of Rac. As Fig. 14c. A. Parkos,C. G. Cochrane,M. Schmin,and A. J. Jesaitis,J. Biol. Chem. 260,6541 (1985). 15T.-H. Chuang,B. P. Bohl, and G. M. Bokoch,J. Biol. Chem. 268, 26206 (1993).
262
CELL EXPRESSION
CYTOSOL
PM
[281
SG ~
100
AG ~
A
80
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0
.
.
.
.
=...,..
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:::::::::= 5
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,
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:. 25
30
Fraction Number FIG. 1. Subcellular distribution of N A D P H oxidase components and superoxide generating activity in resting and PMA-stimulated human neutrophils. Human neutrophils (5 × 108), control unstimulated (A) or stimulated for 5 min at 37 ° with 1/xg/ml P M A (B and C), were fractionated as described. Cytochrome b distribution ((3) was determined spectrophotometrically and was confirmed by densitometric scans of Western blots. R a p l A (@), p47-phox ( • ), p67-phox (¢), GDI (V), and Rac (Vq) distributions were determined by densitometric scans of Western blots, and superoxide dismutase-inhibitable O~ generating activity in fractions from PMA-stimulated cells (11) (C) was determined as described. The percentage of the maximum level of the specific oxidase proteins or oxidase activity (C) recovered from the gradients (including cytosolic fractions) is plotted as a function of fraction number (fraction 1 = 10% sucrose and fraction 29 = 55% sucrose). The specific activity of the peak 02
[28]
MEASUREMENT OF R a c TRANSLOCATION
263
1A shows, GDI is localized completely to the cytosolic compartment (fractions 1-10) in resting cells; however, it remains primarily in the cytosol after activation (fractions 1-10; Fig. 1B). We did, however, observe a very low, but reproducible, amount of GDI associated with membrane fractions in the kinetic studies described below. This level of GDI was far less than the level of translocated Rac (GDI observed was <5% of the total translocated Rac) and was very hard to detect unless high amounts of membrane protein were analyzed (>12/xg protein/lane which is equivalent to 3.4 x 106 cell equivalents/lane). This observation might suggest a transient association of GDI with the membrane. Since the oxidase cofactors p47-phox and p67-phox are known to be translocated during oxidase activation, 2 we also analyzed the subcellular distribution of p47-phox and p67-phox in resting and PMA-stimulated cells to probe the relationship among these translocating Proteins. Figure 1B shows that p47-phox and p67-phox translocated from the cytosol (fractions 1-10) to the plasma membrane (fractions 12-15) after the cells were activated with PMA. Quantitation of this movement using densitometric scans of the Western blots indicated that approximately 10-18% of the total amounts of each of these proteins in the cytosol was translocated. These results are consistent with those previously reported for p47- and p67-phox2 and provide an internal control for experiments looking at translocation of other oxidase proteins such as Rac and GDI.
Kinetics of Rac Translocation As shown in Fig. 2A, O2- generation in PMA-stimulated neutrophils began - 1 min after the addition of PMA and increased steadily over the next 20 rain, although the overall rate of O2- production decreased gradually over the time studied. In cells stimulated with fMLP (Fig. 3A), 02- production began within 15 sec and plateaued approximately 3-4 min after addition of the stimulus. These activation kinetics are typically observed with such nonreceptor- and receptor-mediated stimuli, respectively. Analysis of the plasma membranes from these cells by Western blotting for Rac, p47-phox, and p67-phox showed that all three proteins were stably translocated to the plasma membrane in samples prepared from PMA-stimulated cells (Fig. 2B) or in membranes prepared from fMLP-stimulated cells (Fig. 3B). In
generating fraction was 28.3 nmol O2-/min/mg. Location of the cytosolic fractions and the peak enzyme/marker activities for the plasma membrane (PM), specific granules (SG), and azurophil granules (AG) are indicated at the top of A. The results are representative of four separate experiments (variability in sedimentation profiles between experiments was -<2%). (Reproduced with permission from Quinn et al. 5)
264
CELL EXPRESSION
[28]
Illlll~llrlllll~lrl 1.2
A
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8
0.51 2356 101520
9 10 11 12 13 14 15 16 17 18 19 20 Time (min)
FIG. 2. Kinetics of Rac translocation in P M A - s t i m u l a t e d h u m a n neutrophils. Purified h u m a n neutrophils were treated with 1/xg/ml P M A , and 0 2 - generation (A) was determined
[28]
MEASUREMENTOF Rac TRANSLOCATION
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addition, the time course of Rac, p47-phox, and p67-phox translocation closely paralleled the kinetics of 02- production in both PMA- and fMLPactivated cells. Thus, in PMA-stimulated cells, accumulation at the membrane occurred relatively slowly and increased over the 20 min with a gradual tapering off at the longer time points, whereas all three cytosolic proteins in fMLP-activated cells translocated to a near-maximum membrane-associated level within the first minute of stimulation, and then increased only slightly over the remainder of the time period studied. Discussion Rac (Racl/Rac2) has been shown to be required for activation of the NADPH oxidase in cell-free reconstitution assays3,4 and in intact cells, 16 yet the role of Rac in regulating the oxidase is not yet known. Although the cell-free reconstitution system has provided a significant amount of information about the oxidase, including the identification of all of the known cytosolic components (p47-phox, p67-phox, and Rac), this system does not reflect all of the regulatory processes that are present in the intact cell. The procedures described in this chapter provide a way to assess the activation of Rac and its possible role in the NADPH oxidase in intact cells, and the results show that Rac translocates to the plasma membrane in PMA- and fMLP-stimulated human neutrophils and that the kinetics of Rac translocation parallel the kinetics of 02- production by these cells. Additionally, the Rac regulatory modulator GDI, which binds Rac and maintains it in a cytosolic complex, Iv is not translocated in significant quantities along with Rac, although a low level seems to be present at the membrane after activation. These results begin to elucidate how Rac and GDI may function in the intact cell by showing that cell activation causes dissociation of these proteins, allowing Rac to then associate with the plasma membrane along with the other oxidase cofactors, p47-phox and p67-phox. 16 O. Dorseuil, A. Vazquez, P. Lang, J. Bertoglio, G. Gacon, and G. Leca, J. Biol. Chem. 267, 20540 (1992). 17E. Pick, Y. Gorzalczany, and S. Engel, Eur. 3. Biochem. 217, 441-455 (1993).
as described. In parallel incubations, cells were removed after the indicated time of stimulation and membranes were prepared and analyzed for Rac, p47-phox, and p67-phox using Western blotting as described (see inset in B; --12 /zg/lane). Western blots were scanned using an image analysis system, and the densities of the bands are plotted at each time point for Rac (0), p47-phox (11), and p67-phox ( 0 ) (mean +_ SD; n = 4) (B). The results are representative of four separate experiments (time points above 6 min represent three separate experiments). (Reproduced with permission from Quinn et al. 5)
A i
0.5
0.4
o
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libid,o/ll/o/°~°'t~'l"°"~ o
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I
I
I
I
100
8O E 60 "6 "E ~
p67-phox
40
p47-phox 20 Rac 0.512356
Time (rain)
2
I
I
I
I
3
4
5
6
Time (min)
FIG. 3. Kinetics of Rac translocation in fMLP-stimulated human neutrophils. Purified human neutrophils were treated with 1 tzM fMLP, and 02- generation (A) was determined as described. In parallel incubations, cells were removed after the indicated time of stimulation and membranes were prepared and analyzed for Rac, p47-phox, and p67-phox using Western blotting as described (see inset in B; ~12 /xg/lane). Western blots were scanned using an image analysis system, and the densities of the bands are plotted at each time point for Rac (O), p47-phox ( I ) , and p67-phox ((>) (mean - SD; n = 3) (B). The results are representative of three separate experiments. (Reproduced with permission from Quinn et al. 5)
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MEASUREMENTOF Rac TRANSLOCATION
267
The presence of small amounts of GDI at the membrane suggests the possibility that GDI transiently translocates to deliver or release Rac at its site action on the plasma membrane and, thus, only a very low amount of GDI can be observed at the membrane at any one time. The kinetics of translocation of Rac and other oxidase proteins in response to fMLP stimulation were much more difficult to measure using our procedures than those for PMA-stimulated cells. This was partly due to the lower levels of protein translocated in fMLP-stimulated cells versus PMA-stimulated cells (--5% vs 10-15% of the total Rac translocated, respectively) and also partly because of the sensitivity of many of these proteins to proteolysis. We did find, however, that the amount of translocated proteins observed in fMLP-stimulated cells could be significantly enhanced if phosphatase inhibitors were added to the cells. In particular, incubation of 50 nM calyculin A (a protein phosphatase IA and 2 inhibitor) for 5-15 min with the cells prior to stimulation enhanced Rac translocation two- to fourfold in parallel with enhancement of O2- production. This is a useful way to enhance fMLP-induced translocation, and is consistent with the importance of phosphorylation events, particularly tyrosine phosphorylation, in regulating Rac activation. TM An alternative method to analyze Rac translocation using an in vitro system has been developed by Philips and co-workers 19 where membranes and cytosol are incubated with 100/xM GTPyS, the sample is centrifuged, and Rac can be observed translocated to the membrane pellet. We have confirmed that this procedure works and that this is a specific process regulated by GTP binding to Rac (G. M. Bokoch, unpublished observation 1994). This method is easy to perform and complementary to that described here and is useful in studying the conditions required for translocation of Rac to plasma membranes in a cell-free system. Acknowledgments We thank Drs. Robert A. Clark and William Nauseef from the Department of Medicine, University of Iowa, for their kind gift of anti-p47-phox antibodies and Drs. David J. Uhlinger and J. David Lambeth from the Department of Biochemistry, Emory University School of Medicine, for kindly providing anti-p67-phox antibodies. This work was supported by a National Institutes of Health FIRST Award R40929-01 (Dr. Quinn) and Research Grants GM44428 and HL48008 (Dr. Bokoch). Dr. Quinn is the recipient of an Arthritis Foundation Investigator Award and Biomedical Science Grant.
18 O. Dorseuil, M. T. Quinn, G. M. Bokoch, J. Leukocyte Biol., in press (1995). 19 M. R. Philips, M. H. Pillinger, R. Staud, C. Volker, M. G. Rosenfeld, G. Weissmann, and J. B. Stock, Science 259, 977 (1993).
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[29] R e c o n s t i t u t i o n o f C e l l - F r e e N A D P H O x i d a s e A c t i v i t y by Purified Components
By
ARIE ABO and ANTHONY W. SEGAL
Introduction The assembly of the N A D P H oxidase of phagocytes on activation is essential for the efficient killing of microorganisms. The components of this system are primarily expressed in professional phagocytes which include neutrophils, macrophages, monocytes, and eosinophils. The activated oxidase utilizes N A D P H as an electron donor to catalaze the reduction of oxygen to superoxide (O2- -) which serves as a precursor for the formation of reduced oxygen species, such as H20:, • OH, and HOC1.1 The electron-transporting component of the N A D P H oxidase is an unusual membrane-bound flavocytochrome b composed of two subunits: a 21-kDa ot subunit (also known as p21ph°x) and a 76- to 92-kDa heavily glycosylated/3 subunit (also known as gp91Ph°x). The /3 subunit contains the binding sites for N A D P H and FAD, the location of the heme(s) is as yet uncertain. The activation of the electron transport oxidase is dependent on the assembly of the cytosolic components p47ph°x and p67ph°x and the Rasrelated small GTP-binding protein p21 Rac on the membrane (Fig. 1). Impairment in several components of the oxidase complex is a direct cause of chronic granulomatous disease (CGD), a rare syndrome associated with chronic infections and impaired microbicidal activity. 2 A key to our understanding and identifying the N A D P H oxidase components emerged with the initial discovery of the cell-free system in which in vitro reconstitution was obtained by the combination of cytosol and membranes prepared from resting phagocytes together with optimal concentrations of amphiphiles like arachidonic acid or SDS. 3 Furthermore, by mixing normal membranes and cytosol with those prepared from CGD neutrophils, it was shown that most patients with autosomal recessive CGD exhibit deficiencies in the cytosolic fraction, whereas the membrane fraction is impaired in X-linked patients. 4 t A. W. Segal and A. Abo, Trends Biochem. Sci. 18, 43 (1993). 2 R. M. Smith and J. T. Curnutte, Blood 77, 673 (1991). 3 y . Bromberg and E. Pick, J. Biol. Chem. 260, 13539 (1985). 4 j. T. Curnutte, R. L. Berkow, R. L. Roberts, S. B. Shurin, and P. J. J. Scott, Clin. Invest.
81, 606 (1988). METHODSIN ENZYMOLOGY,VOL.256
Copyright © 1995 by AcademicPress,Inc. All rightsof reproductionin any formreserved.
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Various groups have shown that fractions containing p47 ph°x and p67 ph°x could not alone replace the cytosol in the cell-free assay. We and others then identified an additional cytosolic factor essential for oxidase activity which, on purification, resolved into a heterodimeric complex of proteins subsequently identified as Ras-related small GTP-binding proteins p21 Rac and G D P dissociation inhibitor for R h o ( R h o - G D I ) . 5 R e c o m b i n a n t p21 R a c l or p21 Rac2 loaded with G T P could replace the complex of Rac and G D I . 6-8 Subsequently, it was shown that the oxidase activity was fully reconstituted by purified recombinant p67 ph°x, p47 ph°x, and p21 Rac synthesized in Escherichia coli or Sf9 (Spodoptera frugiperda fall a r m y w o r m ovary) cells together with purified cytochrome b from h u m a n neutrophil or recombinant cytochrome b p r e p a r e d in Sf9 cells. 9-u This chapter describes the preparation of recombinant N A D P H oxidase cytosolic c o m p o n e n t s in E. coli and reconstitution of oxidase activity in the cell-free system with purified cytochrome b or solubilized neutrophil m e m branes.
P r e p a r a t i o n of N e u t r o p h i l M e m b r a n e s a n d Cytosol N o r m a l h u m a n blood is a good source of neutrophils (typical yield is 2 - 3 × 10 s neutrophils/100 ml blood) which are readily purified from the blood and can serve as a source of cytosol and m e m b r a n e fractions for optimization of the cell-free assay. However, this source does not generally provide enough neutrophils for purification of cytochrome b or the cytosolic components. Blood from patients with chronic myeloid leukemia contains up to approximately 100 times the normal neutrophil count and these patients are occasionally leukaphoresed to r e m o v e large n u m b e r of granulocytes. Additional sources of these ceils and purification methods are described in m o r e detail in Segal et al.12 5 A. Abo and E. Pick, J. BioL Chem. 266, 23577 (1991). 6 A. Abo, E. Pick, A. Hall, N. Totty, C. G. Teahan, and A. W. Segal, Nature 353, 668 (1991). 7 U. G. Knaus, P. G. Heyworth, T. Evans, J. T. Curnutte, and G. M. Bokoch, Science 254, 1512 (1991). 8 U. G. Knaus, P. G. Heyworth, B. T. Kinsella, J. T. Curnutte, and G. M. Bokoch, J. Biol. Chem. 267, 23575 (1992). 9 A. Abo, A. Boyhan, I. West, J. Adrian, Thrasher, and A. W. Segal, J. Biol. Chem. 24, 16767 (1992). l0 D. Rotrosen, C. L. Yeung, T. L. Leto, H. L. Malech, and C. H. Kwong, Science 256, 1512 (1992). 11D. Rotrosen, C. L. Yeung, and J. P. Katkin, J. Biol. Chem. 268, 14256 (1993). 12A. W. Segal, A. M. Harper, A. R. Cross, and O. T. G. Jones, in "Methods in Enzymology" (G. Di Sabato and J. Everse, eds.), Vol. 132, p. 378. Academic Press, San Diego, 1986.
270
CELL EXPRESSION
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+
0.,)
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oa~
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[29]
RECONSTITUTION OF NADPH OXIDASEACTIVITY
271
Methods Isolation of Neutrophils from Human Peripheral Blood. This is a brief description of neutrophil isolation. More detailed descriptions can be found in Segal et aL t2 Human blood (100 ml) is drawn from a healthy volunteer into preservative-free heparin (5 U/ml) mixed with 0.15 M NaC1 and sedimented with 10% volume of 10% dextran (w/v, average molecular weight 242,000) for approximately 30 min until erythrocytes have sedimented. The supernatant plasma containing the leukocytes is layered on Ficoll/Hypaque (Lymphoprep, Nycomed, Pharma, 5 vol of plasma on 1 vol of gradient) and is centrifuged at 400g for 15 min. The pellet contains the neutrophils and residual erythrocytes. The erythrocytes are lysed by hypotonic lysis. The pellet, up to 0.2 ml in volume, is resuspended in 20 ml of water and is vigorously mixed on a vortex mixer or pipetted up and down for 10 sec. Osmolarity is restored by the addition of 20 ml 0.31 M NaCI (1.8%) containing 5 IU/ml heparin. Preparation of Membranes and Cytosol. The neutrophils are resuspended at a concentration of 2 × 108 cells/ml in a break buffer consisting of 6 mM PIPES, pH 7.3, 6% (w/w) sucrose, 60 mM KC1, 1.8 mM NaC1, 2.3 mM MgCI2, IIzM diisopropyl fluorophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 /xg/ml TLCK. The cells are sonicated three times at 4° for 15 sec, overlaid on a discontinuous sucrose gradient (in 10 mM Tris, pH 7.4) of 15% (w/w) on top of 34% (w/w) on top of 45% (w/w), and centrifuged at 200,000g at 4°C for 15 rain in a TLX Beckman ultracentrifuge using a TLS55 swing-out rotor. The cytosol is collected above the 15% sucrose, the plasma membranes at the 15/34% interface, and the specific granules at the 34/45% interface. The specific granules and plasma membrane are diluted 1 : 4 with cold break buffer lacking sucrose and are pelleted by centrifugation as described earlier.
F~G. 1. Model of the role p21 Rac might play in the activation complex of the NADPH oxidase. The specialized components of the NADPH oxidase include the a and/3 subunits of flavocytochrome b and the two cytosolic proteins, p47ph°x and p67ph°x. p21 Rac is present in the GDP-bound form in a complex with GDI (GDP dissociation inhibition factor), an association that requires its modification by isoprenylation near its C terminus that could also be required for attachment to the membrane. Activation is associated with separation of p21 Rac from GDI, the exchange of GTP for GDP, and its movement with p47ph°x and p67ph°X in an activation complex into association with the flavocytochrome in the membranes. The bound GTP is then hydrolyzed to GDP as a consequence of endogenous GTPase activity of the rac or through modulation by GAPs that accelerate GTPase activity. The function of a third specialized cytosolic protein, p40ph°x, which is associated with p67ph°x but is not required in the cell-free system,15 remains to be determined.
272
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M e m b r a n e Solubilization and Reconstitution. The membrane and specific granules serve as a good source of cytochrome b for cell-free activation; however, better activity can be obtained if the membrane is solubilized with a detergent such as octylglucoside or deoxycholate which is then removed by dialysis or diluted below the critical micellar concentration to allow reconstitution of the proteins with the lipid vesicles. The pelleted membranes are resuspended to a concentration of 1-2 × 108 cells equivalents/ml in a cold solubilization buffer consisting of 120 mM sodium phosphate, pH 7.4, I mM MgC12, 1 mM EGTA, 1 mM dithiothreitol (DTT), 10-20% glycerol, 1 /xg/ml leupeptin, 1 mM PMSF, and 40 mM octylglucoside, then homogenized with a Dounce homogenizer. The clarified solubilized membranes are stirred on ice for 30 min to allow a complete solubilization. Insoluble material is removed by centrifugation at 200,000g for 15 rain as described earlier. Reconstitution of the soluble proteins into the lipid vesicle can be simply achieved by diluting the solubilized membrane with buffer lacking detergent to reduce the detergent concentration below the critical micelle concentration (CMC). One volume of solubilized membrane is mixed with 4 vol of solubilization buffer lacking octylglucoside which reduces the concentration of octylglucoside to 8 raM, well below the CMC of 25 mM, and is left on ice for 30-60 min. An alternative option is to dialyze the mixture against buffer lacking the detergent.
Purification of Cytochrome b Cytochrome b is purified from the membranes and specific granules of neutrophils as described previously. Briefly, membranes and specific granules are purified from large numbers of neutrophils from normal human blood or by leukapheresis of patients with chronic granulocytic leukemia by scaling up the procedures described earlier. The membranes and granules (approximately 1 vol) are homogenized in (approximately 10-20 vol) buffer A (100 mM Tris-acetate, pH 7.4, 100 mM KC1, 20% (v/v) glycerol, 1 mM DTT, 1 mM EDTA, and protease inhibitors 1 mM PMSF, 1 mg/liter leupeptin, 1 mg/liter TLCK, and 1 mg/ liter pepstatin) containing 0.5% sodium cholate and centrifuged at approximately 100,000g. Cytochrome b remains largely in the pellet which is then washed with buffer without detergent and then extracted with buffer containing 1% Triton N-101 in roughly the same ratio of volumes of pellet to buffer. The Triton extract is then passed through columns containing DEAE-, CM-, and n-aminooctyl-Sepharose, and the cytochrome, which fails to attach to these resins, is trapped on heparin agarose from which it is eluted with a linear gradient of 0-1.0 M NaC1.
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Relipidation of Cytochrome b To obtain functional cytochrome b it is essential to reconstitute purified cytochrome b with lipid. This can be achieved by either preparing artificial lipid vesicles (35% phosphatidylcholine, Sigma) or extracting the endogenous lipid from neutrophil membranes. The lipid is then solubilized and combined with purified solubilized cytochrome b. Cytochrome b and the lipid associate upon removal of the detergent. Prior to relipidation of cytochrome b, the detergent is exchanged from Triton N-101 to solubilization buffer containing 40 mM octylglucoside. Cytochrome b is bound to the heparin agarose column in the Triton buffer, is washed with solubilization buffer containing octylglucoside, and is then eluted in break buffer containing 40 mM octylglucoside with a gradient of 0-1.0 M NaC1. To extracted lipids from neutrophil membranes, 200 ml of the membrane (protein concentration 2 mg/ml) is extracted with 1 ml of chloroform/ methanol (2:1, v/v), vortexed vigorously, and then centrifuged for 5 min at 400g in an Eppendorf centrifuge. The denatured proteins are removed from the interface, and the lower organic layer is evaporated under nitrogen. The dried extract of phospholipids is then solubilized in solubilization buffer. To optimize phospholipid/cytochrome b, 100 tzl of various concentrations of the solubilized phospholipids (100-400 p.g phospholipid) is mixed with 40 pmol of cytochrome b (40 tzl) and the mixture is diluted with 800 tzl solubilization buffer lacking detergent and is left on ice for 3060 min. The relipidated cytochrome is now ready to be used in the cellfree reconstitution assay. Preparation of p67 p~, p47Ph% and p21 Racl Isolation of large amounts of p67 ph°~ and p47ph°x from phagocytes is a laborious process since they are expressed at low abundance and are susceptible to proteolysis, particularly p67eh°~. However, it is possible to prepare small amounts of partially purified fractions enriched with these proteins as a good source for reconstitution in the oxidase. Purification of p21 Rac from phagocytes is described in [5] in this volume. Expression of these proteins in E. coli or in insect cells provides a good source of large amounts of pure proteins.
Preparation of Partially Purified p47ph°x and p67ph°xfrom Phagocyte Cytosol Ammonium sulfate precipitation provides a good method for the preparation of a fraction of cytosol that is enriched in p47ph°x and p67ph°x and depleted of p21 Rac. Ammonium sulfate is added over a period of 30 min
274
CELLEXPRESSION
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to 10-20 ml of neutrophil cytosol (prepared as described earlier) to obtain a final concentration of 37% saturation and the mixture is stirred for 30 min on ice. The mixture is then centrifuged for 15 min at 200,000g in a TLX Beckman ultracentrifuge. The pellet is resuspended to its initial volume in 100 mM sodium phosphate buffer, pH 7.2, containing 1 mM MgC12, 1 mM DTT, 1 mM PMSF, and 1 tzg/ml leupeptin. The ammonium sulfate is removed from the supernatant by dialysis or by exchanging the buffer on a desalting column such as Sephadex G-25M PD10 (Pharmacia LKB Biotechnology Inc.). The presence of these proteins should be confirmed by Western blot analysis with antibodies which can be obtained from several laboratories active in the field. For reconstitution of the oxidase activity, it is also important to check that this fraction can reconstitute the oxidase activity only when supplemented with membrane fraction and p21 Rac protein as discussed below. Partially purified p47ph°x and p67ph°x can also be prepared by gelfiltration chromatography. Neutrophil cytosol is fractionated on a Superose 12 gel filtration column (Pharmacia) and fractions are collected and analyzed by Western blotting with antibodies against p47ph°x, p67ph°x, and p21 Rac. Typically, p47ph°x and p67ph°x coelute as a 260-kDa complex whereas p21 Rac elutes with R h o - G D I as a 45-kDa complex. It is also possible to prepare p47ph°x and p67ph°x by the absorption of neutrophil cytosol on 2',5'-ADP agarose as described. 13
Preparation of Recombinant p47ph°x, p6T 'h°x, and p21 Racl in E. coli Large quantities of p47ph°x, p67ph°x, and p21 Racl can be rapidly prepared in E. coli using the pGEX expression vector. Expression of tagged proteins in Sf9 cells is also a good expression system. Human p47ph°x,p67ph°x,and p21 Racl cDNAs are cloned into a glutathione S-transferase (GST) expression vector pGEX-2T as described previously. Transformed clones with pGEX containing p47ph°x, p67ph°x, or p21 Racl cDNAs were handled as following: 1. Terrific broth (50 ml) supplemented with 100 mg/ml ampicillin is inoculated with E. coli containing the desired clone rotated overnight at 37° in a shaking incubator. 2. The overnight culture is diluted 1 : 10 into fresh Terrific broth containing ampicillin and is grown for 1 hr. 3. Expression of the fusion protein is induced with 200 tzM IPTG and the culture is grown for an additional 4 hr. 13D. Sha'ag and E. Pick, Biochim. Biophys.Acta 1037, 405 (1990).
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OXIDASE ACTIVITY
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4. The cells are harvested by centrifugation at 300g for 15 min, and the obtained cell pellet is resuspended in 5-7 ml of lysis buffer containing 50 mM Tris, pH 7.5, 5 mM MgCI2, 1 mM DTF, and 1 mM PMSF. 5. The cell suspension is sonicated thoroughly (4 × 15 sec) on ice, and the cell debris is removed by centrifugation at 48,000g for 15 min. 6. The supernatant is incubated for 1 hr at 4 ° with 0.5-1 ml of glutathione-Sepharose beads (Pharmacia or Sigma) prewashed with lysis buffer. 7. The glutathione beads are washed three times with 10 ml cold lysis buffer by centrifuging the beads at 400g for 5 min and resuspending them in fresh buffer. 8. The bound proteins are eluted by resuspending the beads in 24 ml of lysis buffer containing 5 mM reduced glutathione (Sigma). The suspension is incubated for 2 rain, and the eluted proteins are recovered in the supernatant obtained by spinning down the beads. 9. To release the desired protein from the GST protein, thrombin cleavage is required. The buffer solution containing the fusion protein is exchanged into thrombin buffer containing 50 mM Tris-HC1, pH 8.0, 150 mM NaC1, 5 mM MgCI2, 2.5 mM CaC12, and 1 mM Dq-T. The buffer exchange can be achieved either by dialysis or by desalting on a PD10 column (Pharmacia). 10. The activity of thrombin obtained from different sources (most commonly human or bovine) varies substantially, making it essential to optimize the cleavage conditions. A small-scale reaction is set by incubating 20 t~g of fusion protein with various amounts of thrombin (1-20 units depending on the source of thrombin) for 1 hr at room temperature. The efficiency of the cleavage is analyzed by S D S - P A G E visualized by Coomassie blue staining. 11. Once the optimal ratio of thrombin to fusion protein is obtained, the reaction is scaled up and the rest of the fusion protein is cleaved. 12. The activity of thrombin is inhibited by the addition of 10 /zM APMSF (Sigma Cat. No. A6664), and the efficiency of the cleavage is again assessed by SDS-PAGE. 13. A further purification is required to separate the fusion protein from GST. This can be achieved either by reabsorbing the GST on glutathioneagarose or by separation by anion-exchange chromatography on a Mono Q column (HR 5/5 Pharmacia) under standard conditions. For instance, p21 Racl and p47ph°xwere separated from GST by fractionation on a Mono Q column; the GST bound to the Mono Q (and can be eluted by 250300 mM NaC1) and p21 Racl and p47ph°xremained in the unbound fractions. Typically, 1 mg of pure recombinant protein is produced using this method from 1 liter of broth culture. The proteins were concentrated by centrifuga-
276
CELL EXPRESSION
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tion on Centricon 10 membrane concentrators (Amicon) to a concentration of 1 mg/ml, aliquoted to 50/xl/vial, snap frozen in liquid nitrogen, and stored at - 7 0 °. R e c o n s t i t u t i o n of Cell-Free NADPH Oxidase
Assay for Superoxide Production The most widely used assay for measuring superoxide production is the reduction of cytochrome c inhibitable by superoxide dismutase (SOD). The assay has been described in detail by Pick. 14 The superoxide ions generated by the activated oxidase reduce cytochrome c, which has an absorption maximum at 550 nm. The rate of superoxide generation is calculated from the slope of the linear range of the absorbance at 550 nm, using e550 = 2.1 × 104 M -1 cm -1 Since cytochrome c can be reduced by reducing sources other then superoxide, it is essential to demonstrate that the reaction is inhibitable by SOD, indicating that the reduction of cytochrome c is specific.
Cell-Free Assay a. Using Cytosol and Membranes. It is advisable to first optimize the reconstitution of the oxidase in the cell-free assay with the solubilized membrane and cytosol. This helps in identifying an inactive recombinant protein. This system can also be used to test cytosol and membranes from patients with C G D to identify the impaired component as described in this series. Solubilized membrane (30-50 ml) (diluted 1:8 to dilute the detergent, 5-20 mg protein) and 10-40/xl cytosol (50-150 mg) are incubated for 90 sec in 0.9 ml assay buffer consisting of 65 m M sodium phosphate buffer, p H 7.0, 1 m M E G T A , 1 m M MgCI2, 1 0 / z M FAD, and 1 0 0 / z M cytochrome c. To determine the optimal concentrations of the SDS activator, various concentrations of SDS are added to the incubation mixture. SDS (6-15 ml) from a 10 m M stock solution is added to the incubation mixture to achieve optimal oxidase activation. A final concentration of SDS is 80-120 mM. The reaction is then initiated by the addition of 200 tzM N A D P H (20 ml from 10 m M stock), and the rate of superoxide generation is measured for 2 min in a double beam spectrophotometer (Uvikon 860, Kontron). The reference sample contains SOD (Sigma; we use at 50/xg/ ml) in addition to reagents mentioned previously. The same procedure can be applied with partially purified components. The solubilized membrane 14E. Pick, in "Methods in Enzymology" (G. Di Sabato and J. Everse, eds.), Vol. 132, p. 407. Academic Press, San Diego, 1986. 15F. B. Wientjes, J. J. Hsuan, N. F. Totty, and A. W. Segal, Biochem. J. 296, 557 (1993).
[29]
RECONSTITUTION ov NADPH OXIDASEACTIVITY
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(as mentioned earlier) is incubated with a fraction enriched in p47ph°x and p67ph°xobtained by the methods described earlier, supplemented with either partially purified or recombinant p21 Racl and an optimal concentration of SDS, incubated for 2 min, and electron transport initiated with NADPH. Note: Figure 2 shows that it is possible to demonstrate that reconstituted activity is dependent on the different components described earlier. b. Reconstitution of Cell-Free NADPH Oxidase from Purified Components. Since the recombinant p21 Racl purified from E. coli is predominantly in the GDP-bound form, to obtain a functional GTPase protein it is essential to exchange the nucleotide to either GTP or GTPyS. This is done by incubating the protein (100/xg/ml) in the presence of 100 mM guanine nucleotide in 5 mM EDTA for 10-15 min at room temperature followed by the addition of 10 mM MgC12 to chelate the EDTA and lock the nucleotide into the protein. Once all the reagents are prepared and the cell-free assay is optimized, the system is ready for reconstitution with purified components. Higher enzymatic activities can be obtained by separating the activation process from the catalysis reaction. Thus, the purified components are first preincubated in an optimal SDS concentration in a small volume (100 txl) of assay buffer containing but lacking cytochrome c and containing FAD
~ l 80O
0
~
L
~
~
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i
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i
20
i
Membrane
+
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.
.
.
.
Cytochromeb p67-phox p47-phox p21rac1 SDS
+ + + +
+
+ +
+ + +
+ + +
+ + +
.
. +
+ + + +
+
+ +
+ + + + +
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+
FIG. 2. Reconstitution of NADPH oxidase activity in the cell-free system by recombinant
p67ph°x, p47ph°x, p21 Racl, and pure cytochrome b. Solubilized neutrophil membrane (6/zg protein, 4 pmol cytochrome b) or pure relipidated cytochrome b (4 pmol) was incubated either with 30 /zl neutrophil cytosol (100 p~g protein) or with p67ph°x (4.5 nM), p47ph°x (43.5 nM), and p21 Racl (48.5 nM) preloaded with GTP and the optimal concentration of SDS (where indicated).
278
CELLEXPRESSION
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and then transferring this to 0.9 ml of catalysis buffer to initiate the reaction with NADPH. Ten microliters of relipidated cytochrome b (0.1-0.5/zg, 1-5 pmol) is preincubated for 2 min at room temperature with 2-40/.d (0.1-4/zg protein) p47ph°x, p67ph°x, and p21 Racl in the presence of an optimal concentration of SDS (100-200/zM, 10-20/zl from 1 mM stock) and 10 t~M FAD, and the volume is complete to 100/xl with assay buffer lacking cytochrome c. The mixture is then transferred to a 1-ml cuvette containing 0.9 ml assay buffer with 100/zM cytochrome c and the reaction is initiated by the addition of 20/xl of NADPH (from 10 mM stock). The superoxide production is monitored for 120 sec in a double-beam spectrophotometer as described earlier. The assay can be performed in a microplate as described in a previous volume of this series.
Calculations Since the reduction of cytochrome c represents the amount of superoxide generated (1 mol of superoxide will reduce 1 tool of cytochrome c), the rate of superoxide generation is calculated from the slope of the linear range of the absorbance at 550 nm, using e550 = 2.1 × 104 M -1 cm 1 of cytochrome c: Absorbance change of 1 unit at 550 n m × 47.6 = nmol 02-. The specific activity is expressed as either nmol O2/min/mg of membrane protein or as mol O2-/mol cytochrome b-245/sec. Typical values should be in the range of 1500-3000 nmol O2-/min/mg protein and 60-120 mol O2-/mol cytochrome b-245/sec, respectively. Acknowledgment We thank the Wellcome Trust for support.
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[30] G e n e t i c a n d B i o c h e m i c a l A n a l y s i s o f C d c 4 2 p Function in Saccharomyces cerevisiae and
Schizosaccharomyces pombe B y JAMES POSADA, PETER J. MILLER, JANET MCCULLOUGH,
MICHAEL ZIMAN, and DOUGLAS I. JOHNSON
Introduction
CDC42 was originally identified in Saccharomyces cerevisiae by isolation of the cdc42-I temperature-sensitive lethal mutant, which displayed a cell cycle arrest as a loss of cell polarity and polarized cell growth when grown at restrictive temperaturesJ Subsequent molecular analyses 2 classified Cdc42p as a member of the Rho/Rac subfamily of the Ras superfamily of low molecular weight GTPases based on the predicted amino acid sequence similarity between Cdc42p and other members of this subfamily. 3,4 Cdc42p homologs have been isolated in Schizosaccharomyces pombe, Caenorhabditis elegans, and Homo sapiens. These proteins exhibit approximately 80% amino acid identity to each other and are able to complement functionally the S. cerevisiae cdc42-P or null mutation. This degree of structural and functional conservation suggests that most true Cdc42p homologs will be functionally interchangeable with the S. cerevisiae protein, an ability that has been used to classify new GTPases as Cdc42p homologs. It also suggests that Cdc42p may have a conserved function in eukaryotic cells. This chapter outlines genetic and biochemical strategies that have been employed to study Cdc42p function in S. cerevisiae and S. pombe. This detailed examination in these distantly related organisms has revealed similarities and important differences in Cdc42p function, a fact that emphasizes the need to study the functions of highly conserved proteins in several experimentally tractable organisms. a A. E. M. Adams, D. I. Johnson, R. M. Longnecker, B. F. Sloat, and J. R. Pringle, J. Cell Biol. 111, 131 (1990). 2 D. I. Johnson and J. R. Pringle, J. Cell Biol. 111, 143 (1990). 3 D. I. Johnson, in "The ras Superfamily of GTPases" (J. C. Lacal and F. McCormick, eds.), p. 297. CRC Press, Inc., Boca Raton, FL, 1993. 4 D. I. Johnson and J. R. Pringle, in "Guidebook to the Small GTPases" (L. A. Huber, M. Zerial, and J. Tooze, eds.). Oxford University Press, Oxford, UK, 1995.
METHODS 1N ENZYMOLOGY, VOL. 256
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M u t a t i o n a l Analysis of C d c 4 2 p F u n c t i o n Saccharomyces cerevisiae and S. p o m b e are especially useful genetic systems because the haploid phase of their life cycles makes the generation of strains with uncovered recessive mutations possible and because wildtype and mutant alleles can be easily introduced episomally or integrated into specific sites in the genome. For a compilation of basic genetic and molecular biology techniques used with S. cerevisiae and S. p o m b e , see Guthrie and Pink s and Alfa et a/. 6 Cells harboring the cdc42-1 loss of function allele were isolated from a population of EMS-mutagenized cells on the basis of the cellular morphology of its temperature-sensitive (ts) cell division cycle arrest. When transferred from the permissive temperature (23-24 ° ) to the restrictive temperature (36-37°), cdc42-1 ts strains arrest uniformly within one cell cycle as enlarged, round, unbudded cells. Although cell division is arrested, D N A replication and nuclear division continue into the next cycle as determined by D N A staining with D A P I and mitotic spindle staining with antitubulin antibodies. The arresting cells incorporate chitin throughout the cell wall instead of localizing it to the incipient bud site, indicating that isotropic incorporation of new cell wall material is not impaired through the loss of Cdc42p function. The polarized organization of the actin cytoskeleton (i.e., cortical actin distribution to the regions of new cell growth and actin cables directed into the enlarging bud) is disrupted in loss of function, dominantactivated, and dominant-negative (see below) cdc42 mutants in both S. cerevisiae and S. p o m b e , indicating that Cdc42p, like other Rho/Rac-like GTPases, functions in the organization and coordination of the actin network during polarized cell growth. The levels of Cdc42 protein in a cdc42Its strain are - 1 0 - f o l d less than in a wild-type strain, even at the permissive temperature, which may contribute to the mutant phenotypes. 7 The structural homology between Ras proteins and Cdc42p suggested critical amino acid residues in Cdc42p important for G T P binding and hydrolysis. Therefore, Ras-like mutations were made in S. cerevisiae and S. p o m b e Cdc42p 7,8 using site-directed mutagenesis protocols. 9 These mutations, G12V, Q61L, and D l l 8 A , are analogous to the dominanttransforming G12V, Q61L, and D l l 9 A mutations of mammalian Ras.
5 c. Guthrie, and G. R. Fink, this series, Vol. 194, 1991. 6 C. Alfa, P. Fantes, J. Hyams, M. McLeod, and E. Warbrick, "Experiments with Fission Yeast: A Laboratory Course Manual." Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1993. 7M. Ziman, J. M. O'Brien, L. A. Ouellette, W. R. Church, and D. I. Johnson, Mot Cell BioL 11, 3537 (1991). 8p. j. Miller and D. I. Johnson, Mol. Cell. Biol. 14, 1075 (1994). 9T. Kunkel, Proc. Natl. Acad. Sci. U.S.A. 82, 488 (1985).
