Rho GTPases (Molecular Biology Intelligence Unit)

MOLECULAR BIOLOGY INTELLIGENCE UNIT

Marc Symons SYMONS

Rho GTPases

MBIU

Rho GTPases 9/8/03, 4:36 PM

MOLECULAR BIOLOGY INTELLIGENCE UNIT

Rho GTPases Marc Symons, Ph.D. Laboratory of Tumor Cell Biology North Shore-LIJ Research Institute Manhasset, New York, U.S.A.

LANDES BIOSCIENCE / EUREKAH.COM GEORGETOWN, TEXAS U.S.A.

KLUWER ACADEMIC / PLENUM PUBLISHERS NEW YORK, NEW YORK U.S.A.

RHO GTPASES Molecular Biology Intelligence Unit Eurekah.com / Landes Bioscience Kluwer Academic / Plenum Publishers Copyright ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system; for exclusive use by the Purchaser of the work. Printed in the U.S.A. Kluwer Academic / Plenum Publishers, 233 Spring Street, New York, New York, U.S.A. 10013 http://www.wkap.nl/ Please address all inquiries to the Publishers: Eurekah.com / Landes Bioscience, 810 South Church Street Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081 www.Eurekah.com www.landesbioscience.com Rho GTPases edited by Marc Symons, Landes / Kluwer dual imprint / Landes series: Molecular Biology Intelligence Unit ISBN: 0-306-47992-3

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

CONTENTS Preface ................................................................................................... x 1. Signalling Networks of Rho GTPases .................................................... 1 Emmanuel Vignal, Anne Blangy and Philippe Fort Abstract ....................................................................................................... 1 Introduction ................................................................................................ 1 Emergence of the Rho Family ...................................................................... 1 Biological Activities of “Unconventional” Rho Proteins ............................... 3 Signalling Networks Controlled by Rho Proteins in Mammals .................... 8 Concluding Remarks ................................................................................. 10

2. Intracellular Targeting of Rho Family GTPases: Implications of Localization on Function ............................................. 17 David Michaelson, Mark Rush and Mark R. Philips Rho Proteins—Diverse Functions Despite Sequence Homology ................ 17 Targeting Motifs—Location, Location, Location ....................................... 19 C-Terminal Targeting Motifs—The Importance of Being Hypervariable ... 25 TC10 and Cdc42—A Tail of Two GTPases .............................................. 26 RhoGDI—The Catcher in the Cytosol ...................................................... 27 Conclusions—You Are Where You Are ..................................................... 28

3. Regulation of the RhoGTPases by RhoGDI ......................................... 32 Gregory R. Hoffman and Richard A. Cerione Abstract ..................................................................................................... 32 Introduction .............................................................................................. 32 GDIs As Multifunctional Regulators of Signalling through Rho GTPases ........................................................................... 33 Structural Insights into GDI Function ....................................................... 35 Future Directions ...................................................................................... 41 Conclusion ................................................................................................ 43

4. Rho Family Guanine Nucleotide Exchange Factors .............................. 46 Ian P. Whitehead Abstract ..................................................................................................... 46 Introduction .............................................................................................. 46 Structure of RhoGEFs ............................................................................... 49 Regulation of RhoGEFs ............................................................................. 50 Physiological Roles for RhoGEFs ............................................................... 56 RhoGEFs and Human Disease .................................................................. 61 Future Perspectives .................................................................................... 63

5. The Superfamily of Rho GTPase-Activating Proteins .......................... 68 Sarah Jenna and Nathalie Lamarche-Vane Abstract ..................................................................................................... 68 RhoGAPs Are Multimodular Proteins ....................................................... 68 Molecular Basis of the GTPase-Stimulating Activity .................................. 72 RhoGAPs Are Multifunctional Proteins ..................................................... 74 RhoGAPs in Host-Pathogen Interaction .................................................... 81 Conclusion ................................................................................................ 85

6. Rnd Proteins: Intriguing Members of the Rho Family ......................... 96 Pierre Chardin Abstract ..................................................................................................... 96 General Features ........................................................................................ 96 Prenylation ................................................................................................ 96 Chromosomal Localization ........................................................................ 98 Biochemical Properties of Rnds ................................................................. 98 Do Rnd Proteins Need GEFs for Activation .............................................. 99 Rnds As Targets of Bacterial Toxins ........................................................... 99 Expression in Human Tissues .................................................................... 99 Localization of Rnd1 in Swiss 3T3 Fibroblasts and Microinjected MDCK Cells ....................................................................................... 101 Rnd1 or Rnd3 Expression Inhibit the Formation of Actin Stress Fibers ... 101 Rnds Inhibit Smooth Muscle Contraction ............................................... 101 Proteins Interacting with Rnds ................................................................ 102 Effects of Rnd1 in Neurons ..................................................................... 102 Induced Expression of Rnd3 Participates in the Transformation of Epithelial Cells by Raf ..................................................................... 103 Rnds in Development .............................................................................. 104 Perspectives .............................................................................................. 104

7. Rho GTPases in the Organization of the Actin Cytoskeleton ............. 107 Ed Manser Abstract ................................................................................................... 107 Introduction ............................................................................................ 107 Different Actin Networks: Membrane Ruffles, Filopodia and Stress Fibres .................................................................................. 108 Actin–Myosin II Contractility and Stress Fibre Formation ...................... 109 Controlling Actin Treadmilling ............................................................... 110 Arp2/3-Mediated Actin Nucleation and Branching ................................. 111 Stimulating Actin Assembly ..................................................................... 111 Tethering Assembled Actin to Membranes .............................................. 112 The Focal Adhesion Complex-Actin Connection ..................................... 112 Conclusions and Future Directions .......................................................... 112

8. Cell-Cell Adhesion ............................................................................. 115 Vania M.M. Braga and Martha Betson Introduction ............................................................................................ 115 Characteristics of Epithelial Cells ............................................................. 115 Cadherin Receptors ................................................................................. 117 Experimental Models to Investigate Cell-Cell Adhesion Regulation ......... 120 Regulation of Cadherin Function ............................................................ 122 Signalling from Cadherin Receptors ........................................................ 130 Conclusions ............................................................................................. 132

9. Rho GTPases and Cell Motility ......................................................... 141 Alexander Beeser and Jonathan Chernoff Abstract ................................................................................................... 141 Rho GTPases and Cell Polarity ................................................................ 142 Actin Polymerization at the Leading Edge ............................................... 145 Remodeling Focal Contacts ..................................................................... 148 The Generation of Contractile Force ....................................................... 150 Summary ................................................................................................. 152

10. The RhoGTPase in Tumor Invasion .................................................. 155 Arthur M. Mercurio Abstract ................................................................................................... 155 Introduction ............................................................................................ 155 RHO Involvement in Tumor Progression................................................ 156 Mechanisms of Rho Involvement in Invasion .......................................... 157 Conclusions ............................................................................................. 159

11. Rho Family GTPases and Cellular Transformation ............................ 163 Antoine E. Karnoub and Channing J. Der Abstract ................................................................................................... 163 Introduction ............................................................................................ 163 The Rho Family of GTPases Is a Major Branch of the Ras Superfamily ......................................................................... 163 Rho GTPases Function As Membrane-Associated GDP/GTP-Regulated Molecular Switches ........................................... 164 Rho Family GTPases Are Critical Components of Diverse Signalling Pathways ............................................................................. 169 Rho GTPases Promote Oncogenic Transformation ................................. 170 Rho GTPases Mediate Transformation through Multiple Effectors ......... 175 Conclusions and Future Directions.......................................................... 177

12. Rho GTPases in Lipid Metabolism .................................................... 185 Maikel P. Peppelenbosch Abstract ................................................................................................... 185 Introduction ............................................................................................ 185 Rho Family GTPases and Phosphatidylintositol-3-Phosphates ................. 185 Rac-Mediated Arachidonic Acid Release .................................................. 188 Rho A As an Effector of Arachidonic Acid Metabolism............................ 190 Rho Family GTPases and Inositol-1,4,5-Triphosphate Generation .......... 190 Phospholipase D As an Effector of Rho Family GTPases ......................... 191 Final Considerations ................................................................................ 192

13. The Role of Rho GTPases in Vesicle Trafficking ............................... 196 Nicole Rusk and Marc Symons Summary ................................................................................................. 196 Introduction ............................................................................................ 196 Role of Rho GTPases in Clathrin-Mediated Endocytosis ......................... 196 Macropinocytosis ..................................................................................... 199 Phagocytosis ............................................................................................ 199 Exocytosis ................................................................................................ 201 Trafficking in Polarized Epithelial Cells ................................................... 202 Trafficking Controlled by Rho GTPases in the Regulation of Cell Motility and Cell-Cell Junctions .............................................. 203 Conclusion .............................................................................................. 203

14. The Dictyostelium Rho Family of Proteins and Effectors .................. 208 David Seastone and James A. Cardelli Abstract ................................................................................................... 208 Choice of Dictyostelium As an Experimental System ............................... 208 An Overview of the Dictyostelium Rho Family of GTPases ..................... 209 Rho Family GTPase Regulators and Effectors .......................................... 212 Other Proteins that Interact with Rho Family GTPases ........................... 215 Summary ................................................................................................. 216

15. Rho GTPases in Plants ...................................................................... 219 Aline H. Valster, Peter K. Hepler and Jonathan Chernoff Abstract ................................................................................................... 219 Introduction ............................................................................................ 219 Rop Identity ............................................................................................ 219 Regulation of Rop ................................................................................... 221 Rop Localization ...................................................................................... 222 Rop-Regulated Processes .......................................................................... 223 Concluding Remarks ............................................................................... 227

Index .................................................................................................. 231

EDITOR Marc Symons, Ph.D. Laboratory of Tumor Cell Biology North Shore-LIJ Research Institute Manhasset, New York, U.S.A. [email protected] Chapter 13

CONTRIBUTORS Alexander Beeser Fox Chase Cancer Center Philadelphia, Pennsylvania, U.S.A. Chapter 9

Martha Betson Harvard Medical School Charlestown, Massachusetts, U.S.A.

James A. Cardelli Department of Microbiology and Immunology The Feist-Weiller Cancer Center LSU Health Sciences Center Shreveport, Louisiana, U.S.A. [email protected] Chapter 14

Chapter 8

Anne Blangy Centre de Recherches en Biochimie des Macromolécules - CNRS UPR1086 Montpellier, France Chapter 1

Vania M.M. Braga Cell and Molecular Biology Section Division of Biomedical Sciences Imperial College School of Medicine London, U.K. [email protected] Chapter 8

Richard A. Cerione Department of Chemistry and Chemical Biology Baker Laboratory Department of Molecular Medicine Veterinary Medical College Cornell University Veterinary Medical Center Ithaca, New York, U.S.A. [email protected] Chapter 3

Pierre Chardin Dynamique moléculaire des compartiments cellulaires Institut de Pharmacologie du CNRS Valbonne, France [email protected] Chapter 6

Jonathan Chernoff Fox Chase Cancer Center Philadelphia, Pennsylvania, U.S.A. [email protected] Chapters 9, 15

Channing J. Der University of North Carolina at Chapel Hill Lineberger Comprehensive Cancer Center Chapel Hill, North Carolina, U.S.A. [email protected] Chapter 11

Philippe Fort Centre de Recherches en Biochimie des Macromolécules - CNRS UPR1086 Montpellier, France [email protected] Chapter 1

Peter K. Hepler Department of Biology 221 Morrill Science Center III University of Massachusetts Amherst, Massachusetts, U.S.A. Chapter 15

Gregory R. Hoffman Department of Cell Biology Harvard Medical School Boston, Massachusetts, U.S.A. Chapter 3

Sarah Jenna Department of Surgery Organelle Signaling Laboratory Royal Victoria Hospital McGill University Montreal, Quebec, Canada Chapter 5

Antoine E. Karnoub Department of Biochemistry and Biophysics University of North Carolina Chapel Hill Chapel Hill, North Carolina, U.S.A. [email protected] Chapter 11

Nathalie Lamarche-Vane Department of Anatomy and Cell Biology McGill University Montreal, Quebec, Canada [email protected] Chapter 5

Ed Manser Institute of Molecular and Cell Biology Glaxo-IMCB Laboratory Singapore [email protected] Chapter 7

Arthur M. Mercurio Deptartment of Pathology Beth Israel Deaconess Medical Center Research North Harvard Medical School Boston, Massachusetts, U.S.A. [email protected] Chapter 10

David Michaelson Department of Cell Biology New York University School of Medicine New York, New York, U.S.A. Chapter 2

Maikel P. Peppelenbosch Laboratory for Experimental Internal Medicine Academic Medical Centre Meibergdreef 9 Amsterdam, The Netherlands [email protected]

David Seastone Department of Biochemistry Pikeville College of Osteopathic Medicine Pikeville, Kentucky, U.S.A.

Chapter 12

Aline H. Valster Laboratory of Tumor Cell Biology North Shore-LIJ Research Institute Manhasset, New York, U.S.A [email protected]

Mark R. Philips Medicine and Cell Biology New York University School of Medicine New York, New York, U.S.A. [email protected] Chapter 2

Mark Rush Department of Biochemistry New York University School of Medicine New York, New York, U.S.A.

Chapter 14

Chapter 15

Emmanuel Vignal Centre de Recherches en Biochimie des Macromolécules - CNRS UPR1086 Universite de Montpellier Montpellier, France Chapter 1

Chapter 2

Nicole Rusk Laboratory of Tumor Cell Biology North Shore-LIJ Research Institute Manhasset, New York, U.S.A. [email protected]

Ian P. Whitehead Dept. of Microbiology and Molecular Genetics New Jersey Medical School Newark, New Jersey, U.S.A. [email protected]

Chapter 13

Chapter 4

PREFACE

M

embers of the Rho family of GTP-binding proteins (GTPases) act as molecular switches that mediate a large number of signals, including growth factors, cytokines and adhesion proteins. Accordingly, these GTPases control a vast array of cellular functions, ranging from the organization of the actin cytoskeleton, microtubule dynamics, cell adhesion, cell motility and cell cycle progression. Over the past several years, our knowledge about Rho proteins has rapidly expanded. Over 20 Rho GTPases have been identified in mammals. Of these, RhoA, Rac1 and Cdc42 have been extensively characterized. Chapter 1 gives an overview of other members of the Rho family and also discusses multiple modes of cross-talk between Rho proteins. The nucleotide state of Rho proteins is regulated by three types of regulatory proteins. Guanine nucleotide exchange factors (GEFs, discussed in Chapter 4) activate Rho proteins by catalyzing the exchange of GDP for GTP. GTPase activating proteins (GAPs, Chapter 5) promote the intrinsic GTP hydrolysis of Rho GTPases, causing their inactivation. Lastly, guanine nucleotide dissociation inhibitors (GDIs, Chapter 3) preferentially bind to GDP-bound GTPases and prevent spontaneous and GEF-catalyzed release of nucleotide, thereby maintaining GTPases in the inactive state. The three members of the Rnd subfamily differ from most other Rho proteins in that they lack GTPase activity and therefore are likely to be constitutively active (reviewed in Chapter 6). A requisite for the proper intracellular targeting of most Rho family GTPases is their post-translational modification by the addition of a prenyl group to the C-terminal CAAX motif. The prenyl group serves as a lipid anchor in the localization of Rho GTPases to their respective membrane compartments (reviewed in Chapter 2). As discussed in Chapters 2 through 5, the three classes of regulators in turn are subject to complex regulation. A critical aspect of these regulatory mechanisms is that they are highly localized. This is especially important for Rho family GTPases such as Cdc42 and Rac, which have been implicated in biological processes that involve directionality, including chemotaxis, neurite outgrowth and cell polarization. Over the past several years, dramatic progress has been made in the elucidation of the signaling pathways that mediate some of the functions that are controlled by Rho proteins. The first biological function that was recognized for RhoA, Rac1 and Cdc42, a little more than a decade ago, was their regulation of distinct features of the actin cytoskeleton. In the intervening time, a concerted effort by a large number of laboratories has elucidated several pathways that mediate the organization of the actin cytoskeleton downstream of these GTPases (reviewed in Chapter 7).