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Briefly, wild-type CDC42 was inserted into M13mp9 to generate a singlestranded D N A template, which was then used with mutagenic oligonucleotide primers and the MUTA-GENE kit (Bio-Rad Laboratories, Richmond, CA) to generate single mutants. After confirmation of the presence of the desired mutations and the absence of any spurious mutations, the mutant genes were inserted into either multicopy or low-copy plasmid vectors for use in yeast transformations. To generate p G A L versions, the wild-type CDC42 promoter sequences were removed by restriction digest and replaced by a D N A fragment containing the divergent G A L l and G A L I O promoters derived from plasmid pBM272.1° In S. cerevisiae, the cdc42 G12v and cdc42 Q6m mutations, when placed under the control of the GAL-inducible promoter system 1° in a CDC42 background, resulted in cell death and heterogenous morphological phenotypes. Seventy percent of the population showed enlarged, amorphous cells with elongated buds and/or cells with multiple buds, suggestive of an activated cell polarity pathway. The actin cytoskeleton is disrupted in these mutants, condensing as actin bars in 60% of cells. In addition, chitin deposition is somewhat perturbed and 20% of cells are multinucleate. These results suggest that constitutively activated Cdc42 proteins have lost the ability to control directed cell wall deposition or polarized cell growth. This phenotype is additionally correlated with disruption of the cellular actin cytoskeleton. The cdc42 Dn8A mutation has a dose- and temperature-dependent dominant lethal phenotype in S. cerevisiae; expression of the cdc42 DnSA allele from a G A L promoter is dominant lethal at 23-27 ° but not at 30-34°. 7,n Cells expressing this allele die with the cdc42 null phenotype of large, round, unbudded cells. Thus, in contrast to the analogous mutation in mammalian Ras which confers a dominant-activated phenotype, the cdc42 DusA mutation is dominant negative, suggesting that the mutant protein interfere with the proper function of endogenous Cdc42p, possibly by improperly sequestering important cellular factors away from it. Multicopy suppression of the dominant lethality associated with the cdc42 DusA allele may identify cellular factors involved in this phenotype (see below). This result may also point to an important difference in the function of Cdc42p and other members of the Ras superfamily, perhaps involving nonconserved amino acids in this region. Similar mutations were generated in S. p o m b e Cdc42p, which was isolated by its ability to complement the S. cerevisiae cdc42-1 mutant, 8 and expressed under the control of the strong, repressible n m t l ÷ promoter in 10 M. Johnston, MicrobioL Rev. 51, 458 (1987). 11 M. Ziman and D. I. Johnson, Yeast 10, 463 (1994).
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multicopy p R E P plasmids. 12 Expression from the nmtl ÷ promoter in S. p o m b e is induced in the absence of thiamine and is repressed by growth in 2/xm thiamine. In contrast to the results with S. cerevisiae, these mutants were not dominant lethal in S. p o m b e but they did exhibit a slower growth rate and a dominant morphological phenotype of large, rounded or misshappen cells and disrupted actin distribution, suggestive of an activated cell polarity pathway. In addition, there was not an obvious difference between the cdc42 DuSA mutant phenotype and the cdc42 G12v and c d c 4 2 Q61L phenotypes in S. pombe. T o further expand the study of Cdc42p in S. pombe, a loss of function cdc42 mutant allele was generated by gene disruption techniques. 13 Again, the morphological phenotype observed in S. p o m b e differed from S. cerevisiae. Both genes are essential for growth and transit through the cell cycle in their respective organisms, but in contrast to the large, round, unbudded cells in S. cerevisiae, loss of function mutants in S. p o m b e resulted in small, round, dense cells, suggesting that Cdc42p function is necessary for incorporation of cellular material into enlarging S. p o m b e cells but is not necessary for isotropic incorporation into S. cerevisiae cells. In addition, D N A replication and the nuclear cycle do not continue in S. p o m b e cdc42 mutants as they do in S. cerevisiae cdc42 mutants. Therefore, through the mutational analysis of a highly conserved protein in two distantly related eukaryotes, important differences in protein function have been revealed.
Genetic A p p r o a c h e s to Identification of I n t e r a c t i n g P r o t e i n s Conditional lethal alleles, either temperature-sensitive or cold-sensitive alleles, such as cdc42-1 ts, or mutant alleles that are expressed under the control of an inducible promoter, such as p G A L - d r i v e n cdc42 alleles, are very useful in genetic screens designed for the identification of new or preexisting genes whose products interact with a gene of interest. Several genetic p h e n o m e n a have been used to identify and characterize proteins that interact with Cdc42p. Analysis of the cellular morphologies of temperature-conditional lethal mutations in S. cerevisiae identified three genes, CDC24, CDC42, and CDC43, that probably acted at a similar stage in the cell polarity pathway. 134 Genetic studies originally suggested a functional interaction between Cdc42p and Cdc43p. In particular, a cdc42 ts cdc43 ts double mutant exhibited synthetic lethality, a p h e n o m e n o n in which a double mutant is inviable at a 12K. Maundrell, Gene 123, 127 (1993). 13S. Moreno, A. Klar, and P. Nurse, this series, Vol. 194, p. 795. 14B. F. Sloat and J. R. Pringle, Science 200, 1171 (1978).
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temperature (i.e., 23 °) that is permissive for each single mutant. 1 Subsequent molecular and biochemical analyses indicated that C D C 4 3 encodes the/3 subunit of the geranylgeranyltransferase type I (GGTase-I) that prenylates the C-terminal Cys-188 residue in Cdc42p. 15-17This prenylation is required for the proper function and cellular localization of Cdc42p (see below). Therefore, this synthetic lethality may reflect the essential role of Cdc43p in the prenylation of Cdc42p, together with a partial loss of function of both mutant proteins at the nominally permissive temperature. Various genetic interactions have also been observed between C D C 4 2 and C D C 2 4 . A cdc42%dc24 ts double mutant displays a synthetic lethal phenotype at 30°.11 In addition, a cdc24 ts mutation can be suppressed by overproduction of Cdc42p on a low-copy number vector18; this multicopy suppression appears to require a partial retention of Cdc24p activity. In a reciprocal interaction, overproduction of Cdc24p on a high-copy number vector or under the control of a G A L promoter can suppress the dominantn e g a t i v e c d c 4 2 D l l s A mutation 11 (see earlier), suggesting that the dominantnegative phenotype may result from sequestration of Cdc24p by the mutant Cdc42p. Finally, although overproduction of Cdc42p or Cdc24p alone is not lethal, simultaneous overproduction of both proteins leads to a lethal phenotype similar to loss of function mutations in either gene. These genetic data are consistent with a role of Cdc24p in stimulation of guanine nucleotide exchange on Cdc42p. Molecular analysis revealed that Cdc24p has 20% sequence identity to a domain of the human dbl oncogene product, 19'2° and biochemical studies have shown that Dbl can stimulate guanine nucleotide exchange on the human Cdc42p. 21 Direct biochemical evidence confirms the genetic data that Cdc24p stimulates guanine nucleotide exchange on Cdc42p in vitro. 22 There is also evidence that the product of B E M 3 , a gene identified through its genetic interaction with another gene ( B E M 2 ) involved in controlling cell polarity, 23 acts as a GTPase-activating protein (GAP) for Cdc42p in vitro. 22 Therefore, through a combination of genetic and biochemical techniques, three necessary components that modulate Cdc42p 15 D. I. Johnson, J. M. O'Brien, and C. W. Jacobs, Gene 98, 149 (1991). 16 A. A. Finegold, D. I. Johnson, C. C. Farnsworth, M. H. Gelb, S. R. Judd, J. A. Glomset, and F. Tamanoi, Proc. Natl. Acad. Sci. U.S.A. 88, 4448 (1991). 17 M. L. Mayer, B. E. Caplin, and M. S. Marshall, J. Biol. Chem. 267, 20589 (1992). 18 A. Bender and J. R. Pringle, Proc. Natl. Acad. Sci. U.S.A. 86, 9976 (1989). 19 S. Miyamoto, Y. Ohya, Y. Ohsumi, and Y. Anraku, Gene 54, 125 (1987). 2o D. Ron, M. Zannini, M. Lewis, R. B. Wickner, L. T. Hunt, G. Graziani, S. R. Tronick, S. A. Aaronson, and A. Eva, N e w Biol. 3, 372 (1991). 21M. J. Hart, A. Eva, T. Evans, S. A. Aaronson, and R. A. Cerione, Nature 354, 311 (1991). 22 y. Zheng, R. Cerione, and A. Bender, J. Biol. Chem. 269, 2369 (1994). 23 A. Bender and J. R. Pringle, Mol. Cell, Biol. 11, 1295 (1991).
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TABLE I CORRELATIONS BETWEEN BIOCHEMICAL FUNCTIONS AND GENETIC INTERACq'IONS
Gene
Biochemical function
Genetic interactions
CDC24
GDP-GTP exchange factor
CDC43 BEM3
/3 subunit of GGTase I GTPase activating protein
Syntheticlethality with cdc4U s at 30° CDC24 multicopy suppressor of cdc42 DllSA CDC42 multicopy suppressor of cdc24-4" Lethal simultaneous overexpression of CDC24 and CDC42 Synthetic lethality with cdc42 ts at 23° Multicopy suppressor of bern2
function have been identified (Table I): one is involved in the subcellular localization of Cdc42p and two are involved in regulating the nucleotidebound state of Cdc42p.
S u b c e l l u l a r Localization of C d c 4 2 p in S a c c h a r o m y c e s
cerevisiae
Affinity-purified C d c 4 2 p - s p e c i f i c antibodies have been used to explore the site and regulation of Cdc42p subcellular localization in S. cerevisiae. 24 Using immunofluorescence microscopy 25 and immunoelectron microscopy, 26 Cdc42p was localized to the plasma m e m b r a n e at sites of new polarized cell growth at the tips of emerging buds during the mitotic cell cycle and to the tips of mating projections in p h e r o m o n e - a r r e s t e d cells. In some serial sections, Cdc42p and actin a p p e a r to colocalize, further suggesting an interaction with the actin cytoskeleton. The C-terminal Cys-188 residue of Cdc42p was shown to be necessary for plasma m e m b r a n e localization, and m e m b r a n e localization was defective in prenylation-minus c d c 4 3 mutants, thereby reinforcing the need for p r o p e r prenylation of Cdc42p for p r o p e r function. Although the order of assembly of Cdc42p and other proteins required at the incipient bud site in S. cerevisiae is not yet defined, the potential of Cdc42p to function as a m e m b r a n e localized switch suggests a key role for Cdc42p in the establishment of cellular polarity during the eukaryotic cell cycle. 24M. Ziman, D. Preuss, J. Mulholland, J. M. O'Brien, D. Botstein, and D. I. Johnson, Mol. Biol. Cell 4, 1307 (1993). 25j. R. Pringle, R. A. Preston, A. E. M. Adams, T. Stearns, D. G. Drubin, B. K. Haarer, and E. W. Jones, Meth. Cell BioL 31, 357 (1989). 26D. Preuss, J. Mulholland, A. Franzusoff, N. Segev, and D. Botstein, MoL BioL Cell 3, 789 (1992).
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E x p r e s s i o n a n d Purification of Cdc42p F u s i o n Proteins Expression in S. cerevisiae of proteins fused to another protein ligand that is specific for a cognate affinity matrix provides a useful way to rapidly isolate large amounts of a given protein of interest. The expression of fusion proteins in S. cerevisiae has several advantages. First, mutant forms of the protein may be expressed at high levels and be rapidly purified independently of the wild-type version of the protein. Another significant advantage over expression of S. cerevisiae proteins in Escherichia coli is that the fusion proteins are expressed in their usual milieu and are therefore subject to post-translational modifications that are often necessary for proper protein function. We will describe the use of glutathione S-transferase (GST) and (His)6 tags to create Cdc42p N-terminal fusion proteins that are readily isolated from yeast cells by chromatography on the appropriate affinity column. C-terminal fusions were avoided because the last four amino acids of Cdc42p comprise the C A A X box that is necessary for prenylation. For expression as a GST fusion protein, the CDC42 D N A was inserted into the SmaI site of pEG(KG). 27 This vector contains an ampicillin resistance marker for selection in E. coli as well as the U R A 3 and L E U 2 - d genes for selection in yeast. This construction resulted in an - 4 3 - k D a protein (Fig. I) with GST being fused in frame to the N terminus of the entire CDC42 coding region. For the (His)6-tagged version of CDC42, a double-stranded oligonucleotide cassette that coded for six tandem His residues along with a Factor Xa cleavage site (kindly provided by S. DeSimone) was linked to the 5' end of CDC42. This construct was subsequently inserted into the B a m H I site of pLGSD5, 28 a multicopy URA3-based plasmid that contains a G A L l - d r i v e n promoter fused to the N terminus of the C Y C I gene. This resulted in an - 2 4 - k D a protein (Fig. i) with an in-frame fusion between the first three amino acids of Cyclp, six histidine residues, a Factor Xa cleavage site, and full-length Cdc42p. Both of these fusion proteins are immunoreactive using affinity-purified anti-Cdc42p antibodies (data not shown). Methods Materials
The fusion protein constructs are transformed into the S. cerevisiae protease-deficient strain BJ5459 (Yeast Genetic Stock Center, Berkeley, CA) to reduce the likelihood of proteolytic degradation of the fusion pro27D. A. Mitchell,T. K. Marshall, and R. J. Deschenes, Yeast 9, 715 (1993). 28L. Guarente, R. R. Yocum, and P. Gifford, Proc. NatL Acad. Sci. U.S.A. 79, 7410 (1982).
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A L,qsate
Flow/Wash
L,qsate
Flow/Wash
Eluate
43 kDa
Eluate
18 kDa
F1G. 1. Coomassie blue visualization of GST-tagged (A) and (His)6-tagged (B) Cdc42p fusion proteins from S. cerevisiae cell extracts. The arrows point to the fusion proteins as determined by Western blot analysis using affinity-purified anti-Cdc42p antibodies.
teins. S. cerevisiae cells are lysed by vortexing with 400- to 500-/xm acidwashed glass beads (Sigma Co., St. Louis, MO) in a Bead-Beater (Biospec Products, Bartlesville, OK). Glutathione-Sepharose (Pharmacia Biotech, Inc., Piscataway, N J) was used to bind the GST fusion proteins and nickelnitrilotriacetic acid (NTA)-Sepharose (Qiagen, Inc., Chatsworth, CA) was used to bind the (His)6-tagged proteins.
Reagents
Raffinose-synthetic complete medium lacking uracil (final concentrations): 0.8% yeast nitrogen base without amino acids, 2% raffinose, 0.0025% stock solution containing all amino acids plus adenine, lacking uracil 20% galactose (filter sterilized) Cell lysis buffer (final concentrations): 30 mM HEPES buffer, 300 mM NaC1, 10 mM 2-Mercaptoethanol, 5 mM MgC12, 10% glycerol, 2% Triton X-100
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Protease inhibitors: 0.5 mM phenylmethylsulfonyl fluoride, 1 ~g/ml aprotinin in water, 1 ~g/ml N-tosyl-L-phenylalanine chloromethyl ketone, 1/~g/ml leupeptin, 1 ~g/ml pepstatin Imidazole gradient buffer (final concentrations): 30 mM HEPES buffer, 300 mM NaC1, 1 mM 2-mercaptoethanol, 5 mM MgC12, 0.1% Triton X-100, 10% glycerol, protease inhibitors (see above), imidazole at various concentrations (step gradient) Glutathione elution buffer (final concentrations): 10 mM glutathione, 50 mM Tris, pH 8.0
Growth of S. cerevisiae Cells Containing Fusion Proteins The same basic growth and cell lysis protocol was followed for the GST fusion protein and (His)6-tagged protein: 1. Grow 1 liter of BJ5459 cells containing the appropriate plasmid in raffinose-synthetic complete medium lacking uracil at 30° to ODsg0 of 0.20.3 (~6 x 109 cells). Add galactose to 2% final concentration and continue growth for 6-8 hr. 2. After harvesting and washing of cells (two times in cold water), cell pellets are weighed and resuspended in cold cell lysis buffer at 2 g/ml. Prechilled glass beads are added and lysates are vortexed in a Bead-Beater for ten 30-sec cycles. The cells are monitored by light microscopy until 8595% cell lysis is achieved. The supernatant is pipetted off and the glass beads are washed twice with an equal volume of lysis buffer. 3. The combined lysate and washes are spun at 10,000g for 1 h at 4 °. Most of the Cdc42p fusion protein remains in the supernatant after this spin. The supernatant is made to 0.2% Triton X-100 and applied to the affinity columns.
GST fusion protein 1. Glutathione-Sepharose beads (200 ~1) are washed three times in cell lysis buffer and then incubated with the cell lysate supernatant in an Eppendorf tube for 1 hr at 4 ° with gentle shaking. The suspension is spun at 500g for 10 min at 4 ° and the supernatant is removed and saved (flowthrough fraction). 2. The beads are washed three times with 2 ml cold lysis buffer and then incubated with glutathione elution buffer (200 ~1) for 10 min at 4 ° with gentle shaking. Beads are spun at 1000g for 10 min at 4 ° and the supernatant is removed and saved. This elution protocol is repeated up to 10 times and the individual eluate fractions are analyzed, along with flowthrough and wash fractions, on a SDS-12% polyacrylamide gel (Fig. 1).
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iI
(His)6- Tagged Protein 1. Nickel-NTA agarose beads (1 ml) are washed two times with lysis buffer and then incubated with the cell lysate supernatant in a 15-ml tube for 1 hr at 4 ° with gentle shaking. The suspension is then poured into a disposable 1-ml column (Bio-Rad, Richmond, CA) and the flow-through is collected and saved. The column is washed with 20 bed volumes of lysis buffer, monitoring the flow-through until the OD590 is less than 0.01. 2. The fusion protein is eluted using a 0-200 mM imidazole gradient (15 ml) in imidazole gradient buffer at no greater than 3-4 bed volumes/ hr. Fractions (1 ml) are collected and analyzed on SDS-12% polyacrylamide gels by Coomassie blue staining (Fig. 1). The peak fractions are pooled and dialyzed. We have found that it is necessary to dialyze away the imidizole because even small amounts significantly decrease the amount of GTP that binds to Cdc42p and another GTPase, Rab3a. Acknowledgments This research was supported by National Science F o u n d a t i o n G ra nt DMB-9105111, A me ri can Cancer Society G r a n t MV 469, and a grant from the Lucille P. M a r k e y Charitable Trust.
[31] L y m p h o c y t e
Aggregation Assay and Inhibition by
Clostridium b o t u l i n u m C 3 A D P - R i b o s y l t r a n s f e r a s e By TOMOKO TOMINAGA and SHUH NARUMIYA The integrin molecules play a major role in the cell adhesion to matrix and mediate some of the cell to cell adhesions. Lymphocyte functionassociated antigen-1 (LFA-1, aL/32, CDlla/CD18) is a member of the leukocyte integrins and binds to its counterreceptors, the intercellular adhesion molecules (ICAMs)-I and -2.1 This binding is involved in such processes as leukocyte adhesion to endothelial and epithelial cells, target cell recognition by cytotoxic T lymphocytes, and binding of T lymphocytes to antigenpresenting cells. LFA-1 is not constitutively avid for ICAMs but requires activation for binding, whereas ICAMs are avid for activated LFA-1 without any stimulation. The LFA-1 activation can be triggered by stimulation of CD2 and CD3 on T lymphocytes or by treatment of cells with phorbol 1 T. A. Springer, Nature (London) 346, 425 (1990).
METHODS IN ENZYMOLOGY,VOL. 256
Copyright© 1995by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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291
myristate acetate (PMA). 2'3 The E p s t e i n - B a r r virus-transformed B lymphoblastoid cell line, JY, expresses high levels of both LFA-1 and ICAM-1, and shows homotypic aggregation in response to PMA. 4 The physiological function of a rho gene product ( R h o p21) has been examined by microinjection of mutant R h o p21 into cells and by inactivation of endogenous R h o p21 by ADP-ribosylation with botulinum C3 exoenzyme. 5-8 Such experiments in cultured fibroblasts 7 and blood platelets 8 have revealed that R h o p21 is involved in the integrin activation pathway and serves as a transducer between cell stimuli and cell-matrix adhesion. This chapter describes the procedure to assess the role of R h o p21 in activation of LFA-1 during PMA-induced homotypic aggregation of JY cells. 9 The R h o p21 responsible for this activation appears to be R h o A p21, because JY cells express high level of r h o A m R N A but little of r h o B and rhoC. This assay is useful in analyzing not only the Rho p21-dependent pathway but the molecular mechanism of integrin activation in general.
Cell C u l t u r e a n d T r e a t m e n t with C3 E x o e n z y m e JY ceils are maintained in R P M I 1640 medium supplemented with 10% fetal calf serum, 50/xg/ml streptomycin, and 50 U/ml penicillin (complete medium) at 37 ° in an atmosphere containing 5% (v/v) CO2. The medium is changed every 2 - 3 days. For experiments, logarithmically growing JY cells are washed with serum-free medium and plated at 7.5 x 105/well in 1.5 ml of the complete medium in a 6-well plate. Various concentrations of recombinant C3 exoenzyme prepared as described l°'u or vehicle are 2 M. L. Dustin and T. A. Springer, Nature (London) 341, 619 (1989). 3y. van Kooyk, P. W. Kemenade, P. Weder, T. W. Kuijpers, and C. G. Figdor, Nature (London) 342, 811 (1989). 4 R. Rothlein and T. A. Springer, J. Exp. Med. 163, 1132 (1986). 5p. Chardin, P. Boquet, P. Madaule, M. R. Popoff, E. J. Rubin, and D. M. Gill, EMBO J. 8, 1087 (1989). 6 H. E. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell Biol. 111, 1001 (1990). 7 A. J. Ridley and A. Hall, Cell 70, 389 (1992). 8N. Morii, T. Teru-uchi, T. Tominaga, N. Kumagai, S. Kozaki, F. Ushikubi, and S. Narumiya, J. Biol. Chem. 267, 20921 (1992). 9T. Tominaga,K. Sugie,M. Hirata, N. Morii, J. Fukata, A. Uchida, H. Imura, and S. Narumiya, J. Cell Biol. 120, 1529 (1993). 10N. Kumagai, N. Morii, K. Fujisawa, Y. Nemoto, and S. Narumiya, J. Biol. Chem. 268, 24535 (1993). 11N. Morii and S. Narumiya, this volume [22].
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added and the cells are cultured for indicated periods. After treatment, the cells are washed twice with serum-free RPMI 1640 medium. An aliquot of cells is suspended in 50/xl of 0.25 M sucrose containing 20 mM Tris-HC1, pH 7.5, 5 mM MgCI2, i mM EDTA, 1 mM dithiothreitol, 2 mM benzamidine hydrochloride, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The suspension is homogenized by sonication (5 sec × three times) on ice and the homogenate is centrifuged at 1000g for 5 min. Supernatants (50/xg protein) are used for the ADP-ribosylation assay as described elsewhere in this volume, u Incubation of cells with 20/~g/ml C3 exoenzyme for 8, 16, and 24 hr typically results in ADP-ribosylation of endogenous Rho p21 of 50, 65, and 78%, and that with 3, 10, and 30/~g/ml of the enzyme for 24 hr results in ADP-ribosylation of 60, 78, and 83% of the cell substrate, respectively (Fig. 1). We use incubation with 20/zg/ml C3 exoenzyme for 24 hr as the standard treatment.
kD 97.4
1
2 3 4
69 46
30
21.5
14.3
F l 6 . 1. In situ ADP-ribosylation in JY cells treated with C3 exoenzyme. JY cells were treated with 0 (lane 1), 3 (lane 2), 10 (lane 3), and 30 (lane 4)/zg/ml of C3 exoenzyme for 24 hr. The cells were washed and homogenized as described in the text, and unmodified Rho p21 was ADP-ribosylated using [32p]NAD. In situ ADP-ribosylation was calculated by subtraction of the [32p]ADP-ribosylation in the treated cells from that in the control cells. Reproduced from Tominaga et aL 9 by copyright permission of the Rockfeller University Press.
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LYMP~qOCVTr~ AGGr~ZGAT~ON[NHIBrrION r~Y C3
~<~
293
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Aggregation Assay in 96-Well Microtiter Plate
Phorbol Ester-Induced Aggregation of J Y Cells Washed JY cells are suspended at a density of 2 × 106 cells/ml in RPMI 1640 medium buffered with 5 mM HEPES-NaOH, pH 7.4 (the experimental medium). The cell suspension (100 ~1) is added to a well of a flat-bottomed 96-well microtiter plate. PMA (Sigma) is added and the volume is adjusted to 200 ~l/well. The cells are allowed to settle spontaneously in a CO2 incubator, and aggregation is observed under a phasecontrast microscope at 30 min, 3 hr, and 16 hr after the PMA addition. The degree of aggregation can be scored semiquantitatively from 0 to +5, where 0 indicates that essentially no cells are in clusters; + 1 indicates less than 10% of the cells in aggregates; +2 indicates less than 50% of the cells are aggregated; +3 indicates that up to 100% of the cells are in irregular, loose clusters; +4 indicates that up to 100% of the cells are in large clusters; and +5 indicates that 100% of cells are in very compact aggregates. PMA
FIG. 3. Time course of PMA-induced JY cell aggregation. JY cell aggregation was evoked by 5 ng/ml of PMA and observed at 0 rain (a), 30 rain (b), 3 hr (c), and 16 hr (d). Bar: 100 ~M.
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induces JY cell aggregation in a concentration-dependent m a n n e r f r o m 0.5 to 500 ng/ml as shown in Figs. 2a-2d. After a 3-hr incubation, the majority of cells aggregate at either concentration, but at 0.5 ng/ml P M A they form only loose clusters of irregular form ( + 3 to +4), whereas the compact ball-like aggregates ( + 5 ) are formed at the higher concentrations, 50 and 500 ng/ml. Aggregation also proceeds in a time-dependent manner; at 5 ng/ml P M A , loose clusters are f o r m e d at 30 min which b e c o m e round ball-like aggregates after 16 hr (Fig. 3). This aggregation is inhibited by monoclonal a n t i - L F A - l a (G-25.2, Becton-Dickinson) and anti-ICAM-1 (LB-2, Becton-Dickinson) antibodies, as previously reported. 4
Inhibition of J Y Cell Aggregation by C3 Exoenzyme Treatment T r e a t m e n t of JY cells with C3 exoenzyme for up to 48 hr does not affect cell viability or cell size. This treatment does not induce change in
FIG. 4. Time-dependent inhibition of PMA-induced JY cell aggregation by C3 exoenzyme treatment. JY cells were treated with 20 tzg/ml of C3 exoenzyme for 0 (a), 8 (b), 16 (c), and 24 (d) hr. PMA (5 ng/ml) was added and aggregation was observed at 16 hr. Bar: 100/xM. Reproduced from Tominaga et al.9by copyright permission of the Rockfeller University Press.
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the expression of LFA-1 and ICAM-1 on JY cells. 9 Inhibition of JY cell aggregation by C3 exoenzyme treatment depends on the extent of A D P ribosylation, in other words, the enzyme amount used in the treatment and the treatment time. Figure 4 shows effects of C3 exoenzyme treatment time on PMA-induced aggregation. The standard stimulation with 5 ng/ml P M A induces aggregation of +5 after 16 hr, which is suppressed to +3 to +4, +2 to +3, and +1 to + 2 aggregation by prior treatment for 8, 16, and 24 hr with 20/~g/ml C3 exoenzyme, respectively. T a k e n together with the results of in situ ADP-ribosylation of R h o p21, inhibition of aggregation does not correlate linearly with the extent of ADP-ribosylation and is observed m o r e clearly and consistently when m o r e than 80% of R h o p21 in the cells is ADP-ribosylated during the enzyme treatment. U n d e r these conditions, inhibition is observed at any of the three observation points, 30 min, 3 hr, and 16 hr, after P M A addition. Inhibition is also observed when the aggregation is induced by higher concentrations of PMA, although the extent of aggregation of C3 exoenzyme-treated cells is increased by much stronger stimuli (Figs. 2e-2h). Aggregation Assay under Shaking Conditions Logarithmically growing J Y cells are washed twice and suspended at 2 x 106/ml in serum-free H E P E S - b u f f e r e d R P M I medium. Two hundred microliters of the cell suspension is transferred into a polystyrene tube (No. TABLE 1 PMA-INDUCED J Y CELL AOGREGATION UNDER SHAKING CONDITIONS a
Cells
Aggregationb (%)
Control cells C3 exoenzyme-treated cells Cytochalasin B-treated cells
62.7 -+ 3.9 27.3 _+ 3.4c 79.3 -_+2 . t d
a j y cells were treated with or without 20/zg/ml of C3 exoenzyme for 24 hr. Cytochalasin B (2 /xM) was added to one group 5 min before the addition of 5 ng/ml of PMA. Reproduced from Tominaga et al. 9 by copyright permission of the Rockfeller University Press. b Values are means + SEM of three independent experiments. Cp < 0.05 for control cells. dp< 0.01 for control cells.
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2054; Falcon Labware, Oxnard, CA). PMA is added at 5 ng/ml, and the tubes are shaken at 200 rpm in a CO2 incubator for 2 hr. The sample is subjected to analysis on a Coulter Multisizer counter (Coulter Scientific Instruments, Hialeah, FL), and free cells in the suspension are counted. The percentage of aggregation of cells is calculated using the formula: 10011 - (number of free cells/number of input cells)]. As shown in Table I, the control cells aggregate more than 60% under these conditions. When cells are pretreated with 20/xg/ml C3 exoenzyme for 24 hr, less than 30% of the cells aggregate. This assay is useful to distinguish the effects on cell adhesion from those on cell motility. Cytochalasin B added at 2 /xM decreases cell motility and inhibits JY cell aggregation in the plate assay. 4 On the other hand, aggregation determined by the shaking method is affected very little as shown in Table I. Rather, it significantly enhances JY cell aggregation.
[32] I n h i b i t i o n o f p 2 1 R h o i n I n t a c t C e l l s b y C 3 Diphtheria Toxin Chimera Proteins
By
PATRICE BOQUET, MICHEL R. POPOFF, MURIELLE GIRY, EMMANUEL LEMICHEZ, and PATRICIA B E R G E Z - A U L L O
Introduction
Clostridium botulinum C and D strains produce a ADP-ribosyltransferase called exoenzyme C31'2 which selectively modifies the p21 GTP-binding protein Rho 3'4 on its asparagine-41. 5 Inactivation of the Rho protein by ADP-ribosylation, on introduction (by microinjection or forced pinocytosis) of exoenzyme C3 into the cytosol, induces the loss of actin filaments in cultured cells. 3'6 The use of exoenzyme C3 was thus of great help in understanding the functional role of the p21 Rho protein which belongs to the p21 Ras superfamily of small GTP-binding proteins. Exoenzyme C3 ADP1 K. Aktories, V. Weller, and G. S. Chhatwal, F E B S Letr 212, 109 (1987). 2 E. J. Rubin, D. M. Gill, P. Boquet, and M. R. Popoff, Mol. Cell Biol. 8, 418 (1988). 3 p. Chardin, P. Boquet, P. Madaule, M. R. Popoff, E. J. Rubin, and D. M. Gill, E M B O J. 8, 1087 (1989). 4 I. Just, C. Mohr, G. Schallehn, L. Menard, J. R. Didsbury, J. Vandekerckove, J. van Damme, and K. Aktories, J. Biol. Chem. 267, 10274 (1992). 5 A. Sekine, N. Fujiwara, and S. Narumiya, J. Biol. Chem. 264, 8602 (1989). 6 W. Wiegers, I. Just, H. Mailer, A. Hellwig, P. Traub, and K. Aktories, Eur. Z Cell BioL 54, 237 (1991).
METHODS IN ENZYMOLOGY, VOL. 256
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ribosylates RhoA, RhoB, and RhoC with great specificity. However, exoenzyme C3 itself is unable to penetrate into the cytosol of cultured cells since, in contrast to bacterial toxins, such as cholera or diphtheria toxins, a binding and membrane-penetrating subunit for C3 has never been found produced by C. b o t u l i n u m C or D strains. In order to use exoenzyme C3 as a reliable and efficient tool to study the in vivo activity of the Rho protein, it is necessary to increase the efficiency of C3 membrane translocation into the cytosol. We have thus linked by genetic fusion the C3 gene to D N A coding for the subunit of diphtheria toxin (B subunit) involved in binding and transport across the membrane of its enzymatic fragment. Diphtheria toxin (DT) is synthesized by toxigenic Corynebacterium diphtheriae as a single-chain protein that is subsequently cleaved into two fragments linked by a disulfide bridge. 7 Fragment A (20 kDa), which is localized at the amino terminus of DT, is inactivated by ADP-ribosylation elongation factor 2 (EF2), thereby blocking protein synthesis. Fragment B (39 kDa) binds, through its 17-kDa carboxy-terminal domain, a cell surface receptor identified as heparin-binding epidermal growth factor-like precursor. 8 Vero cells are the most sensitive species to D T because they have the highest number of D T receptors 9 but mouse and rat cells are unable to bind DT. After binding, D T is taken up by receptor-mediated endocytosis. In the acidic environment of the early endosomal compartment, a conformational change of the amino-terminal portion of the B fragment (called the translocation domain) allows the molecule to enter the lipid core of the membrane. Fragment A translocates through the membrane and, after reduction of the disulfide bond linking A to B, 1° is released into the cytosol where it stops the protein synthesis machinery, thereby killing the cell. The three-dimensional structure of D T shows clearly the three functional domains of diphtheria toxin implicated in the enzymatic, translocation, and cell-binding functions of the molecule, ix D T is also able to translocate additional peptides and polypeptides into the cytosol. 12'13 D T could thus be used to introduce exoenzyme C3 into the cytosol of diphtheria toxinsensitive cellsJ 4 This chapter describes the realization of a fusion protein 7 A. M. Pappenheimer Jr., Annu. Rev. Biochem. 46, 69 (1977). 8j. G. Naglich, J. E. Metherall, D. W. Russell, and L. Eidels, Cell 69, 1051 (1992). 9j. L. Middlebrook, R. B. Dorland, and S. H. Leppla, J. Biol. Chem. 253, 7325 (1978). 10E. Papini, R. Rappuoli, M. Murgia, and C. Montecucco,J. Biol. Chem. 268, 1567 (1993). 11S. Choe, M. J. Benett, G. Fujii, P. M. G. Curmi, K. A. Kantardjieff, R. J. Collier, and D. Eisenberg, Nature 357, 216 (1992). 12H. Stenmark, J. O. Moskaug, I. H. Madshus, K. Sandvig, and S. Olsnes, J. Cell Biol. 113, 1125 (1991). 13A. Wiedlocha,P. O. Falnes, I. H. Madshus, K. Sandvig, and S. Olsnes, Cell 76, 1039 (1994). 14p. Aullo, M. Giry, S. Olsnes,M. R. Popoff, C. Kocks,and P. Boquet, EMBO J. 12, 921 (1993).
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between exoenzyme C3 and diphtheria toxin fragment B and the use of this molecule on cultured cells to inactivate the p21 Rho, thereby producing an alteration of the actin microfilament network. Methods The B subunit of diphtheria toxin contains two domains involved in (1) binding to the cell receptor and (2) translocation of the A fragment across the lipid core of the membranes. The C3/DT chimeric protein described below, called DC3B, corresponds to a fusion between the C3 gene and the codon of the D T gene coding for amino acid 175 which is located 17 residues before the end of DT fragment A, thus retaining the disulfide bond between A and B important for the translocation. Construction, Expression, a nd Purification of Chimeric Toxin Genetic Construction of DC3B The DNA coding for C3 was cloned from C. botulinum C or D in our laboratory. 15 Cloned DNA coding for crm197 (pDT197), a nontoxic mutant of DT, was obtained from Dr. John R. Murphy, Department of Molecular Medicine, The University Hospital, Boston University, Boston, Massachusetts. 1. Amplification by polymerase chain reaction (PCR) of the C3 gene from C. botulinum C or D DNA with creation of a NcoI site in the initiation codon and a SmaI site instead of the stop codon. Design of the oligonucleotide primer for the 5' end of C3: 5'-CCATGGCTTATFCAAATACTTACCAGGAG-Y Design of the oligonucleotide primer for the 3' end of C3: 5'-GCTCCCGGGTATTTAGGATTGATAGCTGTGCC-Y 2. Cloning of the PCR-amplified C3 gene in NcoI-SmaI restriction sites of a KS vector (Stratagene Cloning Systems, La Jolla, CA, modified as to contain a NcoI site in the polylinker). 3. Isolation of the MscI/EcoRI DT B D N A fragment encoding the /3197 B chain and cloning of this insert into the S m a I - E c o R I restriction sites of the KS C3 plasmid. 15M. R. Popoff,D. Hauser, P. Boquet, M. W. Eklund, and D. M. Gill, Infect. lmmun. 59, 3673 (1991).
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4. Isolation of the NcoI-EcoRI insert from the KSDC3B plasmid and cloning of this insert into the NcoI-EcoRI restriction sites of the expression vector pTrc99A (Pharmacia Biotech. Europe Brussel, Belgium) 5. Verification of the DC3B nucleotide sequence in the pTrc99A (see Fig. 1). Expression of DC3B from Escherichia coli The TG1 E. coil strain is transformed with DNA of plasmid pTrc99A containing the DC3B insert. A 200-/zl aliquot of an overnight preculture of the recombinant bacteria (one colony in 5 ml of LB medium containing
KS C3
Ncol
pDT 197
Sinai EcoRI Ncol
I
Mscl
EcoRI
KS DC3B
scl/2
EcoRI
~111¢11/¢.
TRC99A DC3B
Ncol
Mscl/2 Srnal/~
EcoRI
FIG. 1. DC3B genetic construction.
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100/xg/ml ampicillin) is used to inoculate 1 liter of LB medium (preheated at 37 °) containing 100/zg/ml ampicillin to a 3-liter flask which is incubated at 37° with vigorous agitation until an absorbance (A590) of 0.5 is reached. Expression of the recombinant toxin is then induced by adding 1.5 mM isopropyl-/3-D-thiogalactopyranoside (IPTG) for 90 rain at 37 °. The bacteria are then centrifuged for 5 min at 5000 rpm, and the pellet is resuspended in 15 ml of buffer 101 (see the formula below) and lysed by sonication using a sonifier (Branson Smith-Kline equipped with a microtip probe). Parameters of the sonication: output power, 6 set on 50% of the duty cycle; time of sonication, 10 min. To eliminate nucleic acids from the preparation, protamine sulfate (1.5 ml of a stock solution at 20 mg/ml) is then added slowly with gentle mixing until a precipitate is formed. The preparation is then transferred to centrifuge tubes and incubated for 10 min at 0 °. Debris and nucleic acid are removed by centrifugation (18,000g for 15 min at 4°). The supernatant is then filtered through a 0.45-/xm filter (Millipore, Bedford, MA) and loaded on the DT affinity column.
Purification of DC3B by Affinity Chromatography Buffers and Reagents Buffer 101 (pH 8.0) 5× stock: 34.0 g KH2PO4, 9.22 g NaOH, 18.6 g E D T A (50 mM), 219.0 g NaCI, 5.0 ml Tween 20. Final volume 1 liter with doubly distilled water. Dilute 1 : 5 (v/v) before use, Buffer 103 (pH 8.0) 5× stock: 70.5 g KHzPO4, 18.44 g NaOH, 5.0 ml Tween 20. Final volume 1 liter with doubly distilled water. Dilute 1:5 (v/v) before use. Buffer 104 (pH 7.2) 1 x stock: 17.78 g KH2PO4, 500 g guanidine hydrochloride (1 bottle), 1.308 ml Tween 20. Final volume 1308 ml with doubly distilled water. Buffer 105 (pH 8.0) 5 × stock: 6.05 g Tris, 9.3 g EDTA, 5.0 ml Tween 20. Final volume 1 liter with doubly distilled water. Dilute 1:5 (v/v) before use. Buffer 106 (pH 8.0) 1× stock: 1.21 g Tris, 1.86 g EDTA, 20.0 g deoxycholate (DOC), 1.0 ml Tween 20. Final volume 1 liter with doubledistilled water. Diphtheria Toxin Diphtheria toxin (DT) is purified by gel filtration from crude diphtheria toxin (kindly furnished by Institut Pasteur-M6rieux, Lyon, France). Crude diphtheria toxin as a 70% ammonium sufate precipitate is extensively dialyzed against 20 mM Tris-Hcl buffer, pH 7.4, containing 100 mM NaC1
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(buffer A). An aliquot of 20 ml of the toxin preparation (10 mg/ml) is applied to a gel-filtration column (100 × 2.6 cm) (Ultrogel AcA 34 Industrie Biologique Fran~aise IBF, Paris, France) equilibrated and eluted with buffer A. The toxin peak is collected, checked for purity by SDS-PAGE, and kept frozen at - 8 0 ° in small (200/A) aliquots (concentration: 8 mg/ml).
Anti-DT Affinity Column Sera from horses hyperimmunized with diphtheria toxoid (from Institut Pasteur-M6rieux, Lyon, France) are used as a source of antibodies against diphtheria toxin. Specific antidiphtheria toxin antibodies are first isolated from 50 ml of horse serum diluted with 50 ml phosphate-buffered saline (PBS), using a diphtheria toxin affinity column (volume: 20 ml), made by coupling purified DT to cyanogen bromide-activated Sepharose CL-4B (CNBr-Sepharose) (Pharmacia), according to the recommendations of the manufacturer. The column is first washed with PBS until no optical density can be recorded at 280 nm. Glycine buffer (100 mM), pH 3.0, is then applied to the column, and eluted antibodies are collected (fraction of 4 ml) in tubes containing 80/zl of 4 M Tris-HC1 buffer, pH 8.8 (which raises the final pH to 7.0). Fractions containing the DT antibodies are pooled and extensively dialyzed against PBS. Horse antidiphtheria toxin antibodies are then coupled to CNBr-Sepharose according to the manufacturer's recommendations and are used to immunopurify the recombinant DC3B toxin as detailed below.
Purification Method Anti-DT resin (about 50 ml of resin) is poured into a column and allowed to settle to a level of about 1 cm of the lid. The resin is cleaned with the following buffers: 150 ml of buffer 103, 150 ml of buffer 104, 150 ml of buffer 103, and equilibrated in buffer 101. The sample is loaded in buffer 101. The column is then washed with buffer 101 until the flow through is back to an OD280 equal to 0. A volume of 150 ml of buffer 106 is then passed through the column, followed by 100 ml of buffer 105, and then 100 ml of buffer 103. Elution of the recombinant toxin is performed with buffer 104 until a peak of protein is detected. Fractions are collected and finally the column is washed with 100 ml of buffer 103. The fusion toxin is dialyzed for 4 hr at 4 ° against 50 mM Tris-HC1, pH 8.0, and stored in small aliquots (concentration: 40 ~g/ml) at - 8 0 °. Molecules stored at - 8 0 ° are stable for at least 3 months.
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Analysis of Chimeric Toxin b y SDS-PAGE and Immunoblot A 5-ml culture of the bacterial strain pTrc99A DC3B in LB medium is established. Following induction and expression, 1.5 ml of the culture is harvested in a microfuge tube. The pellet is resuspended in 200 ~1 of SDS gel-loading buffer and boiled for 5 min. An aliquot of 25/.d (for Coomassie staining of the gel) or 12/~1 (for immunoblotting) of the sample is loaded on a 12% SDS-polyacrylamide gel and electrophoresed at 50 V until the sample is through the stacking and then at 100 V until the dye front is at the bottom of the gel. For immunoblot analysis, following electrophoresis, soak the SDS gel in H20 for 10 min and then transfer buffer for 3060 min. Presoak 0.20 ~m nitrocellulose acetate (Schleicher and Schuell, Dassel, Germany) in transfer buffer. Proteins are transferred by electrophoresis at 100 V for 1 hr at room tempertaure. The blot is then rinsed in TNB and is incubated for 10 min in TNB buffer with 30/~1 anti-DT antibody (horse antibody, Institut Pasteur-Mdrieux) or 30/~1 anti-C3 antibody (rabbit antibody, from our laboratory) for 30 min. The membrane is washed three times for 10 min in TNB and is then incubated in 10 ml TNB with 10/zl alkaline phosphatase-conjugated anti-equine antibody (Nordic Immunological Laboratories, Norway) or 2 ~tl alkaline phosphatase-conjugated and anti-rabbit antibody (Promega Corp., Madison, WI) for 30 min. The membrane is then washed two times with PBS. The immunoblot is then developed in 10 ml alkaline phosphatase buffer containing 66 ~1 NBT and 33 /xl BCIP. Incubation of the immunoblot in BCIP is performed until the bands appear. The membrane is then rinsed in water, and finally the reaction is stopped by adding trichloroacetic acid (TCA) to a final concentration of 1%.
Buffers for Immunoblotting Transfer buffer: 9.5 g Tris, 45.6 g glycine, 800 ml methanol; dissolve in 3200 ml doubly distilled water. Buffer B: 1.2 g Tris, 8.7 g NaC1, 1.0 g Brij 58; dissolve in 1 liter doubly distilled water. TNB buffer: 1.2 g Tris, 29 g NaCl, 1.0 g Brij 58; dissolve in 1 liter doubly distilled water and adjust to pH 7.4. Alkaline phosphatase buffer: 100 mM Tris-HC1, pH 9.5,100 mM NaCI, 5 mM MgC12. NBT: 50 mg in 1 ml of 70% (w/v) dimethylformamide. BCIP: 50 mg in 1 ml of pure dimethylformamide. Cytopathic Effect of DC3B on Vero Cells Vero cells (African green monkey kidney cells) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and
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antibiotics are seeded on square glass coverslips in 35-mm culture dishes at a concentration of 4 × 104 cells per dish to allow 30% confluence after 48 hr of growth. After exposure to 10 8 M DC3B (about 600 ng/ml) for 6 hr (by which the cytopathic effect induced by DC3B is total and consists in the retraction of the cell bodies with loss of the actin stress fibers), coverslips are washed three times with 2 ml PBS containing calcium and magnesium and fixed with 3% paraformaldehyde (PFA). They are then permeabilized in 2 ml saponin buffer solution (SBS) for 20 min at room temperature. Coverslips are incubated by the Parafilm technique (see below) with 40 /xl of fluorescein isothiocyanate (FITC)-phalloidin (from Sigma, St. Louis, MO) (stock solution: 500/~g/ml in DMSO) at 1 /~g/ml in SBS for 30 min at room temperature to stain F-actin structures. After transfer of the coverslips to their original dishes, they are washed three times with 2 ml SBS (each time for 5 min) followed by one wash in 2 ml PBS and one rapid dip in distilled water. Then the coverslips are mounted on microscope slides in Mowiol mounting medium, put in a 37° dry incubator for 10 min to polymerize the mounting medium, and examined under a conventional or confocal fluorescence microscope (see Fig. 2).
Solutions for Immunofluorescence Paraformaldehyde Solution Paraformaldehyde (3 g) is dissolved in 100 ml PBS by stirring at 80°. Then 10 ~1 of 1 M CaCI2 and 10 /xl of 1 M MgC12 are added to the paraformaldehyde solution under constant stirring. The solution is then cooled to room temperature, and the pH (7.4) is checked. The solution is filtered through a 0.45-~m Millipore filter and divided into convenient aliquots (used only one time) which are stored frozen at - 2 0 °.
Saponin Buffer Solution (SBS) 0.05 g Saponin (Sigma) 0.5 g bovine serum albumin Adjust to 100 ml with PBS
Mounting Medium Mowiol In a 50-ml plastic tube, 6 g of glycerol and 2.4 g of Mowiol 4-88 (Calbiochem, San Diego, CA) are added and mixed. Then 6 ml H20 is subsequently added and incubated for 2 hr at room temperature. A volume of 12 ml 200 mM Tris-HC1, pH 8.5, is then added and incubated in a water bath at 50° (under agitation) until all particles dissolve. The solution is centrifuged for 15 min at 5000g, and the supernatant is aliquoted in 200-/xl fractions which are kept frozen at - 2 0 °.
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FIG. 2. Effect on Vero cells in actin cytoskeleton of the chimeric toxin DC3B. (A) Control cells nontreated with DC3B and stained for F-actin with FITC phalloidin. (B) Ceils treated for 6 hr with 10 8 M DC3B and then stained for F-actin with FITC-phalloidin. Bar: 1/zm.
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Incubation of Coverslips with FITC-Phalloidin by Parafilm Technique A moist chamber is prepared by placing a Whatman 3MM paper (Whatman, Maidstone, England) in a large plastic gel box, wetting it, and placing a strip of Parafilm on the wet paper (do not touch the Parafilm surface with your fingers). The Parafilm should be flat with no air bubbles underneath. The diluted solution of FITC-phalloidin (40 txl) is placed on the Parafilm. The coverslip, on which the cells are grown, is picked up from the dish, excess SBS is drained on a filter paper, and the coverslip is placed (cell side down) over the drop of the FITC-phalloidin solution. Incubation is performed for 30 min at room temperature. The coverslip is lifted by pipetting 200 ~1 SBS underneath it. The coverslip floats, is picked up, and is transferred (cell side up) to its original dish in 2 ml of fresh SBS.
[33]
Growth Factor-Induced Actin Reorganization Swiss 3T3 Cells
in
B y A N N E J. R I D L E Y
One of the earliest cellular responses induced by many extracellular factors is reorganization of the actin cytoskeleton, and this has been studied in a wide variety of cell types. At the plasma membrane, a rapid response to many stimuli is membrane ruffling: the protrusion from the cell surface of moving folds of membrane containing a meshwork of newly polymerized actin filaments.1 These structures are transiently formed, and the response has usually decayed to background levels within 10 to 30 min. Several factors which induce membrane ruffling are also known to be chemoattractants, and cells will subsequently polarize in response to such factors and become motile. The leading edge of motile cells often displays similar membrane ruffling structures to those seen early on in the response. 2,3 In addition to actin reorganization at the plasma membrane, factors can also alter the density and/or organization of actin filaments in the cell interior. In fibroblasts, stress fibers are the principle actin structures observed in the cytosol. Swiss 3T3 cells are well suited to analyzing growth factor-induced changes in actin organization, as they show strong contact inhibition of growth, and density arrest at a low density as a fiat monolayer of cells. Quiescent cells when contact inhibited have very few stress fibers or membrane ruffles, 1 A. J. Ridley, BioEssays 16, 321 (1994). 2 T. P. Stossel, Science 260, 1066 (1993). 3 T. Hasegawa, J. Cell Biol. 120, 1439 (1993).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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and the addition of extracellular factors rapidly induces changes in actin filament organization (Fig. 1). Several extracellular factors have been shown to stimulate the formation of stress fibers and/or membrane ruffles in Swiss 3T3 cells. 4 Furthermore, the small GTP-binding proteins Rho and Rac have been shown to regulate factor-induced formation of stress fibers and membrane ruffles, respectively. 4'5 Sources of Factors Factors were obtained from the following sources: recombinant human epidermal growth factor (EGF) (Collaborative Research); recombinant human platelet-derived growth factor (PDGF), BB homodimer, (Amersham International or Upstate Biotechnology Incorporated); mouse tumor necrosis factor (TNF)-a (Genzyme); recombinant bovine fibroblast growth factor b (FGFb) (Amersham International); bombesin and bovine plasma vitronectin (Calbiochem); porcine platelet transforming growth factor (TGF)-/31 (R and D Systems); L-ot-lysophosphatidic acid (oleoyl), bovine insulin, human fibronectin, bradykinin, bovine thrombin, and phorbol 12myristate 13-acetate (Sigma Chemical Co.). Preparation of Swiss 3T3 Cells for Analysis Swiss 3T3 cells are grown in Dulbecco's modified Eagle's medium (DMEM) containing 0.11 g/liter sodium pyruvate, 4.5 g/liter glucose, 10% bovine fetal calf serum (FCS), 100 U/ml penicillin, and 100/zg/ml streptomycin in a humidified incubator at 37 ° with 10% (v/v) CO2. Cells are passaged two times per week by washing with 137 mM NaCI, 2.7 mM KC1, 8.1 mM Na2PO4, 1.47 mM KH2PO4 (PBS-A), incubating for 2 to 3 rain with 0.05% trypsin and 0.02% EDTA, and collecting the cells in 10% FCS/ DMEM. Cells are seeded at a density of 3.75 × 103/cm 2. Ceils are maintained in culture for approximately 10 passages after thawing and then are discarded, as they gradually alter their properties as they are passaged. They begin to proliferate faster and to grow to a greater density, and the background of stress fibers and membrane ruffles in serum-starved cells increases, making analysis of changes in actin cytoskeletal organization more difficult. For analysis of growth factor-induced actin reorganization in quiescent Swiss 3T3 cells, cells are seeded at a density of between 3 and 5 × 10 4 cells per well on 13-mm-diameter glass coverslips (Chance Propper) in 24-well 4 A. J. Ridley and A. Hall, Cell 70, 389 (1992). A. J. Ridley, H. F. Paterson, C. L. Johnson, D. Diekmann, and A. Hall, Cell 70, 401 (1992).
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[33]
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dishes (Nunc). The density of seeding is dependent on the batch of FCS, as both the rate of cell growth and the density at which cells reach contact inhibited growth arrest vary with different batches (R. Garg and A. J. Ridley, 1994, unpublished data). The ideal density is therefore determined by seeding cells at different densities and analyzing the actin cytoskeleton following serum starvation (see below). In addition, at the correct density the cells reach confluence within 2 to 3 days after seeding. Coverslips are p r e p a r e d for cell culture by washing with nitric acid, then extensively with distilled water and finally with ethanol, and sterilized by baking. Addition of G r o w t h F a c t o r s Five to 7 days after seeding cells on coverslips, they are washed once with D M E M and are then incubated in 1 ml D M E M for 16 hr to obtain serum-free cultures. G r o w t h factors are diluted in PBS-A immediately prior to addition, so that a volume of between 5 and 50/xl per well is added. Initially, a wide range of concentrations of a particular growth factor are added to determine the threshold concentration required to observe a response and the m i n i m u m concentration required to give a maximal response (Table I). In these titrations, growth factors are added, and the cells are incubated at 37 ° for 10 min, then cells are fixed as described below. Once the titration curve has been determined, the time course of the response is investigated, using a concentration that gives a maximal response at 10 min. Factors are added at times between 6 hr to 1 min before fixation. Fixing, Staining, a n d P h o t o g r a p h i n g Cells Following stimulation with growth factors, ceils are washed once with P B S - A containing 0.9 m M CaC12 and 0.5 m M MgCI2 (PBS), and fixed for 20 min at r o o m t e m p e r a t u r e in either 3% (w/v) paraformaldehyde or 3.7% (w/v) formaldehyde diluted in PBS. Paraformaldehyde is p r e p a r e d as a 6% stock solution in P B S - A and is stored frozen in aliquots at - 2 0 °, which are thawed once and then discarded. F o r m a l d e h y d e is diluted fresh from a 37% stock solution and normally gives satisfactory results for analyzing actin filament organization. Following fixation, coverslips are washed extensively
FIG. 1. Growth factor-induced actin reorganization. Quiescent Swiss 3T3 cells were starved for 16 hr (A), then treated with 3 ng/ml PDGF for 10 min (B), or 0.2% FCS (C). Cells were fixed and actin filaments localized with TRITC-conjugated phalloidin. Arrows in (B) indicate typical membrane ruffle structures. Bar: 10/zm. Reproduced, with permission, from Ridley and Hall.4
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with PBS to remove excess fixative, and then permeabilized for 5 min in 0.2% Triton X-100 in PBS. Coverslips are washed twice with PBS and then incubated with 200/xl of 0.1/xg/ml tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (Sigma Chemical Co.) per coverslip for 30-40 min to localize actin filaments. During this step, the coverslips are kept in the dark by wrapping dishes in aluminum foil, as exposure to light diminishes the fluorescence intensity obtained with TRITC-conjugated phalloidin. A stock solution of TRITC-conjugated phalloidin is prepared by dissolving it in H 2 0 at a concentration of 5 0 / z g / m l and is stored at - 2 0 °, in a light-tight container, in small aliquots. Coverslips are then washed extensively with PBS and mounted in Mowiol (Calbiochem) containing 0.1% p-phenylenediamine. This is allowed to set for 1 hr before viewing the cells with a 6 3 x oil immersion objective on a Zeiss axiophot microscope. Cells are photographed using Kodak T - M A X A S A 400 film. Films are developed using Kodak T - M A X developer and Ilford fixative, according to the manufacturers' instructions. Effects of Different E x t r a c e l l u l a r F a c t o r s The responses induced by the extracellular factors we have tested, and the concentrations required for threshold and maximal response, are shown in Table I. The time chosen to measure these responses is set at 10 min TABLE I CONCENTRATIONS OF FACTORS INDUCING ACTIN REORGANIZATION Concentration giving Factora
Major response at 10 minb
FCS LPA Bombesin PDGF EGF Insulin FGFb Thrombin PMA
sf sf sf/mr mr mr mr mr mr mr
Threshold response 0.1% 20 ng/ml 0.4 nM 0.3 ng/ml 1 ng/ml 10 ng/ml 1 ng/ml 8 ng/ml 0.1 nM
Maximal response 1% 200 ng/ml 10 nM 3 ng/ml 10 ng/ml 40 ng/ml 5 ng/ml 40 ng/ml 4 nM
Factors are added to serum-starved, confluent Swiss3T3 cells for 10 min, then cells are fixed, and actin filaments are localized with TRITC-labeled phalloidin. b sf, stress fibers; mr, membrane ruffling.
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following factor addition, because from time course analysis both early stress fiber formation [induced by lysophosphatidic acid (LPA) and bombesin] and membrane ruffling peak around this time point. FCS potently stimulates the formation of stress fibers in Swiss 3T3 cells (Fig. 1C, Table I), and the major component of FCS inducing this response has been shown to be LPA. 4 LPA is the only factor tested which stimulates the formation of stress fibers but no membrane ruffling. Related phospholipids have little effect, except for phosphatidic acid which has approximately 100-fold lower activity than LPA. 4 Using the maximal concentration of LPA (Table I), changes to the actin cytoskeleton are observed within 1 min of adding LPA. Initially, an increase in polymerized actin is detected but with no distinct structures observable. Within 5 min, stress fibers are clearly forming. At low concentrations of LPA (for example, 20 ng/ml), the response has a short time course, reaching a maximum at 10 rain and decaying to background levels by 2 hr after addition. In contrast, high concentrations that are able to stimulate DNA synthesis (35/xg/ml) induce a sustained increase in stress fibers still observed up to 24 hr after addition. This suggests that the decay at low concentrations is not due to downregulation of the response, but to degradation of the LPA. Bombesin is the only other factor tested which rapidly stimulates actin reorganization and the formation of stress fibers with the same time course as LPA. It is not as potent as LPA, even when the concentration used is maximal.4 In addition, it induces the formation of membrane ruffles. Thrombin acts similarly to bombesin in stimulating both membrane ruffling and stress fiber formation, and the maximum response to bombesin and thrombin is comparable. With thrombin, both responses are delayed by approximately 5 min compared to bombesin; however, this difference may reflect the mechanism of thrombin receptor activation, which involves proteolytic cleavage. 6 PDGF, EGF, FGFb, and insulin, all of which act through tyrosine kinase receptors, primarily stimulate membrane ruffling.5 This response is very rapid, detected within 1 min of growth factor addition. In the case of PDGF, ruffles are often observed initially on the dorsal or upper surface of cells, and later at the periphery. The relative potency of different growth factors in inducing membrane ruffling in Swiss 3T3 cells, measured 10 min after addition, is PDGF = FGFb > EGF > insulin > bombesin = thrombin. PDGF, FGFb, EGF, and insulin also stimulate a limited formation of stress fibers as a delayed response: while membrane ruffling peaks between 5 and 10 rain after factor addition, actin reorganization within the cytosol is first detected around this time, and maximum formation of stress fibers is 6 T.-K. Vu, D. T. Hung, V. I. Wheaton, and S. R. Coughlin, Cell 64, 1057 (1991).
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TABLE II FACTORS TESTED WHICH DO NOT STIMULATE ACTIN REORGANIZATION
Factor a TNF-a TGF-/3 Bradykinin Vitronectin Fibronectin
Highest concentration tested 80,000 units 1 ng/ml 50 nM 10/zg/ml 100/zg/ml
"Factors were added to serum-starved Swiss 3T3 cells for 10 min, cells were fixed, and actin filaments were localized by incubation with TRITC-labeled phalloidin.
generally observed at approximately 30 min. Finally, it is notable that the concentration of PD G F is critical for observing membrane ruffling. At concentrations above 20 ng/ml, the stress fiber response progressively decreases, the cytoskeleton becomes disorganized, and there are far fewer membrane ruffles (data not shown). 7 Such changes have not been observed at higher concentrations of EGF, insulin, or bombesin than those listed in Table I. Several factors have been tested which do not have a detectable effect on actin organization within the first 10 min after addition (Table II), although Swiss 3T3 cells have been shown to respond to these factors. TNFoL at concentrations between 5000 and 80,000 units/ml induced the death of serum-starved Swiss 3T3 cells within 2 to 4 hr after addition; however, even at 80,000 units/ml it does not induce actin reorganization after 10 min. Bradykinin activates a number of early signals in Swiss 3T3 cells when added in combination with insulin, s but does not stimulate actin reorganization. Extracellular matrix proteins such as fibronectin and vitronectin promote the formation of stress fibers when cells are seeded on dishes coated with them, in the absence of serum. 9 However, when added to confluent Swiss 3T3 cells already adherent to a surface, they had no effect.
7 S. Rankin and E. Rozengurt, J. Biol. Chem. 269, 704 (1994). 8 M. Issandou and E. Rozengurt, J. Biol. Chem. 265, 11890 (1990). 9 K. Burridge, K. Fath, T. Kelly, G. Nuckolls, and C. Turner, Annu. Rev. Cell Biol. 4, 487 (1988).
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Conclusions Actin reorganization is a very early response induced by many factors in quiescent Swiss 3T3 cells, and changes induced vary between different factors, as does the extent of the response. Analysis of these responses, and the effects of signaling molecules on these responses, will allow the dissection of the signaling pathways leading from receptor activation to increased actin polymerization and the precise organization of new actin filaments into discrete structures. In addition, further studies should shed light on the role of these structures in cellular responses to factors, which is as yet not fully understood.
[34]
Microinjection
of Rho and Rac into Quiescent 3T3 Cells
Swiss
By ANNE J. RIDLEY Microinjection is a technique which rapidly introduces macromolecules into cells, allowing analysis of the immediate cellular changes occurring in response to the injected substance. The technique for microinjecting cultured cells has been described in detail by Graessmann and Graessmann, 1 and has been used to inject protein, RNA, or DNA. The approach has been particularly useful in analyzing the effects of putative signal transduction molecules, either by injecting the proteins themselves or by injecting antibodies to proteins. Responses are generally observed in individual cells by microscopy, often using immunofluorescence techniques; for example, stimulation of D N A synthesis, relocalization of target proteins, induction of protein expression, or reorganization of cytoskeletal elements can all be measured by immunofluorescence. However, it is also possible in some cases to carry out biochemical analysis of microinjected cells; for example, changes in protein phosphorylation have been investigated following injection of fibroblasts with cAMP-dependent protein kinase. 2 Microinjection has been important in defining the functions of several small Ras-related GTP-binding proteins. Initially, microinjection of H-Ras itself showed that it rapidly stimulated membrane ruffling and pinocytosis. 3 1 M. Graessmann and A. Graessmann, in "Methods in Enzymology" (R, Wu, L. Grossman, and K. Moldave, eds.), Vol. 101, p. 482. Academic Press, San Diego, 1983. 2 N. J. C. Lamb, A. Fernandez, M. A. Conti, R. Adelstein, D. B. Glass, W. J. Welch, and J. R. Feramisco, J. Cell BioL 106, 1955 (1988). 3 D. Bar-Sagi and J. R. Feramisco, Science 233, 1061 (1986).
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More recently, the Ras-related proteins Rac and Rho were shown by microinjection to regulate actin reorganization in response to growth factors. Rho rapidly stimulates stress fiber formation when microinjected into quiescent Swiss 3T3 cells. 4,5 A n inhibitor of Rho function, the bacterial exoenzyme C3 transferase which ribosylates Rho proteins, inhibits growth factor-induced stress fiber formation when injected into Swiss 3T3 cells, and ribosylated Rho itself actually acts as a dominant inhibitor of Rho function when injected. Rac, on the other hand, stimulates membrane ruffling when injected and, in addition, a dominant negative inhibitor of Rac function, N17Racl, inhibits growth factor-induced membrane ruffling. 6 P r e p a r a t i o n of Swiss 3"1"3 Cells Swiss 3T3 cells are maintained in culture as described. 7 For microinjection, ceils are seeded at a density of between 3 and 5 × 104 ceils per well on 13-ram-diameter glass coverslips (Chance Propper) in 15-mm-diameter tissue culture wells (Nunc). Prior to seeding the cells, coverslips are marked with a cross using a glass pen to facilitate localization of injected cells, then sterilized by flaming with ethanol. After 5 to 8 days, the medium is removed and replaced with 1 ml Dulbecco's modified Eagle's medium ( D M E M ) without serum, and ceils are incubated overnight (12-16 hr). Before microinjection, coverslips are transferred to 60-mm dishes containing 4 ml DMEM. P r e p a r a t i o n of Proteins for Microinjection Glutathione S-transferase fusion proteins are purified, dialyzed, and concentrated as described. 8 From a l-liter culture of E s c h e r i c h i a coil, proteins are normally concentrated to a volume of approximately 100/zl and stored under liquid nitrogen in 10-/xl aliquots. The concentration of active protein is determined following thawing by measuring binding to [3H]GTP or [3H]GDP. This normally is severalfold lower than the protein concentration measured by the Bradford assay, 8 but gives consistent results when comparing the activity of different protein preparations. It should be noted that the R h o A proteins normally contain a mutation at amino acid 25 from Phe to Asn, which was introduced for technical reasons and increases its 4 H. F. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell Biol. 111, 1001 (1990). • 5A. J. Ridley and A. Hall, Cell 70, 389 (1992). 6m. J. Ridley, H. F. Paterson, C. L. Johnson, D. Diekmann, and A. Hall, Cell 70, 401 (1992). 7A. J. Ridley, this volume [33]. 8A. J. Self and A. Hall, this volume [1].
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stability in E. coli (Table I). 4'8 Immediately prior to microinjection, an aliquot of protein is thawed on ice and centrifuged at 4° for 5 min at 15,000g. Appropriate dilutions of the protein are made in 150 mM NaCI, 50 mM Tris, pH 7.5, 5 mM MgCI2, on ice. Rat immunoglobulin (Pierce) is added as a marker protein at a final concentration of 0.5 mg/ml to allow identification of injected cells. This is also centrifuged for 5 min at 15,000g prior to mixing with the Rac or Rho protein. This centrifugation step is essential to remove any particles from the microinjection mix which will block the tip of the microcapillary, preventing extrusion of the protein solution. Proteins are stored on ice until microinjection. Microinjection of Proteins Glass pipettes (1.2 mm bore; Clark Electroinstruments, Reading, UK) are used to pull microcapillaries of approximately 0.5 /.~m tip diameter, with a programmable Pipette Puller (Campden Instruments Model No. 773). With this instrument, conditions for producing optimal microcapillaries must initially be optimized by varying the temperature of the heating coil, the length of heating time, and the pulling forces. One microliter of protein solution is loaded into a microcapillary using an Eppendorf microloader tip. Cells are observed by phase-contrast microscopy on a Zeiss-inverted microscope, fitted with an enclosed Perspex chamber to maintain the cells at 37 ° in 10% C O 2. The chamber is heated to 37° by a heated stage and an air heater, and the temperature and CO2 concentration are maintained by the temperature regulator TRZ3700 andd CTI controller 3700 [supplied by Zeiss (Oberkochen Ltd]. Humidity is provided by a Perspex dish containing distilled water located within the chamber. Initially, the cross on the coverslip is located using a low-power objective, but cells are observed during microinjection with a 32x/0.4 objective lens and 10x/18 eyepieces. Cells are injected in our laboratory using an Eppendorf microinjector (Model No. 5242) and micromanipulator (Model No. 5170). Prior to microinjection, flow in the microcapillary is initiated with a brief (<5 sec) pulse at high pressure (3000-4000 hPa). Cells are then injected in manual mode, and the pressure is adjusted between 100 and 1000 hPa during microinjection to give a constant flow rate of solution. It has been estimated that between i and 2 x 10 -11 ml is injected per fibroblast cell) Cells in the vicinity of the cross marked on each coverslip are microinjected intracytoplasmically. Between 100 and 150 cells per protein sample are injected in approximately 10 min, and cells are then returned to a humidified 10% (v/v) CO2 incubator at 37 ° for appropriate lengths of time (see below).
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Fixation a n d S t a i n i n g of Cells Cells are fixed and permeabilized as described. 7 They are incubated for 30 to 40 min with 0.1 /~g/ml tetramethylrhodamine isothiocyanate (TRITC)-phalloidin to localize actin filaments, and a 1:400 dilution of fluorescein isothiocyanate (FITC)-labeled goat anti-rat immunoglobulin antibody (Sigma Chemical Co.) to identify microinjected cells. Coverslips are washed, mounted, and observed as described. 7 Activities of Rho P r o t e i n s in Regulating S t r e s s Fiber F o r m a t i o n Rho proteins have been reported to stimulate the formation of stress fibers and focal adhesions following microinjection into quiescent Swiss 3T3 cells (Fig. 1B). 4'5 By titrating the concentration of protein injected, it is possible to determine the critical amount of active protein required to induce this response. Results of such titrations are shown in Table I. It is important to note that the concentrations are based on the level of active protein determined by binding assay (in these cases using [3H]GDP) and are not the total protein concentration. Although the binding assay is not 100% efficient and therefore this is not t h e absolute amount of active protein, we have found that this is a consistent way of comparing the activities of different protein preparations. Titrations of R h o A show that it gives a maximal response at concentrations above 100/zg/ml. At this concentration, a diffuse increase in polymerized actin is observed within 5 min of microinjection, and subsequently these filaments become organized as distinct stress fibers. By 30 rain, cells contain numerous, densely packed stress fibers oriented in predominantly one direction, parallel to each other (Fig. 1B). Between 2 and 6 hr, the surface area of many injected cells decreases significantly compared to control cells, probably as a result of the tension exerted by the stress fibers on cell-matrix interactions. Stress fibers are maintained for at least 24 hr following microinjection, although by this time the response begins to decrease. As the concentration of R h o A protein injected is decreased below maximal response levels, the density of the stress fibers observed at 30 min decreases and the fibers are progressively less organized. At submaximal concentrations, between 10 and 50/~g/ml, the response at 30
FI6.1. Rho- and Rac-induced actin reorganization. Confluent Swiss 3T3 cells were serum starved for 16 hr and injected with 0.1 mg/ml V14RhoA and fixed after 30 min (B) or injected with 0.2 mg/ml V12Racl and fixed after 20 min (C). (A) Control uninjected cells. Bar: 10 /zm. B and C are reproduced with permission from A. J. Ridley, A. J. Self, F. Kasmi, H. F. Paterson, A. Hall, C. J. Marshall, and C. Ellis, E M B O J. 12, 5151 (1993).
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TABLE I CONCENTRATIONS OF R H O AND RAC PROTEINS REQUIRED TO INDUCE RESPONSE
Concentration (/zg/ml)a Protein
Major response
Maximum response
Threshold response
RhoA b V14RhoA b F25RhoA b Racl V12Racl N17Racl V12N17 Racl V12A35 Racl V12 H-Ras
Stress fibers Stress fibers Stress fibers mrc mr Inhibits mr Inhibits mr Inactive mr
100 50 300 ndc 200 200 200 1000 300
10 10 30 300 75 100 100 nac 100
a Proteins were injected into quiescent, serum-starved Swiss 3T3 cells. The concentrations of active protein (determined by binding assay) required to induce a given response, either a maximum response or minimal detectable (threshold) response, are shown. The response was measured at 15 min after microinjection for Racl proteins, at 30 min for RhoA proteins, and at 60 min for H-Ras. The concentrations required to achieve threshold or maximum responses were found to be similar with at least two different protein preparations. b The RhoA and V14RhoA proteins contain a mutation at amino acid 25 from Phe (F) to Asn (N). F25RhoA is the original unmutated protein. c mr, membrane ruffling; nd, not determined; na, not applicable.
m i n r e s e m b l e s the m a x i m u m stress fiber r e s p o n s e i n d u c e d by the n e u r o p e p tide b o m b e s i n , 5,7 w h e r e a s l y s o p h o s p h a t i d i c acid ( L P A ) at c o n c e n t r a t i o n s o v e r 200 n g / m l c a n i n d u c e a stress fiber d e n s i t y a p p r o a c h i n g that o b s e r v e d at m a x i m a l levels of R h o A . This suggests that b o m b e s i n only partially activates e n d o g e n o u s R h o p r o t e i n s , while L P A is a m u c h m o r e p o t e n t activator. B e l o w 5 / z g / m l , little or n o r e s p o n s e to R h o A is d e t e c t e d at 30 m i n after injection. It is i n t e r e s t i n g that the m u t a t i o n of a m i n o acid 14 (a Gly) in R h o A to Val (V) only increases its activity a p p r o x i m a t e l y 2-fold ( T a b l e I) as d e t e r m i n e d by c o m p a r i n g the c o n c e n t r a t i o n s of each p r o t e i n r e q u i r e d to i n d u c e the s a m e level of stress fibers at 30 m i n after injection. F o r e x a m p l e , the r e s p o n s e to V 1 4 R h o A at 5 0 / x g / m l is a p p r o x i m a t e l y e q u i v a l e n t to R h o A at 100 /zg/ml. T h e t h r e s h o l d c o n c e n t r a t i o n r e q u i r e d to give a m i n i m a l d e t e c t a b l e r e s p o n s e is similar for b o t h p r o t e i n s at 10/xg/ml. This contrasts
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to the approximately 10-fold difference in activity observed between H-Ras and the oncogenic V12H-Ras protein following microinjection into REF52 cells? The mutation to V14 renders the protein insensitive to G A P proteins and reduces the endogenous GTPase activity,9 but is not crucial for activity in cells. The mutation of amino acid 25 in R h o A from Phe (F) to Asn (N), introduced for technical reasons, 4'8 appears to increase the activity of the protein approximately threefold (compare R h o A and F25RhoA in Table I). This difference could, however, reflect changes in the stability of the protein and degradation rather than a real change in activity,s A mutation has also been introduced into the protein at amino acid 63, changing it from Gin to Leu. This mutation increases the affinity of the protein for GAPs and the activity of R h o A in cells approximately twofold in comparison to V14RhoA. 1° Mutation of amino acid 37 from Thr to Ala has been reported to inactivate the protein, as does the equivalent mutation in Ras. 4 Finally, no significant difference in the response to V14RhoA and V14RhoB is observed, although it has been reported that they have different intracellular localizations when expressed from exogenous plasmids as tagged proteins. 11 It has not yet been possible to determine whether mutation of amino acid 19 from Thr to Asn creates a dominant inhibitor of Rho activity, equivalent to the dominant inhibitors of Rac and Ras, N17Racl and N17Ras. 6,12This is because this mutation renders the recombinant glutathione S-transferase fusion protein insoluble in E. coli. 8 However, it has been observed that ribosylation of R h o A with C3 transferase prior to microinjection converts it from an activating protein to a dominant inhibitor, as it inhibits stress fiber formation induced by, for example, LPA. 5
Activities of Rac Proteins in Regulating M e m b r a n e Ruffling Racl has been reported to stimulate membrane ruffling when injected into quiescent Swiss 3T3 ceils, followed by a later accumulation of stress fibers which is very limited in comparison to the response to R h o A (Fig. 1C). 6 An increase in actin filaments adjacent to the plasma membrane is observable within 5 rain of injection, and by 10 min these are clearly located in distinct membrane ruffle structures. Maximum membrane ruffling is observed between 15 and 30 rain following injection, and new stress fibers 9 M. D. Garrett, A. J. Self, C. van Oers, and A. Hall, J. Biol. Chem. 264, 10 (1989). 10 C. Nobes and A. Hall, unpublished data (1994). J1 p. Adamson, H. F. Paterson, and A. Hall, J. Cell Biol. 119, 617 (1992). 12 L. A. Feig and G. M. Cooper, Mot. Cell Biol. 8, 3235 (1988).
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are first detected during this time. This response is fairly transient compared to the prolonged response to RhoA and is over by 6 hr following microinjection. This may be due to the relative instability of microinjected Rac proteins. 5 By injecting decreasing concentrations of proteins, it was shown that mutation of amino acid 12 from Gly to Val increases the activity of Racl approximately fourfold. This was determined by comparing the concentrations of Racl and V12Racl required to give a certain level of membrane ruffling 15 min after injection.6 As with RhoA, mutation of amino acid 61 to Leu increases the affinity of Racl for GAPs and also increases its activity in stimulating membrane ruffling two- to fourfold as compared to V12Racl. 1° Mutation of amino acid 35 from Thr to Ala inactivates V12Racl, 6 and even at concentrations of up to 1 mg/ml it does not induce membrane ruffling at 15 min. Mutation of amino acid 17 from Thr to Asn in Racl creates a dominant negative inhibitor of Rac, which inhibits growth factor-induced membrane ruffling when microinjected into quiescent Swiss 3T3 c e l l s . 6 Both V12N17Racl and N17Racl are equally active in inhibiting membrane ruffling, indicating that the N17 mutation dominates over the V12 mutation.
[35] I n h i b i t i o n o f L y m p h o c y t e - M e d i a t e d C y t o t o x i c i t y b y Clostridium botulinum C3 T r a n s f e r a s e
By PAUL LANG and JACQUES BERTOGLIO Cytotoxic lymphocytes are specialized cells of the immune system that mediate the destruction of tumor cells and virus-infected cells. They comprise two classes of cells: the cytotoxic T lymphocytes, which use an antigenspecific receptor and recognize their targets in the context of major histocompatibility complex molecules, and natural killer (NK) cells which do not express polymorphic receptors and do not require preactivation to mediate their effector function. In both systems, target cell recognition involves adhesion molecules expressed at the surface of killer cells and their corresponding ligands on the targets. 1 This initial step triggers transmembrane signaling pathways that lead to the exocytosis of cytolytic granules. 2 1 E. R. Podack and A. Kupfer, Annu. Rev. Cell Biol. 7, 479 (1991). 2 j. R. Yannelli, J. A. Sullivan, G. L. Mandell, and V. H. Engelhard, J. Immunol. 136, 377 (1986).
METHODS IN ENZYMOLOGY, VOL. 256
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Reorganization of the cytoskeleton is an essential c o m p o n e n t in mediating the polarization of granule exocytosis and in regulating the affinity of adhesions molecules involved in the cell-cell interaction. 3 This is particularly true for adhesion molecules in the integrin family such as lymphocyte function-associated antigen ( L F A ) - I [which recognizes intercellular adhesion molecule (ICAM)] and for the T and N K cell m a r k e r CD2 (which binds LFA-3). It has been demonstrated that the intracytoplasmic domains of these molecules are linked to the focal adhesion plaque and directly interact with the actin cytoskeleton. 4'5 Given the known function of Rho proteins in regulating actin cytoskeleton, 6 we hypothesized that R h o might be involved in the process of cytotoxicity. This question was addressed, taking advantage of the inhibitory effect of C3 transferase on the function of the R h o proteins, 7'8 and we showed that C3-induced ADP-ribosylation of R h o A resulted in inhibition of the cytolytic function. 9
C h r o m i u m R e l e a s e A s s a y for Cytotoxic L y m p h o c y t e Activity L y m p h o c y t e cytotoxic activity is p e r f o r m e d as described by Grabstein and Chen, 1° and detailed protocols can be found in various immunology methods books, u Briefly, target cells are first incubated for 1 hr at 37 ° with 100 ~Ci chromium-51, then washed in culture medium. Usually, 5000 target cells are incubated in microtiter plates in quadruplicate, with increasing numbers of cytotoxic effector cells to achieve effector to target ratios varying between 1 : 1 and 100 : 1. The plates are incubated for 4 hr at 37 °. Lysis of the target cells is reflected by release of the 51Cr label into the culture supernatant. Two controls are included for spontaneous 5iCr release by targets alone and for maximal release as induced by 0.1 N H C 1 or a detergent 3 A. Kupfer and S. J. Singer, Annu. Rev. Immunol. 7, 309 (1989). 4 R. O. Hynes, Cell 69, 11 (1992). 5T. A. Springer, Nature 346, 425 (1990). 6 p. Chardin, P. Boquet, P. Madaule, R. M. Popoff, E. J. Rubin, and D. M. Gill, E M B O J. 8, 1027 (1989). 7 A. J. Ridley and A. Hall, Cell 70, 389 (1992). 8 A. J. Ridley, H. F. Paterson, C. L. Johnston, D. Diekmann, and A. Hall, Cell 70, 401 (1992). 9 p. Lang, L. Guizani, I. Vitt6-Mony, R. Stancou, O. Dorseuil, G. Gacon, and J. Bertoglio, J. Biol. Chem. 267, 11677 (1992). 10K. Grabstein and Y. U. Chen, in "Selected Methods in Cellular Immunology" (B. B. Mishell, S. M. Shiigi, C. Henry, and R. I. Mishell, eds.), p. 124. W. H. Freeman, New York, 1980. u j. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds., "Current Protocols in Immunology." John Wiley & Sons, New York, 1991.