The next four contributions review other well-characterized biological functions that are governed by Rho family GTPases. Chapter 8 discusses the role of Rho GTPases in cell-cell adhesion, focusing on adherens junctions in mammalian epithelial cells. Chapter 9 describes how Rho, Rac and Cdc42 direct the sequential steps that lead to cell migration: establishment of cell polarity, induction of cell protrusions, remodeling of cell-substrate contacts and contractile force generation. Two Chapters discuss the multiple roles that are played by Rho family proteins in cancer. Chapter 10 focuses on Rho-mediated tumor cell invasion and Chapter 11 gives a comprehensive overview of the many contributions of Rho proteins to the process of cell transformation, including the stimulation of cell cycle progression and cell survival. The past few years have seen the emergence of additional Rho GTPase-regulated functions. Chapter 12 reviews the impact of Rho GTPases on lipid metabolism, and Chapter 13 discusses recent findings that Rho proteins regulate many steps of vesicular trafficking. In light of the plethora of functions that are regulated by Rho family proteins, it is not surprising that these GTPases also play critical roles during the development of multicellular organisms. A powerful system that is amenable to both genetic and biochemical approaches to study the roles of Rho proteins in development in addition to other biological functions, is provided by Dictyostelium discoideum (reviewed in Chapter 14). In plants, Rho GTPases form a unique group, called Rop GTPases, that appear to be regulated in a manner that is distinct from that of animal Rho proteins. It is therefore expected that the elucidation of the regulatory mechanisms and functions of Rops will shed new light on the core principles that determine the action of Rho GTPases. Rho GTPases have provided students of cell biology and physiology as well as developmental biology with powerful tools to dissect signal transduction pathways. My hope is that this book will serve as a reference point for researchers that are active in these fields and that it will spur the interest in these versatile proteins for new investigators. Marc Symons Laboratory of Tumor Cell Biology North Shore-LIJ Research Institute Manhasset, New York, U.S.A.

CHAPTER 1

Signalling Networks of Rho GTPases Emmanuel Vignal, Anne Blangy and Philippe Fort

Abstract

G

TPases of the Rho family act as molecular switches that convert extracellular signals into multiple cellular effects. Although more than 20 members are known in mammals, only RhoA, Rac1 and Cdc42 have been studied extensively. This chapter gives an overview of the current knowledge on the cellular activities of the other Rho members and describes how multiple Rho activities are coordinated to allow the establishment of complex cellular processes.

Introduction Like other proteins of the Ras-like superfamily, most Rho GTPases are low molecular weight proteins (21-28 kDa) that bind guanine nucleotides GDP or GTP and hydrolyze GTP. These proteins act as signalling gates, which switch on when bound to GTP and switch off when bound to GDP. The first clue of a role of Rho proteins in the control of the actin cytoskeleton came from the correlation between the disappearance of F-actin microfilaments in Vero cells and RhoC ADP-ribosylation upon C. botulinum C3 toxin treatment.1 The causal link between Rho activity and cytoskeletal organization was next concluded from the cellular effects of microinjected recombinant RhoA protein.2 This was followed in 1992 by the demonstration of the pivotal roles of Rac1 and RhoA in remodeling of the cytoskeleton of cells treated by growth factors.3,4 These milestone papers evidenced that Rac1 controls cell spreading through membrane ruffling while RhoA controls cell contraction through F-actin stress fiber assembly. Cdc42 was shown later on to control the formation of filopodia, a third type of F-actin structure also involved in cell motility and spreading.5 Since then, pathways controlled by Rho GTPases have been under intense investigation and have generated over 2000 publications. This wealth of data implicates Rho proteins in a wide variety of fundamental physiological processes, among which cell adhesion and motility, cell proliferation, transformation and apoptosis, cell differentiation or development. Meanwhile, numerous additional Rho proteins have been characterized in mammals and in other eukaryotes. Paradoxically, 95% of the literature has focused only on the cellular activities of RhoA, Rac1 and Cdc42, and the function of most Rho proteins remains poorly known. This review summarizes our current knowledge on "unconventional" Rho members in mammals and emphasizes regulatory crosstalk that coordinate multiple Rho activities in complex physiological processes.

Emergence of the Rho Family The first Rho member identified has been isolated serendipitously in 1985 from a low stringency screen of an Aplysia cDNA library.6 The corresponding mRNA was found to encode a 192 aa protein sharing 35% aminoacid similarity with H-Ras, thus termed Rho, for Ras Homologous. Additional cross hybridization experiments identified human homologues, and also suggested the presence of Rho sequences in the genomes of Drosophila and yeast. This was Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Rho GTPases

Figure 1. Clusters of the Rho family. Aminoacid sequences of mammalian Rho members were aligned and a bootstrapped unrooted tree was constructed. Aminoterminal extensions and heterogenous carboxyterminal regions were omitted from the alignment. Although RhoBTB3 is connected to the RhoBTB cluster, it was excluded from the comparison because of its high level of divergence.

confirmed by the cloning of two yeast homologues.7 Human cDNAs encoding two close relatives to Rho8-10 and two more distantly related sequences, termed rac1 and rac2 (for Ras related C3 botulinum toxin substrate,11) were characterized in 1989. These latter turned out to be in fact poor C3 toxin substrates.12 S. cerevisiae CDC42 protein - involved in the establishment of cell polarity - as well as its human counterpart were next identified as Rho members in 1990.13-15 Strategies for directly hunting new Ras-like sequences16 led to the identification of TC10, a CDC42-related Rho member.17 The Rho family grew bigger during the 90’s: RhoG, a growth-regulated gene product,18 TTF/RhoH, whose gene was found translocated in B cell lymphomas,19 RhoD, cloned by a PCR approach,20,21 Rac322,23 and three Rnd proteins,24 isolated from low stringency screenings. Chp (Cdc42 Homologous Protein) was identified through its ability to bind p21 activated kinases (PAK)25 and recently, Wrch1 (Wnt-1 responsive Cdc42 homolog) was identified from a differential screening for downstream targets of Wnt.26 Meanwhile, the development of genomic projects made it possible to screen the whole human and murine genomes for new Rho members. This allowed the discovery of TCL

Signalling Networks of Rho GTPases

3

(TC10-Like)27 and Rif (Rho in filopodia).28 The hunt for new Rho proteins was not quite over since a new subgroup, termed RhoBTB, was characterized recently. In human, it includes three sequences encoding 600-700 aa proteins, whose N-terminal moiety is bona fide Rho sequence and C-terminal moiety contains BTB protein-protein interacting domains.29 Numerous Rho proteins were also identified in other organisms. To date, six Rho proteins are known in yeast (Cdc42, Rho1-5),30 six in C. elegans (Rac1, Rac2, RhoA, Cdc42, Mig-2 and F22E12) and six in D. melanogaster (Rac1, Rac2, Rho1, Mtl-1, RhoL and RhoBTB),29 thirteen in plants (termed Rop for Rho of plants (for a review, see ref 31) and at least 15 in the slime mold D. discoideum.29

Biological Activities of “Unconventional” Rho Proteins Comparison of the 21 mammalian Rho protein sequences shows that they are distributed in four main sub-groups (Fig. 1): The Rho cluster (RhoA, RhoB and RhoC), the Rnd cluster (Rnd1, Rnd2 and Rnd3), the RhoBTB cluster (RhoBTB1, RhoBTB2 and RhoBTB3) and the Rac/Cdc42 constellation, including the Rac cluster (Rac1, Rac2, Rac3), the Cdc42 cluster (Cdc42, TC10, TCL), RhoG, Chp1 and Chp2/Wrch-1. The remaining proteins (RhoD, Rif and RhoH/TTF) delineate distinct stems in the tree. Given that the complete human genome sequence is now available, this represents probably the final picture of the Rho family in mammals. Since attention has focused mostly on RhoA, Rac1 and Cdc42, the structure of the Rho family raises several questions. Do similar Rho proteins exert similar biological functions? Are they expressed differentially? Are similar proteins regulated by the same factors? Do they bind to similar targets?

The Rho Subgroup In addition to RhoA, this sub-group includes RhoB and RhoC. RhoA and RhoC share 95% amino acid similarity, while RhoB shares about 86% similarity with RhoA and RhoC. Homologues of RhoA, RhoB and RhoC are also found in chicken cDNA databases.29 Amino acid changes between the three Rho proteins are concentrated in the C-terminus, involved mainly in plasma and intracellular membranes targeting.32 Due to their high level of similarity, the technical approaches used in most papers cannot discriminate either protein. For instance, the widely used C. botulinum C3 toxin inactivates all three proteins.33 In addition, most RhoA targets identified so far also interact with RhoB and RhoC. Thus for a large part, activities attributed to RhoA must be considered as a combined "Rho" activity. This is true for actin stress fiber assembly, also triggered by RhoB34 and RhoC.35 However, despite their high level of similarity, there are substantial differences between the three GTPases, not only in the regulation of their expression but also in their cellular activities. RhoB gene is an immediate-early response gene inducible by growth factors or UV,36-38 which encodes a protein mainly distributed in endosomes.39 RhoB is an unstable protein (t1/2 = 2 hours) that accumulates during the S phase of the cell cycle40 and is stabilized in response to TGFβ.41 RhoB expression modulates SRE gene transcription like RhoA does,42 but also affects specific transcription pathways.42,43 RhoB also specifically regulates Epidermal Growth Factor (EGF) receptor traffic through its downstream effector PRK1.44,45 A very particular feature that distinguishes RhoB is its ability to be both farnesylated and geranylgeranylated, whereas RhoA and RhoC are only geranylgeranylated.46 Both types of modifications appear to generate functionally distinct properties, since active RhoB shows weak but significant transforming activity42 and an anti-transforming activity when only geranylgeranylated.47 As far as RhoC is concerned, no information is available yet about specific activities, regulators or targets. However, recent evidence suggests that RhoC controls some pathways distinct from those controlled by RhoA. In particular, the level of expression of RhoC but not RhoA correlates positively with the metastatic properties of melanoma cells and seems to be responsible for their invasive properties.48 Similar findings have been observed in pancreatic adenocarcinoma49 and in inflammatory breast cancers.35

4

Rho GTPases

The Rnd Cluster Rnd proteins represent a distinct group of Rho proteins, which are devoid of GTPase activity. The biology of Rnd proteins is described in Chapter 6.

The RhoBTB Cluster Sequences encoding RhoBTB proteins have been detected in mammals, in the fly and in the slime mold.29 RhoBTB members are also present in chicken (AL587471). Human RhoBTB2 is identical to DBC2 mRNA (AY009093), annotated in databases as homozygously deleted in breast cancer cells. No biological activity of RhoBTB members has been reported so far.

The Rac1/Cdc42 Subgroup The Rac Cluster In addition to Rac1, this cluster includes Rac2 and Rac3, which both share 96% amino acid sequence similarity with Rac1. Rac2 has been extensively analyzed in haemopoietic cells where it is exclusively expressed. Human Rac3 and its chicken homologue cRac1B show preferential expression in brain.23,50 Two Rac3/cRac1B homologues are also present in fish brain cDNA libraries (AW281145 and AW305669). Overexpression of cRac1B in primary neurons elicits neurite outgrowth, whereas no such effect is observed when cRac1A (the homologue of the mammalian Rac1) is expressed.51 This differential effect is cell-type specific, since cRac1A and cRac1B elicit similar phenotypes in fibroblasts. This suggests that Rac3/cRac1b controls a specific signalling pathway in the nervous system. Besides, Rac3 was recently reported to be activated in hyperproliferative human breast cancer-derived cell lines and tumor tissues.52 However, it is not known whether Rac3 deregulation correlates with particular breast tumor stages and has a direct impact on cell proliferation. In addition to Rac3, overexpression of Rac1b - a Rac1 splice variant - has been reported to be associated with breast and colon tumors.53,54

The Cdc42 Cluster

This cluster includes two Cdc42 isoforms,14,15 TC1017 and TCL.27 Cdc42 isoforms result from alternative splicing of 3' terminal exons,55 thereby producing proteins with distinct C-terminal ends (10 aminoacids). One isoform (Cdc42u) is ubiquitously expressed, while the second is brain-specific. Both isoforms were shown recently to have slightly different subcellular distributions.32 It is not known if Cdc42 isoforms exert specific cellular activities, since most studies on Cdc42 function did not mention which isoform was used. TC10 shares 68% aminoacid identity with Cdc42 and shows an extension at its N terminus (NH2 extensions are also found in TCL, Chp1, Wrch1, RhoD and Rif ). TC10 homologues are found in other mammals (mouse BF165256, cow BF044629) and also in fish (BF612628) cDNA databases. Expression of a dominant active TC10 in NIH3T3 cells elicits cell rounding, a reduction in actin stress fibers and the formation of peripheral extensions.56,57 The phenotype was similar but more pronounced than the one elicited by active Cdc42. Screens for downstream effectors showed that TC10 and Cdc42 have the ability to bind similar targets, in particular those containing the consensus CRIB (Cdc42/Rac interactive binding) domain, such as (PAK).56 Nevertheless, additional targets have been identified that specifically bind to TC10 or to Cdc42,56,58,59 indicating that these proteins might control specific pathways. This was documented further by the observations that in contrast to mammalian Cdc42, TC10 does not rescue a yeast Cdc42 defective mutant and is not required for neurite extension in PC12 cells.60 Further differences between both proteins reside in their expression pattern. TC10 mRNA is preferentially expressed in heart and skeletal muscle56 and accumulates in axotomized motoneurons61 and in differentiating adipocytes.62 One physiological role of TC10 in adipocytes might be the control of insulin-stimulated glucose uptake.63 Indeed, glucose uptake is controlled by at least two independent pathways: one activates vesicle trafficking through PI3-Kinase

Signalling Networks of Rho GTPases

5

while the other translocates the GLUT4 glucose transporter to the cell surface. This latter pathway appears to be strictly and specifically dependent on TC10 activity. Recently, we identified TCL (closely related to TC10) from human and mouse databases.27 No strict TCL homologue has been deposited yet in other databases. TCL and TC10 mRNAs are distributed in similar tissues, in particular in heart. TCL and TC10 are also very similar at the biochemical level. However, a particular feature that distinguishes TCL is its ability to trigger the formation of highly dynamic ruffles onto the dorsal membrane of fibroblasts, whereas Cdc42 and TC10 activities are restricted to protrusions at the cell periphery.