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[1% Nonidet P-40 (NP-40) or SDS in water]. Specific cytotoxicity for each concentration of effector cells is calculated according to the formula: % specific lysis = 100 ×
test cpm - spontaneous cpm maximum cpm - spontaneous cpm"
Quantitative comparison of the cytotoxic activity of different effector cells, or cells treated in different ways, is obtained following calculation of lytic units (LU), and is usually expressed in L U per 106 cells. 12A3One lytic unit is defined arbitrarily as the number of effector cells required to yield the selected lysis value (e.g., 30%). This is determined by plotting percent specific lysis versus the logarithm of the effector cell number for each data point. This should yield a straight line that allows calculation of the number of cells that induces 30% specific lysis, and if, for example, this number is 104 cells, then the activity of the preparation is 100 LU30/106 effector cells.
C3 T r a n s f e r a s e T r e a t m e n t of E l e c t r o p e r m e a b i l i z e d Cytotoxic Cells Mammalian cells do not express receptors for C l o s t r i d i u m b o t u l i n u m C3 ADP-ribosyltransferase. Some fibroblast and B lymphoblastoid cell lines have been shown to uptake C3 transferase through spontaneous pinocytosis. 14'15 However, we have been unable to introduce macromolecules, and notably C3 transferase, into cytotoxic T or NK ceils through either spontaneous or stimulated pinocytosis. Microinjection of C3 transferase into cells has been used successfully in fibroblasts and allowed the observation of the involvement of Rho proteins in cytoskeleton organization. 6'16 However, this technique cannot be adapted for treating 1 or 2 million lymphocytes that are required for cytotoxicity assays. We have therefore developed the following protocol using electropermeabilization of cytotoxic cells as outlined in Fig. 1. Cytotoxic cells are washed and resuspended in serum-free culture medium (RPMI 1640, Gibco), buffered to p H 8.0 with 20 mM H E P E S , at a concentration of 5 to 10 × 106 cells/300/zl with the appropriate concentration of recombinant C3 (in the range 0.1 to 5/zg/ml). Ceils are placed on 12H. F. Pross, M. G. Baines, P. Rubin, P. Shragge, and M. S. Patterson, J. Clin. ImmunoL 1, 51 (1981). 13I. Vitt~-Mony, R. Stancou, and J. Bertoglio, J. Immunol. 145, 4272 (1990). 14T. Tominaga,K. Sugie,M. Hirata, N. Morii,J. Fukata, A. Uchida,H. Imura, and S. Narumiya, J. Cell. Biol. 120, 1529 (1993). 15N. Kumaga'i, N. Morii, K. Fujisawa, Y. Nemoto, and S. Narumiya, J. Biol. Chem. 268, 24535 (1993). 16H. F. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell Biol. 111, 1001 (1990).
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Interleukin-2 dependent cytotoxic cells
Electroporation in culture medium (800 V, 40 t~F, 1.7 msec; 100 V, 1500 t~F, 66 msec) with recombinant Clostridium botuIinum C3 transferase
L
1 hr at 37°C in culture medium
washed, adjusted
I Cytotoxicity assay with 51Cr_labeled target cells
3x106 cells lysed in buffer
I
In vitro ADP-ribosylation
with C3 and [32p]NAD F1G. 1. Outline of procedure for C3 transferase inhibition of natural killer cell cytolytic activity.
ice for 10 min in an electrode cuvette (4 mm wide) and submitted to electroporation using a Cellject apparatus (Eurogentec, Belgium) that generates a decaying, exponential waveform, double electric pulse (800 V, 40/zF for 1.7 msec, and then 100 V, 1500 IzF for 66 msec). Immediately after the electric pulse, lymphocytes are resuspended into the cuvette and left on ice for an additional 15 min. Electroporated cells are then resuspended in 10 ml RPMI medium supplemented with serum and antibiotics and incubated at 37 ° for 1 hr in a humidified incubator with 10% CO2. This incubation is required to allow cells to recover their membrane integrity. Under these conditions, over 75% of the cells are able to recover from the electric shock as judged by cell viability and functional assays. Regarding assessment of cell viability, a word of caution is required. If this is done using exclusion of a vital dye such as trypan blue, it must be done after cells have repaired membrane lesions (60 to 90 min). Indeed, cells permeabilized by electroporation will allow entry of trypan blue. This can actually be used to help define the proper conditions for electroporation, alternatively to the Lucifer yellow method described below. Even though we favor the double-pulse method of electroporation, this is not an absolute requirement, and an apparatus that delivers a single electric shock (e.g., Bio-Rad Gene Pulser) can be used as well to introduce macromolecules inside cytotoxic cells. 17 Regardless of the technique used, lV C. Langlet and A. M. Schmitt-Verhulst, J. I m m u n o l . M e t h o d s 151, 107 (1992).
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and depending on each cell line, successful electroporation represents a compromise between effective permeabilization and cell death, and parameters must be carefully chosen. Preliminary determination of appropriate conditions can be performed using uptake of the non-cell-permeant fluorescent probe Lucifer yellow (Sigma, Catalog No. L0259). Cells are submitted to electropermeabilization at different settings in a solution of 1 mM Lucifer yellow in serum-free medium. If using a double-pulse apparatus, the voltage should be varied between 600 and 1000 V with capacitance setting at 40/zF for the first pulse, with the second pulse remaining constant at 100 V and 1500/zF. When using single-pulse electroporation the range of working parameters is 250 ~F and voltage is between 200 and 260 V. Incorporation of Lucifer yellow can then be assessed, following two washes in medium, on a fluorescence microscope or by cytofluorometry using illumination at a 488-nm wavelength (the same as used for fluorescein) and compared to cell viability after 60 to 90 min (or longer) following electroporation. The advantage of using a fluorescent probe over a regular vital dye is that when combined with cytofluorometry, it allows quantification of the marker uptake. A typical experiment is illustrated in Fig. 2, showing that Lucifer yellow does not spontaneously penetrate live cells and that a voltage increase results in a better efficiency of permeabilization as measured by fluorescence intensity, but also decreases the recovery of viable cells.
Controlling for C3 Transferase Uptake by Electroporation Some variability in the technique is introduced depending on different cells and unknown parameters related to cell growth and culture conditions. Furthermore, there is no easy way to control how much C3 transferase is actually delivered inside the cells, yet it is important to determine how efficient the whole process was. Since ADP-ribosylation is a covalent modification, 18 we routinely assess the efficiency of in vivo ADP-ribosylation by measuring how much substrate remains available for further ADPribosylation in vitro. For this purpose, aliquots of electroporated cells are washed in medium, centrifuged at 1500 rpm for 10 rain, and the cell pellet is lysed in 20 mM HEPES, pH 8.0, by three freeze/thaw cycles. The lysates are centrifuged at 15,000 rpm for 10 rain, and the supernatant fractions are quantitated for protein content. Cell extracts (10 /~g proteins) are then ADP-ribosylated using 50 ng/ml recombinant C3 transferase and 5 x 105 18 A. Sekine, M. Fujiwara, and S. Narumiya, J. Biol. Chem. 264, 8602 (1989).
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0V;0 F % CR:100 MFI: 0 600V;401tF % CR:80 MFI: 23 o 800V;40~F % CR:73 MFI: 67 1000V;40~tF % CR:62 MFI: 80
Fluorescence intensity F~o. 2. Setting the electrical parameters for successful electroporation of human natural
killer cells. Human natural killer cells are electroporated using a Cellject apparatus in serumfree culture medium supplemented with 1 mM Lucifer yellow. Settings indicated on the figure represent those used for the first electric pulse, whereas the second pulse was kept constant at 1500/xF and 100 V. Following 2 hr at 37°, cell viability was assessed by trypan blue exclusion (%CR indicates viable cell recovery), and Lucifer yellow uptake was analyzed using an EPICS ELITE cytofluorograph (Coultronics, France). The mean fluorescence intensity (MFI) is expressed as channel number (log scale).
c p m [32p]NAD (30Ci/mmol, D u P o n t - N e w E n g l a n d Nuclear) in A D P ribosylation buffer (20 m M H E P E S , p H 8.0, 1 m M MgCI2, 1 m M A M P , and 15 m M thymidine), in a final v o l u m e of 20 Ixl. A f t e r 30 min at 37 °, T C A - p r e c i p i t a b l e material is r e c o v e r e d and c o u n t e d in a / 3 scintillation counter. T h e results shown in Fig. 3 clearly d e m o n s t r a t e that the decrease in available substrate for C3 A D P - r i b o s y l a t i o n in vitro closely parallels the d o s e - d e p e n d e n t inhibition of cytotoxicity.
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350
5,000
300 4,000
250 3,000
200 150
g
2,000
"el ~.
100 1,000 [-~ 50
0
CTRL
SHAM
4
1
0.4
0
C3 transferase Otg/ml)
FIG. 3. Dose-dependent inhibition of natural killer cell cytolytic activity by C3 transferase and correlation with decreased C3 substrate availability. Human NK cells were either untreated (CTRL) or electropermeabilized in the absence (SHAM) or in the presence of Clostridium botulinum recombinant C3 exoenzyme at the indicated concentrations. Their lytic activity was then assayed against 51Cr-labeled K562 target cells (open bars) in a 4-hr 51Cr release assay at multiple target:ratios, and data are expressed in lytic units (30%) per 106 effector cells. Black shows the reduction in the amount of substrate available for ADP-ribosylation in cell extracts prepared following treatment of NK cells with (or without) C3. Incorporation of 32p into TCA-precipitable material is expressed as cpm per 5-/~g proteins.
Concluding Remarks O n e of the points that is n o t addressed in this c h a p t e r is the question of the potential substrates for C3 transferase in the cells of interest. A s previously reported, a m o n g all m e m b e r s o f the family, m R N A for R h o A appears to be the m o s t a b u n d a n t l y expressed in cytotoxic l y m p h o cytes. F u r t h e r m o r e , using two-dimensional gel analysis of in vitro C3 A D P ribosylated l y m p h o c y t e cell extracts, we could detect only one substrate for C3 transferase that shows the migration characteristics expected for R h o A . 9 M o r e definite identification of C3 transferase substrates in various cell types will be greatly facilitated in the future t h r o u g h the use of antibodies specific for individual m e m b e r s of the R h o family. 19 Little is k n o w n of the role o f R h o family proteins in l y m p h o c y t e functions, and potential directions for research in this area have b e e n discussed. 2° Using the p r o c e d u r e s described in this c h a p t e r we have b e e n able to d e m o n strate that the cytolytic activity of b o t h natural killer cells and allogeneic 19p. Lang, F. Gesbert, J. Thiberge, F. Troalen, H. Dutartre, P. Chavrier, and J. Bertoglio, Biochem. Biophys. Res. Commun. 196, 1522 (1993). 2op. Chavrier, J. P. Gorvel, and J. Bertoglio, ImmunoL Today 14, 440 (1993).
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T lymphocytes requires functional RhoA. Indeed, inhibition of RhoA by C3 transferase leads to inhibition of cytotoxicity. This effect of C3 transferase is clearly mediated at the level of the cytoskeleton and is accompanied by dramatic morphological changes of cytotoxic cells (P. Lang et at, unpublished). How these modifications concur to inhibit cytotoxicity remains a matter of speculation. Both target recognition mediated by adhesion molecules and polarized exocytosis of cytolytic granules could be equally affected, and experiments investigating these specific points are currently in progress.
[36] N e u t r o p h i l
Chemotaxis Assay and Inhibition by C3 ADP-Ribosyltransferase
By MARIE-JOSI~ STASIA and PIERRE V. VIGNAIS
Introduction Neutrophils are professional phagocytes endowed with a number of specific abilities, including chemotaxis, exocytosis, and phagocytosis. Chemotaxis designates the migration of a cell in a concentration gradient of chemical attractant. This migration is accomplished by ameboid movement. Chemotaxis of neutrophils is triggered by specific receptors for chemoattractants, localized in the plasma membrane. Binding of chemoattractants to the receptors generates intracellular signals that are transmitted to the components of the cytoskeleton, resulting in cell motility. Cell motility depends on the coordination of a number of cellular functions, including adhesion and deadhesion, which are integrin-dependent processes, and protrusion and retraction of the cell body, which are mainly actin-dependent processes (for review see Zigmond 1). The role played by small molecular weight GTP-binding proteins (small G proteins) belonging to the Rho group in cell motility has attracted attention (for a review see Narumiya and Morii2). The idea of a function for Rho in cell motility was based on experiments dealing with the effect of ADP-ribosyltransferase from Clostridium botulinum, a protein of 23 kDa often referred to as botulinum C3 exoenzyme, which specifically ADPribosylates the Rho proteins, and also on experiments carried out with 1S. H. Zigmond, Cell MotiL Cytoskel. 25, 309 (1993). 2S. Narumiya and N. Morii, Cell. Signal. 5, 9 (1993). METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Rho mutants. 3-7 Other ADP-ribosyltransferases specific of Rho have been isolated from filtrates of cultures of S t a p h y l o c o c c u s aureus, 8 Bacillus cereus, 9 and Clostridium l i m o s u m . 1° The ADP-ribosyltransferase from S. aureus is referred to as EDIN (epidermis differentiation inhibitor). Among the Rho proteins and structurally related small G protein, only RhoA, RhoB, and RhoC have proved to be efficiently ADP-ribosylated by the C3 exoenzyme. Rac proteins, which are closely related to Rho, are hardly ADP-ribosylated by the C3 exoenzyme, unless they have been previously denatured by sodium dodecyl sulfate (SDS). 1° The amino acid residue of the Rho protein ADP-ribosylated by the C3 exoenzyme is asparagine-41.11 After detachment of the nicotinamide ring, the ADPribose moiety of NAD binds to Asn-41 by N-glycosidation. This chapter describes an electropermeabilization method to load neutrophils with the C3 ADP-ribosyltransferase and analyzes the effect of internalized C3 exoenzyme on chemotaxis and actin organization. Materials The suppliers are Dupont New England Nuclear (Boston, MA) for [3Zp]NAD; Sigma Chemical Co. (St. Louis, MO) for 3-(N-morpholino)propanesulfonic acid (MOPS), EDTA, EGTA, 4-(2-hydroxyethyl) 1-piperazineethanesulfonic acid (HEPES), phenylmethylsulfonyl fluoride (PMSF), leupeptin, soybean trypsin inhibitor, thymidine, ferricytochrome c (horse heart grade VI), isonicotinic hydrazine, Triton X-100, and anti-/3-tubulin (mouse monoclonal antibody, clone 2.1); GIBCO (Gaithersburg, MD) for RPMI and fetal calf serum; Boehringer-Mannheim (Germany) for NADPH, NAD, and ATP; Serva (Heidelberg, Germany) for N-tosyl-Lphenylalanine chloromethyl ketone (TPCK), N-p-tosyl-L-lysine chloromethyl ketone (TLCK), mercaptoethanol, sodium dodecyl sulfate, Coomassie brilliant blue R-250 and G-250, and bromophenol blue; BDH 3 K. Aktories, U. Weller, and G. S. Chhatwal, FEBS Lett. 212, 109 (1987). 4 K. Aktories, U. Braun, S. R6sener, I. Just, and A. Hall, Biochem. Biophys. Res. Commun. 158, 209, 1989. 5 U. Braun, B. Haberman, I. Just, K. Aktories, and J. Vandekerckhove, FEBS Lett. 243, 70 (1989). 6 S. Narumiya, A. Sekine, and N. Fujiwara, J. Biol. Chem. 263, 17255 (1988). 7 E. J. Rubin, D. M. Gill, P. Boquet, and M. R. Popoff, Mol. Cell Biol. 8, 418 (1988). s M. Sugai, T. Enomoto, K. Hashimoto, K. Matsumoto, Y. Matsuo, H. Ohgai, Y. M. Hong, S. Inoue, K. Yoshikawa, and H. Suginaka, Biochem. Biophys. Res. Commun. 173, 92 (1990). 9 I. Just, G. Schallehn, and K. Aktories, Biochem. Biophys. Res. Commun. 183, 931 (1992). 10 I. Just, C. Mohr, G. Schallehn, L. Menard, J. R. Didsbury, J. Vandekerckhove, J. Van Damme, and K. Aktories, J. Biol. Chem. 267, 10274 (1992). 11 A. Sekine, M. Fujiwara, and S. Narumiya, J. Biol. Chem. 264, 8602 (1989).
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Biochemicals (Poole, England) for acrylamide, bisacrylamide, and sucrose; LKB Pharmacia (Uppsala, Sweden) for standard molecular weight markers; Calbiochem (San Diego, CA), for the fluorescein isothiocyanate (FITC)conjugated Fab' fragment of anti-mouse antibodies; and Amersham (Little Chalfont, England) for the antiactin mouse monoclonal antibody. Exoenzyme C3 (C. botulinum ADP-ribosyltransferase) is a gift from Dr. P. Boquet (Pasteur Institute, Paris, France).
Conditions of Electropermeabilization of Bovine Neutrophils in Presence of C3 ADP-Ribosyltransferase for Effective ADP-Ribosylation of Internal Rho Parameters controlling the electric field-mediated transfer of C3 ADPribosyltransferase in bovine neutrophils are investigated through the property of the C3 exoenzyme to ADP-ribosylate specifically the small G protein Rho. Bovine neutrophils are used in this study. However, the method can be applied to human neutrophils as well. One liter of bovine blood is mixed with 10 ml of 0.5 M EDTA. The red cells are lysed by dilution with 1 liter of cold distilled water and mixed by inversion. After 45-60 sec, isotonicity is reestablished by the rapid addition of 0.1 liter of 9% NaC1. The leukocytes are sedimented by centrifugation at 200g for 15 rain at 2-4 °. The pellet is resuspended in 100 ml of 2.7 mM KC1, 136.7 mM NaCI, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4 (phosphate saline medium), and the suspension is filtered through gauze. If necessary, a second hypotonic lysis may be performed to ensure complete removal of red cells. After reestablishment of isotonicity, the leukocytes are again sedimented by centrifugation at 200g for 15 rain and resuspended in a minimal volume of phosphate saline medium. At this stage, the percentage of contaminant eosinophils is determined. When it is higher than 5%, the neutrophil suspension is placed onto 25 ml of a 4060% continuous Percoll gradient in phosphate saline buffer in 50 ml tubes, and centrifuged at 200g for 30 min. Otherwise, the neutrophil suspension is centrifuged through a layer of 40% Percoll at 200g for 30 min. The sedimented neutrophils are resuspended at the concentration of 107 cells/ ml in phosphate saline medium for further use. Conditions for effective loading of bovine neutrophils with external C3 ADP-ribosyltransferase are summarizedJ 2 The suspension of neutrophils in ice-cold phosphate saline medium is supplemented with 10 mM glucose, 1 mM MgC12 and 2/~M NAD (electropermeabilization medium). C3 ADP12M. J. Stasia, A. Jouan, N. Bourmeyster, P. Boquet, and P. V. Vignais, Biochem. Biophys. Res. Commun. 180, 615 (1991),
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ribosyltransferase is added at various concentrations ranging from 10 -1° to 10 -7 M. A 0.8-ml aliquot of this suspension is transferred to a Bio-Rad Pulser cuvette of 0.2 cm width and is subjected to a single discharge of 3 kV/cm (0.6-0.8 msec) from a 25-/.~F capacitor. After the electric discharge, the suspension is brought to 37° for 15 rain. This incubation is required for effective ADP-ribosylation of internal Rho to proceed (nonradioactive ADP-ribosylation step). A control is performed in the absence of exoenzyme C3. In vivo ADP-ribosylation is stopped by adding 5 ml of ice-cold medium containing 120 mM KC1 and 25 mM HEPES, pH 7.0, to the cell suspension. The neutrophils are then sedimented by centrifugation, washed twice to remove externally bound C3, and resuspended at a concentration of 1 to 2 × 107 cells/ml in a sucrose medium consisting of 0.25 M sucrose, 10 mM MOPS, pH 7.5, 2 mM EDTA, 10 mM EGTA, 50 mM 2-mercaptoethanol, 1 mM PMSF, 1 /Mml TLCK, 1 /~g/ml TPCK, 1 /~g/ml soybean trypsin inhibitor, and 10/~g/ml leupeptin. They are then disrupted by ultrasonic irradiation for 4 × 15 sec at 2-4 ° using a Branson sonifer at a 60-W output. Aliquots of the cell homogenate corresponding to 50 to 100/xg protein are then incubated for 1 hr at 37 ° in 0.2 ml of 10 mM HEPES buffer, pH 8.0, containing 15 mM isonicotinic hydrazine, 15 mM thymidine, 1 mM MgCl2, 1 mM ATP, 2/~M [32p]NAD [20 x 106 dpm (disintegrations per minute) per nmol], and 30 ng of C3 exoenzyme (radiolabeled ADPribosylation step). Proteins in the lysate are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following autoradiography of the polyacrylamide gel, radiolabeled Rho is visualized on the autoradiograms as a single spot at 24 kDa. Under our conditions, bovine neutrophils electropermeabilized with one discharge of 3 kV/cm in the presence of 4 x 10 -8 M C3 exoenzyme are able to incorporate C3 to such an extent that all internal Rho is ADP-ribosylated by nonlabeled NAD (nonradioactive ADP-ribosylation step) and none is left for ADP-ribosylation with [32p]NAD in the radioactive ADP-ribosylation step. This assay was systematically carried out in subsequent studies to verify effective permeabilization of neutrophils and access of the added C3 exoenzyme to intracellular Rho. Leakage Although resealing of electropermeabilized cells occurs shortly after the electric pulse, 13 a significant fraction of the cell components is released to the medium during the pulse. Protein release is conveniently assessed by comparing the activity of lactate dehydrogenase in a supernatant of electropermeabilized neutrophils and in a Triton X-100 lysate of neutro13 S. Sixou and J. Teissi6, J. Bioelectrochem. Bioenerg. 31, 237 (1993).
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phils. The lactate dehydrogenase assay is determined spectrophotometrically at 340 nm. Aliquots (100 tzl) of the suspension of electropermeabilized cells or cells treated by 0.1% Triton X-100 (106 cells/ml) are used. After electropermeabilization or Triton X-100 treatment, the cells are sedimented by centrifugation. The assay medium (1 ml) contains the cell supernatant, 0.05 M phosphate buffer, pH 7.2, and 100 tzM NADH. The reaction is started by the addition of pyruvate (0.4 M final concentration). Under conditions of electropermeabilization in the presence of the C3 exoenzyme leading to total inhibition of the Rho ADP-ribosylation (see above), loss of cellular lactate dehydrogenase is between 20 and 30%. Specific Inhibition of Motility of Neutrophils Treated with C3 ADP-Ribosyltransferase The cell motility of bovine neutrophils is tested after electropermeabilization in the presence of C3 ADP-ribosyltransferase added at concentrations ranging between 4 × 10 -11 M and 4 × 10 -8 M, using the electropermeabilization medium containing glucose, NAD, and MgC12 and a single electric discharge of 3 kV/cm (see above). Under these conditions, 40 to 95% of intracellular Rho is ADP-ribosylated. To assay chemotaxis and spontaneous migration of neutrophils, the method described by Nelson e t al. 14 is used with some modifications. This method is based on migration of neutrophils under agarose gel in petri dishes. In our hands the agarose method gives reproducible results. In brief, for four petri dishes (diameter 60 ram), 0.2 g of agarose is dissolved in 10 ml of distilled water by heating in a boiling water bath for 10 rain. After cooling at 48 °, the agarose is mixed with an equal volume of prewarmed RPMI 1640 medium (GIBCO) supplemented with bicarbonate (2 g/liter) and 10% fetal calf serum. Samples of 5 ml of the warmed agarose medium are distributed in the sterile petri dishes. After cooling for about 1 hr at 4°, series of three wells (2.5 mm diameter) spaced 2.5 mm apart are cut in the solid agarose. Zymosan-activated serum (ZAS) is prepared by incubation of 1 ml bovine serum with 4 mg zymosan for 30 rain at 37°. The supernatant (ZAS) recovered by centrifugation is used as such. For each row of three wells (Fig. 1), the center well (c) receives a 10-/zl aliquot of neutrophil suspension (106 cells) in phosphate saline medium, the outer well (o) receives 10/xl of ZAS, and the inner well (i) receives 10/zl of phosphate saline medium. Each experiment is performed in duplicate. The incubation is carried out in a humidified atmosphere containing 5% CO2 for 2 hr at 37°. Neutrophils 14 R. D. Nelson, P. G. Quie, and R. L. Simmons, J. l m m u n o l . 115, 1650 (1975).
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C
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©
I
FIG. 1. (a) Arrangement of four sets of triplicate wells in an agarose gel plate. In each set, the center well (C) receives 10/~1 of the neutrophil suspension (105 cells), the outer well (O) receives 10/xl of chemotactic reagent (ZAS), and the inner well (I) receives 10/zl of nonchemotactic medium (phosphate saline medium). (b) Differential migration of neutrophils electropermeabilized in the absence (plate 1) and in the presence (plate 2) of the C3 exoenzyme. The neutrophil cells placed in the central well (C) are attracted by ZAS placed in the outer well (O) (large arrow on the right, plate 1), but not by the nonchemotactic phosphate saline medium placed on the inner well (I) (small arrow on the left, plate 1).
migrate by insinuating themselves between the agarose and the floor of the petri dish. For fixation of the cells, 3 ml of absolute methanol is added to the agarose plate and left in contact for 30 min, followed by 3 ml of 38% formalin for another 30 min. Following fixation, the agarose gel is removed, and neutrophils adhering to the plates are stained with Giemsa's solution. The distances of migration of cells from the margin of the center well toward the well containing ZAS (A) and toward the well containing PBS (B) are measured under an inverted microscope (Zeiss, Model IM35) equipped with a micrometer and are used as indices of chemotaxis and spontaneous migration, respectively. A chemotactic index A/B was calculated. TM In the experiment illustrated in Fig. 1 (plate 1), the chemotactic index for control neutrophils was 3.2. Loading neutrophils with C3 ADP-ribosyltransferase by electropermeabilization strikingly inhibits the spontaneous motility and also the chemotaxis of neutrophils. In a series of three different experiments, halfinhibition of both spontaneous mobility and chemotaxis is observed with 4 x 10 -11 M C3. When the C3 exoenzyme concentration is raised to 4 x 10 -8 M, the two processes are totally inhibited (Fig. 1, plate 2). Electropermeabilized neutrophils in the absence of the C3 exoenzyme show only a small decrease of motility compared to intact neutrophils (less than 15%). Incorporation of C3 ADP-ribosyltransferase hardly alters other strategic functions of neutrophils, namely exocytosis and production of O2 • These two functions are tested after electropermeabilization in the presence of the C3 exoenzyme in the electropermeabilization medium, followed by
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activation with phorbol myristate acetate. 12 An apparent loss of oxidase activity is due to leakage of pyridine nucleotide since the oxidase activity could be fully restored by the addition of NADPH. Effect of C3 Exoenzyme on Actin Network of Neutrophils Neutrophils (107 cells/ml) in the electropermeabilization medium in the presence of C3 exoenzyme (4 X 10 -8 M) are subjected to a single electric discharge (3 kV/cm). The cell suspension is then diluted to 105 cells/ml by the addition of RMPI 1640 medium supplemented with 10% fetal calf serum. The cells are left to sediment for at least 1 hr at 37° on polylysinecoated coverslips. After a rapid wash with phosphate saline medium, the ceils are fixed with methanol at -20 ° for 6 min. This is followed by three successive washes with phosphate saline medium supplemented with 0.1% Tween 20. Actin and tubulin in the cells are immunodetected after incubation at room temperature for 30 min with mouse monoclonal antibody directed against actin and diluted to 1/500 or mouse monoclonal antibody directed against/3-tubulin and diluted to 1/100, respectively. Then, the cells are washed three times with phosphate saline medium containing 0.1% Tween 20, and are finally incubated for another 30 min with the FITCconjugated Fab' fragment of the anti-mouse antibody. As a control, nuclei are stained with 1% Hoescht solution. The ceils are mounted with aquamount and examined using an epifluorescence Zeiss microscope. As shown in Fig. 2, the actin microfilament network, which appears as a fluorescent ring at the peripheric of control cells (Fig. 2A), is totally disorganized in cells electropermeabilized in the presence of 4 × 10 -8 M C3 ADPribosyltransferase (Fig. 2C). This is in contrast with the absence of an effect of C3 ADP-ribosyltransferase on the tubulin network (Fig. 2E2 vs Fig. 2El). Although cells electropermeabilized in the presence of C3 ADPribosyltransferase (Fig. 2C) are somewhat swollen compared to control cells (Fig. 2A), their multilobed nuclei stained with Hoescht solution (Fig. 2D vs Fig. 2B) appear to be unaltered. A control experiment is made in the absence of anti-/3-tubulin or antiactin antibodies to rule out any aspecific binding of the secondary antibody. Comments
Effects of (?3 ADP-Ribosyltransferase on Cell Morphology In addition to its effect on cell motility, but possibly related to this effect, C3 ADP-ribosyltransferase has been shown to affect cell morphology. For example, permeabilization of 3T3 cells by an osmotic shock in the presence
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G
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m m m I/
D
E1
// // // // E2
FIG. 2. Immunodetection by fluorescence microscopy of the actin filament assembly (A, C) and the tubulin network (El, E2) in bovine neutrophils electropermeabilized in the absence (A and E l ) and in the presence (C and E2) of C3 ADP-ribosyltransferase. The same neutrophil cells in which the actin filament assembly was immunodetected by fluorescence (A and C) were stained with Hoescht solution to reveal their nucleus, The cells electropermeabilized in the presence of C3 ADP-ribosyltransferase displayed the same multilobed nuclei (D) as the control cells (B).
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of the C3 exoenzyme, 6 culture of Vero cells in the presence of the C3 exoenzyme, 15 or microinjection of the C3 exoenzyme in 3T3 cells 16 induced rounding of the cells with disassembly of the actin microfilament network, without alteration of microtubules. It has been reported that some growth factors added to 3T3 cells induced the formation of stress fibers and that this effect was counteracted by the C3 exoenzyme, resulting again in rounding of the cellsJ 7 The relationship between the C3-catalyzed ADP-ribosylation of Rho and the subsequent morphological changes has been discussed (for review see Zigmond a ) in terms of modification of the adhesion plaque, i.e., the zone of contact between cells in culture and their substratum. The adhesion plaque is composed of adhesion molecules such as fibronectin or collagen belonging to the extracellular matrix and membrane receptors of the integrin type belonging to the cell plasma with a number of associated proteins at the intracellular face of the plasma membrane, namely, actin, vinculin, tensin, and a-actinin. TMRho might be involved in the actin polymerization occurring at the level of the adhesion plaquesJ 7 Disruption of the actin microfilaments which bind to the adhesion plaques would lead to cell rounding.
Subcellular Compartmentation of Rho The Rho proteins are geranylgeranylated. 19'2° In bovine neutrophils, the predominant Rho species is Rho A . 21 Most of the Rho A (80%) is located in the cytosol in association with a GDP/GTP exchange inhibitor (GDI), with the remaining Rho being bound to membranes. Both membrane-bound and cytosolic Rho can be ADP-ribosylated by the C3 exoenzyme in the presence of NAD. Through the use of [32p]NAD of known specific radioactivity, it has been possible to determine through the ADP-ribosylation assay the amount of Rho present in bovine neutrophils. During the course of the measurements, it was found that the binding capacity of cytosolic Rho for [32p]ADP-ribose can be nearly doubled, to reach a maximal value, by adding some specific phospholipids, the most 15 p. Chardin, P. Boquet, P. Madaule, M. R. Popoff, E. J. Rubin, and D. M. Gill, EMBO J. 8, 1087 (1989). 16 H. F. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell Biol. 111, 1001 (1990). x7 A. J. Ridley and A. Hall, Cell 70, 389 (1992). 18 K. Burridge, K. Fath, T. Kelly, G. Nuckolls, and C. Turner, Annu. Rev. Cell Biol. 4, 487 (1988). 19 I. G. Macara, Cell Signal. 3, 179 (1991). 20 A. Valencia, P. Chardin, A. Wittinghofer, and C. Sander, Biochemistry 30, 4637 (1991). 2x N. Bourmeyster, M. J. Stasia, J. Garin, J. Gagnon, P. Boquet, and P. V. Vignais, Biochemistry 31, 12863 (1992).
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effective being phosphoinositides, zl Under optimal conditions, the amount of Rho found in neutrophil cytosol is in the range of 30 to 45 pmol/mg protein whereas that found in neutrophil membranes is in the range of 5 to 15 pmol/mg protein. These values are nearly twice those found in brain and three or four times higher than in heart and liver. Neutrophils therefore appear to be by far the richest source of Rho protein in bovine tissues. This peculiar feature might be related to chemotaxis, one of the major functions of neutrophils.
[37] C e l l M o t i l i t y A s s a y a n d I n h i b i t i o n b y R h o - G D P Dissociation Inhibitor
By KENJI
TAKAISHI, TAKUYA SASAKI, and YOSHIMI TAKAI
Introduction Cell motility is essential for inflammatory reactions, tissue repair, and immune system interactions in normal cells. 1 Moreover, cell motility is essential for malignant cancer cells to infiltrate surrounding tissues and for smooth muscle cells in the media to migrate to the intima at the atherosclerotic region of vascular cells. 2'3 The actomyosin system is known to be involved in cell motility. In cultured cells, actin filaments exist principally in three types of structure: the cortical actin network, actin stress fibers, and cell surface protrusions including membrane ruffling and microspikes. Membrane ruffling, which is caused by a polymerization of actin at the inner surface of the plasma membrane, is believed to be associated with cell motility in cultured cells. 4,s The Rho family belongs to the small GTPase superfamily and consists of the Rho, Rac, and G25K families. 6'7 Rho has GDP-bound inactive and GTP-bound active forms which are interconvertible by GDP/GTP exchange and GTPase reactions. The conversion from the GDP-bound form 1 M. Stoker and E. Gherardi, Biochim. Biophys. Acta 1072, 81 (1990). 2 G. Gabbiani, E. Rungger-Br~indle, C. D. Chastonay, and W. W. Franke, Lab. Invest. 47, 265 (1982). 3 F. V. Roy and M. Mareel, Trends Cell Biol. 2, 163 (1992). 4 M. Abercrombie, J. E. M. Heaysman, and S. M. Pegrum, Exp. Cell Res. 60, 437 (1970). 5 A. Dipasquale, Exp. Cell Res. 94, 191 (1975). 6 A. Hall, Science 249, 635 (1990). 7 y. Takai, K. Kaibuchi, A. Kikuehi, and M. Kawata, Int. Rev. Cytol. 133, 187 (1992).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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to the GTP-bound form is regulated by GDP/GTP exchange proteins (GEPs) and the reverse conversion is regulated by GTPase-activating proteins (GAPs). There are two types of GEPs: one is stimulatory and the other is inhibitory. We have found an inhibitory type of GEP for Rho, called R h o - G D P dissociation inhibitor (GDI) (see this volume [6]). 8-1° Only the Rho subfamily is ADP-ribosylated by C3, a Clostridium botulinum ADP-ribosyltransferase, from over 50 members of small GTPases. n-14 C3 ADP-ribosylates Asn 41 of Rho, which is located at the putative effector domain, and the ADP-ribosylation impairs the functions of Rho. 15 By using R h o - G D I or C3, several functions of Rho have been clarified: Rho regulates cell morphology, 16-2° cell motility, 2>23 membrane ruffling, 24 smooth muscle contraction, 25 platelet aggregation, 26 cytokinesis, 27'28 lym8 N. Ohga, A. Kikuchi, T. Ueda, J. Yamamoto, and Y. Takai, Biochem. Biophys. Res. Commun. 163, 1523 (1989). 9 y. Fukumoto, K. Kaibuchi, Y. Hori, H. Fujioka, S. Araki, T. Ueda, A. Kikuchi, and Y. Takai, Oncogene 5, 1321 (1990). 10T. Ueda, A. Kikuchi, N. Ohga, J. Yamamoto, and Y. Takai, J. Biol. Chem. 265, 9373 (1990). 11 K. Aktories, S. R6sener, U. Blaschke, and G. S. Chhatwal, Eur. J. Biochem. 172, 445 (1988). 12 A. Kikuchi, K. Yamamoto, T. Fujita, and Y. Takai, J. Biol. Chem. 263, 16303 (1988). 13 S. Narumiya, A. Sekine, and M. Fujiwara, J. Biol. Chem. 263, 17255 (1988). 14 U. Braun, B. Habermann, I. Just, K. Aktories, and J. Vandekerckhove, FEBS Lett. 243, 70 (1989). 15 A. Sekine, M. Fujiwara, and S. Narumiya, J. Biol. Chem. 264, 8602 (1989). 16 E. J. Rubin, D. M. Gill, P. Boquet, and M. R. Popoff, Mol. Cell. Biol. 8, 418 (1988). 17 p. Chardin, P. Boquet, P. Madaule, M. R. Popoff, E. J. Rubin, and D. M. Gill, EMBO J. 8, 1087 (1989). 18H. F. Paterson, A. J. Self, M. D. Garrett, I. Just, K. Aktories, and A. Hall, J. Cell Biol. 111, 1001 (1990). 19 A. J. Ridley and A. Hall, Cell 70, 389 (1992). 2o y. Miura, A. Kikuchi, T. Musha, S. Kuroda, H. Yaku, T. Sasaki, and Y. Takai, J. Biol. Chem. 268, 510 (1993). 21 M.-J. Stasia, A. Jouan, N. Bourmeyster, P. Boquet, and P. V. Vignais, Biochem. Biophys. Res. Commun. 180, 615 (1991). 22 K. Takaishi, A. Kikuchi, S. Kuroda, K. Kotani, T. Sasaki, and Y. Takai, Mol. Cell. Biol. 13, 72 (1993). 23 K. Takaishi, T. Sasaki, M. Kato, W. Yamochi, S. Kuroda, T. Nakamura, M. Takeichi, and Y. Takai, Oncogene 9, 273 (1994). 24 T. Nishiyama, T. Sasaki, K. Takaishi, M. Kato, H. Yaku, K. Araki, Y. Matsuura, and Y. Takai, Mol. Cell. Biol. 14, 2447 (1994). 25 K. Hirata, A. Kikuchi, T. Sasaki, S. Kuroda, K. Kaibuchi, Y. Matsuura, H. Seki, K. Saida, and Y. Takai, J. Biol. Chem. 267, 8719 (1992). 26 N. Morii, T. Teru-uchi, T. Tominaga, N. Kumagai, S. Kozaki, F. Ushikubi, and S. Narumiya, J. Biol. Chem. 267, 20921 (1992). 27 K. Kishi, T. Sasaki, S. Kuroda, T. Itoh, and Y. Takai, J. Cell Biol. 120, 1187 (1993). 28 I. Mabuchi, Y. Hamaguchi, H. Fujimoto, N. Morii, M. Mishima, and S. Narumiya, Zygote 1, 325 (1993).
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phocyte toxicity,29 and lymphocyte aggregation. 3° All of these cell functions are dependent on the actomyosin system. As for cell motility and membrane ruffling, we have shown by microinjection of R h o - G D I or C3 into cultured cells that Rho is involved in the fetal calf serum (FCS)-induced cell motility in Swiss 3T3 cells,22 in the hepatocyte growth factor (HGF)-induced and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell motility,23 and in membrane rufflings24 in epithelial cell lines. This chapter first describes the cell motility assays based on phagokinetic and scattering activities. Phagokinetic activity is estimated by measuring cell tracks that are formed by phagocytosis of colloidal gold particles due to migrating cultured cells, 22,31,32 and scattering activity is estimated by observing the scattering of the epithelial cells growing in colonies by phasecontrast microscopy.23 The chapter then describes the methods for microinjection of R h o - G D I or C3, both of which inhibit cell motility by the cell motility assays.22'23
Materials Swiss 3T3 cells and the mouse keratinocyte cell line, 308R cells, 33 a r e kindly supplied by Dr. E. Rozengurt (Imperial Cancer Research Fund, London, England) and by S. H. Yuspa (National Cancer Institute, Maryland), respectively. Human recombinant HGF purified from the culture fluid of C-127 cells is kindly supplied by T. Nakamura (Osaka University, Suita, Japan). C3 is kindly provided by S. Narumiya (Kyoto University, Kyoto, Japan) (see this volume [24]). Anti-E-cadherin ( E C C D - 2 ) 34 and anti-P-cadherin (PCD-1) 35 monoclonal antibodies are kindly provided by M. Takeichi (Kyoto University, Kyoto, Japan). R h o - G D I is purified as a glutathione S-transferase (GST) fusion protein from Escherichia coli overexpressing G S T - R h o - G D I (see this volume [6]). H A u C I 4 and TPA are obtained from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), Ham's F12 medium (F12), and FCS are purchased from GIBCO-BRL (Gaithersburg, MD). All other chemicals are reagent grade. 29 p. Lang, L. Guizani, I. Vitt6-Mony, R, Stancou, O. Dorseuil, G. Gacon, and J. Bertoglio, J. Biol. Chem. 267, 11677 (1992). 30 T. Tominaga, K. Sugie, M. Hirata, N. Morii, J. Fukata, A. Uchida, H. Imura, and S. Narumiya, J. Cell Biol. 120, 1529 (1993). 31 G. Albrecht-Buehler and R. D. Goldman, Exp. Cell Res. 97, 329 (1976). 32 G. Albrecht-Buehler and R. M. Lancaster, J. Cell Biol. 71, 370 (1976). 33 S. n . Yuspa and D. L. Morgan, Nature 293, 72 (1981). 34 y. Shirayoshi, A. Nose, K. Iwasaki, and M. Takeichi, Cell Struct. Funct. 11, 245 (1986). 35 A. Nose and M. Takeichi, J. Cell Biol. 103, 2649 (1986).
[37]
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Methods
1. Cell Culture Stock cultures of Swiss 3T3 cells are maintained at 37° in a humidified atmosphere of 10% CO2 and 90% air (v/v) in DMEM containing 10% FCS, penicillin (100 U/ml), and streptomycin (100 ~g/ml). Stock cultures of 308R cells are maintained at 37° in a humidified atmosphere of 5% CO2 and 95% air in a 1:1 mixture of DMEM and F12 (DMEM/F12) containing 10% FCS, penicillin (100 U/ml), and streptomycin (100 ~g/ml).
2. Cell Motility Assay by Measuring Cell Tracks by Phagokinesis a. Gold Particle Preparation and Gold Coating. To coat 35-mm grid tissue culture dishes (Nunc Inc., Naperville, IL) with bovine serum albumin (BSA), 1 ml of 10 mg/ml BSA is added to the dishes. After incubating for a few minutes, BSA is removed and the dishes are quickly rinsed with 1 ml of 100% ethanol and dried by a hair dryer. To prepare colloidal gold particles, 1.8 ml of 14.5 mM HAuCI4 and 6 ml of 36.5 mM Na2CO3 are added to 11 ml of H20, and the solution is heated in a glass beaker. Immediately after reaching the boiling point, the solution is removed from the heat and 1.8 ml of a 0.1% formaldehyde solution is quickly added, resulting in formation of a dark brown solution. Colloidal gold is formed within a minute. The solution is cooled to about 50° and 1-3 ml of the solution is poured into each BSA-coated 35-mm grid tissue culture dish. The density of colloidal gold particles on the dishes is dependent on the volume of the solution poured into the dishes. After a 30-min incubation, the solution is removed and 1 ml of phosphate-buffered saline (PBS) is added. PBS must be removed immediately before the cells are seeded on the dishes. b. Inoculation of Swiss 3 T3 Cells and Estimation of Phagokinetic Activity. Growing Swiss 3T3 cells in 10-cm culture dishes are harvested by the following procedures. After the medium is removed, 1 ml of 0.02% EDTA/ 0.025% trypsin in PBS is added and the cells are incubated for 5 min at 37 ° in a humidified atmosphere of 10% CO2 and 90% air to detach the cells from the dishes. The cells are harvested and suspended in an appropriate volume of DMEM containing 10% FCS at a density of 2 × 103 cells per ml of the medium. One milliliter of the cell suspension is seeded on each colloidal gold-coated dish. The gold colloidal particles are observed as a homogeneous layer of fine black particles (Fig. 1). When Swiss 3T3 cells are seeded on the colloidal gold-coated dish, they migrate on this substrate, phagocytize, and remove the gold particles to produce a white track free of the particles in a time-
340
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FIG. 2. Time course of cell motility of Swiss 3T3 cells. After Swiss 3T3 cells were microinjected with buffer A, R h o - G D I , or C3 and incubated for various periods of time as indicated, the cell motility was examined. (3, with buffer A; O, with 5 mg/ml of R h o - G D I ; II, with 40/zg/ml of C3. The results shown are the means _+SE of three independent experiments. (Reproduced from Takaishi et a/. 22)
dependent manner (Fig. 1A). One migrating cell is visible inside the white track as a black body. For the cell motility assay, photographs are taken, and cell motility is evaluated by measuring the areas free of the gold particles. The area of the track increases linearly until 30 hr after seeding the cells on the colloidal gold-coated dishes (Fig. 2). Effects of various drugs on cell motility can be examined by adding each drug into the medium at 4-6 hr after inoculation of the cells on the colloidal gold-coated dishes.
3. Cell Motility Assay by Observing Scattering Activity Epithelial cell lines including 308R cells grow forming colonies of the cells in culture dishes. Some cell motility-inducing growth factors or agents
FlG. 1. Inhibition of cell motility by microinjection of R h o - G D I or C3 into Swiss 3T3 cells. After Swiss 3T3 cells were microinjected with buffer A, R h o - G D I , or C3 and incubated for various periods of time as indicated, the cell motility was analyzed by phase-contrast microscopy. (A) With buffer A; (B) with 5 mg/ml of Rho-GDI; and (C) with 40/zg/ml of C3. (a) Gold particles only; (b) before microinjection; (c) 2 hr after microinjection; (d) 6 hr after microinjection; and (e) 12 hr after microinjection. Results shown are representative of three independent experiments. Bar: 30 /xm. All photographs were taken with the same magnification. (Reproduced from Takaishi et al. 2z)
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such as HGF or TPA induce scattering of the colonies.23 For analysis of scattering activity in 308R cells, 308R cells are seeded and incubated by the following procedures. Growing 308R cells in 10-cm culture dishes are harvested by incubation with 1 ml of 0.02% EDTA/0.025% trypsin in PBS for 5-10 min at 37° in a humidified atmosphere of 10% CO2 and 90% air to detach the cells from the dishes. The cells are harvested and suspended roughly in an appropriate volume of DMEM/F12 containing 10% FCS at a density of 8 × ]04 cells per ml of the medium. One milliliter of the cell suspension is seeded on each 35-mm grid tissue culture dish, and the cells are incubated for 20 hr. The medium is changed to DMEM/F12 containing 1% FCS and the cells are further incubated for 4 hr. After incubation, growth factors or agents are added and cell motility is analyzed by phasecontrast microscopy. HGF and TPA induce scattering of the colonies in a time-dependent manner. It is believed that epithelial cell-cell adhesions are caused by cadherins, a family of Ca2+-dependent homophilic cell adhesion molecules. 36 It is better to incubate the cells with both the anti-E-cadherin (ECCD-2) 34 and anti-P-cadherin (PCD-1) 35 monoclonal antibodies for a short time (until 6 hr) because cell motility-inducing growth factors or agents such as HGF or TPA induce scattering of the colonies more readily in the presence of both ECCD-2 and PCD-1 monoclonal antibodies. 23HGF induces complete dissociation of the colonies in the presence of both ECCD2 and PCD-1 monoclonal antibodies in a time-dependent manner (Fig. 3).
4. Microinjection The general procedure utilizes a glass capillary needle filled with the samples to be injected into the cells, a micromanipulator to place the needle into the cells, a microinjector to transfer the samples from the needle into the cells, and phase-contrast microscopy to allow visualization of the injection process. The general techniques of microinjection have been described in detail elsewhere 37 and there are various kinds of apparatus for microinjection. This chapter describes the methods of microinjection that we practice. a. Preparation of Capillary Needles. The glass capillary needles are generated by a one-stage horizontally pulling process for glass capillaries using a glass microelectrode puller (PN-3, Narishige, Tokyo, Japan). We use borosilicate glass capillaries with inner filaments (1.0 mm o.d., 0.78 mm i.d.) (GC100TF-10, Clark Electromedical Instruments, England). The glass capillaries are clamped into the glass microelectrode puller, and then the 36 M. Takeichi, Annu. Rev. Biochem. 59, 237 (1990). 37 M. Graessmann and A. Graessman, this series, Vol. 101, p. 482.
[371
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O
FIG.3. Time course of HGF-induced motility of 308R cells. 308R cells were seeded and incubated for 4 hr with DMEM/F12 containing 1% FCS. After incubation, HGF (1 x 10 u M), ECCD-2 (x 100), and PCD-1 (x 1000) monoclonal antibodies were added. Photographs were taken at 0 hr (A), 2 hr (B), or 6 hr (C) after the addition of HGF. Results shown are representative of three independent experiments. (Reproduced from Takaishi et al.23) center of the capillary within the heater filament is heated while being pulled horizontally, resulting in generation of glass capillary needles with a tip diameter of less than 1 /xm. The tip diameter can be controlled by both heating t e m p e r a t u r e and pulling force. Glass capillary needles should be p r e p a r e d immediately before each experiment. b. Preparation of Microinjected Samples. The samples to be microinjected into the cells must be in a microinjection buffer that does not have deleterious effects on the cells. A variety of buffers have been used with success. We use buffer A (20 m M Tris/HCl at p H 7.4, 20 m M NaC1, 2 m M MgC12, 100/xM ATP, 0.1 m M E D T A , and 1 m M 2-mercaptoethanol) for microinjection of R h o - G D I or C3 into the cells. R h o - G D I is purified as a G S T fusion protein from E. coli-overexpressing G S T - R h o - G D I (see this volume [6]). G S T - R h o - G D I is concentrated in Centricon-10 (Amicon) to the concentration of over 10 mg/ml. Protein concentrations are determined with B S A as a standard by densitometric tracing of protein bands
344
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stained with Coomassie brilliant blue on a sodium dodecyl sulfate-polyacrylamide gel. During the concentration, the buffers contained in the sample preparation are replaced by buffer A. The microinjected samples are stored in 2-3/zl of each tube at -80 °. Recombinant C3 is purified from E. coli-overexpressing C3 (see this volume [21]) and is stored in 2-3/zl of each tube at - 8 0 °. The protein concentration of C3 is recommended over 520/zg/ml. When microinjection is performed, R h o - G D I and C3 are diluted with buffer A to indicated concentrations. About 5 and 1.5 × 10 -14 liters of samples are microinjected by one injection into Swiss 3T3 cells and 308R cells, respectively. G S T - R h o GDI is microinjected at 5 mg/ml, and the intracellular concentrations of microinjected R h o - G D I are calculated to be about 13 /zM in both cell lines. C3 is microinjected at 40 and 130/zg/ml into Swiss 3T3 cells and 308R cells, respectively, and the intracellular concentrations of the microinjected C3 are about 0.21 and 0.74/zM, respectively. c. Microinjection into Cultured Cells. The samples are loaded into the glass capillary needles by capillary action at 4°. The cells are immediately removed from the incubator and the samples are microinjected with the microinjection system (Micromanipulator; combined with a 3-dimensional joystick-type hydraulic micromanipulator, MO102, Narishige. Microinjector; IM4B, Narishige. Phase-contrast microscopy; IMT-2, Olympus, Tokyo, Japan). Generally, more than 100 cells can be microinjected within 15 min. The appropriate volume microinjected into each cell can be controlled by a visualized analysis because the cells swell slightly during the injection process and the cytoplasm appears to lose contrast momentarily. When Swiss 3T3 cells are microinjected on colloidal gold particles, the microinjection is performed at 4-6 hr after the cells are seeded on colloidal gold particles. The cells must be microinjected carefully, and 30 cells can be maximally microinjected within 15 rain because the cells on colloidal gold particles are easily detached from the dishes. About 30-40% and 2030% of the microinjected cells with R h o - G D I and C3, respectively, still attach to the dishes after a 24-hr incubation. Microinjection of R h o - G D I into Swiss 3T3 cells inhibits the cell motility for 6 hr after the microinjection (Figs. 1B and 2). After 6 hr, the cell begins to move again and the migration area reaches about 80% of that of the control cell at 24 hr (Fig. 2). Thus, the R h o - G D I action is reversible and microinjection of R h o - G D I does not kill the cells. The reversible action of R h o - G D I may be due to degradation of the microinjected Rho-GDI. Microinjection of C3 into Swiss 3T3 cells also inhibits the cell motility for 6 hr after the microinjection (Figs. 1C and 2), but in this case, the cell does not begin to move again even at 24 hr because the ADP-ribosylation of Rho by C3 is an irreversible reaction (Fig. 2).
[371
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When 308R cells are microinjected, each colony consisting of 10-20 cells is selected for microinjection. About 10 colonies can be microinjected within 15 min. More than 90% of the microinjected cells usually survive. The HGF- and TPA-induced cell motility is inhibited by microinjection of R h o - G D I or C3 (Fig. 4). After 6 hr, the cells microinjected with R h o GDI begin to move again and dissociate completely at 24 hr after the microinjection, whereas the cells microinjected with C3 do not begin to move again or dissociate even at 24 hr after the microinjection. Thus, the R h o - G D I action is reversible, but the C3 action continues, at least up to 24 hr. Comments For analysis of cell tracks by phagokinesis, it is important to seed the cells at an appropriate density. If the density of the cells is too high, cell tracks by one migrating cell overlap each other and cannot be measured. For analysis of scattering activity, it is necessary to obtain an appropriate density and size of the colonies of the cells. When 308R cells are harvested and suspended in the medium, the cells must not be suspended too intensely because then the cells will be too separated to form smaller colonies. To observe the effects of the microinjected samples, the bottom of grid dishes is numbered and the place of the microinjected cells or the microinjected colonies of the cells is checked by the number on the grid. Isolated cells or isolated colonies of the cells must be selected for microinjection in these cell motility assays. When effects of the samples on cell motility are examined by microinjection into the cells, the control cells must be microinjected with a microinjection buffer only to exclude the possibility of the deleterious effects by microinjection or a microinjection buffer. If the cells are easily detached from the dishes by microinjection, the dishes should be coated with polylysine, fibronectin, or collagen. Protein concentrations for the microinjection should be adjusted according to the purpose of the experiment. When fluorescently labeled structural proteins are introduced into the cells, the protein concentrations can be relatively lower compared with the endogenous amounts. When antibodies or inhibitory proteins directed against components of the cells are introduced into the cells, the overwhelming amounts should be microinjected compared with the endogenous amounts. When R h o - G D I is microinjected at 5 mg/ml in Swiss 3T3 cells and 308R cellls, the intracellular concentrations of microinjected R h o - G D I are about 80- and 40-fold more than their endogenous levels in Swiss 3T3 cells and 308R cells, respectively. R h o - G D I inhibits the cell motility of Swiss 3T3 cells and 308R cells when R h o - G D I is microinjected minimally at 2.5 mg/ml. Generally, when
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the protein concentrations of microinjected samples are higher, it is relatively difficult to microinject appropriate volumes of the samples into the cells due to their viscosity. If those effects are observed, the samples should be microinjected at lower protein concentrations or mixed with BSA at 0.1 mg/ml as a final concentration.
[38] By
Cell Transformation
by dbl Oncogene
DANIELA ZANGRILLI and ALESSANDRA EVA
Introduction The dbl oncogene was identified by transfection analysis of D N A from the affected spleen of a patient with diffuse B-cell lymphoma. 1 A cosmid genomic library was constructed from a third cycle transfectant DNA, and four overlapping clones were isolated whose 40- to 45-kb inserts contained human repetitive sequences. One of these clones, containing a 45-kb human D N A fragment, efficiently induced focus formation (600 focus-forming units per pmol). We identified a second activated dbl gene in D N A of a human nodular poorly differentiated B cell lymphoma (NPDL-dbl). 2 The m c f 2 oncogene 3 was isolated by D N A transfection from the human mammary carcinoma cell line MCF-7, and its transforming sequences were subsequently found to be derived from the same genetic locus as the dbl oncogene. Each isolate contains different 5' rearrangements likely generated by the transfection procedure. To further characterize the dbl oncogene and define its transcriptional unit, a c D N A library was constructed in Agt11 with poly(A) + R N A purified from a dbl transfectant. The complete nucleotide sequence of the dbl c D N A was determined, and its single long open reading flame (ORF) of 1434 bp was predicted to encode a 478 amino acid polypeptide. 4 In order to clone the proto-dbl cDNA, libraries were prepared from cells known to be positive for the proto-dbl transcript (human brain stem and fetal tissue). A single O R F was identified encoding a predicted primary translational product of 925 1A. Eva and S. A. Aaronson, Nature 316, 273 (1985). 2 A. Eva, G. Vecchio,M. Diamond, S. R. Tronick, D. Ron, G. M. Cooper, and S. A. Aaronson, Oncogene 1, 355 (1987). 3 O. Fasano, D. Birnbaum, L. Edlund, J. Fogh, and M. Wigler, MoL Cell Biol. 4, 1695 (1984). 4A. Eva, G. Vecchio, C. D. Rao, S. R. Tronick, and S. A. Aaronson, Proc. Natl. Acad. U.S.A. 85, 2061 (1988). METHODSIN ENZYMOLOGY,VOL.256
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-
ANpdbl
FIG. 1. Structure of dbl and proto-dbl cDNA clones and their respective amino-terminal truncated mutants. Open reading frames are indicated by large boxes. Lines represent 5'and 3'-untranslated regions. White boxes denote dbl locus sequences. Black areas represent the 5' structural rearrangement of the dbl gene involving a recombinational event with unrelated human sequences. The single amino acid difference within the C terminus of both proteins (A/V) is also indicated. Restriction sites are: E, EcoRI; HII, HinclI; S, SaclI; B, BamHI; Ac, AccI; and St, StyI. The sites indicated in parentheses were created with linkers for subcloning purposes.
amino acids. 5 A schematic diagram and a physical map of the dbl genomic clone and both cDNAs are shown in Fig. 1. Comparison of the c D N A sequence of the proto-dbl with that of the dbl oncogene indicated that the dbl oncogene represents a truncated version of its normal counterpart. The breakpoint was located at codon 497 of the predicted proto-dbl ORF. The nucleotide sequence from the breakpoint through the 3' end of proto-dbl was identical to that of the dbl oncogene except for three nucleotide changes, one of which involved a C (proto-dbl) to T (dbl) transition resulting in a conservative change from Ala in proto-dbl to Val in the dbl oncogene. The entire region of proto-dbl upstream from the breakpoint was completely deleted from the dbl oncogene and substituted by 476 nucleotides of unrelated human sequences which provided a new amino terminus of 50 amino acids derived from a different locus. Subsequently, we found 5 D. Ron, S. R. Tronick, S. A. Aaronson, and A. Eva, EMBO J. 7, 2465 (1988).
[38]
dbl TRANSFORMATION
349
that the dbl protooncogene resides on chromosome Xq27-q28, while the heterologous sequences found in the dbl oncogene were derived from chromosome 3 (p13q-ter). 6
T r a n s f o r m i n g Activity of dbl and Proto-dbl Oncogene In order to determine the molecular basis of the transforming activity of the dbl oncogene, to decipher the normal function of the dbl protooncogene, and to study its mechanism of activation, we constructed eukaryotic expression vectors containing the coding sequences of dbl and proto-dbl and analyzed them for transforming activity by the NIH 3T3 transfection assay. The transfectants were analyzed for acquisition of known properties of malignant cells. We established that both dbl and proto-dbl can induce transformation of NIH 3T3 cells but that the activity of proto-dbl is significantly lower than that of the dbl oncogene.
Subcloning of dbl and Proto-dbl cDNAs in Eukaryotic Expression Vector To characterize the structural features that contribute toward the transforming activity of proto-dbl and dbl, cDNA sequences are subcloned into the pZIP-Neo SV(X) vector 7 by standard techniques. The dbl cDNA is assembled and subcloned in the EcoRI site of the pUC13 vector. The insert can then be released from the plasmid by digestion with SacII and StyI with resultant removal of most of the 5'- and Y-untranslated regions (see Fig. 1). The SacII and StyI sites are rendered blunt-ended by filling the ends with Klenow fragment, converted to BamHI sites with BamHI linkers, and ligated to the BamHI sites of pZIP-Neo SV(X) vector. The same procedure is used for subcloning proto-dbl cDNA, assembled and subcloned in pUC18, except that the insert is released from the plasmid by digestion with HincII and StyI (Fig. 1).
Transforming Activity of Proto-dbl and dbl Oncogenes The transforming potential of the expression vectors is ascertained by introducing the plasmid into NIH 3T3 cells using the calcium phosphate transfection procedure of Graham and van der Eb. 8 Foci induced by the dbl oncogene construct appear about 7-10 days after transfection whereas foci induced by the proto-dbl become detectable a few days later. As shown 6 S. R. Tronick, O. W. McBride, N. C. Popescu, and A. Eva, Genomics5, 546 (1989). 7 C. L. Cepko, B. E. Roberts, and R. C. Mulligan, Cell37, 1053 (1984). 8F. L. Graham and A. J. van der Eb, J. Virol. 52, 456 (1973).
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in Table I and discussed below, the dbl protooncogene transforms with 50to 100-fold lower efficiency than the dbl oncogene. Following transfection and marker selection, a mass population of cells is suspended at different dilutions and grown in 0.5% agar solution (Noble Agar, Difco) in DMEM supplemented with 10% bovine serum. Colonies start to appear in 10-15 days. Usually dbl-transformed NIH 3T3 display more dramatic anchorage-independent growth than NIH 3T3 cells transformed by the dbl protooncogene. To determine dbl transfectants tumorigenicity, 0.5 ml of cell suspensions containing 1 × 10 3, 1 X 10 4, and 1 × 105 cells is inoculated into the intramuscular region of NFR nu/nu weanling mice (nude NCR or BALB/ c nu/nu mice can also be used). Cells should be trypsinized, washed in 20% serum-containing DMEM to neutralize trypsin, counted, resuspended at the appropriate concentration in serum-free media, and inoculated. In our h a n d s , 10 3 dbl-transformed cells are sufficient to produce tumors in all injected nude mice within 2 weeks of inoculation, whereas 105 dbl protooncogene-transformed cells are required for 100% tumor incidence. Construction of Proto-dbl M u t a n t s and Generation of dbl-Specific Antibodies for S t r u c t u r e / F u n c t i o n S t u d y As described earlier and indicated in Table I, the dbl protooncogene can transform NIH 3T3 cells when overexpressed, but its transforming
TABLE I TRANSFORMING ACTIVITY AND BIOCHEMICAL CHARACTERISTICS OF PRODUCTS OF AMINO-TERMINAL TRUNCATED dbl MUTANTS AND THEIR PARENTAL c D N A s
Biochemical characteristics of dbl products b
DNA transfected
Proto-dbl dbl ANpdbl z~Ndbl pZipNeo
Transforming activity a (ffu/pmol D N A ) 5 2 4 3 <1
× × × × ×
103 105 105 10 5 10 °
Localization c N
P
S
Phosphorylation
Half-life
NA
+ + + + NA
+ + + + NA
+ + + + NA
1 hr 5/6 hr 5/6 hr 5/6 hr NA
Transfection assays were carried out by titration of each cloned D N A on recipient N I H 3T3 cultures, and foci were scored from 7 to 14 days posttransfection. b The products of dbl, proto-dbl, and their respective N-terminal deletion mutants were analyzed and compared for their biochemical characteristics, as described in the text. c N, nuclei; S, soluble fraction (S-100); P, insoluble fraction (P-100); NA, not applicable.
[38]
dbl TRANSFORMATION
351
activity is about 50- to 70-fold lower than that of the dbl oncogene. The genesis of dbl involved the loss of the first 497 amino acids of proto-dbl and the acquisition of a new N terminus from another human locus. The last 428 amino acids of proto-dbl and dbl products are identical with the exception of a single conservative amino acid change (Fig. 1). Thus, any of these alterations could be responsible for the greater transforming activity of dbl. The first study we undertook to investigate how these alterations modulate proto-dbl transforming activity involved the generation of Nterminal truncated mutants from both proto-dbl and the dbl oncogene and the evaluation of their transforming activities. 9 Second, on generation of dbl-specific antibodies, we analyzed and compared the biochemical properties of dbl and proto-dbl products as well as the products of their respective N-terminal deletion mutants. 9-11 We generated three kinds of antibodies which specifically recognize the dbl and proto-dbl products. The first were derived from mice bearing dbl-induced tumors. Polyclonal rabbit sera were generated to synthetic peptides corresponding to specific residues of the predicted primary amino acid sequence of the proto-dbl gene product. Finally, we expressed the dbl product into the p G E X prokaryotic expression vector (Pharmacia) and used the SDS-PAGE-purified fusion protein to immunize rabbits.
Construction of dbl and Proto-dbl Mutants To construct the N-terminal truncated mutants of proto-dbl (designated ANpdbl) and dbl (designated ANdbl), the AccI-BamHI fragment of each parental cDNA is isolated (the BamHI site replaced the previous StyI site) (Fig. 1). Each of these fragments is then ligated to a 66-mer oligonucleotide containing a 53 bp corresponding to the nucleotide sequence between the breakpoint of the dbl oncogene (nucleotide 1665 of proto-dbl sequence 5) and the AccI site (nucleotide 1718 of proto-dbl sequence 5), an in-frame A T G to initiate translation of the truncated protein and an adaptor BamHI site 5' to the ATG.
Tumor-Bearing Mouse Antisera Two- to 6-week-old NFS mice are inoculated with 107 NIH 3T3 dbltransfected cells by intramuscular, intraperitoneal, or subcutaneous routes. Sera from tumor-bearing mice are collected 3-4 weeks later. Control sera 9 D. Ron, G. Graziani, S. A. Aaronson, and A. Eva, Oncogene4, 1067 (1989). 10S. Srivastava,R. H. P. Wheelock,S. A. Aaronson, and A. Eva, Proc.Natl.Acad. Sci. U.S.A. 83, 8868 (1986). 11G. Graziani, D. Ron, A. Eva, and S. Srivastava, Oncogene4, 823 (1989).
352
BIOLOGICALACTIVITY
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are obtained from mice injected with similar numbers of untransfected NIH 3T3 cells.
Peptide Antisera Peptide antisera are obtained by injecting rabbits with synthetic peptides corresponding to amino acids 3-16, 453-467, 592-606, and 818-831 of the predicted proto-dbl gene product. 5 Each peptide is conjugated with thyroglobulin (bovine type 1, Sigma) by suspending 24 mg of thyroglobulin with 2 mg of peptide in 2 ml of 0.1 M NaPO4, pH 7.5, and then adding 1 ml of 20 mM glutaraldehyde (80 ml of 2.5 M stock solution in 10 ml H20) dropwise with stirring. Following incubation at room temperature for 30 min, 1.5 ml of this reaction is diluted with 3.5 ml of H20, aliquoted in 10 sterile vials, and frozen at - 7 0 °. Rabbits are injected with a 0.5-ml aliquot of conjugated peptide intradermally every other week. A double injection is administered the first week. Animal are bled every other week, following the fourth injection.
Antisera against dbl Fusion Protein Glutathione S-transferase (GST)-dbl fusion protein is prepared by first introducing a BamHI site at the start codon of dbl by the polymerase chain reaction in order to fuse the dbl cDNA in-frame with the 3' end of the GST cDNA in the pGEX-2T vector (Pharmacia). After induction of expression of the dbl fusion protein with 1 mM IPTG, bacteria cells are collected, lysed in 8 M urea, and clarified by ultracentrifugation at 100,000g for 30 min at 4 °. The supernatant is boiled in 1% SDS, 1 mM 2-mercaptoethanol and is subjected to SDS/8% PAGE. The area containing the fusion protein is sliced from the gel, weighed, minced by running it through a syringe three or four times, and aliquoted in 11 equal amounts (by polyacrylamide weight). Between 200 and 300/xg of fusion protein should be contained in each aliquot. Initial injection is done with two aliquots of antigen which is mixed with an amount of Freund's complete adjuvant equal to the weight of the acrylamide, emulsified, and then injected subcutaneously into the rabbit. Subsequent injections are done by mixing the antigen with an amount of Freund's incomplete adjuvant equal to the weight of acrylamide, emulsifying, and injecting subcutaneously into shaved areas along the back of the rabbit. The first boost is made 2 weeks after the initial injection; second and subsequent injections are made at 2-week intervals. The first test bleed is obtained 1 week after the second boost, and second and subsequent test bleeds are obtained at 1-week intervals. Before starting the immunization procedure, a preimmune bleed is taken.
[381
dbl TRANSFORMATION
353
Biochemical Analysis of dbl and Proto-dbl Products The translational product of the dbl oncogene is a 66-kDa (p66) 1° protein, whereas the human dbl protooncogene encodes a translational product of 115 kDa (pl15). u pl15 and p66 are both cytoplasmic phosphoproteins present in both the cytosol and crude membrane preparations. Membrane fractionation studies revealed that the proteins are primarily associated with plasma membranes and that the membrane-associated forms of pl15 and p66 are fairly resistant to solubilization by nonionic detergents, suggesting that they associate with the cytoskeletal matrix. The half-life of proto-dbl pl15 is significantly shorter (1 hr) than that of dbl p66 (5-6 hr). When the N-terminal deletion mutants were analyzed, we found that the transforming activity of each was similar to that of the dbl oncogene (Table I). These findings suggest that the loss of the first 497 amino acids of protodbl, rather than the acquisition of a new N terminus, is crucial to the enhanced transforming activity of the dbl oncogene. Analysis of the mutant proteins indicated that they are equally distributed between the membrane and cytosolic fractions, like their parental proteins, but neither mutant was phosphorylated. Each mutant protein has a half-life of 5-6 hr. These data suggest that the N-terminal domain down-regulates transforming activity and that sequences within this region are responsible for rapid turnover of the protein.
Identification of dbl Proteins Most of the antibodies generated recognize dbl products both in immunoprecipitation and in immunoblot analyses. For immunoprecipitation, subconfluent cultures of NIH 3T3 cells are labeled with 250-500/xCi each of [35S]methionine and cysteine for 3 to 4 hr in 2 to 4 ml of Met-Cys-serumfree DMEM. Cells are lysed in 1 ml of lysis buffer [10 mM sodium phosphate, pH 7.4, 0.1 M NaC1, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 tzg/ml aprotinin, 10/xg/ml leupeptin, and 10/xg/ml pepstatin]. Lysates are clarified at 10,000g for 15 min at 4 °, immunoprecipitated with specific antibodies for 1 hr at 4 °, recovered on protein A-Sepharose beads by shaking for 30 min at 4 °, washed three times with 1 ml of lysis buffer, boiled for 5 rain in S D S - P A G E loading buffer (50 mM Tris/HC1, pH 7.5, 2% SDS, 250 mM mercaptoethanol, 10% glycerol, 0.1% bromophenol blue), and resolved by SDS/8% PAGE. For immunoblot analysis, unlabeled cell lysates are processed for immunoprecipitation as described earlier. Alternatively, a 100-/xg amount of total cell lysate proteins is subjected to S D S - P A G E and then transferred onto nitrocellulose membranes for probing with antisera. 125I-labeled protein A is used to localize immunocomplexes.
354
BIOLOgiCAL ACTIVITY
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Subcellular Fractionation
Unlabeled or metabolically labeled cells are swollen in hypotonic buffer containing 1 m M Tris/HC1, p H 7.5, 1 m M PMSF, 10/xg/ml each aprotinin, leupeptin, and pepstatin and are disrupted by 30 strokes in a tight-fitting Dounce homogenizer until 80% of the nuclei are released as observed by phase-contrast microscopy. After low-speed centrifugation (600g for 15 min) to remove nuclei and cell debris, soluble (S-100) and particulate (P-100) fractions are prepared from the resultant supernatant by centrifugation at 100,000g for 1 hr at 4 °. The low-speed pellet containing nuclei, undisrupted cells, and large cellular fragments is further centrifuged at 100,000g for 1 hr through 1.2 M sucrose in order to purify the nuclear fraction, t2 The purity of cellular fractions is monitored by the distribution of lactate dehydrogenase, 13N A D H diaphorase, 14and 5'-nucleotidase) 5 The integrity of the nuclear fraction can be established by labeling the cells with [3H]thymidine and analyzing trichloroacetic acid precipitable 3H in the various fractions.
DH Domain: Biologic a n d B i o c h e m i c a l Activity A region between residues 498-738 of proto-dbl ( D H for dbl homology) shares significant sequence similarity with the product of CDC24, a Saccharomyces cerevisiae cell division cycle protein required for correct budding and establishment of cell polarity and bcr, a gene implicated in the pathogenesis of chronic myelogenous leukemia. 16 We thus examined whether this conserved region is required for dbl-transforming activity. We made deletions within and outside the D H domain. Deletions within D H domains were targeted to either the most highly or less well conserved stretches. Deletions outside the D H domain involved internal deletions as well as truncations of up to 100 residues from the COOH-terminal region of dbl. The extent of the deletions and their localization with respect to the protodbl open reading frame are summarized in Fig. 2. Each of the deletion mutants was cloned into pZipNeoSV(X)I, and the focus-forming as well as the colony-forming activity of each construct was tested following transfection of N I H 3T3 cells. As shown in Fig. 2, every deletion within the 12A. J. Hay, Virology 60, 398 (1974). 13F. Stolzenback, Enzymology 9, 278 (1966). 14j. Arvuch and D. F. H. Wallach, Biochim. Biophys. Acta 233, 334 (1971). 15K. Radke, V. C. Carter, P. Moss, P. Dehazya, M. Schliwa, and G. S. Martin, J. Cell Biol. 97, 1601 (1983). 16D. Ron, M. Zannini, M. Lewis, R. B. Wickner, L. T. Hunt, G. Graziani, S. R. Tronick, S. A. Aaronson, and A. Eva, New Biol. 3, 372 (1991).
dbl TRANSFORMATION
[38]
355 ffu/pmol DNA % of control
Oncogenic dbl
735 l!ii!i!i!i!~.!iiii!ii!!iii!ii!i!iliiiiiii!ii!!~!i!~!~i~I
925
t,505-518
V |ii~iiiiii~i!iiiiiiiiliiii~ilHilililiiiiiii~ililit
I
<0.001
,~628-634
ti!i'i iiiiiill ~!! i ~iii ii
I
<0.001
~640-.646
V t!iiiiiiii!i!]il!ii~i~ili!iiii~ilili!i!i!~ili~i~i!i!i!il
1
<0.001
4664-670
V lililijiiiiiii]iiiiiiiiiJi~iiljQiiiiiiiiiiiii~iJi~i!i~g
]
<0.001
A676-687
|i!Jilililili!i!ii!i~!i!i!i!i!!i!ili!i!i!!:!i!i!i!i!i!l
I
<0.001
I
97
498
~,807-813
|~iiiiiiiiiliiiiiiiiiiiiii.=.iii.=.iiiiiiii!iiiiiiii!iiiiil
A876-925
Iiiiii~!~i~i~!~i~iii~i!iiiiiiiiiiii!i!ii~!~ii.?..iiiiiiiit
Substitution 640-646
lili~iiiiiii~!~!~i~i~iiiiiii~iii~iiiil
_ _ ]
v
100
I
95
IIIDRII
I
1.4
F~G. 2. Structure and transforming activity of ANpdbl mutants. Numbering of the residues deleted corresponds to the amino acids of the predicted proto-dbl protein. Each deletion (V), substitution (t_~), or truncation is schematically indicated. Each box represents the open reading frame of the mutant and the shaded area represents the DH domain. Focus-forming units, flu.
D H domain completely abolished the transforming activity of dbl whereas deletions outside the conserved region had no effect on the specific transforming efficiency of the parental molecule. We also generated a mutant in which the stretch of seven amino acids, 640-646, whose deletion abolished transforming activity, was substituted with residues of similar charge. This mutant (Fig. 2) was expressed at levels comparable to that of the wildtype dbl product, but its focus-forming activity was reduced 50- to 70-fold. These results suggest that even changes of only a few residues within the conserved region are sufficient to significantly decrease dbl-transforming activity.
Construction of Mutant and Expression Vectors The parental molecule used in these studies is the NHz-terminal truncated mutant containing residues 498-925 of proto-dbl described earlier (Fig. 1). dbl cDNA is released from the pZipNeo SV(X)I vector by BamHI digestion and is subcloned into the BamHI site of M13p19. Deletion mutagenesis is performed according to the instructions provided by the supplier with the MUTA-GENE kit (Bio-Rad). 18-mer oligonucleotide primers that flank the 5' and 3' ends of the region to be deleted
356
BIOLOGICAL ACTIVITY
[38]
are used. Each oligonucleotide (2.4 mg) is phosphorylated in 0.1 M Tris/ HC1, pH 8.0, 10 mM MgC12, 5 mM dithiothreitol, 0.4 mM ATP, and 2.5 U of T4 kinase in a total volume of 30 ml at 37 ° for 45 rain. Carboxyterminal truncations are made by inserting stop codons in the desired location. All the mutations are confirmed by nucleotide sequence analysis and then each mutant D N A is recloned into the mammalian expression vector pZIPNeoSV(X)I.
Transforming Activity and Protein Detection All the mutants generated are assayed for transforming activity by transfection of NIH 3T3 cells as described earlier. For immunodetection of the mutant proteins, two peptide antisera are used. The first one, directed against residues 592-606, is suitable for all the mutants in which the D H domain is not affected. In our hands this antiserum reacts only in immunoprecipitation so that cells have to be metabolically labeled before lysis. The second peptide antiserum is directed against amino acids 818-831 of protodbl and can be used to detect the translational product of mutants which carry deletions or substitutions within the D H domain.
Morphology of dbl-transformed Cells NIH 3T3 cells transformed by the dbl oncogene as well as by each of the mutants retaining transforming activity show a very peculiar and characteristic phenotype. Densely growing spindle-shaped fibroblast-like cells are present together with giant polynueleated cells (Fig. 3). The unusual phenotype appears to be a genetically stable property since clonally derived lines retain the complex morphology. Time-lapse cinematography 17 of dbl transformants seems to indicate that the large multinucleated cells form as a result of several cycles of nuclear division with no apparent cytokinesis (A. Eva, unpublished observation, 1986). This unique phenotype is helpful for recognizing dbl-induced loci.
Conclusion We have employed several approaches to analyze the biological and biochemical properties of dbL Each method has provided complementary data that support the following conclusions: (1) biologic activity resides within the region starting at residue 498 of proto-dbl and extending down17 K. K. Sanford, G. M. Jones, R. E. Tarone, and C. H. Fox, Exp. Cell. Res. 109, 454 (1977).
[381
dbl TRANSFORMATION
357
FIG. 3, Morphology of a transformed focus induced in NIH 3T3 cells by the dbl oncogene. Magnification: (A) x170; (B) x425.
stream for approximately 327 amino acids. (2) The 497 residues which constitute the N-terminal domain of proto-dbl and which are deleted in the oncogenic dbl can down-regulate proto-dbl transforming activity. We have shown that dbl catalyzes the dissociation of G D P from the human
358
BIOLOGICALACTIVITY
[391
CDC42 protein (CDC42Hs) and RhoA. 18'19 Interestingly, we have determined that the minimal unit on dbl that is critical to its transforming function is also the one that regulates G D P - G T P exchange activity on Cdc42Hs and R h o A J 9 Details of these experiments are described in [9] in this volume. 18M. J. Hart, A. Eva, T. Evans, S. A. Aaronson, and R. A. Cerione, Nature 354, 31l (1991). 19M. J. Hart, A. Eva, D. Zangrilli, S. A. Aaronson, T. Evans, R. A. Cerione, and Y. Zheng, Z Biol. Chem. 269, 62 (1994).