RhoG

RhoG was first identified as a growth-activated gene.18 The RhoG protein is distantly related to the Rac cluster and to the fly Mtl-1 and the worm Mig-2, which has been implicated in axon guidance.64 RhoG homologues are found in frog and fish databases (BF615856 and BF158461). RhoG mRNA is ubiquitously expressed in mammalian tissues, with preferential expression in haemopoietic tissues, lung and heart.18 Expression of a dominant active RhoG mutant in fibroblasts triggers cellular effects similar to those elicited by active Rac1 and Cdc42: low transforming activity, JNK activation65 and formation of filopodia and membrane ruffling.66 Wild-type RhoG triggers similar effects although to a lower efficiency, whereas dominant negative RhoG has opposite activities. In addition, RhoG is mainly distributed in intracytoplasmic membranes and its activity requires the microtubule network. This feature is probably mediated by kinectin, a protein suspected to bridge cytoplasmic vesicles to the microtubule motor kinesin.67 RhoG was also shown to mediate neurite outgrowth in PC12 cells stimulated by nerve growth factor (NGF).68

Chp and Wrch

Chp has been isolated from a screen for proteins that interact with PAK2,25 while Wrch (Wnt-1 responsive Cdc42 homolog) has been found in a differential screen for Wnt-1 downstream targets.26 Chp and Wrch proteins differ from Rac1 and Cdc42 in that they display N-terminal extensions containing a potential SH3-binding site and in the case of Chp, that it is devoid of CAAX box. Both proteins are preferentially expressed in brain. Sequences homologues to Wrch but not Chp are found in frog (BF611792) and fish (AU167900) cDNA databases. Expression of constitutively active Chp or Wrch activates the JNK pathway, similar to Rac1, Cdc42 and RhoG. Chp however fails to activate the p38 pathway. Active Chp also triggers the formation of lamellipodia, albeit to a much lower efficiency than Rac1 or RhoG, whereas active Wrch elicits the formation of filopodia and promotes G1 to S phase progression of the cell cycle like many Rho GTPases do.69

Other Rho Members RhoH RhoH or TTF (translocation three four) was identified from the analysis of translocation breakpoint between chromosomes 3 and 4, associated with B-diffuse large cell non Hodgkin’s lymphomas.19 As a result of the translocation, the expression of the LAZ3/BCL6 transcription factor is driven by haemopoietic-specific RhoH promoting sequences. A second type of translocation involving RhoH and the immunoglobulin heavy chain locus was shown recently to be associated with multiple myeloma.70 The RhoH amino acid sequence shares 50-60% similarity with other Rho GTPases and is slightly closer to Chp, Wrch and Cdc42. RhoH homologues are only found in mouse databases (BF182503). No information is available on pathways controlled by RhoH.

RhoD and Rif RhoD and Rif are 210-211 aa in length and share 75% sequence similarity. Both proteins show a 15 aa NH2 terminal extension (with respect to e.g., RhoA) and share approximately

6

Rho GTPases

Figure 2. Different types of connections link Rho GTPases activities. Parallel connections: the input signal activates Rho regulators (RhoGEF or RhoGAP) that control several GTPases at once. This allows the simultaneous activation of multiple downstream targets. Series connections: the input signal activates a Rho regulator that controls one GTPase, which in turn initiates a second regulator/RhoGTPase activation. This allows the sequential activation of downstream targets. Redundancy: one particular target is activated by two or more distinct Rho GTPases. Thus, activation of either GTPase elicits identical cellular effects mediated by the common target.

65% similarity with all other Rho GTPases. Homologues of RhoD and Rif are found in pig and cow cDNA databases (BE235803/BE232109 and AW428134/AW652620, respectively). No matches have been hitherto detected in other vertebrate databases. Expression of active RhoD triggers the formation of peripheral extensions and a reduction in actin stress fibers.21 Interestingly, morphological changes are associated with a reduction of early endosome motil-

Signalling Networks of Rho GTPases

7

Table 1. Main biological features of mammalian Rho members mRNA Distribution

Protein Localization

Cytoskeletal Effects

Binding Targets

Downstream Rho req’d.

Activity Opposed with

Ubiq.123

PM, cytosol32,39

SF3

REM-1, RKH124

Rac178

Lung,125 neurons50 RhoC ND

Endosomes39

SF34

ND

PM, cytosol39

SF35

ND

ND

Rac1

Ubiq.11,87

PM, cytosol4

RhoA4

RhoA80,83

Rac2 Rac3

Hemopoietic11 Brain23,50

PM, cytosol 32 ND

lamellipodia, ruffles, SF4 ? filopodia ruffles51 filopodia, lammelipodia, SF5 filopodia56

REM-1, RKH124 REM-1, RKH124 CRIB, coiledcoil116,124 CRIB 124

Rac1, Cdc42, RhoG66,80 ND

ND ND

ND ND

CRIB116

Rac15

RhoA80

CRIB56

ND

ND

CRIB27

Rac1, Cdc4227 Rac1, Cdc4266 ND

ND

RhoA

RhoB

Cdc42 Ubiq.87

TC10

Heart, skeletal muscle56 Heart27

Endomembranes PM32 PM, vesicles56

Chp

Brain, testes25

PM, vesicles27 dorsal ruffles27 PM, ER, lamellipodia vesicles66 filopodia66 PM, Golgi25 lamellipodia25

Wrch RhoH RhoD Rif

Ubiq.26 Hemopoietic19 Ubiq.21 Ubiq.28

ND ND PM, vesicles21 PM28

TCL

RhoG Ubiq.18

filopodia26 ND filopodia21 filopodia28

Not CRIB66 Coiled-coil67 CRIB25 CRIB26 ND ND Not CRIB28

ND ND ND Independent of Rac1 and Cdc4228

RhoA66 ND ND ND RhoA71 ND

Abbreviations: Ubiq : ubiquitously expressed, PM: plasma membrane, ER: endoplasmic reticulum, SF: stress fibers, REM-1: Rho Effector Motif type I, RKH: Rho Kinase Homology, CRIB: Cdc42/Rac Interactive Binding. ND: not determined.

ity. However, no effect on endocytic trafficking was observed when wild-type or dominant negative RhoD were expressed. RhoD activation has also been reported to alter cell migration and cytokinesis.71 Here again, no effect was observed when the dominant negative mutant was expressed. Additional data are clearly needed to confirm RhoD cellular activities and to understand the underlying mechanisms. Like RhoD, Rif triggers the formation of filopodia-like extensions, all around the cell but also on the apical membrane, eliciting a «hairy» phenotype.28 Rif activity is lowered when NH2 tagged, suggesting that the N-terminal extension bears sequence determinants crucial for downstream signalling. Rif appears to shuttle between the plasma membrane, where the GTP bound form preferentially accumulates, and endosomes, when bound to GDP. RhoD and Rif mRNAs are ubiquitously expressed in human tissues, with higher levels in kidney, liver, intestine and lung for RhoD, and in colon, stomach and spleen for Rif.

8

Rho GTPases

Signalling Networks Controlled by Rho Proteins in Mammals As mentioned above, members of the Rho family have been shown to be inserted in a wide range of signalling pathways controlling basic cellular events as diverse as membrane protrusions, actin polymerization, kinase activation, gene transcription or protein targeting. Rho members have partially similar cellular activities, which indicate that i) different Rho GTPases can share one or several pathways in common, ii) several Rho GTPases can be coregulated and iii) Rho GTPases mutually influence their activities. Rho GTPases are therefore thought to be inserted in a global regulatory network in which positive and negative signals are transmitted through multiple parallel and series connections. Parallel connection implies that several GTPases can be controlled by the same regulators, while series connection implies that one GTPase can be controlled by another GTPase (Fig. 2). According to this scheme, different basic cellular events, each controlled by one Rho GTPase, would then be properly coordinated, thus allowing the establishment of more complex physiological processes. For example, the activities of Cdc42, Rac and Rho are coordinated during CSF-1-dependent macrophage chemotaxis (for a review, see ref 72): extensions controlled by Cdc42 are projected towards the CSF-1 gradient. This is associated with the formation of oriented lamellipodia and new adhesions to the extracellular matrix, controlled by Rac. Movement of the whole cell body requires intracellular tension, generated by Rho-dependent actomyosin contractility. Interestingly, in the same cell system, Cdc42, Rac and Rho activities are uncoupled during phagocytosis : only Rac and Cdc42 are necessary to extend membrane projections towards immunoglobulin-coated particles, while uptake of complement C3b-coated particles only requires Rho.73 In addition, Cdc42 but not Rac is necessary for Salmonella invasion of epithelial cells, an endocytic process comparable to phagocytosis.74 Thus, depending on the cell system and the physiological process, activities of Rho proteins can be interconnected according to different branching patterns.

Series Connections Most studies on Rho members have focused mainly on the control of cell morphology. As summarized in (Table 1), Rho GTPases basically elicit the formation of a limited number of cellular structures: stress fiber bundles (RhoA, B and C), lamellipodia and peripheral ruffles (Rac1 and Chp), dorsal ruffles (TCL), microspikes and filopodia - (Rac3, Cdc42, TC10, RhoD, Rif, Wrch, Rnd) or a mixed phenotype (lamellipodia and filopodia (RhoG). This opens the possibility that the cellular effects of a given GTPase are mediated by another GTPase acting downstream.

Positive Signalling Such linear cascades have been identified in fibroblastic cells. For example, Cdc42 elicits the formation of filopodia, followed by the formation of Rac-dependent lamellipodia.5 Similarly, Rnd promotes the formation of Rac-dependent neuritic processes in PC12 cells.75 Despite the high number of Rho GTPases that trigger formation of extensions at the cell periphery, little is known about functional crosstalk. Only Rif activity was shown to be independent of that of Cdc42 and Rac1.28 Another type of cascade is controlled by Rac1, which promotes membrane ruffling and threafter triggers stress fiber assembly through the activation of RhoA.4 Rac1-dependent activation of RhoA might be mediated by Ost, an exchange factor that binds to Rac1 but catalyzes GTP exchange on RhoA.76 The sequential activation of Rac1 and RhoA is also required for PIP5 kinase activation upon G-protein coupled receptor stimulation.77 Interestingly, a reverse cascade has been described in Ras-transformed Rat-1 cells, in which Rac activation by PI3-K requires Rho activity.78 Signalling cascades might also involve more than two GTPases : RhoG was found to activate independently the formation of Rac1-dependent lamellipodia and Cdc42-dependent filopodia and microvilli.66 This is also the case for TCL, whose activity is partially and differently inhibited by dominant negative Rac1 and Cdc42.27

Signalling Networks of Rho GTPases

9

Negative Signalling Although Rac1 or Rif can activate RhoA in particular cell systems, in most situations RhoA is opposed to many other Rho GTPases. In exponentially growing Swiss3T3, REF-52, MDCK or BHK cells, activation of Rac1, Cdc42, RhoG, TCL or Rnd trigger stress fiber disassembly.27,66,79-82 Conversely, RhoA inhibition mimics the cytoskeletal effects of active Rac1 and Cdc42. As shown in NIH3T3 fibroblasts, the antagonistic effects of RhoA and Rac1/Cdc42 correlate with changes in the levels of their GTP-bound forms, suggesting that the balance is mediated through downregulation of GEFs or activation of GAPs.83 A second level of control is potentially mediated by PAK, a target positively activated by at least Rac1 and Cdc42 and shown to inhibit myosin light chain kinase (MLCK) involved in stress fiber bundling and contraction.84 Rac1 and RhoA also have opposed effects in other physiological processes such as macrophage spreading,85 endothelial permeability86 or skeletal muscle differentiation.87 Antagonistic effects have also been evidenced in neuron cells, in which RhoA promotes neurite retraction whereas Rac1 elicits neurite outgrowth.88-90 Recent studies in integrated neuronal systems suggest that differences between RhoA, Rac1 and Cdc42 are not just opposed activities: Rac1 positively controls the level of branching of Xenopus optic tectal neurons and negatively controls axogenesis,91,92 whereas RhoA negatively controls branch extension but has no effect on branch addition or retraction.91 Similar specific activities of Rac1 and RhoA have also been reported in pyramidal neurons of rat hippocampal slice cultures.93 The antagonistic activities of RhoG and RhoA are mediated by at least two mechanisms. First, RhoG might counteract RhoA indirectly through downstream activation of Rac1 and Cdc42. In addition, RhoG and RhoA antagonism might be mediated by kinectin, which binds to RhoG and RhoA via two distinct domains.67 Kinectin overexpression triggers stress fiber disassembly in cells with intact microtubules whereas it triggers a dramatic stress fiber formation when microtubules are disrupted or when RhoG is inhibited. This suggests that kinectin can cooperate with RhoA in stress fiber formation.

Parallel Connections Concomitant activation or inhibition of different Rho activities is also mediated by RhoGEFs, RhoGAPs and RhoGDIs. As far as RhoGEFs are concerned, about 50 proteins can be identified in databases. In brief, RhoGEFs contain a Dbl-Homology (DH) domain usually associated with a Pleckstrin Homology (PH) domain (see Chapter 4). As shown for Dbl, Vav or p115, RhoGEFs are thought to be activated by phosphorylation or translocation upon extracellular stimuli, thereby converting the initial upstream signal into downstream Rho activation.94-100 Only few RhoGEFs have been examined for their exchange specificities and their activities were mostly assayed on RhoA, Rac1 and Cdc42 only. Some RhoGEFs seem to have a narrow specificity (e.g., Lbc, Lfc, GEF-H1, p190, p114 for RhoA, Tiam1 for Rac1, β-PIX for Cdc42), while others have a much broader specificity. This is the case for Dbl and Vav-2, which in vitro promote GTP exchange on at least Rac1, Cdc42, RhoG and RhoA.99,101,102 That Dbl acts on several GTPases was confirmed in vivo using focus formation assays, which showed that Dbl needs activation of at least RhoA, Rac1 and Cdc42 for establishing cell transformation.103 These GTPases are likely to be activated in parallel, since each of them shows low transforming activity whereas they efficiently cooperate in cell transformation.104 Contrarily to RhoGEFs, RhoGAPs downregulate Rho GTPases by enhancing their intrinsic GTPase activity, which favors the inactive GDP-bound form. Also here, at least 50 proteins containing a RhoGAP domain can be identified in databases (see Chapter 5). Most RhoGAPs analyzed so far appear to have a broad specificity, suggesting that RhoGAPs control several Rho-dependent pathways in parallel. Recent reports suggest that like RhoGEFs, the activities of RhoGAPs can be regulated by extracellular signalling.105,106 Parallel Rho regulation can also be achieved by multifunctional regulators such as Trio or Bcr/Abr. Trio contains two GEF domains, one acting on RhoG and Rac1, and the other on RhoA.107,108 Although is not known how the activities of both GEF domains are regulated,

10

Rho GTPases

Trio is crucial for developmental organization of neural tissues, which requires coordinated activities of RhoG, Rac1 and RhoA.109 Bcr/Abr contains a GEF domain, acting on Cdc42, Rac1 and RhoA, and a GAP domain, acting on Rac1 and Cdc42.110 As for Trio, how the activities of both Bcr domains are regulated remains to be characterized. RhoGDI proteins represent a last class of Rho regulators (see Chapter xx). RhoGDIs interfere with the actions of both RhoGEFs and RhoGAPs, and in addition can retrieve Rho GTPases from membranes. Three RhoGDIs have been identified, which act on several Rho GTPases and might therefore represent another regulatory level for controlling simultaneously several Rho-dependent pathways.