[39] I n h i b i t i o n Antisense
of Rac Function Oligonucleotides
Using
B y OLIVIER DORSEUIL, Gt~RALD LECA, AIMt~ V A Z Q U E Z ,
and G~r~ARD GACON Introduction R a c l and Rac2 proteins are small GTP-binding proteins highly related to each other (92% homologous) and belonging to the Rho family of the Ras-related proteins. 1 Biochemical studies have shown that both of these proteins could regulate, in a cell-free system, the superoxide-generating enzyme, N A D P H oxidase. Racl and Rac2 proteins have been purified from phagocyte extracts, guinea pig macrophages 2 and human neutrophils, 3 respectively, as the G T P - d e p e n d e n t factor involved in N A D P H oxidase. It has also been shown that when added to phagocyte extracts, Rac proteins stimulate the N A D P H oxidase activity. 2'3 This enzymatic activity is of crucial importance for the physiology of the phagocytic cells (monocytes, macrophages, neutrophils, eosinophils). On cell stimulation it generates the microbicidal agent superoxide anion, which is required for host defense against infection by bacteria, fungi, or certain parasites. B lymphocytes express both racl and rac2 genes, and whereas these cells are nonphagocytic, they are also able to produce superoxide anions upon cell stimulation by cross-linking of surface immunoglobulins (Ig) or
1j. Didsbury, R. F. Weber, G. M. Bokoch, T. Evans, and R. Snydennan, J. Biol. Chem. 264, 16378 (1989). 2 A. Abo, E. Pick, A. Hall, N. Totty, C. G. Teahan, and A. W. Segal, Nature 353, 668 (1991). 3U. G. Knaus, P. G. Heyworth, T. Evans, J. T. Curnutte, and G. M. Bokoch, Science 254, 1512 (1991).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
[391
INHIBITIONOF Rac USINGANTISENSE
359
phorbol ester treatment. 4-7 The NADPH oxidase involved in B ceils is similar, if not identical, to the phagocyte enzyme5'8 and we observed in B cells that surface Ig-mediated superoxide production was enhanced in the presence of guanosine 5'-(3-O-thio)triphosphate (GTPyS). Epstein-Barr virus (EBV)-transformed B lymphocytes can produce superoxide anions and constitute, as cell lines, a convenient model for study of the NADPH oxidase.4'7 Antisense Technology In order to investigate the function of Rac proteins in living cells and to confirm the observations made at the biochemical level in the cell-free systems, the ideal approach would be to inhibit specifically Rac function inside the cell and look at the cellular effects, particularly the NADPH oxidase. This can be attempted using antisense technology as a way to inhibit the expression of the rac genes, thereby affecting the biological function regulated by the Rac proteins. This strategy relies on the premise that an oligonucleotide, the antisense form of the racI and rac2 messenger RNA, is going to form a complex with its complementary sequence and thereby interfere with Rac protein production. The action of an antisense oligonucleotide is mainly due to the intracellular ribonuclease H, which recognizes the duplex R N A - D N A and then catalyzes the hydrolysis of the phosphodiester bonds in the RNA molecule. Other mechanisms seem also involved in the action of the antisense oligonucleotide; depending on the targeted sequence inside the messenger RNA, the duplex oligo-RNA can interfere with the 5' capping process, affect the binding of the ribosomal subunits, inhibit the RNA splicing, or even alter the RNA stability by affecting its superstructure. In all these cases, the consequence is the inhibition of the RNA translation and the decrease of the targeted protein content inside the cell. 9,m Therefore, in an attempt to investigate the involvement of Rac proteins during the oxidative burst of B cells, we used a Racl and Rac2 specific 4 D. J. Volkman, E. S. Buescher, J. I. Gallin, and A. S. Fauci, J. Immunol. 133, 3006 (1984). 5 F. E. Maly, A. R. Cross, O. T. Jones, G. Wolf-Vorbeck, C. Walker, C. A. Dahinden, and A. L. De Weck, 3". Immunol. 140, 2334 (1988). 6 j. T. Hancock, F. E. Maly, and O. T. Jones, Biochem. J. 262, 373 (1989). 7 G. Leca, G. Benichou, A. Bensussan, F. Mitenne, P. Galanaud, and A. Vazquez, J. ImmunoL 146, 3542 (1991). 8 L. Cohen-Tanugi, F. Morel, M. C. Pilloud-Dagher, J. M. Seigneurin, P. Franqois, M. Bost, and P. V. Vignais, Eur. J. Biochem. 2112, 649 (1991). 9 C. H61~ne and J. J. Toulm6, Biochim. Biophys. Acta 11149, 99 (1990). 10 R. P. Erikson and J. G. Izant, in "Gene Regulation: Biology of Antisense RNA and DNA." Raven Press, New York, 1992.
360
BIOLOGICALACTIVITY
[39]
antisense oligomer to down-regulate Rac proteins in EBV-transformed B cells. We show that the superoxide production by B cells in response to stimulation of cell surface receptors or after protein kinase C (PKC) activation can be clearly reduced by a Rac-specific antisense oligomer, concomitantly with a decrease in Rac protein content. This provides evidence for the physiological involvement of Rac in the NADPH oxidase. 11 Methods
Cell Culture The B cell line used is the lymphoblastoid B cell line, GL1, previously described, 7 which was obtained by infection of normal human B cells with the B95.8 (EBV-producing marmoset lymphoblastoid cell line) supernatant. GL1 cells are then selected by successive subcloning. Cells are cultured at 37° in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS).
Oligonucleotides Nonmodified oligodeoxyribonucleotides, synthesized by GENSET (Paris, France), are purified by HPLC, ether extracted, and lyophilized. A 16-mer antisense (AS) oligonucleotide was chosen as complementary to a sequence shared by racl and rac2 genes in order to affect both protein synthesis. It starts at the ATG initiation codon (5'-ACTTGATGGCCTGCAT-3'). A sense (S) oligonucleotide is also used as a control (5'-ATGCAGGCCATCAAGT-3' ). Oligonucleotides are dissolved in RPM! 1640 medium at a concentration of 4 raM.
Oligomer Treatment of Cells All the experiments have been done with a single lot of FCS, heat inactivated at 65° for 20 min, in order to reduce the nuclease activities toward the oligonucleotides. Different concentrations of FCS are used to determine the lowest concentration allowing cell growth for several days while decreasing the nuclease action. Cells at a starting density of 106/ml are cultured in RPMI 1640 medium supplemented with 1% heated FCS and, as indicated, in the presence of 10 to 40/xM (final concentration) of oligomer for 72 hr. Oligomers are added directly in the cell culture medium at 0 and 48 hr. Control cell cultures are left untreated. After 3 days, cells are washed with phosphate11 0 . Dorseuil, A. Vasquez, P. Lang, J. Bertoglio, G. Gacon, and G. Leca, J. Biol. Chem. 267~ 20540 (1992).
[391
INHIBITIONOF Rac USINGANTISENSE
361
buffered saline and processed for immunoblotting or measurement of superoxide production.
Antibodies and Immunoblotting Antibodies to Rac proteins are raised in rabbits against a Rac2 peptide corresponding to the residues 122-144 of the human Rac2 sequence. Antibodies are affinity purified on a peptide CNBr column and are shown to detect Racl and Rac2 proteins as a single 25-kDa band. For Western blot analysis, GL1 cells are lysed for 30 min on ice in 10 mM Tris, pH 7.6, 50 mM NaC1, 5 mM EDTA, 1% Nonidet P-40, 1% aprotinin, and 1 mM phenylmethylsulfonyl fluoride. The lysate is centrifuged for 30 rain at 13,000g, and 100/zg of protein from the supernatant is resolved on a 15% SDS-polyacrylamide gel and with Western blotting as previously described) 1
Oligonucleotide Effect on Protein Content To evaluate the efficiency of the AS oligonucleotide action on Rac synthesis, we determined the amount of Rac proteins in GL1 cells incubated for 72 hr with 40/xM of S or AS oligonucleotide as compared to untreated cells. This was achieved by Western blotting using the antibody recognizing both Racl and Rac2 proteins, followed by densitometric scanning of the autoradiographs. Data presented in Fig. 1 show a decrease of 60% in the level of expression of the Rac proteins in the AS-pretreated cells as compared to the S-pretreated or untreated cells. To check the specificity of the oligonucleotide-induced inhibition of Rac synthesis, the amount of the surface immunoglobulin is determined as a control and is found not to be significantly modified by the S or AS oligonucleotide treatment (96%) in comparison to the untreated cells (100%) (data not shown).
Measurements of Superoxide Production Lucigenin-dependent chemiluminescence according to the procedure described by Allen 12 is adopted as being a sensitive assay for superoxide dismutase-inhibitable superoxide production during the respiratory burst, as discussed previously.7 Assays are performed at 37°, with 106 GL1 cells in suspension in MEM chemiluminescence medium without phenol red (Boehringer-Mannheim, Indianapolis, IN). Cells, in the presence of lucigenin solution (bis-N-methylacridinium nitrate, 100 /zM), are stimulated as R. C. Allen, in "Methods in Enzymology" (M. Deluca and W. D. McElroy, eds.), Vol. 133, p. 449, Academic Press, San Diego, 1986.
362
BIOLOGICALACTIVITY
[391
A
B
•
1
1.1
0.4
,,A_,.A. A C
S
AS
FIG. 1. Inhibition of Rac protein expression by antisense oligonucleotides. The Rac protein content from extracts of GL1 cells treated with 40 tzM of antisense (AS) or sense (S) oligonucleotides as compared to untreated (C) cells was determined by immunoblotting with anti-Rac antibodies (A). Densitometric scans of Rac-specific bands are shown in (B). Numbers refer to relative units of the integrated peaks.
by phorbol 12-myristate 13-acetate (PMA) (0.1/xg/ml) or by cross-linking of the surface Ig using the Staphylococcus aureus Cowan I strain (SAC) (Pansorbin, Calbiochem, La Jolla, CA) at 1/1000 final dilution, and chemiluminescence is measured on Picolite luminometer (Packard 6100, Packard Instrument Co., Inc., Downers, Growe, IL). Superoxide anion synthesis occurs within minutes following cell stimulation and reaches a maximal rate after 30-35 min (Fig. 2). The maximal rate is taken as an index to evaluate the effect of oligonucleotide pretreatment. Cell stimulations and chemiluminescence measurements are carried out in duplicate, and the results are expressed in counts per minute as the difference between the sample and the blank (cell-free medium). Oligonucleotide Effect on Superoxide Production Under oligonucleotide pretreatment conditions in which we observed a 60% decrease of the Rac protein content, we examined whether the N A D P H oxidase activity was affected or not. Superoxide production induced by surface Ig cross-linking is strongly inhibited (60%) by Rac AS oligonucleotide pretreatment, whereas S oligonucleotide pretreatment does not induce any significant difference as compared to untreated cells (Fig.
[39]
INHIBITION OF R a c USING ANTISENSE
363
2). Similar inhibitory effects by oligonucleotide pretreatment are observed in phorbol ester-induced superoxide production (Fig. 3b). A dose-response curve using different oligonucleotide concentrations shows that superoxide production in GL1 cells stimulated by PMA (Fig. 3b) or surface Ig cross-linking (Fig. 3a) is Rac dependent and inhibitable in a dose-dependent manner by Rac AS oligonucleotides. To assess the specificity of the AS action, several other cellular functions were checked. Cell growth and immunoglobulin M production are unaffected by AS oligonucleotide pretreatment in comparison to S oligonucleotide-pretreated cells. In addition, cell viability is similar for oligonucleotide-pretreated and untreated cells. Inhibition of PMA- and SAC-induced superoxide production by Rac-specific AS oligonucleotide was reproduced in three separate experiments which gave similar results.
Summary Using a Rac antisense oligonucleotide we were able, in a whole cell system, to decrease the Rac protein content and to inhibit superoxide 14000
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Fie. 2. Inhibition of superoxide production induced by surface Ig cross-linking using Rac antisense oligonucleotides. GL1 cells, untreated or pretreated for 72 hr with 40 p,M of sense or antisense oligonucleotides, were incubated in the presence of SAC (1/1000 dilution), and lucigenin-dependent chemiluminescence was recorded for 40 min after stimulation (see methods). Results in cpm were calculated by subtracting the blank (chemiluminescence of cell-free medium) from sample values. Data are representative of two experiments. ~, control; e , sense; O, antisense.
364
BIOLOGICAL ACTIVITY
A
[39]
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[OIIgonueleotide] ~tM Ft6. 3. Effect of different concentrations of sense and antisense Rac oligonucleotides on superoxide production. GL1 cells were cultured in the presence or absence of various concentrations of sense and antisense oligonucleotides for 72 hr. Curves of chemiluminescence induced by SAC (A) and PMA (B) were recorded in each case; percentage of inhibition was determined by comparing maximal chemiluminescence of oligonucleotide-pretreated cells to that of control untreated cells. Each point represents the mean of two experiments. O, antisense; O, sense. p r o d u c t i o n . P r e v i o u s studies d o n e in cell-flee systems suggested the involvem e n t of R a c p r o t e i n s in N A D P H oxidase activation. This c h a p t e r p r o v i d e d e v i d e n c e that R a c is i m p l i c a t e d n o t only in s u p e r o x i d e p r o d u c t i o n f r o m cell-flee systems b u t also in a n intact cell system. This c h a p t e r also showed
[391
INHIBITIONOF Rac USINGANTISENSE
365
that the Rac dependency of the N A D P H oxidase exists not only in phagocytic cells but also in B lymphocytes. We observed a clear correlation between the decrease of the Rac protein content and the inhibition of superoxide production, thus demonstrating the physiological involvement of Rac proteins in the N A D P H oxidase activity of B lymphocytes.
Concluding Remarks In this study we described the use of the antisense technology as a means to study Rac protein function in B ceils. Although simple, this method necessitates some controls in order to check the specificity of the antisense oligonucleotide action. Here, we used a 16-met antisense oligonucleotide whose sequence has been selected to be unique for the r a c genes among the data banks. We also used as a control a sense oligonucleotide whose sequence did not match any known sequence in the data banks. This control oligomer is also a 16-met and its base content is very similar to the antisense oligomer, but it has no action in comparison to untreated cells. In addition, different complex cellular functions such as proliferation, IgM production, and cell viability were not affected by the antisense oligomer as compared to the sense oligomer. Altogether, this suggests that the inhibition of the N A D P H oxidase by the antisense oligomer is specific. The antisense approach has already been used numerous times 9'1° and should also be useful in the field of the small GTP-binding proteins. Although simple, this approach has its own limitations and one should be aware of some of the parameters affecting its efficiency. The uptake of the oligomer by the ceils is a limiting step and depends mainly on the pinocytosis rate. H e r e we chose to work with B cells, as they are a model with a high pinocytosis rate. t3 The pinocytosis rate varies from one cell type to another and could account for the difficulties encountered by some people attempting to use the antisense technology. Artificial ways to introduce the oligomers into cells, like osmotic shock or receptormediated endocytosis using an oligomer-polylysine-transferrin complex, have also been used with success. TM The oligomer size is important, it should be large enough to be specific for its target, but not too long, as it could by partial interaction with a nontargeted sequence induce the inhibition of an unspecific gene. Another reason to use small oligomers is that after the ribonuclease H action on the duplex m R N A - o l i g o m e r , the smaller oligomers are released and are 13R. K. Cheung and H. M. Dosch, J. Biol. Chem. 266, 8667 (1991). t4 G. Citro, D. Perroti, C. Cucco, I. D'Agnano, A. Sacchi, G. Zupi, and B. Calabretta, Proc. Natl. Acad. Sci. U.S.A. 89, 7031 (1992).
366
BIOLOGICALACTIVITY
[391
able to potentially bind to other molecules, then acting very efficiently in a catalytic way. The oligomer we chose was a 16-mer, and in most of the studies described the oligonucleotides are 15- to 20-mers. The site targeted by the oligomer is of importance too, as in addition to the ribonuclease H action the oligomer could also interfere with the mRNA-interacting factors. The ATG-starting codon area chosen for this study is a relatively commonly used area, as the oligomer may also interfere with ribosomal subunit binding. It should be mentioned that the nuclease action toward the oligomers can be minimized by heat treatment of the serum or, even better, by using the currently available nuclease-resistant modified oligonucleotides, such as the phosphorothioates, which are still recognized by ribonuclease H ) 5 The lifetime of the protein studied has to be taken into consideration as well, as the approach inhibits mRNA translation during the course of renewal of the protein. So, for stable proteins the oligonucleotide treatment will likely have to be longer in order to see any effects on protein levels. Another limitation of this approach is the potential functional redundancy of certain proteins, enabling them to complement the function of a lost protein, thus masking any detectable biological effects. In conclusion, the antisense strategy is an attractive approach that is worth trying, but, for all the previously mentioned reasons, it is still difficult to predict the success of a particular attempt. Acknowledgments This work was supported in part by the Association pour la Recherche sur le Cancer and the Foundation pour la recherche M6dicale.
is j. Goodfellow, Bioconj. Chem. 1, 165 (1990).
AUTHOR INDEX
367
Author Index
Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.
A Aaronson, S. A., 12, 69, 77-78, 80(8), 81(8), 82(9), 84(9), 85, 90-91, 93(9), 97(9), 126, 139, 285, 347-348, 351, 351(5), 352(5), 353(10), 354, 358 Abercrombie, M., 336 Abo, A., 16, 17(5), 24(5), 25-26, 33, 36, 139, 207, 213, 213(4), 215(4, 12), 227, 257, 265(4), 268-269, 358 Adams, A. E. M., 215,281,284(1), 285(1), 286 Adams, J. M., 91 Adamson, P., 4, 17, 43, 162, 164-165, 170(5), 229-230, 319 Adam-Vizi, V., 193 Adelstein, R., 313 Adra, C. N., 36, 41,105 Adrian, J., 269 Aepfelbacher, M., 226 Afar, D. E. H., 125, 127-128 Agnel, M., 115 Agwu, D. E., 246 Ahmed, S., 114, 115(2, 3, 5), 116(3-7), 118(46), 120(3), 124(7), 125(3), 133, 134(16) Akhiro, S., 174 Aktories, K., 4, 9(15), 26, 167, 174, 175(6, 11), 178(11), 184-185, 185(2, 4), 186, 186(9, 10), 187, 187(12, 17), 188, 188(10, 17), 191, 191(10), 192, 192(32), 193, 193(17, 32), 194, 194(39), 195-196, 197(10), 200, 203(10), 291, 297, 314, 315(4), 317(4), 319(4), 322, 328, 335, 337 Albrecht-Buehler, G., 338 Albright, C. F., 68, 106, 112(4), 134, 138(20) Alessandra, E., 16, 41(13), 42, 48(13) Alfa, C., 282 Algrain, M., 164 Allen, J., 91
Allen, R. C., 361 Almendral, J. M., 151 Aloug, J. E., 184(5), 185 Altman, A., 84 Altschul, S. F., 158 Amberg, D. C., 92 Amrein, P. C., 248 Ando, S., 16, 17(7, 9), 18(9), 21(7), 23(7, 9), 32-33, 41, 41(10, 11), 42, 85, 207, 213(6), 256 Anraku, Y., 285 Anthes, J. C., 246 Antoniades, H. N., 151 Araki, K., 42, 48(23), 197, 203(15), 337, 338(24) Araki, S., 16, 41, 98, 100, 337 Arlinghaus, R., 129 Arlinghaus, R. B., 128 Arpin, M., 164 Arvuch, J., 354 Asada, M., 16, 17(7, 9), 18(9), 21(7), 23(7, 9), 32-33, 41, 41(10, 11), 42, 85, 207, 213(6) Ashby, M. N., 54 Ashkenazi, A., 12, 68, 138 Aspenstr6m, P., 228 Atherton, A. J., 166, 171 Audigier, Y., 159 Aullo, P., 175, 193, 298 Ausubel, F., 141 Axel, R., 3 Axell, R., 196
B Babior, B.M., 249 Backer, J.M., 54 Baier, G., 84 Baines, M.G., 322
368
AUTHORINDEX
Ballester, R., 67 Baltimore, D., 68, 140, 147(1), 148, 219 Barbacid, M., 76, 91 Barfod, E. T., 12, 68, 138 B~irmann, M., 185, 187 Bar-Sagi, D., 77, 313, 319(3) Bartel, P. L., 228 Bartram, C. R., 84 Baserga, R., 151 Becherer, K., 231 Beeler, J. F., 91 Behrens, T., 41 Belyi, Y. F., 174 Bender, A., 84, 91, 138, 215, 216(5), 285 Benett, M. J., 298 Benichou, G., 359 Bensussan, A., 359 Bergez-Aullo, P., 297 Berkow, R. L., 268 Berns, A., 91 Bertoglio, J., 151,182, 203(30), 206, 265,320322, 326, 326(9), 338, 360 Best, A., 114, 115(3), 116(3, 7), 120(3), 124(7), 125(3) Billah, M. M., 246, 248 Birnbaum, D., 69, 347 Bishop, J. M., 164 Blam, S. B., 90 Blanchard, J. M., 152-153, 159 Blaschke, U., 185, 186(10), 188(10), 191(10), 337 Bligh, E. G., 250 Bockoch, G. M., 16 Bocock, G. M., 3 Boguski, M., 67, 85 Boguski, M. S., 77, 85 Bohl, B. P., 26, 32(3), 261 Bokoch, G. M., 17, 25, 25(21), 26, 26(2), 27, 32(3, 11), 33-34, 68-69, 91, 207, 213(5), 256-258, 261, 263(5), 265(3, 5), 266(5), 267, 269, 358 Bollag, G., 67(8), 68, 76, 209 Bonnieu, A., 152, 160 Booker, G. W., 148 Boquet, P., 33, 174, 175(5, 8), 176, 178(19), 181,181(20), 185-186, 191(11), 192-193, 193(16), 194(11, 40), 195-196, 200, 202, 203(7, 25, 29), 206, 291, 297-299, 321, 322(6), 328-329, 333(12), 335, 336(21), 337
Boschek, C. B., 193 Bost, M., 359, 360(7), 361(7) Botstein, D., 286 Bottaro, D. P., 90 Bourmeyster, N., 33, 181, 192-193, 194(40), 200, 203(29), 206, 329, 333(12), 335, 336(21), 337 Bourne, H. R., 187 Bowman, E. P., 246-247, 250(3) Bowtell, D., 148 Boyartchuk, V. L., 54 Boyhan, A., 213, 269 Boyum, A., 50 Braun, U., 174, 328, 337 Bravo, R., 151,158(7) Breathnach, R., 153 Breeden, L., 236 Brill, S., 68, 73(12), 75(12), 91, 114, 120(1), 126, 130, 132, 138(15), 139(15), 207, 208(7), 240 Broek, D., 85 Bromberg, Y., 22, 23(24), 268 Brooks, M. W., 84 Brown, A. M., 67, 67(7), 68, 226 Brown, M. L., 11 Brown, M. S., 49 Brugge, J. S., 140 Buday, L., 67, 84 Buescher, E. S., 359 Burckardt, J., 151 Burgess, W. H., 90 Burn, P., 84 Burridge, K., 312, 335 Burstein, E. S., 130 Bush, J., 131 Bustelo, X. R., 91
C Cadwallader, K., 49 Calabretta, B., 365 Campbell, I. D., 148 Campbell, M., 129 Campbell, M. L., 128 Campisi, J., 151 Canaani, E., 129 Cal~ada, F. J., 50, 54(8) Cantley, L., 244 Cantrell, D. A., 88
AUTHORINDEX Caplin, B. E., 285 Caput, D., 36, 41, 105, 156 Cardelli, J., 131 Carraway, K. L. III, 13, 103 Carter, A. N., 242 Carter, V. C., 354 Casey, P. J., 49, 175 Catalano, G., 91 Cathala, G., 159 Cepko, C. L., 349 Cerione, R., 131,138, 215-216, 216(5), 285 Cerione, R. A., 3, 11-13, 14(1, 4, 9), 16, 34, 41(13), 42, 48(13), 68-69, 77-78, 80(8), 81(8), 82(9), 84, 84(9), 85, 91, 93(9), 97(9), 98, 99(1), 101,103, 103(6), 104(1), 105(1), 130, 138-139, 285, 358 Chabot, M. C., 246 Chambard, J.-C., 154 Chan, A. M.-L., 90-91 Chant, J., 215 Chardin, P., 76, 163, 174, 175(5), 182, 185, 191(11), 194(1l), 196, 203(7), 291, 297, 321,322(6), 335, 337 Chastonay, C. D., 336 Chavrier, P., 182, 326 Chelsky, D., 50 Chen, P.-L., 231 Chen, W., 131,216 Chen, Y. T., 164 Chen, Y. U., 321 Cheung, R. K., 365 Chhatwal, G. S., 174, 185, 186(9, 10), 188(10), 191(10), 297, 328, 337 Chien, C.-T., 228 Chilton, F. H., 248 Choe, S., 298 Chuang, T. H., 26, 32(3), 34, 69, 258, 261 Church, W. R., 282, 283(7) Cicchetti, P., 68, 140, 147(1), 148, 219 Citro, G., 365 Clark, R., 3, 11, 67(7), 68, 76, 130-131,216, 226 Clark, R. A., 16, 257-258, 263(2) Clark, S. G., 3 Clarke, C., 166, 171 Clarke, S., 4, 49, 50(1), 52, 54, 59(17) Clayton, D. F., 115 Clerc, G., 142, 146(7) Coburn, J., 183 Cochran, B. H., 156
369
Cochrane, C. G., 27, 260-261 Coggeshall, K. M., 84 Cohen-Tanugi, L., 359, 360(7), 361(7) Coligan, J. E., 321 Collard, J. G., 91 Collier, R. J., 298 Collins, F., 67 Conray, L., 17, 25(21) Conroy, L., 67(7), 68, 130, 226 Conti, M. A., 313 Cooke, F. T., 246 Cooper, G. M., 10, 319, 347 Cooper, J. A., 228, 240(3) Corcoran, L. M., 176, 178(22) Corrado, K., 215 Coughlin, S. R., 311 Courtneidge, S. A., 68, 246 Coutavas, E., 3 Crescenzi, M., 90 Crews, C. M., 151 Crosier, W. J., 130 Cross, A. R., 269, 271(12), 359 Cucco, C., 365 Curmi, P. M. G., 298 Curnutte, J. T., 16-17, 25(21), 26-27, 32(11), 33, 207, 213(5), 257-258, 265(3), 268269, 358 "Current Protocols in Molecular Biology," 251,252(13), 253(13)
D D'Agnano, I., 365 Dahinden, C. A., 359 Dani, C., 153, 159 Daniel, L. W., 246 Das, P., 148 Davis, R. W., 227 Dehazya, P., 354 Deikmann, D., 164 de Klein, A., 84 Della-Bianca, V., 213 Deschenes, R. J., 287 D'Eustachio, P., 3 De Vos, A. M., 3 De Weck, A. L., 359 Dhand, R., 68, 148, 246 Diamond, M., 347 Didsbury, J., 3, 16, 25, 33, 41(11), 42, 358
370
AUTHOR INDEX
Didsbury, J. R., 33,174-175,175(11), 178(11), 186, 187(17), 188(17), 193(17), 196, 297, 328 Diekmann, D., 68, 91, 114, 120(1), 126, 130, 207, 213, 215(12), 226, 307, 311(5), 314, 319(6), 320(6), 321 Dignard, D., 216, 227(12) Dillon, S., 181,242, 244(9), 245(9) Dillon, S. T., 174, 182, 243 Dinauer, M. C., 258, 260(6) Dipasquale, A., 336 Dixon, J. E., 117 Doll, T., 164 Donelson, J. E., 258 Dong, J.-M., 114, 115(9), 116(9), 132, 134(14), 135(14) Doreuil, O., 203(30), 206 Dorland, R. B., 298 Dorseuil, O., 26, 151, 265, 267, 321, 326(9), 338, 358, 360 Dosch, H. M., 365 Downes, C. P., 242, 244 Downward, J., 67, 88, 212, 216 Drivas, G. T., 3 Drubin, D. G., 77, 286 Durfee, T., 231 Dusi, S., 213 Dustin, M. L., 291 Dutartre, H., 182, 326 Dyer, W. J., 250
E Eberle, M., 242 Eccleston, J. F., 67 Eckel, S., 248 Edlund, L., 347 Egan, R. W., 246, 248 Egan, S. E., 84 Eidels, L., 298 Eisenberg, D., 298 Eklund, M. W., 175-176, 178(19), 181(20), 186, 193, 299 Elledge, S. J., 231 Ellis, C., 68, 105, 113, 139, 317 Ellis, J. M., 248 El Sabrouty, S., 153, 159 Engel, S., 33, 39(10) Engelhard, V. EI., 320 Enomoto, T., 328
Erdman, R. A., 4 Erikson, R. L., 151 Erikson, R. P., 359, 365(10) Errada, P. R., 54 Eva, A., 12, 14(9), 69, 77-78, 80(8), 81(8), 82(9), 84, 84(9), 85, 90-91, 91(4), 93(4, 9), 97(9), 98, 99(1), 104(1), 105(1), 126, 139, 285, 347-349, 351, 351(5), 352(5), 353(10, 11), 354, 358 Evan, G. I., 164 Evans, T., 3-4, 11-12, 14(1, 4, 9), 16, 25-26, 26(2), 33-34, 41(13), 42, 48(13), 68-69, 77-78, 80(8), 81(8), 82(9), 84(9), 85, 91, 93(9), 97(9), 98, 99(1), 101, 103(6), 104(1), 105(1), 130-131, 138-139, 207, 213(5), 215-216, 257, 263(5), 265(3, 5), 266(5), 269, 285, 358 Everhart, L. P., 153
F Falnes, P. O., 298 Fantes, P., 282 Farnsworth, C. C., 4, 285 Farnsworth, C. L., 84, 91, 94(12) Fasano, O., 347 Fath, K., 312, 335 Fauci, A. S., 359 Feig, L., 181,242, 244(9), 245(9) Feig, L. A., 10, 84, 91, 94(12), 174, 243, 319 Feramisco, J. R., 313, 319(3) Ferguson, K. M., 27 Fernandez, A., 313 Fernley, R., 148 Field, S., 228 Fields, S., 228 Figdor, C. G., 291 Finegold, A. A., 285 Fink, G. R., 282 Fiorentini, C., 195 FitzGerald, D., 184, 185(1) Fleming, T., 84 Fleming, T. P., 69, 90-91, 91(4), 93(4) Fogh, J., 347 Fort, P., 3, 151-152, 152(14), 153, 154(14), 158, 158(14), 159-160 Foster, L. C., 68, 105-106, 109(3), 112(4), 113(3), 134, 138(20) Foster, R., 105, 110 Fox, C. H., 356
AUTHOR INDEX Fox, J. E. B., 241-242, 244(8) Franqois, P., 359, 360(7), 361(7) Franek, K., 131 Franke, W. W., 336 Franzusoff, A., 286 Fraser, E. D., 11 Frech, M., 76, 131 Freer, J. H., 184(5), 185 Fritsch, E. F., 152, 157(18), 229 Fritz, G., 195,200 Fry, M. J., 68, 148, 242, 244(8), 246 Fujii, G., 298 Fujii, H., 18 Fujimoto, H., 196(14), 197, 198(14), 200(14), 203(14), 337 Fujioka, H., 16, 18(11), 34, 41, 43, 69, 85, 100, 337 Fujisawa, K., 197, 201(19), 203(19), 291,322 Fujita, T., 337 Fujiwara, M., 174, 186, 196-197,199, 199(16), 200(16), 201(I), 206(9), 324, 337 Fujiwara, N., 297, 328, 335(6) Fukata, J., 175, 196(13), 197, 203(13), 291, 292(9), 293(9), 295(9), 296(9), 322, 338 Fukumoto, Y., 16, 18(11), 41, 100, 337 Fung, B. K. K., 4
G Gabbiani, G., 336 Gacon, G., 26, 151, 203(30), 206, 265, 321, 326(9), 338, 358, 360 Gadba, R., 22, 23(24) Gagnon, J., 33, 181, 192, 200, 335, 336(21) Galanaud, P., 359 Galanti, N., 151 Gale, N. W., 77 Galland, F., 69 Gallin, J. I., 16, 359 Garg, R., 309 Garin, J., 33, 181, 192, 200, 335, 336(21) Garrels, J. I., 181 Garrett, M. C., 18 Garrett, M. D., 4, 9(15), 68, 91, 114, 120(1), 126, 130, 167, 174, 175(6), 185, 187(12), 196, 197(10), 203(10), 291, 314, 315(4), 317(4), 319, 319(4), 322, 335, 337 Garsky, V. M., 49 Geipel, U., 185
371
Gelb, M. H., 4, 285 George, J. M., 115 Germain, R. N., 88 Gesbert, F., 182, 326 Gherardi, E., 336 Gibbs, J. B., 49 Gibson, T., 83, 91, 140 Giddings, B. W., 84 Gietz, D., 233, 234(9) Gifford, P., 287 Gill, D. M., 174-175,175(5, 8), 176, 178(19), 181(20), 183, 185-186, 191(11), 193, 193(16), 194(11), 196, 202, 203(7, 25), 291,297, 299, 321,322(6), 328, 335, 337 Gilman, A. G., 27 Giry, M., 175, 193, 195, 297-298 Gish, W., 158 Glass, D. B., 313 Glass, G. A., 249 Glomset, J. A., 4, 226, 285 Goldman, R. D., 338 Goldstein, J. L., 49 Goodfellow, J., 366 Goody, R. S., 131 Gorvel, J. P., 326 Gorzalczany, Y., 33, 39(10) Goto, Y., 174, 187 Goud, B., 175 Gout, I., 68, 148, 246 Grabstein, K., 321 Graessmann, A., 166, 167(10), 313, 342 Graessmann, M., 166, 167(10), 313,342 Graham, F. L., 349 Grasveld, G., 84 Graves, J. D., 88 Graziani, G., 69, 77, 91, 126, 285, 351, 353(11), 354 Greenberg, J. R., 160 Greenberg, M. E., 160 Griffin-Shea, R., 115 Groffen, J., 68, 84, 91,129, 138 Grogan, A., 25, 33 Grussenmeyer, T., 86 Grzeskowiak, M., 213 Guan, K., 117 Guarente, L., 287 Guillemot, J.-C., 36, 41,105 Guizani, L., 203(30), 206, 321,326(9), 338 Gulbins, E., 84 Guthrie, C., 282
372
AUTHORINDEX
Gutterman, J., 125 Guy, P. M., 13 H
Haarer, B. K., 286 Habermann, B., 174, 191-192, 192(32), 193(32), 194, 328, 337 Habermann, E., 185 Habets, G. G. M., 91 Halenbeck, R., 67 Hall, A., 3-4, 9(15), 12, 16-17, 17(5), 24(5), 33-34, 35(14), 43, 67-69, 70(2), 73(12), 75(12), 77, 91,101,103(6), 106, 113-114, 120(1), 126, 130, 132, 138(15), 139, 139(15), 164-165, 167, 170(5), 174, 175(6), 181, 185, 187(12), 196, 197(10), 203(10, 11), 207, 208(7), 209, 212-213, 213(4), 215(4, 12), 226-227, 229-230, 238, 240-242, 244, 244(9), 245(9), 257, 265(4), 269, 291, 307, 309(4), 311(4, 5), 314, 315(4, 8), 317, 317(4, 5), 318(5), 319, 319(4-6, 8), 320(5, 6, 10), 321-322, 328, 335-337, 358 Hall, C., 68, 91, 114, 115(2, 9), 116(6, 9), 118(6), 120(1), 126, 130, 132, 132(7), 133, 134(14, 16), 135(14), 216 Hamaguchi, Y., 196(14), 197, 198(14), 200(14), 203(14), 337 Hamers, M. N., 260 Hancock, J. F., 34, 35(14), 49, 69, 85 Hancock, J. T., 359 Hanks, S. K., 126 Hansen, P. F., 84 Harcus, D., 216, 227(12) Harden, N., 114, 115(5), 116(5), 118(5) Harlow, E., 141 Harper, A. M., 269, 271(12) Hart, M. J., 3, 11-12, 14(1, 4, 9), 16, 34, 41(13), 42, 48(13), 68-69, 77-78, 80(8), 81(8), 82(9), 84(9), 85, 91, 93(9), 97(9), 98, 99(1), 101, 103(6), 104(1), 105(1), 130131, 138-139, 215-216, 258, 285, 358 Hartwell, L. H., 227 Harvey, R., 91 Hasegawa, T., 306 Hashimoto, K., 174, 187, 328 Hata, Y., 98 Hauschka, P. V., 153 Hauser, D., 176, 181(20), 193, 299
Havlik, M. H., 126, 127(4, 5), 128(4, 5) Hawkins, P. T., 246 Hay, A. J., 354 Heaysman, J. E. M., 336 Hegenbarth, S., 195 Heimer, G. V., 168 Heisterkamp, N., 68, 84, 91, 129, 138 Helene, C., 359, 365(9) Hellwig, A., 193, 194(39), 297 Hengeveld, T., 203(28), 206 Henzel, W., 12, 14(9), 16, 41(13), 42, 48(13), 69, 77, 98, 99(1), 104(1), 105(1) Herschman, H. R., 151 Herskowitz, L, 215 Heyworth, P. G., 16-17, 25(21), 26-27, 32(11), 33, 207, 213(5), 257-258, 265(3), 269, 358 Hieter, P., 231 Higashijima, T., 27 Hiles, I., 68, 246 Hiles, I. D., 148 Hinsch, E., 194 Hinsch, K.-D., 194 Hiraoka, K., 16, 17(7, 9), 18(9), 21(7), 23(7, 9), 32-33, 41, 41(10, 11), 42, 85, 207, 213(6) Hirata, K., 42, 181, 191,203(31), 206, 337 Hirata, M., 175, 196(13), 197, 203(13), 291, 292(9), 293(9), 295(9), 296(9), 322, 338 Hiroi, T., 203(27), 205 Hiroyoshi, H., 16 Hiroyoshi, M., 85 Ho, J., 114, 115(3), 116(3), 120(3), 125(3) Holeomb, C., 164 Hollenberg, S. M., 228, 240(3) Holmes, K. C., 3 Hong, Y.-M., 187, 328 Hopp, T. P., 92 Hori, Y., 16, 18(11), 34, 41, 43, 69, 100, 337 Horii, Y., 91 Horiuchi, H., 4, 17, 43 Hoshijima, M., 196, 200(6) Houston, H., 91 How, B.-E., 114, 114(10), 115, 115(8), 133134, 134(17), 137(17), 138(17) Howald, W. N., 4 Hryeyna, C. A., 54 Hsuan, J., 68, 91, 114, 120(1), 126, 130, 148, 246 Hsuan, J. J., 271(15), 276 Hu, P., 91
AUTHOR INDEX Huang, R., 242 Hung, D. T., 311 Hunt, L. T., 69, 77, 91, 126, 285, 354 Hunter, T., 126, 151,152(13) Hutchinson, M.A., 86 Hyams, J., 282 Hynes, R.O.,321
Ikai, K., 203(26), 204(26), 205 Ikeda, K., 47 Imamura, S., 203(26), 204(26), 205 Imura, H., 175, 196(13), 197,199(16),200(16), 203(13), 291, 292(9), 293(9), 295(9), 296(9), 322, 338 Inge, K. L., 17, 25(22) Innis, M.A., 3, 11, 131,216 Inoue, S., 174, 187, 328 Isomura, M., 34, 42-43, 69, 98 Issandou, M., 312 Itoh, T., 42, 48(20), 203(33), 206, 337 Iwasaki, K., 338, 342(34) Izant, J.G., 359, 365(10)