Redundancy Different Rho GTPases also elicit similar responses independently, which suggests partially redundant functions. For example, at least Rac1, Cdc42 and TC10 modulate the activity of c-Jun N-terminal Kinase (JNK), p38 or PAK,111,112 as well as the activation of the Serum Response Factor (SRF) and Nuclear Factor κB (NF-κB) transcription factors.60,113,114 A similar situation may hold for focal complexes controlled by Rac1 and Cdc42 and focal adhesions controlled by RhoA. Indeed, these focal structures are very similar in composition and differ mainly in size (for a review, see ref. 115). In most situations, functional redundancy between Rho GTPases is likely to rely on their ability to bind to identical targets. During the last decade, extensive search for Rho targets have allowed identification of numerous effectors of RhoA, Rac1, Cdc42 and TC10. No information is available yet for RhoD, Rif, RhoH, RhoBTBs. Most partners of Rac1 and Cdc42 contain a consensus Rho binding domain, termed CRIB domain.116 CRIB domain-containing proteins are also targets of TC10, TCL, Chp,25,27,56 but not RhoA, Rnd or Rif.28,82,116 CRIB-containing proteins are not targets of all Rac1/Cdc42 members, since RhoG does not bind CRIB motifs,66 and all CRIB proteins do not bind to GTPases with the same affinity. For example, WASP and ACK bind to Cdc42 preferentially,117,118 while PAK1 binds to Rac1, Cdc42 TC10 and TCL with similar apparent affinity. Another group of CRIB proteins termed binders of Rho GTPases (Borg) show preferential binding to Cdc42 and TC10 (and probably to TCL) with the same efficiency but not to Rac1.58 Two types of consensus Rho binding domains have been evidenced among the targets of RhoA : Rho effector motif (REM) and ROK Kinase Homology (RKH).119,120 Although poorly documented, both types of motifs also are likely to bind to RhoB and RhoC: for example, PRK-1 binds to RhoB,44 and p160ROCK binds to RhoB and RhoC (our unpublished results). This might explain the similar effects of RhoA, RhoB and RhoC on actin stress fiber assembly.34,35

Concluding Remarks Although the Rho family contains 21 members in mammals, most of the literature has focused on Rac1, Cdc42 and RhoA. This supported unambiguously the notion that Rho GTPases control many basic aspects of cell physiology (e.g., membrane activity, kinase activation or gene transcription) and cooperate in complex processes (e.g., motility, apoptosis, differentiation). However, we still do not know the full repertoire of basic functions of each of the GTPases. In addition, in many instances the precise relationship between the GTPases that underlies the orchestration of these complex functions remains to be defined. Several reasons account for this ambiguity:

Lack of Accuracy of Tools Used Most conclusions on the cellular functions of Rho GTPases are derived from expression of dominant active or dominant negative mutants. These mutants were first identified from studies on Ras proteins, then extended to Rho proteins. It is not totally clear whether Rho mutants behave as expected and several inconsistencies suggest that some of these might indeed gain new functions (for a review, see ref. 121). In many cases, convergent results using wild-type or

Signalling Networks of Rho GTPases

11

various other mutant proteins could help support the conclusions. Furthermore, given that different Rho members share identical regulators and targets, the expression of a particular mutant is likely to affect pathways controlled by other GTPases. For example, the widely used T17N dominant negative mutants, thought to exert their inhibitory effect by sequestering exchange factors, might inhibit several Rho GTPases simultaneously. This is particularly crucial for closely related GTPases. Indeed, cellular functions attributed for instance to Rac1 or RhoA might be mediated respectively by Rac3 or RhoC.

Fragmentary Data As mentioned above, most data accumulated on pathways controlled by Rho GTPases have focused on RhoA, Rac1 and Cdc42. This represents a clear limitation when specificities of regulators are addressed. Given the high number of potential RhoGEFs and RhoGAPs identified so far (more than fifty of each), high throughput techniques such as the Yeast Exchange Assay we developed recently are clearly needed.122 This is also the case for Rho targets, whose binding specificity remain to be fully analyzed.

Readout Inaccuracy Studies on cellular effects controlled by Rho GTPases have examined mostly changes in cell morphology. These latter fall into three main classes of cell structures : i) ruffles and peripheral lamellipodia, ii) microvilli and peripheral filopodia and iii) stress fibers. In general, these structures are described by low resolution parameters (e.g., presence or absence). It would be certainly valuable to increase the number of parameters which describe these cell structures. For example, the impact of Rho GTPases on the highly dynamic ruffles, lamellipodia and filopodia has been mostly examined on fixed cells. Extending the comparison between Rho GTPases by dynamic studies such as time-lapse videomicroscopy would thus be particularly informative. Many of these limitations will undoubtly be solved in the next few years and completed by gene inactivation studies in mice. This will give a better picture of the cellular functions of each member of the Rho family as well as their crosstalk in more complex processes, in particular cell motility, apoptosis and transformation.

Acknowledgments We wish to thank C. Gauthier-Rouvière for helpful discussions and constant support. This work was supported by institutional grants from the INSERM and the CNRS, and grants from the Ligue Nationale Contre le Cancer («Equipe Labellisée») and the Association pour la Recherche contre le Cancer (n°5284).

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64. Zipkin ID, Kindt RM, Kenyon CJ. Role of a new Rho family member in cell migration and axon guidance in C. elegans. Cell 1997; 90(5):883-894. 65. Roux P, Gauthier- Rouvière C, Doucet-Brutin S et al. The small GTPases Cdc42Hs, rac1 and RhoG delineate raf-independent pathways that cooperate to transform NIH3T3 cells. Curr Biol 1997; 7(9):629-637. 66. Gauthier-Rouvière C, Vignal E, Mériane M et al. RhoG GTPase controls a pathway that independently activates Rac1 and Cdc42Hs. Mol Biol Cell 1998; 9(6):1379-1394. 67. Vignal E, Blangy A, Martin M et al. Kinectin is a key effector of microtubule-dependent RhoG activity. Mol Cell Biol. In press. 68. Katoh H, Yasui H, Yamaguchi Y et al. Small GTPase RhoG is a key regulator for neurite outgrowth in PC12 cells. Mol Cell Biol 2000; 20(19):7378-7387. 69. Olson MF, Ashworth A, Hall A. An essential role for rho, rac, and cdc42 GTPases in cell cycle progression through G1. Science 1995; 269(5228):1270-1272. 70. Preudhomme C, Roumier C, Hildebrand MP et al. Nonrandom 4p13 rearrangements of the RhoH/ TTF gene, encoding a GTP- binding protein, in nonHodgkin’s lymphoma and multiple myeloma. Oncogene 2000; 19(16):2023-2032. 71. Tsubakimoto K, Matsumoto K, Abe H et al. Small GTPase RhoD suppresses cell migration and cytokinesis. Oncogene 1999; 18(15):2431-2440. 72. Ridley AJ. Rho proteins, PI 3-kinases, and monocyte/macrophage motility. FEBS Lett 2001; 498:168-171. 73. Caron E, Hall A. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 1998; 282(5394):1717-1721. 74. Chen LM, Hobbie S, Galan JE. Requirement of CDC42 for Salmonella-induced cytoskeletal and nuclear responses. Science 1996; 274(5295):2115-2118. 75. Aoki J, Katoh H, Mori K et al. Rnd1, a novel rho family GTPase, induces the formation of neuritic processes in PC12 cells. Biochem Biophys Res Commun 2000; 278(3):604-608. 76. Horii Y, Beeler JF, Sakaguchi K et al. A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links Rho and Rac signalling pathways. EMBO J 1994; 13(20):4776-4786. 77. Chatah NE, Abrams CS. G-protein coupled receptor activation induces the membrane translocation and activation of PIP5K Ia by a RAC and RHO-dependent pathway. J Biol Chem 2001; 28:28. 78. Tang Y, Yu J, Field J. Signals from the Ras, Rac and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts. Mol Cell Biol 1999; 19(3):1881-1891. 79. Kozma R, Ahmed S, Best A et al. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol Cell Biol 1995; 15(4):1942-1952. 80. Moorman JP, Luu D, Wickham J et al. A balance of signalling by Rho family small GTPases RhoA, Rac1 and Cdc42 coordinates cytoskeletal morphology but not cell survival. Oncogene 1999; 18(1):47-57. 81. Zondag GC, Evers EE, ten Klooster JP et al. Oncogenic Ras downregulates Rac activity, which leads to increased Rho activity and epithelial-mesenchymal transition. J Cell Biol 2000; 149(4):775-782. 82. Chardin P. Rnd proteins: A new family of Rho-related proteins that interfere with the assembly of filamentous actin structures and cell adhesion. Prog Mol Subcell Biol 1999; 22:39-50. 83. Sander EE, ten Klooster JP, van Delft S et al. Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J Cell Biol 1999; 147(5):1009-1022. 84. Sanders LC, Matsumura F, Bokoch GM et al. Inhibition of myosin light chain kinase by p21-activated kinase. Science 1999; 283(5410):2083-2085. 85. Ory S, Munari-Silem Y, Fort P et al. Rho and Rac exert antagonistic functions on spreading of macrophage- derived multinucleated cells and are not required for actin fiber formation. J Cell Sci 2000; 113(Pt 7):1177-1188. 86. Wojciak-Stothard B, Potempa S, Eichholtz T et al. Rho and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci 2001; 114(Pt 7):1343-1355. 87. Meriane M, Roux P, Primig M et al. Critical activities of Rac1 and Cdc42Hs in skeletal myogenesis: Antagonistic effects of JNK and p38 pathways. Mol Biol Cell 2000; 11(8):2513-2528. 88. Kozma R, Sarner S, Ahmed S et al. Rho family GTPases and neuronal growth cone remodelling: Relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol Cell Biol 1997; 17(3):1201-1211.

Signalling Networks of Rho GTPases

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89. Leeuwen FN, Kain HE, Kammen RA et al. The guanine nucleotide exchange factor Tiam1 affects neuronal morphology; Opposing roles for the small GTPases Rac and Rho. J Cell Biol 1997; 139(3):797-807. 90. Yamaguchi Y, Katoh H, Yasui H et al. RhoA Inhibits the Nerve Growth Factor-induced Rac1 Activation through Rho-associated Kinase-dependent Pathway. J Biol Chem 2001; 276(22):18977-18983. 91. Li Z, Van Aelst L, Cline HT. Rho GTPases regulate distinct aspects of dendritic arbor growth in Xenopus central neurons in vivo. Nat Neurosci 2000; 3(3):217-225. 92. Ruchhoeft ML, Ohnuma S, McNeill L et al. The neuronal architecture of Xenopus retinal ganglion cells is sculpted by rho-family GTPases in vivo. J Neurosci 1999; 19(19):8454-8463. 93. Nakayama AY, Harms MB, Luo L. Small GTPases Rac and Rho in the maintenance of dendritic spines and branches in hippocampal pyramidal neurons. J Neurosci 2000; 20(14):5329-5338. 94. Kozasa T. Regulation of G protein-mediated signal transduction by RGS proteins. Life Sci 2001; 68(19-20):2309-2317. 95. Zhu K, Debreceni B, Bi F et al. Oligomerization of DH domain is essential for Dbl-induced transformation. Mol Cell Biol 2001; 21(2):425-437. 96. Hart MJ, Roscoe W, Bollag G. Activation of Rho GEF activity by G alpha 13. Methods Enzymol 2000; 325:61-71 97. Kato J, Kaziro Y, Satoh T. Activation of the guanine nucleotide exchange factor Dbl following ACK1- dependent tyrosine phosphorylation. Biochem Biophys Res Commun 2000; 268(1):141-147. 98. Majumdar M, Seasholtz TM, Buckmaster C et al. A rho exchange factor mediates thrombin and Galpha(12)-induced cytoskeletal responses. J Biol Chem 1999; 274(38):26815-26821. 99. Schuebel KE, Movilla N, Rosa JL et al. Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2. Embo J 1998; 17(22):6608-6621. 100. Bustelo XR. Regulatory and Signalling Properties of the Vav Family. Mol Cell Biol 2000; 20:1461-1477. 101. Yaku H, Sasaki T, Takai Y. The Dbl oncogene product as a GDP/GTP exchange protein for the Rho family: Its properties in comparison with those of Smg GDS. Biochem Biophys Res Commun 1994; 198(2):811-817. 102. Abe K, Rossman KL, Liu B et al. Vav2 is an activator of Cdc42, Rac1, and RhoA. J Biol Chem 2000; 275(14):10141-10149. 103. Lin R, Cerione RA, Manor D. Specific contributions of the small GTPases Rho, Rac, and Cdc42 to Dbl transformation. J Biol Chem 1999; 274(33):23633-23641. 104. Fort P. Small GTPases of the Rho family and cell transformation. Prog Mol Subcell Biol 1999; 22:159-181. 105. Fincham VJ, Chudleigh A, Frame MC. Regulation of p190 Rho-GAP by v-Src is linked to cytoskeletal disruption during transformation. J Cell Sci 1999; 112(Pt 6):947-956. 106. Ren XR, Du QS, Huang YZ et al. Regulation of CDC42 GTPase by proline-rich tyrosine kinase 2 interacting with PSGAP, a novel pleckstrin homology and Src homology 3 domain containing rhoGAP protein. J Cell Biol 2001; 152(5):971-984. 107. Bellanger JM, Lazaro JB, Diriong S et al. The two guanine nucleotide exchange factor domains of Trio link the Rac1 and the RhoA pathways in vivo. Oncogene 1998; 16(2):147-152. 108. Blangy A, Vignal E, Schmidt S et al. TrioGEF1 controls Rac- and Cdc42-dependent cell structures through the direct activation of RhoG. J Cell Sci 2000; 113:729-739. 109. O’Brien SP, Seipel K, Medley QG et al. Skeletal muscle deformity and neuronal disorder in Trio exchange factor- deficient mouse embryos. Proc Natl Acad Sci USA 2000; 97(22):12074-12078. 110. Chuang TH, Xu X, Kaartinen V et al. Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family. Proc Natl Acad Sci USA 1995; 92(22):10282-10286. 111. Manser E, Leung T, Salihuddin H et al. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature 1994; 367(6458):40-46. 112. Minden A, Lin A, McMahon M et al. Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. Science 1994; 266(5191):1719-1723. 113. Hill CS, Wynne J, Treisman R. The rho family GTPases RhoA, rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 1995; 81(7):1159-1170. 114. Perona R, Montaner S, Saniger L et al. Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins. Genes Dev 1997; 11(4):463-475. 115. Schwartz MA, Shattil SJ. Signalling networks linking integrins and rho family GTPases. Trends Biochem Sci 2000; 25(8):388-391. 116. Burbelo PD, Drechsel D, Hall A. A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. J Biol Chem 1995; 270(49):29071-29074.

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117. Symons M, Derry JM, Karlak B et al. Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization. Cell 1996; 84(5):723-734. 118. Manser E, Leung T, Salihuddin H et al. A nonreceptor tyrosine kinase that inhibits the GTPase activity of p21cdc42. Nature 1993; 363(6427):364-367. 119. Reid T, Furuyashiki T, Ishizaki T et al. Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain. J Biol Chem 1996; 71(23):13556-13560. 120. Leung T, Chen XQ, Manser E et al. The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol 1996; 16(10):5313-5327. 121. Symons M, Settleman J. Rho family GTPases: More than simple switches. Trends Cell Biol 2000; 10(10):415-419. 122. De Toledo M, Colombo K, Nagase T et al. The yeast exchange assay, a new complementary method to screen for Dbl- like protein specificity: Identification of a novel RhoA exchange factor. FEBS Lett 2000; 480(2-3):287-292. 123. Moscow JA, He R, Gnarra JR et al. Examination of human tumors for rhoA mutations. Oncogene 1994; 9(1):189-194. 124. Bishop AL, Hall A. Rho GTPases and their effector proteins. Biochem J 2000; 348:241-255. 125. Fritz G, Gnad R, Kaina B. Cell and tissue-type specific expression of Ras-related GTPase RhoB. Anticancer Res 1999; 19(3A):1681-1688.

CHAPTER 2

Intracellular Targeting of Rho Family GTPases: Implications of Localization on Function David Michaelson, Mark Rush and Mark R. Philips

Rho Proteins—Diverse Functions Despite Sequence Homology

R

ho proteins are Ras-related GTPases that regulate a wide variety of cellular processes. More than fifteen mammalian Rho proteins have been described including RhoA-E, G and H, Rac1-3, two isoforms of Cdc42hs, and TC10. Originally identified based on their sequence homology to Ras, a great deal of interest in Rho proteins was awakened when Ridley and Hall discovered that RhoA, Rac1 and Cdc42hs differentially regulate the actin cytoskeleton.1-3 Subsequently it was found that Rho family GTPases were also involved in signalling pathways, including the JNK/stress-activated protein kinase4,5 and p38 6 MAP kinase cascades and the serum response factor pathway7 that lead to transcriptional activation. Moreover, Ras-mediated cellular transformation is dependent on Rho GTPases.8 Other cellular processes regulated by Rho GTPases include the phagocyte NADPH oxidase, 9,10 insulin-stimulated recruitment of the GLUT4 glucose transporter to the plasma membrane,11,12 macrophage phagocytosis,13 endocytosis,14-16 epithelial cell polarization,17 and morphogenesis.18 The diversity of functions among Rho proteins belies the extensive sequence homology found among them (Fig. 1). The majority of the amino acid sequences of the Rho proteins are conserved, including those found in the GTP binding domains and the effector loops, giving an overall amino acid identity among family members of 50% or greater with further sequence similarities. Many of the amino acid differences among the Rho proteins occur within the C-terminal 20 amino acids, the so-called hypervariable region. In fact, some Rho proteins are virtually identical except for their hypervariable regions (e.g., Rac 1, 2 and 3; Rho A and B; the two isoforms of Cdc42hs). This region is involved neither in the GTPase activity of Rho proteins nor in the interaction of Rho GTPases with effectors, but it plays a critical role in localization (see below). Of all the various protein-protein interactions in which Rho proteins engage, only the interaction with GDP Dissociation Inhibitors (GDIs) has been found to involve amino acids in the hypervariable region.19 The Guanine nucleotide Exchange Factor (GEF), GTPase Activating Protein (GAP) and effector interactions of Rho proteins depend primarily on sequences outside of the hypervariable region where sequence homology is quite high. As expected, this leads to considerable, though not complete, overlap of affinities shown by Rho proteins for GEFs, GAPs and effectors.20 For example, many Dbl-related GEFs interact with multiple Rho proteins (reviewed in21,22). Similarly, p50RhoGAP has GAP activity for TC10 and Cdc42hs, and somewhat lower activity for RhoA and Rac1.23,24 Among effectors, PAK1-3 interact with Rac1, TC10 and Cdc42hs,24,25 while MLK3 interacts with Rac1 and Cdc42hs, but not TC10.24,26 RhoA and Rac1 also share Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Rho GTPases

Figure 1. Alignment of Rho family amino acid sequences. Amino acid sequences of Rho proteins were aligned using the CLUSTAL method with MacVector software. The resulting consensus sequence is shown beneath the protein sequences. Dark grey boxed residues show regions of high sequence identity among Rho proteins. Light grey boxed residues show regions of sequence similarity. The hypervariable region is underscored with a magenta box. The CAAX motif is underscored with a cyan box.

Intracellular Targeting of Rho Family GTPases

19

Figure 2. Posttranslational modifications of the CAAX motif. CAAX motif processing proceeds in three enzymatic steps: 1) addition of the prenyl group by one of two prenyltransferases (geranylgeranylation is shown), 2) proteolysis of the terminal AAX tripeptide, and 3) methyl esterification of the new C-terminal α carboxyl group by prenylcysteine carboxylmethyltransferase (pcCMT).

effectors such as Citron kinase.27 Given their strong sequence homology and the resulting overlap of protein-protein interactions, how does activation of a specific Rho protein regulate a specific pathway? Some explanation for the diverse functions of Rho family proteins may indeed be based on differences in affinities for GEFs, GAPs and effectors due to subtle differences in the amino acid sequences of the GTPases. However, the considerable sequence divergence within the hypervariable region suggests that at least some of the diversity in function is due to diversity of sequence within this region. What effect does the hypervariable region have on Rho family proteins?

Targeting Motifs—Location, Location, Location Like Ras proteins and G protein γ subunits, Rho GTPases are synthesized as cytosolic proteins that acquire the capacity to associate with membranes by virtue of a series of posttranslational modifications. These modifications occur at the C-terminal CAAX motif and include prenylation, proteolysis, and carboxyl methylation (Fig. 2 and ref. 28). These modifications render the otherwise hydrophilic molecule hydrophobic at its C-terminus, allowing interaction with membrane. The CAAX motif signals for prenylation which in turn targets the protein to the ER where it encounters the RCE1 protease29 and the prenylcysteine carboxyl methyltransferase (pcCMT). 30 In the case of Ras proteins, localization beyond the endomembrane requires a second targeting signal that lies adjacent to the CAAX motif and consists of either cysteines that are sites of palmitoylation (N-Ras and H-Ras), or a polybasic region (K-Ras4B). Whereas N-Ras and H-Ras then traffic to the plasma membrane (PM) on

20

Rho GTPases

Figure 3. Classification of Ras and Rho family hypervariable regions. The hypervariable regions of Ras and Rho family proteins are divided into four classes based on their amino acid sequence immediately upstream of the CAAX motif (underlined). Class I proteins have a hypervariable region with 6 or more positively charged residues (bold) adjacent to the CAAX motif. Class II proteins have one or two palmitoylated cysteine residues (outlined). Class I/II proteins also have one or two palmitoylated cysteines adjacent to the CAAX motif as well as several positively charged residues near the palmitoylation sites and thus are hybrids of the Class I and Class II hypervariable regions. Class III proteins have weakly basic hypervariable domains (less than 6 positive residues adjacent to the CAAX motif ) which in the case of Cdc42 isoforms are neutralized by adjacent acidic residues (shown in grey).

Intracellular Targeting of Rho Family GTPases

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Figure 4. Localization of GFP-tagged full length Ras and Rho proteins in live MDCK cells. The Ras and Rho family proteins are also divided into four classes based on their intracellular localization. The classes defined in (Fig. 3) based on sequence similarity within the hypervariable region match well with the classes shown here based on localization. A.) Class I proteins, with strongly basic hypervariable domains, are expressed almost exclusively on PM. B.) Class II, palmitoylated proteins are found on PM and Golgi. C.) Class I/II proteins, with both palmitoylation and basic residues, localize on PM and on vesicles that colocalize with internalized transferrin. D.) Class III proteins, which have weakly basic or neutral hypervariable domains and no palmitoylation sites, localize like GFP-CAAX, on ER and Golgi. Note the single exception of RhoA. Bars indicate 10 µM.

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Rho GTPases

Figure 5. Localization of Rac Isoforms: correlation of PM localization with positive charge of hypervariable region. The localization of Rac1, 2 and 3 in living MDCK cells are shown to indicate that as the positive charge of the hypervariable region increases, the proportion of protein found in the PM also increases. This shows the distinction between Class I and Class III GTPases as a continuum. Bars indicate 10 µM.

the cytoplasmic face of secretory granules via the Golgi, K-Ras4B takes an alternative, as yet uncharacterized path that displays faster kinetics and is unaffected by inhibitors of the secretory pathway.31,32 This simple dichotomy of Ras targeting mechanisms is also seen among Rho proteins, though a greater variety of sequence within the hypervariable region leads to a greater diversity of membrane localization patterns. Using subcellular fractionation and indirect immunofluorescence, Rho proteins have been localized in cytosol,9,10,33 PM,34 including cholesterol-rich microdomains,12,35 subplasmalemmal actin mesh,36 Golgi,37 endosomes,33,38 multivesicular bodies,36 and nuclei.39 The membrane localization of many Rho family proteins is further complicated by the ability of GDIs to bind and sequester them in the cytosol prior to activation.40-42 Indeed, upon activation, Rho proteins have been shown to translocate from cytosol to membranes.34,43-46 A comparison of hypervariable region sequences of the Ras and Rho family proteins reveals at least four different classes of hypervariable regions (Fig. 3). K-Ras4B, RhoC and Rac1 all have polybasic domains consisting of six or more positively charged residues in close proximity to the CAAX motif. We refer to this as a Class I hypervariable region. H-Ras, N-Ras (not shown) and RhoB all have one or two palmitoylated Cys residues adjacent to the CAAX. We refer to this as a Class II hypervariable region. TC10 has six basic residues just upstream of two palmitoylated Cys residues that are, in turn, immediately upstream of the CAAX motif. We refer to this as a ClassI/II hybrid hypervariable region. TC10 is the only GTPase yet described that has two second signals adjacent to the ER-targeting CAAX motif. RhoD, however, has four positive residues followed by a single palmitoylated cysteine. We tentatively also classify this as a ClassI/II hypervariable region. Finally, Rac2, Rac3, RhoA and Cdc42hs have weak polybasic domains or no obvious second signal at all. This kind of hypervariable region, which we refer to as Class III, would be predicted to target proteins in a manner similar to a CAAX motif alone, predominantly to ER and Golgi. When a hypervariable domain of a Rho protein is fused to the C-terminus of the normally cytosolic green fluorescent protein (GFP), it is sufficient to direct targeting of the fusion protein to membranes (see below). Interestingly, the four classes of hypervariable regions defined above, when fused to the C-terminus of GFP, result in four distinct patterns of membrane localization.42 Thus, the sequence differences within the hypervariable domains can be used to reliably predict the fusion protein’s ultimate subcellular destination. When GFP is fused to the N-terminus of a full length Rho protein the situation is more complicated due to the affinity of most Rho proteins for RhoGDIα, a unbiquitously expressed

Intracellular Targeting of Rho Family GTPases

23

isoform of GDI. At lower levels of expression there is enough endogenous RhoGDIα present in the cell to sequester the GFP-tagged Rho protein in the cytosol. However, at higher levels of expression, membrane localization is dominant since in many cell types the cellular level of endogenous RhoGDIα is equal to the combined levels of endogenous Rho proteins.42 Thus when GFP fused to a full length Rho protein is expressed in live cells at levels sufficient to overwhelm endogenous RhoGDIα, the pattern of membrane localization is similar to that seen with the hypervariable domains alone (see below). As can be seen in (Fig. 4A), GFP fused to full length GTPases with a Class I hypervariable region has a prominent PM localization. In many cell types, GFP fused to either K-Ras4B or Rac1 give indistinguishable localization patterns with predominantly PM fluorescence. This localization of Rac1 determined by tracking the GFP-fused protein is consistent with earlier studies of Rac1 localization. Endogenous Rac1 has been localized by cell fractionation to both cytosol and crude membranes34,35 and has been shown to translocate from cytosol to membranes in response to PDGF.43 Furthermore, indirect immunofluorescence of myc-tagged Rac1V12, a constitutively active mutant of Rac1, shows expression on PM.47 GFP fused to full length GTPases with a Class II hypervariable region localizes in a manner distinct from the predominantly PM localization seen for Class I hypervariable domains. Figure 4B shows that Class II hypervariable region GTPases localize to both PM and Golgi. The perinuclear fluorescence seen with GFP-H-Ras, -N-Ras (not shown) and -RhoB all colocalize with a variety of Golgi-specific markers.32,42 Endogenous RhoB, as well as RhoD, has been previously localized, using indirect immunoflourescence after Triton X-100 permeablization, to endosomes. Colocalization with endosomal markers was extensive for RhoD38 but only partial for RhoB.33 We found that vesicular localization of endogenous RhoB was highly dependent on fixation and permeablization conditions. When saponin was used instead of Triton X-100 to permeablize cells, endogenous RhoB colocalized precisely with Manosidase II, a Golgi marker. This endogenous localization for RhoB in saponin permeablized cells was identical to that of GFP-RhoB seen in live, unfixed cells.42 Interestingly, in saponin permeablized cells RhoD colocalized precisely with internalized transferrin, an endosomal marker38 while RhoB did not. 42 The stronger colocalization of RhoD with endosomal markers under both permeablization conditions, combined with the Golgi localization of RhoB in saponin permeablized cells and of GFP-RhoB in live cells, together suggest that RhoB, like H- and N-Ras, localizes primarily to PM and Golgi and does not localize strongly to endosomes. In contrast, GFP fused to full length TC10 with a Class I/II hybrid hypervariable region localizes to both PM and distinct cytoplasmic, often perinuclear, vesicles that do not colocalize with Golgi markers (Fig. 4C). Instead, these vesicles colocalize with endosomal markers such as internalized Texas-red conjugated transferrin in live cells and the endosomal/lysosomal marker LAMP in fixed cells.42 Interestingly, RhoD has a similar endosomal localization as TC10 38 and, as shown in (Fig. 3), it also has several basic residues upstream of a palmitoylation site near the CAAX motif. The similarity in localization on endosomes suggests that the Class I/II dual second signal may function in localizing both RhoD and TC10. Finally, as expected if CAAX processing alone directs localization, the full length Class III GTPases Rac2 and both the brain and placental isoforms of Cdc42hs (bCdc42 and pCdc42, respectively) are predominantly localized on ER and Golgi (Fig. 4D). Although some GFP-Rac2 and GFP-Cdc42hs localize on PM in some cells, it is always to a lesser extent than their endomembrane localization. This shows that the Class III hypervariable region is inefficient at localizing proteins to PM. Cell fractionation studies have shown that endogenous Cdc42hs localizes to both the cytosol and membrane pellet34,48,49 while indirect immunofluorescence has localized it to Golgi.37 The localization of GFP-pCdc42hs in live cells to ER and nuclear envelope in addition to Golgi demonstrates the superior sensitivity of imaging in live cells. Golgi localization is consistent with the recent observation that Cdc42hs binds the γ-subunit (γCOP) of a coatomer complex50 since γCOP also localizes to the Golgi.