3 Jackson, C. L., 227 Jackson, P., 140 Jackson, T.R., 246 Jacobs, C.W., 285 Jahner, D., 151,152(13) Jaken, S., 242, 244(8) Jakobs, K.H., 185 Jalink, K., 203(28), 206 Jancarik, J., 3 Jeanteur, P., 3, 151-152, 152(14), 153, 154(14), 158, 158(14), 159-160 Jesaitis, A.J.,25,26(2),27,33,257-258,260, 260(6, 11), 261, 261(11), 263(5), 265(5), 266(5) John, J.,3, 76 Johnson, C.L.,307,311(5),314,319(6),320(6) Johnson, D.I., 11, 14(4), 131,215-216, 281283,283(7,8),284(1),285,285(1,11),286 Johnson, D.J., 3 Johnson, J. S., 175 JohnsomK. S.,4,6(16),86,92,93(23),116,128 Johnson, M, 54, 56(19) Johnson, R.A., 244
373
Johnston, C. L., 164, 207, 213, 215(12), 226, 321 Johnston, M., 283 Jones, E.W., 286 Jones, G.M.,356 Jones, O. T., 359 Jones, O. T.G., 269, 271(12) Jones, T. A., 114, 115(9), 116(9), 132, 134(14), 135(14) Jouan, A., 193, 194(40), 203(29), 206, 329, 333(12), 337 Judd, S.R., 285 Jung, M.,188 Just, I., 4, 9(15), 26, 167, 174, 175(6, 11), 178(11), 184-185, 185(2), 186-187, 187(12, 17), 188, 188(17), 191-192, 192(32), 193, 193(17, 32), 194, 194(39), 195-196,197(10),203(10),291,297,314, 315(4),317(4),319(4),322,328,335,337
K Kaartinen, V., 68, 91 Kabsch, W., 3 Kahan, C., 152, 153(16) Kahn, C. R., 152 Kaibuchi, K., 16, 17(7, 9), 18(9, 11), 21(7), 23(7, 9), 32-34, 41, 41(10, 11), 42-43, 69, 85, 98,100,203(31), 206-207,213(6), 226, 256, 336-337 Kamata, Y., 196-197, 203(27), 205, 206(9) Kantardjieff, K. A., 298 Kaplan, S., 77 Kasmi, F., 68, 113, 139, 317 Katada, T., 192, 200 Katayama, M., 4, 17, 34, 42-43, 69, 181, 191 Katkin, J. P., 269 Kato, K., 16 Kato, M., 25, 41(12), 42, 47(18), 48(22, 23), 197, 203(15), 337, 338(23, 24), 342(23), 343(23), 346(23) Katzav, S., 69, 84, 91 Kawamura, M., 16, 43, 85 Kawata, K., 17 Kawata, M., 4, 43, 256, 336 Keeler, M., 244 Keller, T., 244 Kelly, T., 312, 335 Kemenade, P. W., 291 Khagad, M., 36, 41,105
374
AUTHOR INDEX
Kikuchi, A., 16, 18(15), 34, 41-43, 48(19, 21), 69, 98-100, 105, 174, 181, 187, 191, 196, 200(6), 203(31, 32), 206, 256, 336-337, 338(22), 341(22) Kilburn, A. E., 231 Kim, S., 3 King, W. G., 181,242, 244(9), 245(9) Kinkade, J. M., Jr., 247 Kinsella, B. T., 4, 27, 32(11), 258, 269 Kishi, K., 16, 18(11), 42, 48(20), 203(33), 206, 337 Kishida, S., 226 Klar, A., 284 Knaus, U. G., 16-17, 25, 25(21), 26-27, 32(11), 33-34, 69, 207, 213(5), 257-258, 265(3), 269, 358 Knight, D. E., 193 Koch, G., 188, 192-193 Kocks, C., 175, 193, 298 Koland, J. G., 3, 11, 14(4), 103, 131,216 Kondo, J., 196, 200(6) Koshland, D. E., 50, 52 Kotani, K., 42, 48(21), 181, 191,203(32), 206, 256, 337, 338(22), 341(22) Kozaki, S., 175,186,196-197,201(18), 203(12, 26, 27), 204(26), 205,206(9), 241,291,337 Kozma, R., 114, 115(2, 3, 5), 116(3, 5-7), 118(5, 6), 120(3), 124(7), 125(3), 133, 134(16) Kreck, M. L., 17, 25(22) Krengal, U., 3 Kroizman, T., 36 Kruisbeek, A. M., 321 Kuang, W., 68 Kuang, W.-J., 12, 138 Kucera, G. L., 242 Kuijpers, T. W., 291 Kumagai, N., 175, 196-197, 201(19), 203(12, 19), 241,291,322, 337 Kunkel, T., 282 Kupfer, A., 320-321 Kuroda, S., 42, 48(19-22), 181, 191,203(3133), 206, 256, 337, 338(22, 23), 341(22), 342(23), 343(23), 346(23) Kurzrock, R., 125 Kuver, R., 27 Kwee, C., 54, 56(19) Kwong, C., 213 Kwong, C. H., 16, 17(8), 24(8), 32-33, 269
L Laemmli, U. K., 180, 189, 243 Lai, C. C., 85 Lamb, N. J. C., 313 Lambeth, J. D., 17, 25(22), 246-247, 250(3) Lancaster, A. C., 240 Lancaster, C. A., 68, 73(12), 75(12), 132, 138(15), 139(15), 207, 208(7) Lancaster, R. M., 338 Landau, N. R., 125, 126(2) Lane, D., 141 Lane, P., 54, 56(19) Lang, P., 182, 203(30), 206, 265,320-321,326, 326(9), 327, 338, 360 Langlet, C., 324 Lapetina, E. G., 265 Lau, L. F., 151 Leberer, E., 216, 227(12) Le Bowitz, J. H., 142, 146(7) Leca, G., 26, 265, 358-360 Ledbetter, J. A., 91 Lee, J., 52, 53(13), 114, 115(3, 5), 116(3, 5, 7), 118(5), 120(3), 124(7), 125(3) Lee, W.-H., 231 Leevers, S. J., 151 Le Gallic, L., 158 Lehto, V.-P., 140 Leidal, K. G., 257, 263(2) Lelias, J.-M., 41,105 Lelias, J. M., 36 Lemichez, E., 297 Leonard, D., 11-12, 14(9), 16, 34, 41(13), 42, 48(13), 69, 77, 105, 139 Leonard, D. A., 98, 99(1), 104(1), 105(1) Leppla, S. H., 298 Letcher, R., 67 Leto, T., 213 Leto, T. L., 16, 17(8), 18, 24(8), 32-33, 269 Leung, T., 114, 114(10), 115, 115(2, 8), 130, 132(7), 133-134, 134(16, 17), 137, 137(17), 138, 138(17), 139, 139(21), 215216, 217(11, 15), 219(11), 220(15), 221(11, 15), 225(11) Levine, R. A., 151 Levinson, A. D., 3 Lewis, G. K., 164 Lewis, M., 69, 77, 91, 126, 285, 354 Li, W., 91, 128
AUT~ORINDEX
Liao, L., 242, 244(8) Lim, B., 36, 41, 105 Lim, H.-H., 114, 115(2, 9), 116(6, 9), 118(6), 131-133, 134(14, 16), 135(14), 216 Lira, L., 68, 91, 114, 114(10), 115, 115(2, 3, 5, 8, 9), 116(3, 6, 7, 9), 118(5, 6), 120(3), 124(7), 125(3), 126, 130-132, 132(7), 133-134, 134(14, 16, 17), 135(14), 137, 137(17), 138, 138(17), 139, 139(21), 215216, 217(11, 15), 219(11), 220(15), 221(11, 15), 225(11) Lints, T., 91 Lipman, D. J., 158 Liri, T., 200 Lisanti, M. P., 58 Liscovitch, M., 250 Littman, D. R., 125, 126(2) Liu, J., 129 Liu, T., 187 Liu, T.-Y., 187 Lloyd, C. W., 103 Loetterle, L. R., 25, 26(2), 33, 257, 263(5), 265(5), 266(5) Lomax, K. J., 16 Long, J. E., 69, 84, 90, 91(4), 93(4) Longnecker, R. M., 215, 281,284(1), 285(1) Louvard, D., 164 Lowe, P. N., 3 Lowenstein, E. J., 77 Lowy, D. R., 105 Lu, D., 129 Lu, K.. 151 Lutter, R., 260
M Mabuchi, I., 196(14), 197, 198(14), 200(14), 203(14), 337 Macara, I. G., 130, 335 MacDonald-Bravo, H., 151 Madaule, P., 3,163,174-175,175(5), 182,185, 191(11), 194(11), 196, 203(7), 291, 297, 321,322(6), 335,337 Madshus, I. H., 298 Maeda, A., 41 Maehama, T., 192 Magun, B. E., 153 Major, G. N., 68, 130
375
Malech, H., 213 Malech, H. L., 16, 17(8), 24(8), 32-33,269 Maltese, W. A., 4 Maly, F. E., 359 Manclark, C. R., 187 Mandell, G. L., 320 Maniatis, T., 152, 157(18), 229 Manser, E., 114, 114(10), 115, 115(8, 9), 116(9), 130,132, 132(7), 133-134,134(14, 17), 135(14), 137, 137(17), 138, 138(17), 139, 139(21), 215-216, 217(11, 15), 219(11), 220(15), 221(11, 15), 225(11) Marchuk, D., 67 Mareel, M., 336 Margolis, B., 91 Margulies, D. H., 321 Markert, M., 249 Marshall, C. J,, 4, 17, 43, 49, 68, 113, 139, 151, 165,229, 317 Marshall, M. S., 49, 285 Marshall, T. K., 287 Martin, G. A., 67(7), 68, 226 Martin, G. S., 354 Marty, L., 153, 158-159 Maru, Y., 34, 69, 91, 126, 127(3, 4), 128, 128(4), 139 Marui, N., 203(26), 204(26), 205 Masuda, T., 41-42 Matias, P. M., 3 Matrisian, L. M., 153 Matsuda, 1., 16, 17(7, 9), 18(9), 21(7), 23(7, 9), 32, 41, 41(10), 42, 85,207, 213(6) Matsui, Y., 131,215 Matsumoto, K., 174, 187, 328 Matsumur, K., 203(27), 205 Matsumura, Y., 197, 203(15) Matsuo, Y., 328 Matsuura, Y., 4, 16-17, 17(9), 18(9), 23(9), 41(10), 42-43, 48(23), 85, 203(31), 206, 337, 338(24) Matus, A., 164 Maundrell, K., 284 Mayer, B. J., 68, 140, 147(1), 148, 219 Mayer, M. L., 285 Mayo, L. A., 26 McBride, O. W., 349 McCaffrey, M., 175 McCall, C. E., 246 McCleary, W. R., 54
376
AUTHOR INDEX
McCormick, F., 3, 11, 16, 17(9), 18(9), 23(9), 41(10), 42, 67, 67(7, 8), 68, 76, 76(4), 77, 85, 105, 130-131,209, 216, 226 McCullough, J., 281 McGovern, E. S., 91 McGrath, J. P., 3 McLeod, M., 282 McPhail, L. C., 246 Meichsner, M., 164 Menard, L., 16, 33, 41(11), 42, 174-175, 175(11), 178(11), 186, 187(17), 188(17), 193(17), 196, 297, 328 Mercola, M., 156 Metherall, J. E., 298 Meyer, B. E., 140 Mhr, C., 196 Michael, G. J., 114, 115(9), 116(9), 132, 134(14), 135(14) Middlebrook, J. L., 298 Miki, T., 69, 84, 90-91, 91(4), 93(4) Milburn, M. V., 3 Miller, J., 88 Miller, J. H., 236 Miller, P. J., 215, 281-282, 283(8) Miller, R. A., 54, 56, 57(21), 58(21) Miller, W., 158 Mishima, M., 196(14), 197, 198(14), 200(14), 203(14), 337 Mitchell, D. A., 287 Mitenne, F., 359 Mitra, K., 3 Miura, Y., 34, 42-43, 48(19), 337 Miyamoto, S., 285 Miyazaki, M., 226 Mizoguchi, A., 47 Mizoguchi, H., 47 Mizuno, T., 15-16, 17(7, 9), 18(9, 11), 21(7), 23(7, 9), 32-33, 41, 41(10, 11), 42-43, 85, 207, 213(6), 256 Mohr, C., 174, 175(11), 178(11), 186, 187(17), 188, 188(17), 191-192, 192(32), 193(17, 32), 297, 328 Molloy, C. J., 90 Monaghan, P., 166, 171 Monfries, C., 68, 114, 115(2, 5), 116(5-7), 118(5, 6), 120(1), 124(7), 126, 130, 132(7), 133, 134(16), 216 Monfries, D., 91 Montecucco, C., 298 Moolenaar, W. H., 203(28), 206
Moore, H. P., 164 Moores, S. L., 49 Moran, M., 105 Morel, F., 359, 360(7), 361(7) Moreno, S., 284 Morgan, D. L., 338 Morgan, S. J., 68, 246 Morii, N., 175, 196, 196(13, 14), 197, 198(14), 199, 199(16), 200, 200(14, 16), 201(19), 203(12-14, 19, 26, 28), 204(26), 205-206, 206(9), 241, 291, 292(9, 11), 293(9), 295(9), 296(9), 322, 327, 337-338 Moriishi, K., 187 Morishi, K., 174 Morris, C., 138 Moser, D. R., 258 Moskaug, J. O., 298 Moss, J., 181, 184, 185(3), 186-187, 191(18), 192(18) Moss, P., 354 Mosser, S. D., 49 Mulholland, J., 286 Mullen, M. L., 258 Muller, A. J., 125-126, 126(2), 127(4, 5), 128(4, 5) Miiller, H., 193, 194(39), 297 Mtiller, R., 152 Mulligan, R. C., 349 Mullmann, T. J., 248 Munemitsu, S., 3, 11,130-131,216 Murgia, M., 298 Murooka, Y., 187 Murphy, V., 3 Musaechio, A., 83, 91, 140 Musha, T., 16, 17(7), 21(7), 23(7), 32-33, 41, 41(11), 42, 48(19), 207, 213(6), 337 Myers, A. M., 175 Myers, E. W., 158
N Naemura, J. R., 27, 260 Nagata, K.-I., 200 Naglich, J. G., 298 Nakamura, T., 42, 48(22), 337, 338(23), 342(23), 343(23), 346(23) Nakanishi, H., 15 Nakao, K., 197, 199(16), 200(16) Namba, T., 175, 186, 197, 200, 201(18)
AUTHOR INDEX Narasimhan, V., 3, 11, 14(4), 68, 105, 109(3), 113(3), 131,216 Narumiya, S., 174-175,186, 196, 196(13, 14), 197, 198(14), 199, 199(16), 200, 200(14, 16), 201(1, 18, 19), 203(12-14, 19, 26, 28), 204(26), 205-206, 206(9), 241,290-291, 292(9, 11), 293(9), 295(9), 296(9), 297, 322, 324, 327-328, 335(6), 337-338 Nasmyth, K., 236 Nathans, D., 151 Nauseef, W. M., 16, 257-258, 263(2) Neal, S. E., 67 Neel, B. G., 84, 91, 94(12) Nelson, R. D., 331,332(14) Nemoto, Y., 175, 186, 197, 200, 201(18, 19), 203(19), 291,322 Nevins, B., 33 Nishiki, T., 196-197, 203(27), 205,206(9) Nishimura, S., 3 Nishiyarna, Y., 16, 17(9), 18(9), 23(9), 41(10, 12), 42, 48(22), 85, 197, 203(15), 337, 338(24) Nobes, C., 319, 320(10) Noguchi, S., 3 Nonaka, H., 41 Norgauer, J., 193 Norgauer, T., 242 Northup, J. K., 11 Nose, A., 338, 342(34, 35) Nozawa, Y., 200 Nuckolls, G., 312, 335 Nunoi, H., 16, 17(7, 9), 18, 18(9), 21(7), 23(7, 9), 32, 41, 41(10), 42, 85, 207, 213(6) Nurse, P., 284
O O'Brien, J. M., 282, 283(7), 285-286 O'Flaherty, J. T., 248 O'Hara, M. B., 49 Ohashi, Y., 197, 199, 199(16), 200(16) Ohga, N., 16, 18(15), 41-42, 69, 98, 105,337 Ohgai, H., 174, 187, 328 Ohishi, I., 185 Ohnishi, Y., 197 Ohno, K., 199 Ohoka, Y., 192 Ohsumi, Y., 285 Ohtsuka, E., 3 Ohtsuka, T., 192, 200
377
Ohya, Y., 285 Okabe, K., 67 Okumura, H., 174, 187 Oliver, J. M., 91 Olsnes, S., 175, 193, 298 Olson, M. F., 228 Olson, S. C., 246, 250(3) Orkin, S. H., 258, 260(6) Otsu, M., 68, 246 Ouellette, L. A., 282, 283(7)
P Page, M. J., 3 Pai, E. F., 3 Paik, S.-Y., 187 Painter, R. G., 27, 260 Panayotou, G., 68, 148, 246 Papini, E., 298 Pappenheimer, A. M., Jr., 298 Pardee, A. B., 151 Parker, P., 246 Parker, P. J., 68 Parkos, C. A., 258, 260(6, 11), 261,261(11) Pastan, I., 184, 185(1) Paterson, H., 162 Paterson, H. F., 4, 9(15), 68, 113, 139, 164, 167, 170(5), 174, 175(6), 185, 187(12), 196, 197(10), 203(10), 207,212,226, 230, 291,307, 311(5), 314, 315(4), 317,317(4), 319, 319(4, 6), 320(6), 321-322, 335, 337 Patterson, M. S., 322 Pawson, T., 105, 126, 127(5), 128(5), 148 Pearson, 158 Pegrum, S. M., 336 Pelech, S. L., 151 Pember, S. O., 247 Pendergast, A. M., 125-126, 126(2), 127(4, 5), 128(4, 5) Perera, J., 151 P~rez-Sala, D., 50, 54(8) Perroti, D., 365 Peters, K. L., 128 Pfisterer, S., 194 Philips, M. R., 49-50, 53, 54(10), 56(9, 10), 59(10), 267 Pick, E., 16, 17(5), 22, 23(24), 24(5), 33, 36, 39(10), 139, 207, 213(4), 215(4), 227,257, 265(4), 268-269, 274, 276, 358 Piechaczyk, M., 152-153, 159
378
AUTHOR INDEX
Pillinger, M. H., 49-50, 53, 54(10), 56(9, 10), 59(i0), 267 Pilloud-Dagher, M. C., 359, 360(7), 361(7) Piwnica-Worms, H., 78 Pizon, V., 76 Platko, J. V., 12, 14(9), 16, 41(13), 42, 48(13), 69, 77, 98, 99(1), 104(1), 105(1) Podack, E. R., 320 Poenie, M., 54 Polakis, P., 3, 11, 16, 17(9), 18(9), 23(9), 41(10), 42, 67, 67(7), 68, 85, 105,130-131, 216, 226 Polakis, P. G., 11, 14(1), 33 Pollard, T. D., 176 Pompliano, D. L., 49 Pondel, M., 125, 126(2) Popescu, N. C., 349 Popoff, M., 175, 178(19) Popoff, M. R., 174-175, 175(5, 8), 176, 181(20), 185-186, 191(11), 193, 193(16), 194(11), 195-196, 202, 203(7, 25), 291, 297-299, 321,322(6), 328, 335, 337 Porfiri, E., 85 Posada, J., 281 Pouyss6gur, J., 152, 153(16), 154 Powell, A. J., 103 Powers, S., 85 Prescott, D. M., 153 Presek, P., 193 Preston, R. A., 286 Preuss, D., 286 Prickett, K. S., 92 Pringle, J. R., 11, 91, 215, 281, 284, 284(1), 285, 285(1), 286 Prosorovskii, S. V., 174 Pross, H. F., 322 Puil, L., 126, 127(5), 128(5)
Q Qiang Guo, J., 129 Quie, P. G., 331,332(14) Quinn, A. M., 126 Quinn, M. T., 25, 26(2), 33, 256-258, 260(6, 11), 261(11), 263(5), 265(5), 266(5)
R Racker, E., 61 Radke, K., 354
Ramer, S. W., 227 Ramsay, G., 164 Rando, R. R., 50, 54(8) Rands, E., 49 Rankin, S., 312 Rao, A., 54 Rao, C. D., 78, 347 Rappuoli, R., 298 Rautman, G., 153 Rayter, S., 88 Rech, J., 152, 160 Reddy, D., 164 Rees, D. A., 103 Regazzi, R., 99, 181 Reibel, L., 151 Reiss, Y., 49 Ren, R., 148 Reuner, K. H., 193 Reynet, C., 152 Reynolds, S. H., 90 Rhodes, S., 3 Rice, P., 83, 91 Ridley, A., 10 Ridley, A. J., 68, 113, 139, 164, 185, 196, 203(11), 207, 226, 241,291,306-307, 309, 309(4), 311(4, 5), 313-314, 317, 317(5, 7), 318(5, 7), 319(5, 6), 320(5, 6), 321, 335, 337 Rimm, D. L., 176 Rine, J., 54 Rittenhouse, S. E., 179, 181,241-242, 244(8, 9), 245(9, 12) Roberts, B. E., 349 Roberts, R. L., 268 Robertson, D., 162, 166, 171 Roder, L., 115 Rollins, B., 156 Ron, D., 69, 77, 90-91,126, 285,347-348,351, 351(5), 352(5), 353(11), 354 Roos, D., 260 Rose, M. D., 231 Rosen, C. L., 58 R6sener, S., 174, 185, 186(10), 188(10), 191(10), 193, 328, 337 Rosenfeld, M. G., 50, 56(9), 267 Rosenthal, W., 187 Rossi, F., 213 Rossier, J., 175 Rothlein, R., 291,295(4), 297(4) Rotrosen, D., 16, 17(8), 24(8), 32-33,213,269
AUTHOR INDEX Roy, F. V., 336 Rozakis-Adcock, M., 148 Rozengurt, E., 312 Rubin, E. J., 174, 175(5, 8), 185-186,191(11), 193(16), 194(11), 196, 202, 203(7, 25), 291,297, 321,322(6), 328, 335, 337 Rubin, J. S., 90 Rubin, P., 322 Rubinfeld, R., 130 Ruiz-Larrea, F., 68, 246 Rungger-Br~indle, E., 336 Rush, M. G., 3 Ruskin, B., 50 Russell, D. W., 298 Riither, U., 152
S Sacchi, A., 365 Safer, D., 189 Saida, K., 203(31), 206, 337 St. Jean, A., 233, 234(9) Sakaguchi, G., 196-197, 206(9) Sakaguchi, K., 91 Sakai, T., 203(26), 203(31), 204(26), 205-206 Sakoda, T., 16, 43, 85, 226 Salihuddin, H., 137,139, 139(21), 216,217(11, 15), 219(11), 220(15), 221(11, 15), 225(11) Salzer, J. L., 58 Sambrook, J., 152, 157(18), 229 Sander, C., 335 Sandvig, K., 298 Sanford, K. K., 356 Sanghera, J. S., 151 Saraste, M., 83, 91, 140 Sasaki, Y., 16, 17(9), 18(9), 23(9), 25, 41,41 (10, 12), 42, 47(18), 48(19-23), 85, 98, 181, 191,197, 203(15, 32, 33), 206, 256, 336337, 338(22-24), 341(22), 342(23), 343(23), 346(23) Saulino, A., 67 Schaber, M. D., 49 Schallehn, G., 174, 175(11), 178(11), 186, 187(17), 188, 188(17), 193(17), 196, 297, 328 Scheidtmann, K. H., 86 Scher, C. D., 151 Schering, B., 185, 187 Scherle, P., 41
379
Schiestl, R. H., 233, 234(9) Schill, W.-B., 194 Schlessinger, J., 77, 91 Schliwa, M., 354 Schmitt, M., 261 Schmitt-Verhulst, A. M., 324 Scholtes, E. H. M., 91 Scott, P. J., 26-27 Scott, P. J. J., 268 Seabra, M. C., 49 Sechler, J. M. G., 16 Segal, A., 25, 227 Segal, A. W., 16, 17(5), 24(5), 26, 33, 139, 207, 213, 213(4), 215(4, 12), 257, 265(4), 268-269, 271(12, 15), 276, 358 Segev, N., 286 Seidel-Dugan, C., 140 Seigneurin, J. M., 359, 360(7), 361(7) Seki, H., 203(31), 206, 337 Sekine, A., 186, 196-197, 199(16), 200(16), 201(1), 297, 324, 328, 335(6), 337 Sekura, R. D., 187 Self, A., 244 Self, A. J., 3-4, 9(15), 67-68, 70(2), 73(12), 75(12), 113, 132, 138(15), 139, 139(15), 167, 174, 175(6), 185, 187(12), 196, 197(10), 203(10), 207, 208(7), 209, 212, 238, 240, 291,314, 315(4, 8), 317,317(4), 319, 319(4, 8), 322, 335, 337 Selzer, J., 188 Settleman, J., 68, 105-106, 109, 109(3), 110, 112(4), 113(3), 134, 138(20) Seuwen, K., 152, 153(16) Sha'ag, D., 274 Shaltiel, S., 30 Shapira, R., 247 8hepard, R. C., 151 Shevach, E. M., 321 Shibuya, M., 128 Shih, A., 3 Shiku, H., 47 Shimizu, K., 41 Shinjo, K., 3, 1.1-12, 14(4), 77, 101, 103(6), 130-131, 138, 215-216 Shirataki, H., 16, 85 Shirayoshi, Y., 338, 342(34) Shiritaki, H., 226 Shoshan, M. C, 195 Shou, C., 84, 91, 94(12) Shpungin, S., 22, 23(24)
380
AUTHOR INDEX
Shragge, P., 322 Shurin, S. B., 268 Siegel, M. I., 246, 248 Simmons, R. L., 331, 332(14) Sin, W.-C., 114, 115(9), 116(9), 132, 134(14), 135(14) Singer, S. J., 321 Singh, R., 142, 146(7) Sixou, S., 330 Sizeland, A. M., 84 Skinner, R. H., 3 Sklar, L. A., 27, 242, 260 Sloat, B. F., 215, 281,284, 284(1), 285(1) Smigel, M. D., 27 Smith, A. D., 68, 246 Smith, C., 90 Smith, C. L., 69, 84, 90, 91(4), 93(4) Smith, D. B., 4, 6(16), 86, 92, 93(23), 116, 128, 176, 178(22) Smith, G. E., 92 Smith, L. A., 181, 186, 191(18), 192(18) Smith, P., 114, 115(2, 9), 116(6, 9), 118(6), 132-133, 134(14, 16), 135(14) Smith, R. M., 268 Smithgall, T. E., 128 Smrcka, A., 246 Snyderman, R., 3, 11, 16, 25, 33, 41(11), 42, 175, 358 Sommer, D., 151 Song, O., 228 Soprano, K. J., 151 Sorisky, A., 242 Spangler, B. D., 184(6), 185 Springer, T. A., 290-291,295(4), 297(4), 321 Spurr, N. K., 114, 115(9), 116(9), 132, 134(14), 135(14) Srivastava, S., 351,353(10, 11) Stam, J. C., 91 Stancou, R., 151, 203(30), 206, 321-322, 326(9), 338 Stasia, M.-J., 33, 181, 192-193, 194(40), 200, 203(29), 206, 327, 329, 333(12), 335, 336(21), 337 Staud, R., 50, 53, 56(9), 267 Staudt, L. M., 41 Stearns, T., 286 Steinberg, F., 187 Stenmark, H., 298 Stephens, L., 246 Stephenson, J. R., 84
Stephenson, R. C., 54, 59(17) Sternglanz, R., 228 Sternweis, P. C., 27, 246 Stiles, C. D., 151, 156 Stock, J., 52, 53(13) Stock, J. B., 50, 52-54, 54(10), 56, 56(9, 10, 19), 57(21), 58(21), 59(10), 267 Stoker, M., 336 Stolzenback, F., 354 Stossel, T. P., 248, 306 Strober, W., 321 Stryer, L., 187 Siidhof, T. C., 49 Sugai, M., 174, 187, 242, 245(12), 328 Sugie, K., 175, 196(13), 197, 203(13), 291, 292(9), 293(9), 295(9), 296(9), 322, 338 Suginaka, H., 174, 187, 328 Sullivan, J. A., 320 Summers, M. D., 92 Swendsen, C. L., 248 Sydenham, M., 3 Syuto, B., 174, 187
T Tachibana, M., 91 Takahashi, K., 192 Takai, S., 91 Takai, Y., 4, 15-17, 17(7, 9), 18(9, 11, 15), 21(7), 23(7, 9), 25, 32-34, 41, 41(10-12), 42-43, 47, 47(18), 48(19-23), 69, 85, 98100, 105, 174, 181, 187, 191, 196-197, 200(6), 203(15, 31-33), 206-207, 213(6), 226, 256, 336-337, 338(22-24), 341(22), 342(23), 343(23), 346(23) Takaishi, K., 16, 17(7), 21(7), 23(7), 32-33, 41, 41(11), 42, 48(21-23), 197, 203(15, 32), 206-207, 213(6), 256, 336-337, 338(22-24), 341(22), 342(23), 343(23), 346(23) Takeichi, M., 42, 48(22), 337-338, 338(23), 342, 342(23, 34, 35), 343(23), 346(23) Talpaz, M., 125 Tamanoi, F., 285 Tan, E.-C., 138 Tan, E. W., 50, 54(8) Tan, L., 137, 139(21), 216, 217(15), 220(15), 221(15) Tanaka, K., 41 Tarone, R. E., 356
AUTHOR INDEX Tartakovskii, I. S., 174 Tavitian, A., 76, 163, 182, 196 Taylor, C. E., 168 Taylor-Harris, P. M., 68, 73(12), 75(12), 132, 138(15), 139(15), 207, 208(7), 240 Teahan, C. G., 16, 17(5), 24(5), 33, 139, 207, 213(4), 215(4), 227,257,265(4), 269, 358 Teissid, J., 330 Teo, M., 114, 115(9), 116(9), 130, 132, 132(7), 134(14), 135(14), 216 Teru-uchi, T., 175, 196, 200, 203(12), 241, 291,337 Thelestam, M., 195 Thiberge, J., 326 Thiberge, J.-M., 182 Thiel, G., 68, 140, 147(1), 219 Thom, D., 103 Thomas, D. Y., 216, 227(12) Thomas, P. S., 152 Thomas, S. M., 140 Thompson, A., 68, 246 Thompson, J., 83, 91 Thrasher, 269 Thrasher, A. J., 213 Tilbrook, P. A., 4, 17, 43, 165, 229 Timmons, M. S., 128, 134 Toh, E. A., 215 Toh-e, A., 131 Toksoz, D., 84, 91 Tomhave, E., 16, 33, 41(11), 42, 175 Tominaga, T., 175, 196, 196(13), 197, 203(12, 13), 241,290-291,292(9), 293(9), 295(9), 296(9), 322, 337-338 Tong, L., 3 Torti, M., 265 Totty, H., 207, 213(4), 215(4) Totty, N., 16, 17(5), 24(5), 33, 68, 91, 114, 120(1), 126, 130, 139, 227, 246, 257, 265(4), 269, 358 Totty, N. F., 148, 271(15), 276 Toulme, J., 359, 365(9) Towbin, 190 Trahey, M., 67, 76(4), 130 Traub, P., 193, 194(39), 297 Traynor-Kaplan, A. E., 242 Troalen, F., 182, 326 Tronick, S. R., 69, 77-78, 90-91, 126, 285, 347-349, 351(5), 352(5), 354 Truong, O., 148 Tsuyama, S., 185
381
Turner, C., 312, 335 Turunen, O., 164 Tyagi, S. R., 17, 25(22)
U Uchida, A., 175, 196(13), 197, 203(13), 291, 292(9), 293(9), 295(9), 296(9), 322, 338 Ueda, T., 16, 18(15), 41, 47, 69, 100, 105, 337 Ueno, K., 200 Uhing, R. J., 175 Uhlinger, D. J., 17, 25(21, 22), 246-247 Ui, M., 192, 200 Ullrich, A., 3, 11, 91,131,216 Ushikubi, F., 175, 196, 200, 203(12), 241, 291,337
V Vaheri, A., 164 Valencia, A., 335 van Corven, E. J., 203(28), 206 van Damme, J., 174, 175(11), 178(11), 186, 187(17), 188, 188(17), 193(17), 196, 328 Vandekerckhove, J., 174, 175(11), 178(11), 186-187, 187(17), 188, 188(17), 193(17), 196, 297, 328, 337 van der Eb, A. J., 349 van der Kammen, R. A., 91 Van Dop, C., 187 van Erp, H. E., 68, 73(12), 75(12), 132, 138(15), 139(15), 207, 208(7), 240 van Kooyk, Y., 291 Vanniasingham, V., 114, 115(2), 133, 134(16) van Oers, C., 319 van Schaik, M. L. J., 260 van Soest, S., 68, 91 Van Zwieten, R., 260 Vaughan, M., 181, 184, 185(3), 186-187, 191(18), 192(18) Vauti, F., 226 Vazquez, A., 26, 265, 358-360 Vecchio, G., 78, 347 Vertiev, Y. V., 174 Viciana, P. R., 212, 216 Vi6, A., 152 Vignais, P. V., 33, 181,192-193, 194(40), 200, 203(29), 206, 327, 329, 333(12), 335, 336(21), 337, 359, 360(7), 361(7)
382
AUTHORINDEX
Vincent, S., 3, 151, 152(14), 153, 154(14), 158, 158(14) Vittd-Mony, I., 203(30), 206, 321-322, 326(9), 338 Vojtek, A. B., 228, 240(3) Vola, C., 115 Volker, C., 50, 53-54, 54(10), 56, 56(9, 10, 19), 57(21), 58(21), 59(10), 267 Volkman, D. J., 359 Volpp, B. D., 16, 257-258, 263(2) Von Eichel-Streiber, C., 195 Vu, T.-K., 311 W Wada, K., 226 Wade, J., 148 Waechter, D. E., 151 Wagner, E. F., 152 Walker, C., 359 Walker, L., 258, 260(6) Wallach, D. F. H., 354 Walseth, T. F., 244 Walter, G., 86 Wang, P., 246 Warbrick, E., 282 Warne, P. H., 88, 212, 216 Waterfield, M. D., 68, 148, 242, 244(8), 246 Watt, K., 130 Webb, M. R., 25, 33, 67 Weber, P. C., 226 Weber, R. F., 3, 25, 33, 358 Weder, P., 291 Weinberg, R. A., 68, 84, 105-106, 109(3), 112(4), 113(3), 134, 138(20) Weiss, A., 91 Weisshaar, B., 164 Weissmann, G., 50, 53, 54(10), 56(9, 10), 59(10), 267 Welch, W. J., 313 Weller, U., 174, 185, 186(9), 297, 328 Wen, L.-P., 114, 115(3), 116(3), 120(3), 125(3) West, I., 213, 269 West, R. E., 187 Wever, R., 260 Wheaton, V. I., 311 Wheelock, R. H. P., 351,353(10) Whiteway, M., 216, 227(12) Whitman, M., 244
Wickner, R. B., 69, 77, 91,126, 285,354 Wiedlocha, A., 298 Wiegers, W., 193, 194(39), 297 Wientjes, F. B., 271(15), 276 Wigler, M., 67, 347 Williams, D. A., 84, 91 Williamson, K. C., 181,186, 191(18), 192(18) Willumsen, B. M., 105 Winston, F., 231 Witte, O. N., 34, 69, 91, 125-126, 126(2), 127, 127(3, 4, 5), 128, 128(4, 5), 134, 139 Wittinghofer, A., 3, 76, 131,335 Wolf-Vorbeck, G., 359 Wolheim, C. B., 181 Wollheim, C. B., 99 Woods, R. A., 233, 234(9) Woolkalis, M. J., 183 Wulf, G. M., 36, 41, 105 Wykle, R. L., 246, 248 X Xiec, H., 4 Xu, X., 17, 25(21), 34, 69, 258 Y Yaku, H., 42, 48(19, 23), 197, 203(15), 337, 338(24) Yamada, K., 91 Yamaguchi, T., 226 Yamamoto, J., 16, 18(15), 41, 69, 105, 337 Yamamoto, K., 196, 200(6), 337 Yamamoto, M., 196, 203(26), 204(26), 205, 206(9) Yamamoto, T., 4, 16-17, 18(11), 41, 43, 85 Yamanaka, G., 187 Yamane, H., 4 Yamochi, W., 41-42, 48(22), 337, 338(23), 342(23), 343(23), 346(23) Yang, Y., 231 Yannelli, J. R., 320 Yantani, A., 67(7), 68 Yatani, A., 67, 226 Yeh, S.-H., 231 Yeramian, P., 163, 182, 196 Yeung, C. L., 213, 269 Yocum, R. R., 287
AUTHOR INDEX Yoshida, Y., 4, 17, 43 Yoshikawa, K., 187,328 Young, J. C., 125, 126(2) Yuspa, S. H., 338
Z Zangrilli, D., 12, 78, 82(9), 84(9), 347, 358 Zannini, M., 69, 77, 91, 126, 285, 354 Zhang, J., 181,242, 244(8, 9), 245(9, 12) Zhao, Z.,114, 115(3),116(3), 120(3), 125(3)
383
Zhao, Z.-S., 139, 216, 217(11), 219(11), 221(11), 225(11) Zheng, Y., 11-12, 68, 77-78, 82(9), 84, 84(9), 138, 215,216(5), 285, 358 Ziff, E. B., 160 Zigmond, S. H., 327, 335(1) Ziman, M., 281-283, 283(7), 285(11), 286 Zullo, J., 156 Zumstein, P., 156 Zupi, G., 365 Zuydgeest, D., 91
SUBJECT INDEX
385
Subject Index
A Abelson nonreceptor tyrosine kinase, SH3 domain fusion protein with glutathione S-transferase, biotinylation, 141-142 proteins binding, detection, 140-148 Actin disruption in yeast cdc42 mutants, 282, 283 growth factor-induced reorganization in Swiss 3T3 cells, 306-313 network in neutrophils, effect of C3 exoenzyme, 333 stress fibers, formation regulation by Rho, 317-319 stimulation by growth factors, 311-312 Actomyosin, RhoA functions dependent on, inhibition by Rho-GDI, 48 ADP-ribosylation in assay of Rho, 243-244 C3-catalyzed applications, 195 assay, 188-191, 197-200 detection in intact cells, 193-195 Rho at asparagine-41, 186-187, 196 by bacterial ADP-ribosyltransferases in vitro, 184-195 C3 ADP-ribosyltransferase for, loading into neutrophils, 329-330 detergent effects, 192 guanine nucleotide effects, 191 lipid effects, 192 Mg2÷ effects, 191 temperature effects, 191 ADP-ribosyltransferases bacterial, ADP-ribosylation of Rho in vitro, 184-195 C3, see C3 transferase
Affinity chromatography C3 exoenzyme-diphtheria toxin fragment B, 301,302 Rho-GDI-glutathione S-transferase, 45 Amino acids, aceeptor, in C3-1ike transferases, testing, 192-93 Amino acid sequence analysis 3BP-1 and 3BP-2, 141 Rho-GTPase-activating protein, alignment of conserved residues, 137-138 Ammonium sulfate, precipitation of RacGDI, 36-37 Antibodies dbl-specific, generation, 350-352 monoclonal, see Monoclonal antibodies to l~ac, production, 361 to Ras, for immunolabeling studies, 162-163 Antisense oligonucleotides, inhibitory effects Rac function, 358-366 specificity, controls, 361,365 Rac synthesis, 361 superoxide production, 362-365 Arachidonic acid, in assay of NADPH oxidase, 22, 24 Autoradiography, Rac-GTPase-activating protein, reverse image, 136 Avidin, blocking of plaque-containing filters, 144
B Bacillus cereus, C3-1ike exoenzyme,
187-188 Bacteriophages high titer stocks, preparation, 146 recombinant lysogens, isolation, 146
386
SUBJECT INDEX
Baculoviruses Cdc42Hs expressed by, purification, 11-15 insect cells infected with, p190 purification, 106-109 p190, production, 106 B cells, GL1 culture, 360 pinocytosis rate, 365 Rac synthesis, inhibition by antisense oligonucleotide, 361 superoxide production during respiratory burst effect of Rac antisense oligonucleotides, 362-364 measurements, 361-362 treatment with oligomers, 360-361 Bern3 gene product, function as GTPase-activating protein for Cdc42p, 285-286 Binding constants, R h o - G A P binding to GDP- and GTP-bound Rho, Rac, and G25K, 76 Biotin, blocking of plaque-containing filters, 144 Biotinylation, Abl SH3-glutathione S-transferase fusion protein, 141-142 Blotting, see also specific techniques lysogen extract, 146-147 Bombesin, induction of actin reorganization in Swiss 3T3 cells, 311 3BP-1, identification in cDNA expression library, 140-148 Bradykinin, effect on actin organization in Swiss 3T3 cells, 312 Brain bovine, Rho-GDI purification, 99-101 chimaerin identification, 116 murine, membrane and cytosol distribution of RhoA, B, and C, 184 rat cytosol, ADP-ribosylation of Rho, assay, 189 GTPase-activating protein identification, 132-137 GTP-Cdc42-associated kinase p65PAK purification, 221-224 Breakpoint cluster region kinase assay, 126-128 functional domains, 126
purification, 127 SH2-binding activity, assay, 128-129 Bromodeoxyuridine, incorporation by cells, 153-154
C C3 ADP-ribosyltransferase, see C3 transferase Carboxyl methylation, Ras-related proteins in intact neutrophils, 51-52 Carboxyl methyltransferase, prenylcysteinedirected, in human neutrophil membranes activity, 53-58 detergent effects, 53-54 detergent-extracted partial purification, 61-62 reconstitution in liposomes, 59-61 endogenous Ras-related protein substrates, 53-54 kinetics, 57 localization, 58-59 prenylcysteine analogs as substrates, 55-58 recombinant Ras-related protein substrates, 54-55 Cdc24p, role in guanine nucleotide exchange on Cdc42p, 285 Cdc42 activation of purified p65-PAK, 225-226 association with Dbl, effect of guanine nucleotides, 97 -GTP, associated kinase p65-PAK, purification, 221-224 mammalian, identification, 11 Cdc42Hs baculovirus-expressed, purification, 11-15 n-chimaerin identification as GTPase-activating protein for, 120-122 -Dbl interaction, dependence on nucleotide-bound state, 84 expression in Sf21 cells via baculovirus infection, 12-13 GDP dissociation inhibition by Rho-GDI, assay, 101 in presence of Dbl, assay, 80-83 -GDP-dissociation inhibitor interaction, 15
SUBJECT INDEX GTPyS binding in presence of Dbl, assay, 79-80 membranes with, purification, 103-104 purification from Sf21 cell membrane fractions, 13-15 purity assessment, 14-15 regulatory proteins for, 12 solubilization from membranes by RhoGDI, 98-105 assay, 104-105 Cdc42p Bern3 gene product as GTPase for, 285-286 function, mutational analysis, 282-284 -glutathione S-transferase, expression and purification, 287-289 guanine nucleotide exchange, role of Cdc24p, 285 (His)6-tagged, expression and purification, 287-289, 290 prenylation, role of Cdc43p, 285 proteins interacting with, identification, 284-286 Saccharomyces cerevisiae functional analysis, 281-290 subcellular localization, 286 Schizosaccharomyces pombe, function, 281-290 Cdc43p, role in prenylation of Cdc42p, 285 Cell cultures GL1 cells, 360 growth stimulation, 152 JY cells, 291-292 for microinjection preparation, 165-166 selection, 165 Swiss 3T3 cells, 339 Cell cycle gene expression during, 151 late G1 phase, genes induced in, isolation, 156-157 Cell lines, C3 exoenzyme effects, 202-203 Cell motility assays cell track measurement by phagokinesis, 339-341,345 scattering activity, 341-342, 345 neutrophils, inhibition by C3 ADP-ribosyltransferase, 331-333
387
C3 exoenzyme, see C3 transferase Chemiluminescent assay, superoxide production, 361-362 Chemotaxis, neutrophils, assay and inhibition by C3 ADP-ribosyltransferase, 327-336 n-Chimaerin expression in Escherichia coli, 116 as GTPase-activating protein for Racl and Cdc42Hs, 120-122 modulation by lipids, 123-124 substrate specificity, 121 metalloprotein function, 118 as phospholipid-dependent phorbol ester receptor, 118-120 purification, 117 refolded, phorbol ester binding, effect of zinc, 119-120 Chimaerins identification in brain, 116 structural characteristics, 114-115 Chromatography, see also specific techniques Cdc42Hs, 14-15 GTP-Cdc42-associated kinase p65-PAK, 221-224 p190, 107, 108 Rac2, 19-22, 28-31 Rho-GDP dissociation inhibitor, 100 Chromium release assay, for cytotoxic lymphocyte activity, 321-322 Cloning, see also Subcloning GTPase-activating proteins detected by overlay assay, 137-138 Clostridium botulinum, culture filtrate, C3 exoenzyme purification, 200-201 Clostridium limosum, C3-1ike exoenzyme, 187 Colloidal gold coating of tissue culture dishes, 339 particle preparation, 339 Competition assay, Rho-GAP binding to GDP- and GTP-bound Rho, Rac, and G25K, 76 Complementary DNA dbl and proto-dbl clone structure, 348 subcloning in eukaryotic expression vectors, 349
388
SUBJECT INDEX
differentially expressed, selection, 157-158 growth-stimulated genes characterization, 158 cloning, 154-156 library screening for proteins binding Abl SH3, 142-144 Rho, epitope-tagged, microinjection and immunolabeling, 162-173 Confocal microscopy myc-tagged Racl in Rat2 cells, 170 myc-tagged Rho in Swiss 3T3 cells, 168-170 COS1 cells, transfection by electroporation, 88-89 C3 transferase applications in biological systems, 202-206 catalyzed ADP-ribosylation applications, 195 assay, 188-191,197-200 detection in intact cells, 193-195 -diphtheria toxin fragment B cytopathic effect on Vero cells, 303 -306 expression from Escherichia coli, 300-301 genetic construction, 299-300 immunoblotting, 303 purification, 301, 302 SDS-PAGE, 303 effect on actin network of neutrophils, 333 effect on cell morphology, 333-335 -glutathione S-transferase cleavage from glutathione beads by thrombin, 178 expression in Escherichia coli, 175-176 precipitation on glutathione beads, 177-178 purification from bacterial cell lysate, 176-179 inhibitory effects JY cell aggregation, 295-296 lymphocyte-mediated cytotoxicity, 320-327 neutrophil motility, 331-333 loading into neutrophils by electropermeabilization, 329-330