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Rho GTPases

Figure 6. Localization of most Rho GTPases is determined by the hypervariable region. A) Comparision between the localizations of GFP fused to the hypervariable regions of Cdc42hs or Rac1, or to the respective full length proteins indicates that the hypervariable region contains all of the membrane targeting signals for these proteins. RhoA is an exception wherein the full length protein has additional information that leads to cytosolic localization. B) Repalcement of the TC10 hypervariable region with that of pCdc42hs (left panel) targets the hybrid protein to the same membranes as pCdc42hs itself, again indicating that the hypervariable domain contains the targeting signals. Localization of full length TC10 is shown in the right panel for comparison. Bars indicate 10 µM.

Intracellular Targeting of Rho Family GTPases

25

In neutrophils Rac2 regulates assembly of the NADPH oxidase that is essential for host defense. In resting cells Rac2 is in the cytosol in a 1:1 complex with RhoGDIα.10 Upon activation in vitro,46 and upon activation of neutroplasts,51 Rac2 translocates to a membrane fraction that has been interpreted as PM. However, a method for separating PM from other light membranes by fractionation of neutrophils has not been developed. Given the predominantly endomembrane localization of GFP-Rac2 expressed in excess of RhoGDIα, this will have to be reevaluated. The NADPH oxidase is thought to be assembled in vivo on phagosome membranes, derived from PM. However, given the diffusing capacity of reactive oxygen species, assembly on endomembrane may be effective. Comparison of the localization of GFP-Rac1, 2 and 3 reveals an interesting trend (Fig. 5 and Chan, et. al., manuscript in press). The hypervariable domains of these proteins vary in terms of their positive charge, from Rac2 (+3) to Rac1 (+6) with Rac3 (+4) being intermediate between the two. GFP-Rac2 localizes almost exclusively on endomembrane, GFP-Rac1 localizes almost exclusively on PM and GFP-Rac3 localizes partly on endomembrane and partly on PM. Thus the Class I and Class III hypervariable regions represent something of a continuum, with the degree of PM localization being largely predictable by the degree of postitve charge. The only full length GTPase whose localization cannot be predicted by its hypervariable region sequence is RhoA. This GTPase is always cytosolic even when overexpressed at a high enough level to overwhelm endogenous GDI (Fig. 4D). This is despite its having a hypervariable region with a +5 charge which would suggest it might localize like Rac3 (Fig. 5). Although RhoA has been reported in both cytosol and membrane pellets34 and has been colocalized with caveolin-1 in PM microdomains by immunogold electron microscopy,35 indirect immunofluorescence analysis, in agreement with live cell GFP fusion protein analysis, has shown RhoA to be cytosolic.33,42,44 We shall address this point in further detail below.

C-Terminal Targeting Motifs—The Importance of Being Hypervariable In order to avoid the influence of RhoGDI and to test if the hypervariable region is sufficient to drive the targeting of Rho proteins to specific membranes, it is helpful to fuse GFP to the hypervariable regions alone. The hypervariable regions of Rac1 and Cdc42hs, as shown in (Fig. 6A), target GFP to the same membranes as the full length proteins. This observation shows that, for many Rho proteins, the hypervariable region contains all the necessary information to dictate membrane targeting.42 This is similar to the situation with Ras proteins.32 However, one protein, RhoA is an exception. GFP fused to full length RhoA is always cytosolic no matter how highly overexpressed while, as seen in (Fig. 6A), the RhoA hypervariable region targets GFP to the same membranes as Rac2, Rac3 and Cdc42hs full length proteins or the Cdc42hs hypervariable region alone. In fact, as might be predicted by its +5 charge, the RhoA hypervariable region localizes similarly to Rac3 full length, whose hypervariable region has a +4 charge (Fig. 5). Since the intracellular levels of RhoGDIα are insufficient to account for the exclusively cytosolic localization of over-expressed full length RhoA, and since the RhoA hypervariable region alone gives GFP a typical Class III localization, sequences upstream of the hypervariable region must give additional targeting information that keeps the protein in the cytosol. Cytosolic binding proteins other than RhoGDIα or intramolecular sequestration of the prenyl group, are possible explanations for the exclusively cytosolic localization of full length RhoA. It has been shown that GFP fused with a truncated version of RhoA lacking amino acids 1-73 of RhoA localizes primarily on endomembrane rather than cytosol, indicating that sequestration in the cytosol depends on the N-terminus of RhoA.42 Another way to demonstrate the importance of the hypervariable region is to make hybrid molecules consisting of hypervariable sequences of one GTPase fused to the bulk of any other GTPase. The localization of such chimeras is usually determined by the hypervariable sequence. For example, fusion of the last 20 amino acids of Cdc42hs with the first 188 amino acids of TC10 yields a protein that has the Class III localization of Cdc42hs rather than the PM and

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Rho GTPases

endosomal localization of TC10 itself (Fig. 6B and ref. 42). Thus, with the exception of RhoA, sequences upstream of the hypervariable region appear to be irrelevant with regard to the membrane targeting of Rho family GTPases.

TC10 and Cdc42—A Tail of Two GTPases TC10 and CDC42hs are 69% identical and 83% similar, yet they have very different hypervariable regions and correspondingly different localizations. How do these differences affect the function of these two GTPases? To some degree these two proteins have similar, though not identical, functions. Both stimulate filopodia formation and participate in similar signalling pathways.52 Nevertheless, these highly homologous proteins are not functionally identical. TC10 is not able to rescue the growth defect of a Cdc42 mutant in yeast nor does it participate in neurite outgrowth stimulated by nerve growth factor as does Cdc42hs.52 Additionally, as will be discussed below, inactive Cdc42hs can be sequestered in the cytosol by RhoGDIα while TC10 does not interact with RhoGDIα, suggesting that the regulation of these two molecules differs.42 The most interesting difference between TC10 and Cdc42hs so far determined is in their involvement in insulin signalling. Although Cdc42hs plays no apparent role in insulin signalling, TC10 is required along with the PI3K pathway for insulin-stimulated translocation of the glucose tranporter, GLUT4, from an intracellular compartment to the PM.11,12 PI3K activation is necessary but not sufficient for efficient translocation of GLUT4 to the PM. In addition, the insulin receptor recruits the Cbl protooncogene product to the PM via the adaptors APS and CAP. Cbl in turn recruits the CrkII-C3G complex, which then acts as a GEF for TC10 activation. TC10 activation cooperates with the PI3K pathway to bring GLUT4 to the PM11 probably acting through N-WASP to effect necessary actin rearrangements.53 Cdc42 is unaffected by insulin binding and does not participate in GLUT4 translocation. Since GLUT4 localizes to intracellular vesicles in unstimulated cells,54,55 the ER/Golgi localization of Cdc42hs may prevent it from participating in the vesicle-to-PM targeting event. By contrast, TC10 is present on PM and intracellular vesicles12,42 and so may be optimally positioned for participation in GLUT4 transport. Although no function has yet been reported for TC10 localized to internal vesicles, an interesting possibility is that the bridging of the CrkII-C3G complex at the site of the activated insulin receptor and TC10 present on the GLUT4-containing vesicle may be a critical event in insulin signalling. However, it has not yet been determined if the GLUT4-containing vesicles and the TC10 vesicles are the same. Localization of TC10 to particular microdomains of the PM is critical for its ability to be activated by insulin and to function in GLUT4 translocation. TC10, like H-Ras, is a palmitoylated protein, and the combination of prenylation and palmitoylation targets TC10 and H-Ras to cholesterol-rich lipid raft domains of the PM, the same regions where the insulin receptor associated CrkII-C3G complex is localized. Replacement of the TC10 hypervariable region with that of H-Ras does not alter this localization or the ability of the chimera to be activated by insulin, and the chimera has the same effect as overexpressed wild type TC10 on GLUT4 translocation.12 By contrast, replacement of the TC10 hypervariable region with that of nonpalmitoylated K-Ras4B targets the hybrid protein to the nonlipid raft region of the plasma membrane. This hybrid is then unable to be activated by insulin or to affect insulin-stimulated GLUT4 translocation.12 Thus localization of TC10 to particular intracellular vesicles as well as to particular microdomains of the PM may be critical for its function in insulin signalling. Similarly, Cdc42hs, by virtue of its endomembrane localization, may be optimally localized for participation in trafficking of intracellular vesicles (reviewed in ref. 56). As noted previously, Cdc42hs has been found to bind the γ-subunit of the coatomer complex, which is involved in intracellular vesicle trafficking as part of the coat assembled on Golgi membranes.50 Cdc42hs has also been found to be involved in Golgi-to-ER transport57 and sorting of proteins from the Golgi to apical and basolateral membranes.58 Data from experiments using yeast

Intracellular Targeting of Rho Family GTPases

27

Figure 7. GDI binding modifies the localization of some, but not all, Rho proteins. Rac1 and Cdc42hs localize to the cytosol when expressed at low levels in MDCK cells (left column). When expressed at levels that exceed the buffering capacity of endogenous RhoGDIα, these proteins localize to membranes based on their hypervariable region sequence (middle column). When RhoGDIα is coexpressed, cytosolic localization is restored (right column). In contrast, RhoB and TC10 do not bind GDI and thus never localize to the cytosol even when RhoGDIα is coexpressed. Bars indicate 10 µM.

Cdc42 indicate that Cdc42 may participate in the docking of vesicles to target membranes.59 Thus the differential localizations of Cdc42hs and TC10 may be one key factor in their differential activity with Cdc42hs acting within the endomembrane system and TC10 acting to bring vesicles to and from the PM.

RhoGDI—The Catcher in the Cytosol Although Ras protein localization is almost exclusively determined by the nature of its hypervariable domain, Rho family proteins have an additional level of regulation due to their interaction with GDIs. Three main GDIs have been identified. RhoGDIα is ubiquitous and has been found to bind most, but not all, Rho family proteins.42 RhoGDIβ is expressed only in hematopoietic cells and has a 10-fold lower affinity for RhoA, Rac1 and Cdc42hs compared with RhoGDIα.60 RhoGDIγ is expressed mainly in brain and pancreas, binds RhoA with low affinity, and is itself targeted to membranes by virtue of a hydrophobic N-terminal domain.61,62 As mentioned above, GFP-tagged Rho family proteins often localize differently depending on their level of expression.42 This is shown in Figure 7 Low levels of expression of GFP-Rac1, GFP-Rac2 (not shown) and GFP-Cdc42hs result in largely cytosolic localizations. At higher levels of expression, all of these proteins localize to membranes in a fashion identical to the pattern seen when GFP is attached to the hypervariable regions alone. If these GFP tagged

28

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molecules are coexpressed with RhoGDIα, then the cytosolic localization is restored, indicating that RhoGDIα binding is responsible for the cytosolic localization of these Rho GTPases. In contrast to these molecules, GFP-RhoB and GFP-TC10 are never cytosolic even when coexpressed with RhoGDIα (Fig. 7) due to the inability of these GTPases to bind RhoGDIα.42 RhoB and TC10 are unable to bind RhoGDIα both because they are palmitoylated, sterically hindering GDI binding to the prenyl group of the GTPase,42 and because they lack critical amino acids that form specific hydrogen bonds with RhoGDIα.19 In this sense RhoB and TC10 localization follows the pattern of H- or N-Ras rather than that of other Rho family proteins. The stoichiometry of RhoGDIα in cells is nearly 1:1 with the combined concentrations of RhoA, Rac1 and Cdc42hs.42 This emphasizes the very tight regulation of Rho protein localization. Under normal circumstances, most Rho proteins in the cell will be bound to RhoGDIα and thus kept away from the membranes where they are activated. Yet the system is balanced so closely that slight perturbations may result in release of Rho proteins from RhoGDIα and subsequent translocation to membranes. This balance allows a rapid response to signalling since there is a large pool of available Rho GTPase sequestered in the cytosol poised to respond. No new protein synthesis is required to achieve rapid signalling. Conversely, Rho protein signalling could be rapidly turned off since RhoGDIα is available to bind the GTPase as it becomes inactivated. Thus in addition to their different membrane localizations, determined by their hypervariable regions, Rho proteins are also regulated by GDIs. RhoA, Rac1 and Cdc42hs would not only individually be regulated by RhoGDIα, but, given the tight stoichiometry, they might also affect each other since increased or decreased levels of one GTPase would affect the amount of RhoGDIα available to bind the other GTPases. TC10 and RhoB are unaffected by this system.

Conclusions—You Are Where You Are Rho family proteins look a great deal alike in terms of their amino acid sequences. Despite this, they are capable of considerable specificity of function. The most striking result of the analysis of GFP-tagged Rho proteins in live cells is the diversity of localizations despite their shared pathway of CAAX modification. This diversity of localization is likely to contribute significantly to the functional diversity of these proteins. The diversity in localization is regulated primarily by determinants in the hypervariable region upstream of the CAAX motif and by the capacity to bind RhoGDI. Localization might be critical for function for a number of reasons. Localization to different membrane compartments would allow interaction with different activators and effectors. Furthermore, many GTPases act by targeting binding partners to membranes or by bringing together two different membrane surfaces in preparation for fusion. The activation of Ras proteins, for example, leads to their binding the protein kinase Raf at the plasma membrane where Raf then acts to stimulate the MAP Kinase cascade. In mammalian cells, both RhoA and the Rho-effector Citron kinase relocalize during anaphase to the cleavage furrow, and disruption of RhoA function disrupts the relocalization of Citron-kinase.63 The functional significance of the cell-cycle dependent colocalization of RhoA and Citron kinase has not yet been determined. In budding yeast, the Ras family protein Rsr1, when activated at the site of a previous budding, will bind Cdc24, a GEF for Cdc42, thus activating Cdc42 at a nascent bud site adjacent to a previous bud site.64-66 Cdc42 can then reorganize the actin cytoskeleton to set up appropriate cell polarity. Additionally, Cdc42 also recruits other GTPases such as Rho1 and Sec4 to the new bud site to facilitate bud enlargement and cell wall synthesis.67,68 Cdc42 acts as a key regulator of cell polarity in budding yeast and its subcellular localization is key to this regulation.69 Localization of GTPases to specific membranes allows for specific intracellular localization of effectors and thus for very precise spatial signalling. In some cases localization suggests function. For example, the endosomal and PM localization of TC10 places it in an ideal position to regulate GLUT4 translocation to the PM after

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insulin signalling. Golgi localization of pCdc42hs is consistent with its ability to bind γCOP and to regulate sorting of proteins from the Golgi to apical and basolateral membanes. Rac1 localization primarily to the PM is consistent with its role in assembling the actin cytoskeleton into membrane ruffles. However, the localization of Cdc42hs primarily in the endomembrane and only weakly in the plasma membrane is less consistent with its role in filopodial formation at the PM. Thus not all Rho protein functions are easily predictable from localization. Many of these correlations between function and localization remain speculative and the precise relationship between localization and function remains to be determined. Elucidation of the biology of Rho GTPases will require a more detailed understanding of their intracellular targeting.