lymphocyte treatment with electropermeabilized cytotoxic cells, 322-324 JY cells, 291-293 PC-12 cell treatment with, 206 properties, 185-186, 201-202 purification from culture filtrate of Clostridium botulinum, 200-201 reaction in broken cell extracts, inhibitors, 181 recombinant activity on Rho proteins, 179-181 assay, 179-180 ion-exchange chromatography, 178-179 as probe for Rho proteins, 179-184 purification from Escherichia coli, 201 related exoenzymes, 187-188 acceptor amino acid, testing, 192-193 Swiss 3T3 cell treatment with, 205-206 uptake by electroporation, control, 324-325 Cytochrome b neutrophil, spectrophotometric assay, 260 purification, 272-273 relipidation, 273 Cytosol cerebral extracts, RhoA, B, and C distribution, 184 granulocyte, fraction, isolation, 248-249 HL-60 cell, fraction, preparation, 19 neutrophil from activated cells, Rac in, translocation to membranes, 256-267 preparation, 26-27, 269-271 phagocyte cell-free assay of NADPH oxidase, 276-277 preparation of partially purified p47phox and p67-phox, 273-274 platelet preparation, 242-243 Rho-dependent phosphoinositide 3-kinase, assay, 241-246
D dbl oncogene antibodies specific to, generation, 350-352
SUBJECT INDEX cDNA clone structure, 348 subcloning in eukaryotic expression vector, 349 cell transformation by, 347-358 DH domain, 77, 78, 83 biologic and biochemical activity, 354-356 transforming activity, 349-350 Dbl protein association with Racl or Cdc42, effect of guanine nucleotides, 97 biochemical analysis, 353-354 -Cdc42Hs interaction, dependence on nucleotide-bound state, 84 expression in Escherichia coli, 84 in Spodoptera frugiperda cells, 78-79 family members, common structural features, 83 GDP dissociation from Cdc42Hs in presence of, assay, 80-83 -glutathione S-transferase antisera, 352 effects on GDP dissociation from Cdc42Hs, 81-83 preparation, 78 GTPyS binding to Cdc42Hs in presence of, assay, 79-80 identification, 353 purification from Spodoptera frugiperda cells, 78-79 -Rho-related GTPase interaction, 90-98 De(ADP-ribosylation), in testing of acceptor amino acid of C3-1ike transferases, 192-93 Detergent effects ADP-ribosylation of Rho, 192 prenylcysteine-directed carboxyl methyltransferase, 53-54 Diacylglycerol kinase, refolded, phorbol ester binding, effect of zinc, 119-120 Diphtheria toxin fragment B binding to receptor, 298 -C3 exoenzyme fusion protein cytopathic effect on Vero cells, 303-306 expression from Escherichia coli, 300-301
389
genetic construction, 299-300 immunoblotting, 303 purification, 301, 302 SDS-PAGE, 303 purification, 301-302 DNA, complementary, see Complementary DNA DNA probes, immobilization on membranes, 161 Dot-blot immunoassay, p190, 107 Dot-blot immunotitration, pl90, 110
E Ect2, interaction with Rho-related GTPases, 90-98 Electron microscopy, see Immunoelectron microscopy Electropermeabilization, bovine neutrophils leakage during, assay, 330-331 in presence of C3 ADP-ribosyltransferase, 329-330 Electroporation C3 transferase uptake by, control, 324-325 cytotoxic lymphocytes, 323-324 transfection of COS1 cells, 88-89 Epidermal growth factor, induction of actin reorganization in Swiss 3T3 cells, 311-312 Epithelial cells MDCK, immunogold localization of myctagged RhoB, 173 308R microinjection of Rho-GDI, 344-345 motility inhibition by Rho-GDI, 336-347 scattering activity assay, 341-342, 345 Epitopes plasmids tagged with, construction, 164-165 Rho family cDNAs tagged with, microinjection and immunolabeling, 162-173 tags, selection, 164 Escherichia coli C3 exoenzyme-diphtheria toxin fragment B expression, 300-301 n-chimaerin-expressing, extract preparation, 116-117
390
SUBJECT INDEX
n-chimaerin expression, 116 crude supernatant, preparation, 44 C3 transferase-glutathione S-transferase expression, 175-176 purification, 176-177 cultivation, 44 Dbl expression, 84 lysate preparation, 176-177 recombinant C3 exoenzyme purification, 201 recombinant p47-phox, p67-phox, and p21 Racl preparation, 274-276 recombinant Rho-GDI-glutathione Stransferase purification, 101-103 recombinant Rho/Rac/G25K purification, 1-10 Rho-GD1 expression and purification, 250-253 Rho-related GTPase expression, 93 smgGDS expression, 86, 253-254 smgGDS purification, 253-254 Exoenzymes, C3, see C3 transferase Extraction, Ras-bound guanine nucleotides, 89
F Fibroblast growth factor, induction of actin reorganization in Swiss 3T3 cells, 311-312 Fibroblasts CCL39, growth factor-stimulated, assay, 153-154 NIH3T3 dbl-transformed, morphology, 356 growth factor-stimulated, assay, 153-154 Swiss 3T3 actin reorganization induced by growth factors, 306-313 culture, 339 fixation, 309, 317 growth factor addition, 309 microinjection with Rho-GDI, 344 motility assay by cell track measurement by phagokinesis, 339-341, 345 inhibition by Rho-GDI, 336-347
myc-tagged Rho distribution, 168-170 phagokinetic activity, estimation, 339-341 photography, 310 preparation, 307-309, 314 Rac microinjection into quiescent cells, 313-320 Rho microinjection into quiescent cells, 313-320 staining, 310, 317 treatment with C3 exoenzyme, 205-206 3T3, morphology, effect of C3 ADP-ribosyltransferase, 333-335 Fibronectin, effect on actin organization in Swiss 3T3 cells, 312 Filter transfer assay,/3-galactosidase production by yeast two-hybrid system, 236 Fixation fibroblasts for bromodeoxyuridine labeling, 153-154 Swiss 3T3 cells, 309, 317 Fusion proteins Abl SH3-glutathione S-transferase, biotinylation, 141-142 Cdc42p, expression and purification, 287-290 C3 exoenzyme-diphtheria toxin fragment B, inhibition of p21 Rho in intact cells, 297-306 C3 transferase-glutathione S-transferase cleavage from glutathione beads by thrombin, 178 expression in Escherichia coli, 175-176 precipitation on glutathione beads, 177-178 purification from bacterial cell lysates, 176-179 dbl-glutathione S-transferase, antisera, 352 G25K-glutathione S-transferase, recovery, 6-7 Racl-glutathione S-transferase, recovery, 6-7 RhoA-glutathione S-transferase, recovery, 6-7 SH2-glutathione S-transferase, in assay of SH2-binding reactions, 128-129
SUBJECT INDEX
G /3-Galactosidase, production by yeast twohybrid system, 236-237 GDI, see GDP dissociation inhibitors GDP dissociation from Cdc42Hs inhibition by Rho-GDI, assay, 101 in presence of Dbl, assay, 80-83 dissociation of DH domain-containing protein/GTPase complex, 97 - G T P exchange off rates, 69-7l in RhoA, R h o - G D I regulating, assay, 45-46 - R h o A complex formation, R h o - G D I activity for, assay, 46-47 smgGDS-catalyzed release, assay, 86-87 GDP dissociation inhibitors interaction with Cdc42tts, 15 -Rac complex detection assays, 35-36 purification from phagocyte cytosol, 33-41 -Rac dissociation, 265-267 regulation of GTP-binding protein localization, 99 Rho-GDI, see Rho-GDP dissociation inhibitor small GTP-binding protein, see Small GTP-binding protein GDP dissociation inhibitor subcellular distribution in activated neutrophils, 261-263 GDP dissociation stimulator, smgGDS, see Small GTP-binding protein GDP dissociation stimulator Gel electrophoresis, see also Isoelectric focusing C3 exoenzyme-diphtheria toxin fragment B, 303 GTPase-activating proteins, subsequent detection by overlay assay, 130-139 GTP-p21-binding proteins, subsequent detection, 218-219 p190, 107 Gel filtration p190, 109 Rac2, 28-29 Rac-GDI, 38-41
391
Genes growth-stimulated clones, regulatory analysis, 158-162 isolation, 154-158 Northern analysis, 152-153 induced in late G1 phase, isolation, 156-157 G25K assay, 7-8 dialysis, 7 GDP- and GTP-bound, Rho-GAP binding constants for, 76 guanine nucleotide off rates, 69-71 intrinsic GTPase activity, 72-75 L61 mutant, affinity for Rho-GAP, 240 mutant proteins, 10 recombinant, purification from Escherichia coli, 1-10 Rho-GAP-stimulated GTPase activity, 74-76 stability, 8-9 storage, 7 wild-type, purification, 5-7 Glutathione beads C3 transferase-glutathione S-transferase cleavage from, 178 C3 transferase-glutathione S-transferase precipitation on, 177-178 Glutathione S-transferase -Abl SH3, biotinylation, 141-142 -Cdc42p, expression and purification, 287-289 -C3 transferase cleavage from glutathione beads by thrombin, 178 expression in Escherichia coli, 175-176 precipitation on glutathione beads, 177-178 purification from bacterial cell lysates, 176-179 - d b l , antisera, 352 fusion proteins with Rho/Rac/G25K, recovery, 6-7 fusion vector pGEX-2T, construction, 4-5 -Rho-GDI purification, 44-45 recombinant, purification from Escherichia coli, 101-103
392
SUBJECT INDEX
-SH2, in assay of SH2-binding reactions, 128-129 Gold, colloidal, see Colloidal gold Granulocytes, see Neutrophils Growth factors addition to Swiss 3T3 ceils, 309 cells stimulated by, assay, 153-154 induction of actin reorganization in Swiss 3T3 cells, 306-313 mRNA turnover in response to, 159-160 GTP -Cdc42, associated kinase p65-PAK, purification, 221-224 dissociation of DH domain-containing protein/GTPase complex, 97 - G D P exchange off rates, 69-71 in RhoA, R h o - G D I regulating, assay, 45-46 -p21, proteins binding, detection, 218-219 smgGDS-catalyzed binding, assay, 87-88 GTPase-activating proteins detected by overlay assay, cloning, 137-138 expression screening, 137 functional roles, 139 identification in cell and tissue extracts, 132-137 interactions with Rho and Rac, in vitro binding assay, 207-215 GTPase inhibitors, detection, 217 GTPases complex with DH domain-containing protein, dissociation by guanine nucleotides, 97 intrinsic and Rho-GAP-stimulated activities of Rho, Rac, and G25K, assays, 72-76 loading with tritiated guanine nucleotide, 70 Rho-related expression in Escherichia coli, 93 interaction with Ect2 and Dbl, 90-98 small, association with regulators, detection, 92 GTPyS binding to Cdc42Hs in presence of Dbl, 79-80 35S-labeled, binding activity, 23, 27
Guanine nucleotides Dbl-catalyzed exchange, 77-84 dissociation of DH domain-containing protein/GTPase complex, 97 effect on ADP-ribosylation of Rho, 191 exchange on Cdc42p, role of Cdc24p, 285 Ras-bound, labeling and extraction, 89 smgGDS-promoted exchange in vitro, assays, 86-88 tritiated, loading of GTPase, 70
H Highfive cells, baculovirus-infected, p190 purification, 106-109 High-performance liquid chromatography, in assay of Rho-dependent phosphoinositide 3-kinase, 244-245 HIS3 gene, in yeast two-hybrid system for detection of protein-protein interactions, 228 Hybridization in analysis of growth-regulated clones, 162 differential, isolation of genes induced in late G1 phase, 156-157 Hydrophobic interaction chromatography, Rac-GDI, 37-38
I Immunoassay breakpoint cluster region kinase, 127-128 dot-blot, p190, 107 Immunoblot analysis C3 exoenzyme-diphtheria toxin fragment B, 303 dbl proteins, 353 Immunoelectron microscopy cells microinjected with epitope-tagged Rho cDNAs, 170-173 myc-tagged Racl in Rat2 cells, 171-173 myc-tagged RhoB in MDCK cells, 173 Immunofluorescence cells microinjected with epitope-tagged Rho cDNAs, 167-170 growth factor-stimulated cells, 153-154 Vero cells treated with C3 exoenzymediphtheria toxin fragment B, 304-306
svBJZCa" INDzx
Immunolabeling, epitope-tagged Rho cDNAs, 162-173 Immunoprecipitation, dbl proteins, 353 Immunotitration, dot-blot, p190, 110 Inclusion bodies, proteins from, refolding, 119-120 Insect cells, see also Highfive cells; Spodoptera frugiperda
lysates, p190 Rho-GAP activity in, assay, 113 Insulin, induction of actin reorganization in Swiss 3T3 cells, 311-312 Ion-exchange chromatography breakpoint cluster region kinase, 127 detergent-extracted prenylcysteine-directed carboxyl methyltransferase, 61-62 Rac2, 28, 29 Rac-GDI, 38 recombinant C3 transferase, 178-179 Rho-GDP dissociation inhibitor, 100 Ionic strength, effect on binding assay for Rho and Rac interactions with GAPs, 211-212 Isoelectric focusing, see also Gel electrophoresis two-dimensional, RhoA, B, and C, 181-183
K Kinetics prenylcysteine-directed carboxyl methyltransferase, 57 Rac translocation from cytosol to membranes in activated neutrophils, 259260, 263-265, 267
L
Labeling, see also Immunolabeling; Radiolabeling cells with bromodeoxyuridine, 153-154 Lactate dehydrogenase, in assessment of leakage during neutrophil electropermeabilization, 330-331 lacZ gene, in yeast two-hybrid system for detection of protein-protein interactions, 228
393
Lipids effects on ADP-ribosylation of Rho, 192 effects on n-chimaerin GAP activity, 124 enzyme-regulating, classification, 123 preparation, 123 Liposomes preparation, 123 reconstitution of detergent-extracted prenylcysteine-directed carboxyl methyltransferase in, 59-61 sedimentation, for assay of lipid interaction with proteins, 123-124 Liquid culture assay,/3-galactosidase production by yeast two-hybrid system, 236-237, 239-240 Lucifer Yellow, in definition of conditions for lymphocyte electroporation, 324 Lymphocytes, see also B cells aggregation assay under shaking conditions, 296-297 inhibition by C3 exoenzyme, 295-296 microtiter plate assay, 294-296 phorbol ester-induced, 294-295 culture, 291-292 cytotoxic C3 transferase uptake by electroporation, control, 324-325 cytotoxic activity chromium release assay, 321-322 inhibition by Clostridium botulinum C3 transferase, 320-327 electropermeabilized, C3 transferase treatment, 322-324 viability after electroporation, assessment, 323 treatment with C3 exoenzyme, 291-293 Lysogens extracts blot, 146-147 preparation, 146 recombinant phage, isolation, 146 Lysophosphatidic acid, induction of actin reorganization in Swiss 3T3 cells, 311
M Macrophages, peritoneal, isolation from guinea pig, 34-35
394
SUBJECT INDEX
Magnesium effects ADP-ribosylation of Rho, 191 guanine nucleotide off rates, 70-71 Membranes associated Cdc42Hs, solubilization by Rho-GDI, 98-105 binding of RhoA, Rho-GD1 activity inhibiting, assay, 47 Cdc42Hs-containing, purification, 103-104 cerebral extracts, RhoA, B, and C distribution, 184 dissociation of exogenous RhoA, RhoGDI stimulating, assay, 48 granulocytc, fraction, isolation, 248-249 HL-60 cell, fraction, preparation, 19 neutrophil from activated cells, Rac translocation to, 256-267 from cell surface, localization of prenylcysteine-directed carboxyl methyltransferase, 58-59 prenylcysteine-directed carboxyl methyltransferase activity, 53-58 preparation, 269-272 reconstitution, 272 solubilization, 272 phagocyte, cell-free assay of NADPH oxidase, 276-277 ruffling induction by growth factors, 306-307, 311-312 regulation by Rac, 319-320 Sf21 cell, fractions, Cdc42Hs purification, 13-15 Metalloproteins, n-chimaerin as, assessment, 118 Methylation, Ras-related proteins in intact neutrophils, 51-52 Microinjection capillary needles for, preparation, 342-343 cell cultures for preparation, 165-166 selection, 165 epitope-tagged Rho cDNAs, 162-173 protein concentrations for, 345 Rac and Rho into quiescent Swiss 3T3 cells, 313-320 Rho-GDI
into epithelial 308R cells, 344-345 into Swiss 3T3 cells, 344 Microscopy, s e e Confocal microscopy; Immunoelectron microscopy Microtiter plate assay, lymphocyte aggregation, 294-296 Monoclonal antibodies anti-FLAG in detection of small GTPase association with regulators, 92 DH domain-containing protein detection, 94 Y13-259, coupling to Protein A Sepharose, 89-90 Mowiol 4.88 solution, preparation, 154, 304 Mutations, Ras-like, in analysis of Cdc42p function, 282-284
N NADase inhibitors, 183-184 NADPH oxidase assay, 24 cell-free activity assay, 22-23 cell-free assay, 276-277 reconstitution by purified components, 268-278 superoxide production, assay, 276 in phagocytes, constituents, 15-16 Rac2-mediated regulation, 16-17, 23-25 regulation by Rac proteins, 358-359 analysis with Rac antisense oligonucleotides, 362-365 regulatory components subcellular distribution in activated neutrophils, 258-259 translocation from neutrophil cytosol to membranes, 256-257 Neutrophils actin network, effect of C3 exoenzyme, 333 activated, Rac translocation from cytosol to membranes assay, 256-267 kinetics, 259-260, 263-265, 267 carboxyl methylation of Ras-related proteins, 51-52 chemotaxis, assay and inhibition by C3 ADP-ribosyltransferase, 327-336
SUBJECT INDEX cytosol, preparation, 26-27, 248-249, 269-271 electropermeabilization leakage during, assay, 330-331 in presence of C3 ADP-ribosyltransferase, 329-330 fractionation, 258-260 GDP dissociation inhibitors, subcellular distribution, 261-263 isolation, 50-51,247, 271 labeling, 247-248 membranes prenylcysteine-directed carboxyl methyltransferase activity, 53-58 preparation, 248-249, 269-272 reconstitution, 272 solubilization, 272 motility, inhibition by C3 ADP-ribosyltransferase, 331-333 phospholipase D, inhibition by Rho-GDI and stimulation by smgGDS, 246-256 p47-phox and p67-phox, subcellular distribution, 263 preparation, 258 Rac, subcellular distribution, 261-263 Rac2, purification, 25-32 Rho, subcellular compartmentation, 335-336 stimulation by phorbol myristate acetate, 258 subcellular fractions localization of prenylcysteine-directed carboxyl methyltransferase, 58-59 preparation, 51 surface membranes, localization of prenylcysteine-directed carboxyl methyltransferase, 58-59 Nitrocellulose filters, probing, 144-146 Northern blot analysis, growth-stimulated genes, 152-153 NRK cells, ADP-ribosylation of Rho, assay, 189 Nuclei isolated, run-on assays, 160-162 purified, isolation from cell culture, 160-161 Nucleotide sequence analysis, cDNA clones, 158
395
O Oligonucleotides, antisense, see Antisense oligonucleotides Overlay assay advantages, 131 GTPase-activating protein identification, 130-139 GTPase inhibitors, 217 sensitivity, 130
P p21 -GTP, proteins binding, detection, 218-219 [y-32p]GTP-labeled, expression screening with, 219-221 p190 antigenic activity, recovery after purification, 110 complex with Ras-GAP, 105, 113 partitioning between cellular subfractions, 109-110 purification from baculovirus-infected insect cells, 106-109 R h o - G A P activity, assay, 112-113 Paraformaldehyde, solution for immunofluorescence, 304 Peptides, synthetic, in generation of dbl-specific antibodies, 352 Phagocytes, see also Macrophages; Neutrophils cytosol cell-free assay for NADPH oxidase, 276-277 preparation of partially purified p47phox and p67-phox, 273-274 Rac-GDI purification, 33-41 source, 34 Phagokinesis, measurement of cell tracks for cell motility assay, 339-341, 345 Phorbol ester receptors, phospholipid-dependent, n-chimaerin as, 118-120 Phorbol esters binding to n-chimaerin, assay, 118 binding to extracts of n-chimaerin-expressing cells, 119 induced aggregation of JY cells, 294-295 Phorbol myristate acetate, stimulation of neutrophils, 258
396
SUBJECT I N D E X
Phosphoinositide 3-kinase, Rho-dependent, assay in platelet cytosol, 241-246 Phosphoinositides, metabolism in platelets, 242 Phospholipase D assay, 249-250 GTP-activated, stimulation by smgGDS, 255 GTPyS-stimulated, inhibition by RhoGDI, 254 Photography, Swiss 3T3 cells, 310 Pinocytosis, rate in GL1 cells, 365 Plaques filters with, biotin/avidin blocking, 144 positive, isolation, 146 Plasmids epitope-tagged, construction, 164-165 pACTII, for yeast two-hybrid system, construction, 229-231 pAS, for yeast two-hybrid system, construction, 229-231 pEV55, in production of p190 baculovirus, 106 pGEX-2T, construction, 4-5 pGEX2T-C3, construction, 175-176 pGEX-2T-Rho-GDI, construction, 44 pYTH6, for yeast two-hybrid system, construction, 229-231 pZIP-Neo SV(X), subcloning dbl and proto-dbl cDNAs into, 349 Platelet-derived growth factor, induction of actin reorganization in Swiss 3T3 cells, 311-312 Platelets aggregation, 241 cytosol preparation, 242-243 Rho-dependent phosphoinositide 3-kinase, assay, 241-246 phosphoinositide metabolism, 242 p47-phox partially purified, preparation from phagocyte cytosol, 273-274 recombinant, preparation in Escherichia coli, 274-276 subcellular distribution in activated neutrophils, 263 p67-phox partially purified, preparation from phagocyte cytosol, 273-274
recombinant, preparation in Escherichia coli, 274-276 subcellular distribution in activated neutrophils, 263 Precipitation C3 transferase-glutathione S-transferase on glutathione beads, 177-178 dbl proteins, 353 Rac-GDI, 36-37 Prenylation, Cdc42p, role of Cdc43p, 285 Prenylcysteine analogs, as substrates for prenylcysteine-directed carboxyl methyltransferase, 55-58 Protein A-Sepharose, coupling to monoclonal antibody Y13-259, 89-90 Protein kinase C, refolded, phorbol ester binding, effect of zinc, 119-120 Protein kinases, Rac/Cdc42-associated, purification and assay, 215-227 Proteins Abl SH3-binding, detection, 140-148 DH domain-containing applications, 97-98 complex with GTPase, dissociation by guanine nucleotides, 97 expression in insect cells, 93-94 interaction with Rho family proteins, detection, 94-96 GTPase-activating, see GTPase-activating proteins GTP-binding, functions regulated by, analysis with Rho-GDI and smgGDS, 254-255 GTP-p21-binding, detection, 218-219 from inclusion bodies, refolding, 119-120 interacting with Cdc42p, identification, 284-286 -protein interactions detection with yeast two-hybrid system, 228-241 screening with biotinylated glutathione S-transferase fusion protein, 140-148 Proto-dbl oncogene cDNA clone structure, 348 subcloning in eukaryotic expression vectors, 349 mutants, construction, 350-351
SUBJECT INDEX products, biochemical analysis, 353-354 transforming activity, 349-350
R Rac in activated neutrophils subcellular distribution, 261-263 translocation from cytosol to membranes assay, 256-267 kinetics, 259-260, 263-265, 267 antibodies, production, 361 effector protein for, detection, 212-215 function, inhibition by antisense oligonucleotides, 358-366 - G D P dissociation inhibitor complex detection assays, 35-36 dissociation, 265-267 purification from phagocyte cytosol, 33-41 GDP- and GTP-bound, R h o - G A P binding constants for, 76 GTPase-activating protein interactions with, in vitro binding assay, 207-215 kinases interacting with, purification and assay, 215-227 L61 mutant, interaction with Rho-GAP, 209 microinjection into quiescent Swiss 3T3 cells, 313-320 recombinant, purification from Escherichia coli, 1-10 regulation of membrane ruffling, 319-320 regulatory functions, 207 synthesis, inhibition by antisense oligonucleotide, 36l Racl activation of purified p65-PAK, 225-226 assay, 7-8 association with Dbl, effect of guanine nucleotides, 97 n-chimaerin identification as GTPase-activating protein for, 120-122 dialysis, 7 guanine nucleotide off rates, 69-71 intrinsic GTPase activity, 72-75 lipid modifications, 17 L61 mutant, affinity for Rho-GAP, 240
397
mutant proteins, 10 myc-tagged, in Rat2 cells immunoelectron microscopy, 171-173 intracellular localization, 170 recombinant, preparation in Escherichia coli, 274-276 Rho-GAP-stimulated GTPase activity, 74-76 stability, 8-9 storage, 7 wild-type, purification, 5-7 Rac2 activation of NADPH oxidase, 23 biologically active, detection, 26 lipid modifications, 17 properties, 23 purification from differentiated HL-60 cells, 18-22 from human neutrophils, 25-32 purity, 31-32 -[rho]GDI complex, separation, 32 role in phagocyte function, 26 yield from human neutrophils, 32 Rac-GTPase-activating protein, in tissue extracts identification, 132-137 reverse autoradiographic imaging, 136 Radiolabeling human granulocytes, 247-248 Ras-bound guanine nucleotides, 89 recombinant p21 proteins, 131-132 Ras antibodies, for immunolabeling studies, evaluation, 162-163 guanine nucleotides bound to, labeling and extraction, 89 smgGDS-catalyzed GDP release, assay, 86-87 smgGDS-catalyzed GTP binding, assay, 87-88 Ras-GRF, DH domain, 93-94 Ras-GTPase-activating protein, complex with p190, 105, 113 Ras-related proteins carboxyl methylation in intact neutrophils, 51-52 in neutrophil membranes, 53-55 post-translational modifications, 49-50 Rat2 cells, immunoeleetron microscopy of myc-tagged Racl, 171-173
398
SUBJECT INDEX
Reconstitution cell-free NADPH oxidase by purified components, 268-278 neutrophil membranes, 272 neutrophil prenylcysteine-directed carboxyl methyltransferase after detergent extraction, 59-61 Respiratory burst, associated superoxide production effect of Rac antisense oligonucleotides, 362-365 measurements, 361-362 Rho ADP-ribosylation at asparagine-41, 186-187, 196 assay, 188-191 by bacterial ADP-ribosyltransferases in vitro, 256~ 184-195 C3 ADP-ribosyltransferase for, loading into neutrophils, 329-330 detergent effects, 192 guanine nucleotide effects, 191 lipid effects, 192 Mg2÷ effects, 191 temperature effects, 191 assay by ADP-ribosylation, 197-200, 243-244 cDNAs, epitope-tagged, microinjection and immunolabeling, 162-173 cellular functions, 202 C-terminal post-translational modification, 42-43 C3 transferase as probe, 179-184 DH domain-containing protein interactions with, detection, 94-96 GDP- and GTP-bound, R h o - G A P binding constants for, 76 GTPase-activating protein interactions with, in vitro binding assay, 207-215 inhibition in intact cells by C3 exoenzyme-diphtheria toxin fragment B, 297-306 L63 mutant, interaction with Rho-GAP, 209 microinjection into quiescent Swiss 3T3 cells, 313-320 myc-tagged, localization in Swiss 3T3 cells, 168-170 recombinant labeling, 131-132
purification from Escherichia coli, 1-10 regulation, 4 regulation of stress fiber formation, 317-319 related GTPases expression in Escherichia coli, 93 interaction with Ect2 and Dbl, 90-98 role in lymphocyte cytolytic activity, 326-327 smgGDS-catalyzed GTP binding, assay, 87-88 subcellular compartmentation in neutrophils, 335-336 subfamily members, structural similarity, 3-4 RhoA actomyosin-dependent functions, RhoGDI inhibiting, assay, 48 assay, 7-8 cerebral, membrane and cytosol distribution, 184 dialysis, 7 - G D P complex formation, Rho-GDI activity for, assay, 46-47 GDP/GTP exchange, R h o - G D I regulating, assay, 45-46 guanine nucleotide off rates, 69-71 intrinsic GTPase activity, 72-75 L63 mutant, interaction with R h o - G A P comparison with L61Racl and L61G25K, 240 comparison with wild-type RhoA, 237-238 mutant proteins, 10 Rho-GAP-stimulated GTPase activity, 74-76 smgGDS-catalyzed GDP release, assay, 86-87 stability, 8-9 storage, 7 translocation, R h o - G D I regulating, assay, 47-48 two-dimensional isoelectric focusing SDS-PAGE, 181-183 wild-type interaction with Rho-GAP, 237-238 purification, 5-7 RhoB cerebral, membrane and cytosol distribution, 184
SUBJECT INDEX
myc-tagged, in MDCK cells, immunoelectron microscopy, 173 two-dimensional isoelectric focusing SDS-PAGE, 181-183 RhoC cerebral, membrane and cytosol distribution, 184 two-dimensional isoelectric focusing SDS-PAGE, 181-183 RhoG, expression, induction by serum, 151-162 R h o - G D P dissociation inhibitor bovine brain, purification, 99-101 complex with Rac2, separation, 32 effects, interpretation, 256 expression in Escherichia coli, 250-252 -glutathione S-transferase, recombinant, purification from Escherichia coli, 101-103 inhibitory effects cell motility, 336-347 GDP dissociation from Cdc42Hs, 101 G D P - R a c conversion to GTP-Rac, 16-17, 24 GTPyS-stimulated phospholipase D, 254 interaction with Rac, role of Rac lipid modifications, 17 microinjection into cultured cells, 344-345 preparation for microinjection, 343-344 purification, 99-101,252-253 recombinant complex formation with G D P - R h o A , activity for, assay, 46-47 induction, 44 inhibition of actomyosin-dependent RhoA functions, 48 properties, 45-48 purification, 44-45 regulation of GDP/GTP exchange reaction of RhoA, 45-46 RhoA translocating activity, assay, 47-48 role in NADPH oxidase inhibition, 23, 24-25 solubilization of Cdc42Hs from membranes, 98-105 assay, 104-105
399
Rho-GTPase-activating protein binding constants for GDP- and GTPbound Rho, Rac, and G25K, 76 conserved residues, alignment, 137-138 L61G25K, L61Racl, and L63RhoA affinities for, comparison, 240 L63RhoA and wild-type RhoA interactions with, comparison, 237-238 related activity of p190, assay, 112-113 Rho/Rac mutant interactions with, 209 stimulated GTPase activities of Rho, Rac, and G25K, assays, 74-76 Ribonuclease H, in analysis of Rac function with antisense oligonucleotides, 359, 366 RNA accumulation in absence of protein translation, time course, 158-159 messenger, turnover in response to growth factors, 159-160 Run-on assays, on isolated nuclei, 160-162
S Saccharomyces cerevisiae actin organization, disruption in cdc42 mutants, 282, 283 with cdc42-1 loss of function allele, 282 Cdc42p function, analysis, 281-290 fusion protein-containing cells, growth, 289 Ras-like mutations, 282-284 two-hybrid system for detection of protein-protein interactions, 228-241 media, 231-232 plates, 232-233 solutions for, 233-234 strains, 231 Y190, transformation with pAS and pACTII, 235-236 with pAS and pYTH6, 234-235 Y190:pAS RhoA and Y190:pYTH6 RhoA, transformation, 235 Schizosaccharomyces pombe actin organization, disruption in cdc42 mutants, 282 with cdc42 loss of function allele, 282 Cdc42p function, analysis, 281-290 Ras-like mutations, 282-284
400
SUBJECT INDEX
Sedimentation assay, lipid interaction with proteins, 123-124 Sepharose-Protein A, coupling to monoclonal antibody Y13-259, 89-90 Serine/threonine kinase, p65-PAK GTP-Cdc42-associated, purification, 221-224 purified, activation by Cdc42 and Racl, 225 -226 Serum, induction of RhoG expression, 151-162 Small GTP-binding protein GDP dissociation inhibitor analysis in vivo, 88-90 effects, interpretation, 256 expression in Escherichia coli, 86, 253 interaction with prenoid group of target, 85 promoted guanine nucleotide exchange in vitro, assays, 86-88 purification, 86, 253-254 stimulation of GTP-activated phospholipase D, 255 Small GTP-binding protein GDP dissociation stimulator interaction with Rac, role of Rac lipid modifications, 17 role in NADPH oxidase stimulation, 23, 24-25 stimulation of GDP-Rac conversion to GTP-Rac, 16, 24 SmgGDS, see Small GTP-binding protein GDP dissociation stimulator Solubilization Cdc42Hs from membrane fractions of Sf21 cells, 13 from membranes by Rho-GDI, 98-105 assay, 104-105 neutrophil membranes, 272 Spectrophotometric assay, neutrophil cytochrome b, 260 Spodoptera frugiperda Sf9 cells baculovirus-infected, p190 purification, 106-109 breakpoint cluster region kinase purification, 127
Cdc42Hs-containing membranes, purification, 103-104 D B L oncogene product expression and purification, 78-79 p190 baculovirus production, 106 Sf21 cells Cdc42Hs expression via baculovirus infection, 12-13 membrane fractions Cdc42Hs purification, 13-15 preparation, 13-14 Src homology 2 domain binding by breakpoint cluster region kinase, assay, 128-129 breakpoint cluster region kinase motifs binding, 126 Src homology 3 domain function, 140 screening, in identification of 3BP-1 in cDNA expression library, 140-148 Staining, Swiss 3T3 cells, 310, 317 Staphylococcus aureus, C3-1ike exoenzyme, 187 Subcloning, dbl and proto-dbl cDNAs in eukaryotic expression vectors, 349 Superoxide production assay, 276 by neutrophil cells and fractions, assay, 260 during respiratory burst effect of Rac antisense oligonucleotides, 362-365 measurements, 361-362
T Temperature effects ADP-ribosylation of Rho, 191 binding assay for Rho and Rac interactions with GAPs, 211 Testis, rat, extracts, GTPase-activating protein identification, 132-137 Thin-layer chromatography assay neutrophil phospholipase D, 250 p190 R h o - G A P activity, 112 Thrombin cleavage of C3 transferase-glutathione S-transferase from glutathione beads, 178
SUBJECTINDZX cleavage of glutathione S-transferaseRho-GDI, 45 induction of actin reorganization in Swiss 3T3 cells, 311-312 Transcription, on isolated nuclei, 161 Transcription factors, GAL4, in yeast twohybrid system for detection of proteinprotein interactions, 228 Transfection, COS1 cells by electroporation, 88-89 Transformation, by dbl oncogene, 347-358 Transforming growth factor-/3, effect on actin organization in Swiss 3T3 cells, 312 Trypan blue, in assessment of lymphocyte viability after electroporation, 323 Tumor cells hepatoma, FAO, C3-catalyzed ADP-ribosylation, detection, 194-195 pheochromocytoma, PC-12, treatment with C3 exoenzyme, 206 promyelocytic leukemia, HL-60 cytosol fractions, preparation, 19 differentiated, preparation, 19 membranes fractions, preparation, 19 solubilized components, preparation, 22 preparation, 35 Rac2 purification, 18-22
401
Tumor necrosis factor-a, effect on actin organization in Swiss 3T3 cells, 312 Tumors, dbl-induced, mice bearing, derived antibodies, 351-352 V Vero cells cytopathic effect of C3 exoenzymediphtheria toxin fragment B, 303-306 morphology, effect of C3 ADP-ribosyltransferase, 335 Vitronectin, effect on actin organization in Swiss 3T3 cells, 312
W Western blotting Rac, 261,361 Rac-GDI, 35-36 Y Yeast, see Saccharomyces cerevisiae; Schizosaccharomyces pornbe
Z Zinc, effect on phorbol ester binding to refolded protein, 119-120