References 1. Nobes C, Hall A. Rho, Rac and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995; 81:53-62. 2. Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992; 70:389-399. 3. Ridley AJ, Paterson HF, Johnston CL et al. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992; 70:401-410. 4. Coso OA et al. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signalling pathway. Cell 1995; 81:1137-1146. 5. Minden A, Lin A, Claret FX et al. Selective activation of the JNK signalling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 1995; 81:1147-1157. 6. Minden A, Lin A, Claret FX et al. Selective activation of the JNK signalling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 1995; 81:1147-57. 7. Hill CS, Wynne J, Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 1995; 81:1159-70. 8. Qiu RG, Chen J, McCormick F et al. A role for Rho in Ras transformation. Proc Natl Acad Sci USA 1995; 92:11781-5. 9. Abo A et al. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature 1991; 353:668-670. 10. Knaus UG, Heyworth PG, Evans T et al. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science 1991; 254:1512-1515. 11. Chiang SH et al. Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10. Nature 2001; 410:944-948. 12. Watson RT et al. Lipid raft microdomain compartmentalization of TC10 is required for insulin signalling and GLUT4 translocation. J Cell Biol 2001; 154:829-840. 13. Caron E, Hall A. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 1998; 282:1717-1721. 14. Jou TS et al. Selective alterations in biosynthetic and endocytic protein traffic in Madin-Darby canine kidney epithelial cells expressing mutants of the small GTPase Rac1. Mol Biol Cell 2000; 11:287-304. 15. Lamaze C, Chuang TH, Terlecky LJ et al. Regulation of receptor-mediated endocytosis by Rho and Rac. Nature 1996; 382:177-9. 16. Leung SM et al. Modulation of endocytic traffic in polarized Madin-Darby canine kidney cells by the small GTPase RhoA. Mol Biol Cell 1999; 10:4369-84. 17. Kroschewski R, Hall A, Mellman I. Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells. Nat Cell Biol 1999; 1:8-13. 18. Barrett K, Leptin M, Settleman J. The Rho GTPase and a putative RhoGEF mediate a signalling pathway for the cell shape changes in Drosophila gastrulation. Cell 1997; 91:905-15. 19. Hoffman GR, Nassar N, Cerione RA. Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 2000; 100:345-356. 20. Ridley AJ. Rho family proteins: Coordinating cell responses. Trends Cell Biol 2001; 11:471-7. 21. Cerione RA, Zheng Y. The Dbl family of oncogenes. Current Opinion in Cell Biology 1996; 8:216-222. 22. Zheng Y. Dbl family guanine nucleotide exchange factors. Trends Biochem Sci 2001; 26:724-32. 23. Lamarche N, Hall A. GAPs for rho-related GTPases. Trends Genet 1994; 10:436-40. 24. Neudauer CL, Joberty G, Tatsis N et al. Distinct cellular effects and interactions of the Rho-family GTPase TC10. Curr Biol 1998; 8:1151-60. 25. Bagrodia S, Cerione RA. Pak to the future. Trends Cell Biol 1999; 9:350-5.

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26. Teramoto H et al. Signalling from the small GTP-binding proteins Rac1 and Cdc42 to the c- Jun N-terminal kinase/stress-activated protein kinase pathway. A role for mixed lineage kinase 3/ protein-tyrosine kinase 1, a novel member of the mixed lineage kinase family. J Biol Chem 1996; 271:27225-8. 27. Madaule P et al. A novel partner for the GTP-bound forms of rho and rac. FEBS Lett 1995; 377:243-8. 28. Clarke S. Protein isoprenylation and methylation at carboxyl terminal cysteine residues. Annual Review of Biochemistry 1992; 61:355-386. 29. Schmidt WK, Tam A, Fujimura-Kamada K et al. Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage. Proc Natl Acad Sci USA 1998; 95:11175-11180. 30. Dai Q et al. Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum. J Biol Chem 1998; 273:15030-15034. 31. Apolloni A, Prior IA, Lindsay M et al. H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol Cell Biol 2000; 20:2475-87. 32. Choy E et al. Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell 1999; 98:69-80. 33. Adamson P, Paterson HF, Hall A. Intracellular localization of the P21rho proteins. J Cell Biol 1992; 119:617-627. 34. Boivin D, Beliveau R. Subcellular distribution and membrane association of Rho-related small GFP-binding proteins in kidney cortex. Am J Physiol 1995; 269:F180-F189. 35. Michaely PA, Mineo C, Ying Y et al. Polarized distribution of endogenous Rac1 and RhoA at the cell surface. J Biol Chem 1999; 274:21430-21436. 36. Robertson D, Paterson HF, Adamson P et al. Ultrastructural localization of ras-related proteins using epitope- tagged plasmids. J Histochem Cytochem 1995; 43:471-80. 37. Erickson JW, Zhang C, Kahn RA et al. Mammalian Cdc42 is a Brefeldin A-sensitive component of the Golgi apparatus. J Biol Chem 1996; 271:26850-26854. 38. Murphy C et al. Endosome dynamics regulated by a Rho protein. Nature 1996; 384:427-32. 39. Baldassare JJ, Jarpe MB, Alferes L et al. Nuclear translocation of RhoA mediates the mitogen-induced activation of phospholipase D involved in nuclear envelope signal transduction. J Biol Chem 1997; 272:4911-4. 40. Fukumoto Y et al. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins. Oncogene 1990; 5:1321-1328. 41. Isomura M, Kikuchi A, Ohga N et al. Regulation of binding of rhoB p20 to membranes by its specific regulatory protein, GDP dissociation inhibitor. Oncogene 1991; 6:119-124. 42. Michaelson D. et al. Differential Localization of Rho GTPases in Live Cells. Regulation by hypervariable regions and rhogdi binding. J Cell Biol 2001; 152:111-126. 43. Fleming IN, Elliott CM, Exton JH. Differential translocation of Rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor. J Biol Chem 1996; 271:33067-33073. 44. Kranenburg O, Poland M, Gebbink M et al. Dissociation of LPA-induced cytoskeletal contraction from stress fiber formation by differential localization of RhoA. J Cell Sci 1997; 110:2417-27. 45. Kraynov VS et al. Localized Rac activation dynamics visualized in living cells. Science 2000; 290:333-7. 46. Philips MR, Feoktistov AS, Pillinger MH et al. Activation Dependent Translocation of p22rac and p22CDC42hs from Neutrophil Cytosol to Plasma Membrane. Clin Res 1993; 40(2):351A. 47. Jou TS et al. Selective alterations in biosynthetic and endocytic protein traffic in Madin-Darby canine kidney epithelial cells expressing mutants of the small GTPase Rac1. Mol Biol Cell 2000; 11:287-304. 48. Bilodeau D, Lamy S, Desrosiers RR et al. Regulation of Rho protein binding to membranes by rhoGDI: Inhibition of releasing activity by physiological ionic conditions. Biochem & Cell Biol 1999; 77:59-69. 49. Bokoch GM, Bohl BP, Chuang T. Guanine nucleotide exchange regulates membrane translocation of rac/rho GTP-binding proteins. J Biol Chem 1994; 269:31674-31679. 50. Wu WJ, Erickson JW, Lin R et al. The g-subunit of the coatomer complex binds Cdc42 to mediate transformation. Nature 2000; 405:800-804. 51. Philips MR, Feoktistov A, Pillinger MH et al. Translocation of p21rac2 from cytosol to plasma membrane is neither necessary nor sufficient for neutrophil nadph oxidase activity. J Biol Chem 1995; 270:11514-11521. 52. Murphy GA et al. Signalling mediated by the closely related mammalian Rho family GTPases TC10 and Cdc42 suggests distinct functional pathways. Cell Growth Differ 2001; 12:157-67.

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53. Jiang ZY, Chawla A, Bose A et al. A phosphatidylinositol 3-kinase-independent insulin signalling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling. J Biol Chem 2002; 277:509-15. 54. Millar CA, Shewan A, Hickson GR et al. Differential regulation of secretory compartments containing the insulin-responsive glucose transporter 4 in 3T3-L1 adipocytes. Mol Biol Cell 1999; 10:3675-88. 55. Simpson F, Whitehead JP, James DE. GLUT4—at the cross roads between membrane trafficking and signal transduction. Traffic 2001; 2:2-11. 56. Ridley AJ. Rho proteins: Linking signalling with membrane trafficking. Traffic 2001; 2:303-10. 57. Luna A et al. Regulation of Protein Transport from the Golgi Complex to the Endoplasmic Reticulum by CDC42 and N-WASP. Mol Biol Cell 2002; 13:866-79. 58. Musch A, Cohen D, Kreitzer G et al. cdc42 regulates the exit of apical and basolateral proteins from the trans-Golgi network. Embo J 2001; 20:2171-9. 59. Muller O, Johnson DI, Mayer A. Cdc42p functions at the docking stage of yeast vacuole membrane fusion. Embo J 2001; 20:5657-65. 60. Gorvel JP, Chang TC, Boretto J et al. Differential properties of D4/LyGDI versus RhoGDI: phosphorylation and rho GTPase selectivity. FEBS Lett 1998; 422:269-73. 61. Adra CN et al. RhoGDIgamma: A GDP-dissociation inhibitor for Rho proteins with preferential expression in brain and pancreas. Proc Natl Acad Sci USA 1997; 94:4279-84. 62. Zalcman G et al. RhoGDI-3 is a new GDP dissociation inhibitor (GDI). Identification of a noncytosolic GDI protein interacting with the small GTP-binding proteins RhoB and RhoG. J Biol Chem 1996; 271:30366-74. 63. Eda M et al. Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividing cells. J Cell Sci 2001; 114:3273-84. 64. Bender A, Pringle JR. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci USA 1989; 86:9976-80. 65. Chant J, Herskowitz I. Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell 1991; 65:1203-12. 66. Zheng Y, Bender A, Cerione RA et al. Interactions among proteins involved in bud-site selection and bud-site assembly in Saccharomyces cerevisiae. J Biol Chem 1995; 270:626-30. 67. Drgonova J et al. Rho1p, a yeast protein at the interface between cell polarization and morphogenesis. Science 1996; 272:277-9. 68. Cabib E, Drgonova J, Drgon T. Role of small G proteins in yeast cell polarization and wall biosynthesis. Annu Rev Biochem 1998; 67:307-33. 69. Drees BL et al. A protein interaction map for cell polarity development. J Cell Biol 2001; 154:549-71.

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CHAPTER 3

Regulation of the RhoGTPases by RhoGDI Gregory R. Hoffman and Richard A. Cerione

Abstract

S

ignal transduction pathways mediated by the Rho-family GTPases require tight temporal and spatial control. The GDI proteins are key components of the regulatory machinery controlling both the timing and the localization of Rho GTPase activity. Recent structural work has provided significant insight into the mechanisms by which the GDI proteins control Rho-family signalling. In this review, the basis of the three distinct biochemical activities of the GDI toward the Rho GTPases, namely (1) inhibition of nucleotide dissociation, (2) inhibition of GTP hydrolysis, and (3) membrane release, are described in the context of this structural information. Understanding the biochemistry of the GDI provides a starting point for exploring the cell biology of this important class of regulatory molecules and recent progress in understanding the roll of the GDI in the cell is also discussed.

Introduction Members of the Rho family of GTPases are remarkable in their ability to regulate an enormous range of cellular responses (described in reviews of this series and in references 1, 2). The ability to bind and hydrolyze GTP lies at the heart of the biological activity of the Rho GTPases, allowing them to function as molecular switches cycling between an active, GTP-bound conformation and an inactive, GDP-bound conformation. In the GTP-bound state, Rho GTPases are able to specifically couple to downstream effector proteins and activate their biological activities, giving rise to signals that control important cellular processes including organization of the actin cytoskeleton, maintenance of cell polarity, lipid signalling, membrane trafficking, gene expression, and oncogenic transformation. Given the complex signalling networks governed by Rho-family proteins, it is clear that both the timing and the localization of the GTP binding and hydrolytic cycle must be tightly controlled. To this end, the cell has evolved a series of regulatory factors responsible for controlling signalling events mediated by the Rho GTPases. Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) catalyze activation via GDP/GTP exchange and inactivation via GTP hydrolysis, respectively (reviewed in this series and in refs. 3-5). Guanine nucleotide dissociation inhibitors (GDIs) represent a third class of regulatory proteins that are critical to the control of signalling events mediated by the Rho GTPases. The GDIs are unique among regulatory proteins in that they exhibit multiple effects on their Rho-family substrates, controlling both the nucleotide state of the GTP-binding proteins as well as their cellular location. Recently, significant progress has been made toward understanding the structural basis of the interaction of the Rho GTPases with GDIs. As described in the following review, this structural information provides important insights into the signalling events mediated by the Rho GTPases and provides a context for understanding the cellular function of the GDIs.

Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Figure 1. Biochemical activities of the GDI. The GDI serves as a multi-functional regulator of signalling by Rho GTPases. The three distinct biochemical activities of the GDI (1) inhibition of nucleotide dissociation, (2) inhibition of GTP hydrolysis, and (3) membrane release, are illustrated in relation to the GTP-binding and hydrolytic cycle of the Rho proteins.

GDIs As Multifunctional Regulators of Signalling through Rho GTPases The GDI proteins comprise a family of regulatory factors that control signalling by the Rho proteins (previously reviewed in ref. 6) and exhibit three distinct biochemical activities toward these GTPases (Fig. 1). The GDI proteins were initially identified based on their ability to bind to the inactive, GDP-bound form of Rho proteins and block nucleotide dissociation.7-9 In this capacity, the GDI acts as a negative regulator of Rho-family signalling, blocking GEF-mediated

Figure 2. The RhoGDI family. Three Rho family-specific GDI proteins have been identified, each exhibiting a high degree of primary sequence conservation (indicated as percent identity). The domain architecture of the GDIs described in the text is illustrated in relation to the primary sequence, with the amino-terminal regulatory arm shown in orange and the immunoglobulin-like domain in blue (residue 74 marks the beginning of the first β-strand in the immunoglobulin like domain of RhoGDI and is marked by the arrow). The approximate locations of important residues described in the text are indicated. The unique helical region at the amino-terminus of GDI3 is shown in pink.

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Figure 3. Structure of the GTPase/GDI complex. (A) A ribbon diagram of the Cdc42/GDI complex is shown with Cdc42 in yellow, with the switch I in red and switch II in orange, and GDI in blue. The Mg2+ ion and GDP are shown in the nucleotide-binding pocket of Cdc42 as a ball and stick model. A transparent molecular surface of the GDI is shown to emphasize the domain architecture of the GDI. (B) A similar depiction of the complex is shown with the molecular surface colored by electrostatic potential with regions of net negative potential shaded red and regions with net positive potential shown in blue.

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exchange and maintaining the GTPase in an inactive GDP-bound state. Subsequently, it was shown that RhoGDI is also able to bind with high affinity to the GTP-bound form of the Rho family member Cdc42 to act as a GTPase inhibitory protein (GIP),10 blocking both intrinsic and GAP-catalyzed GTP-hydrolysis and thus maintaining Cdc42 in a GTP-bound conformation. The third, and perhaps the most important cellular activity of the GDI, is its ability to solubilize Cdc42 from cellular membranes.11,12 In the absence of a GDI, Rho proteins are stably associated with cellular membranes by virtue of a series of post-translational modifications directed toward their carboxy-terminal CAAX motif13 (see also Chapter 2). The most significant of these modifications is the covalent attachment of a prenyl moiety to the CAAX box cysteine. In most cases this lipid group is a 20-carbon geranylgeranyl group attached by geranylgeranyl transferase I14 (RhoB, RhoD and RhoE are modified with the smaller 15-carbon farnesyl group). Following prenylation, a specific protease cleaves the last three residues of the CAAX motif, and the new carboxyl-terminal prenylated cysteine is carboxy-methylated. The hydrophobic prenyl moiety, along with a polybasic region proximal to the modified cysteine, directs the Rho family proteins to specific membrane-bound locations where they carry out their cellular function.15 RhoGDI binds only to the prenylated form of Rho proteins, and GDI binding sequesters the lipid moiety from the membrane, creating a soluble cytosolic GDI/GTPase complex. Appropriate localization of Rho proteins is critical for their biological function, and regulation of this localization by RhoGDI is likely to play an important role in signalling through Rho GTPases (see also Chapter 2). A GDI activity acting on Rho and Cdc42 was independently purified from bovine brain cytosol by two groups.7-9 The sequencing and cloning of the protein responsible for this activity lead to the identification of RhoGDI (also known as GDI1 or αGDI ), the first Rho family specific GDI protein. In addition to RhoGDI, two related proteins have been described (Fig. 2). The Ly- or D4-GDI (also referred to as GDI2 and βGDI) was found as a GDI protein preferentially expressed in hematopoietic cells16-18 with 74% sequence identity to RhoGDI. In spite of the high degree of conservation at the level of primary sequence, GDI2 interacts with a 20-fold lower affinity toward Cdc42. This decreased binding affinity has been attributed to a single amino-acid change from Ile177 in RhoGDI to an asparagine at the corresponding position in GDI2.19 Two groups have recently described a third member of the GDI-family, referred to as GDI3 (also called γGDI).20,21 This protein is enriched in brain and appears to have a high degree of specificity to RhoB and RhoG and also binds more weakly to Cdc42 and RhoA but not Rac. GDI3 has a unique, extended amino-terminal region comprised of an amphipathic α-helix. Unlike the other members of this family, which are entirely cytosolic, GDI3 is associated with a Triton-X insoluble fraction and appears to localize to vesicular structures in the vicinity of the ER. While the conserved nature of the primary sequence of GDI3 suggests a common mode of interaction with Rho-family substrates, the distinctive noncytosolic localization and substrate specificity point to a specialized role, possibly in the regulation of RhoB signalling.6

Structural Insights into GDI Function

We have recently described the structure of the Cdc42/RhoGDI complex22 which, along with the structure of the Rac1/GDI2 complex,23 the recently described Rac2/RhoGDI structure,24,25 and a low resolution structure of the RhoA/RhoGDI complex,26 provides a consistent mechanistic picture of the regulation of Rho GTPases by the GDIs. In addition, these structures reveal important differences between the RhoGDI and GDI2 proteins and provide important insights into the cellular function of these regulatory proteins. The structure of the post-translationally modified form of Cdc42 bound to RhoGDI is shown in (Fig. 3). The molecular surface of the GDI is drawn to emphasize the domain architecture of the GDI, which is comprised of two distinct domains, a well-ordered carboxy-terminal domain and a more flexible amino-terminal region.

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Earlier structural work on the carboxy-terminal domain of the GDI27,28 showed that this region adopts an immunoglobulin-like fold, comprised of two anti-parallel β-sheets that pack against one another to form a β-sandwich. The most striking feature of the GDI immunoglobulin-like domain is the presence of a unique hydrophobic cavity between the two sheets of the β-sandwich, which provides a binding site for the geranylgeranyl moiety. In the absence of Cdc42 binding, the hydrophobic pocket of the GDI is too small to accommodate the lipid. Upon complex formation, the base of the pocket dramatically expands to allow insertion of the geranylgeranyl group. In the structure of the Cdc42/GDI complex, the geranylgeranyl moiety of Cdc42 is completely sequestered from the solvent, and the binding of the lipid group within this hydrophobic cavity provides a mechanism for release of the GTPase from cellular membranes. In contrast, the geranylgeranyl moiety was not visible in the electron density map of the Rac1/GDI2 structure.23 The lack of an ordered geranylgeranyl group in the Rac1/GDI2 complex reflects a weak affinity of the GDI2 for binding to the lipid moiety. As noted above, a single amino acid change from isoleucine at position 177 of RhoGDI to an asparagine at the corresponding position in GDI2 accounts for a 20-fold difference in binding affinity for Cdc42.19 Isoleucine 177 in RhoGDI forms hydrophobic contacts with the geranylgeranyl group important in stabilizing the lipid in the binding pocket. The introduction of a polar asparagine residue at this position is likely to disrupt geranylgeranyl binding as seen by the disordered nature of the lipid in the Rac1/GDI2 complex. Importantly, the structure of Rac2 bound to RhoGDI24 clearly shows the presence of the geranyl-geranyl group in the hydrophobic pocket with a conformation nearly identical to that seen for the Cdc42/RhoGDI complex. This structure confirms that lack of a well ordered geranylgeranyl moiety in the Rac2/GDI2 structure is due to the differences in lipid binding between the two GDI proteins, rather than differences in the GTPases, and may reflect functional differences between these GDIs. In each of these structures, the amino-terminal region of the GDI folds into a well-ordered helix-loop-helix, and this ‘regulatory-arm’ of the GDI interacts with the switch I and switch II regions of the Rho protein to influence the nucleotide state of the GTPase. Importantly, NMR spectroscopy of the full-length GDI shows that the amino-terminal region of the GDI is disordered in the absence of Cdc42 binding.27-29 As predicted by these NMR studies, the structure of the complex clearly shows that this flexible region adopts an ordered structure upon binding to the GTPase. The amino-terminal region of the GDI is essential for its function, and biochemical studies show that while deletion of the first 22 residues from the GDI has only slight affects on its interaction with Rho proteins, deletions of 45 or 60 residues completely abrogates binding and blocks GDI activity.30 The amino-terminal region of the GDI contributes a significant portion of the protein-protein interactions at the complex interface, and the structural data demonstrates that this region is responsible for blocking guanine nucleotide dissociation and inhibition of GTP hydrolysis (described in detail below). A number of recent NMR studies have investigated the functional importance of the disordered amino-terminal region of the GDI. These studies show that the free GDI is in equilibrium between two conformations, one in which the amino-terminus is in a random coil and a second in which the amino-terminal domain transiently adopts a helical structure similar to that seen in complex with Rho GTPases.29,31 Mutations in the amino-terminal domain that favor the random coil structure dramatically interfere with GDI binding to Rac1.32 The structure of the Cdc42/GDI complex shows that the extreme amino-terminal region of RhoGDI (residues 10-15) forms a short helical region that caps the geranylgeranyl binding pocket, further sequestering the lipid moiety from the solvent. In the Rac1/GDI2 structure this region is disordered, consistent with NMR data showing that this region of RhoGDI exhibits transient helical structure while GDI2 does not. These differences in the helical nature of the amino-terminal region may also contribute to the disparity in lipid binding and the divergent function of these two GDIs.

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Figure 4. The involvement of threonine 35 in the regulation of nucleotide binding. Threonine 35 provides an important site for the regulation of nucleotide binding through the action of GEFs and GDIs. The Cdc42/GDI complex and the Rac/Tiam1 complex are shown oriented relative to the GTPase to illustrate that both regulatory proteins engage the switch residues of the GTPase. The contacts surrounding Thr35 in each structure are shown in detail below the ribbon diagram. In the more detailed description of the Rac/ Tiam1 nucleotide binding pocket, the approximate position of the GDP in the Cdc42/GDI complex is indicated by the pink oval and the position of the Mg2+ ion in the Cdc42/GDI is indicated by the X.

As emphasized by these NMR studies, a critical aspect of complex formation is stabilization of the flexible amino-terminal region of the GDI. A universally conserved arginine residue in switch II (Arg66 in Cdc42) is crucial to the GTPase/GDI interaction and is involved in a series of interactions that buttress the flexible regulatory arm of the GDI against the stable immunoglobulin like domain.22 In the absence of these bridging interactions, the amino-terminal region of the GDI will not adopt the stable fold required for interacting with the switch regions of the GTPase. Based on the central importance of the conserved arginine in switch II, we predicted from the structural data that mutation of this residue would render Rho GTPases unable to interact with the GDI. The importance of this arginine in the Cdc42/GDI interaction was recently demonstrated biochemically by Gibson and Wilson-Delfosse, showing that the Cdc42(R66A) mutant is unable to couple to the GDI and, thus, localizes exclusively to the membrane fraction.33 Importantly, this mutation appears to be specific to the GDI interaction, as other known effectors and regulatory factors do not rely on this arginine residue for binding. As described in the following sections, the structural data provides a mechanistic understanding of the three biochemical functions of the GDI; however, the cellular role of these regulatory

38

Rho GTPases

interactions remains less clear. GDI-insensitive mutants, such as the Cdc42(R66A) mentioned above, will be a valuable reagent in unraveling the role of the GDI in signalling events mediated by the Rho GTPases.

Mechanism of the GDI Activity A primary function of the GDI is to block the dissociation of the bound nucleotide and inhibit the exchange activity of Dbl-family GEFs. Recently the structure of Rac in complex with its specific GEF Tiam1 was determined,34 and comparison of this structure with that of the Cdc42/GDI complex provides important insight into the antagonistic activities of these two proteins. On a basic level, both the GDI and Tiam1 form extensive contacts with the switch regions of the Rho protein (Fig. 4), and the binding of GDI will clearly be competitive with that of exchange factors. More fundamentally, these proteins engage a similar set of residues to exert opposing biochemical effects. All GTP-binding proteins require a Mg2+ ion for high affinity nucleotide binding. This Mg2+ ion offsets the negative charge of the phosphate groups to stabilize the bound nucleotide. Rho proteins exhibit unique coordination of this critical Mg2+ ion in the GDP-bound state,35 with the main-chain carbonyl of Thr35 directly coordinating the Mg2+ ion, replacing a water molecule found in the corresponding position of Ras. Importantly, the unique coordination of the Mg2+ ion by the main chain carbonyl of Thr35 provides access to the bound Mg2+ ion, allowing GDIs and GEFs to control nucleotide binding by influencing Mg2+ ion coordination. Specifically, the GDI interacts directly with the Thr35 side chain to stabilize the interaction of this residue with the Mg2+ ion and lock the nucleotide in the binding pocket, contacts mediated by a conserved aspartic acid and serine residues in the regulatory arm of the GDI (T47 and S47 in RhoGDI). As shown in (Fig. 4), Tiam1 forms main chain hydrogen bonding contacts on either side of this threonine residue, ratcheting switch I laterally along the nucleotide binding cleft, to pull the main chain carbonyl of Thr35 away from its position in the Cdc42/GDI complex. Disruption of the favorable interactions between Thr35 and the Mg2+ ion contributes to release of the bound nucleotide. In addition, Tiam1 disrupts the structure of the switch II domain of Rac such that the side chain of Ala59 protrudes into the Mg2+ ion binding site to assist in its ejection from the binding-pocket. Again, the GDI engages a similar set of residues in the switch II region to achieve the opposite effect, specifically fixing a conformation of switch II consistent with Mg2+ ion binding and stabilization of the associated GDP.

Mechanism of GIP Activity The GDI is unique among effectors and regulatory proteins in its ability to associate with both nucleotide states of Rho-family proteins. In the case of Cdc42, the affinity of the GDI for the GTP- and GDP-bound forms of the GTPase is identical.30 This ability to bind both conformations of the GTPase with equal affinity relies in part on the extensive contacts between the regulatory arm of the GDI and switch II. Relative to other members of the Ras superfamily, the nucleotide-dependent conformational changes in switch II are relatively subtle, thus allowing the GDI to bind to Cdc42 and presumably other Rho proteins in both nucleotide states.22 In the GTP-bound complex, the GDI is able to inhibit GTP hydrolysis. All of the GTPase/ GDI structures solved to date involve a GDP-bound Rho protein, and while definitive proof of the mechanism of the GDI’s GIP activity awaits a GTP-bound complex, some clues to the ability of the GDI to block GTP hydrolysis can be gleaned from comparison with GTPase/ GAP complexes. Structures of Rho proteins in complex with their specific GAP proteins have been solved in both the ground state (GTP-bound)36 and transition state (aluminum fluoride-bound) conformations.37,38 These data demonstrate that GAP-catalyzed GTP hydrolysis proceeds by a two-pronged attack. First, the GAP introduces a catalytic arginine, the so-called ‘arginine-finger’, into the active site. Second, the interaction of the GAP protein stabilizes a catalytically active conformation of the conserved glutamine residue in switch II (Gln61 in Cdc42). This glutamine residue is universally conserved in all GTP binding proteins and is

Regulation of the RhoGTPases by RhoGDI

39

Figure 5. Structural model for membrane release. A) A model, which accounts for the available structural and kinetic data,12 for GDI-mediated release of Rho family GTPases from cellular membranes is shown. The GTPase is localized to membranes by the insertion of the geranylgeranyl group into the bilayer as well as interactions between the poly-basic tail and the acidic phospholipid head groups. The GDI first binds rapidly to the GTPase at the membrane where specific interactions with the polybasic tail and the geranylgeranyl group subsequently cause release of the GTPase/GDI complex into the cytosol. B) The structure shown in Figure 3B is rotated 90º showing a view looking directly into the geranylgeranyl binding pocket. The polybasic tail of Cdc42 (shown in magenta) interacts with an acidic patch on the surface of the GDI and may provide some of the driving force for membrane release.

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Rho GTPases

absolutely required for GTP hydrolysis. In the Cdc42/GDI complex, GDI-binding rotates the side chain of the catalytic glutamine residue out of the nucleotide-binding pocket into an orientation that can no longer stabilize the transition state for GTP-hydrolysis. Thus, the binding of the GDI has essentially the same effect as mutating this critical catalytic residue. By subtly perturbing the structure of switch II, the GDI removes the catalytic glutamine from the active site into a position where it can no longer stabilize the transition state for GTP hydrolysis. Without this glutamine residue, hydrolysis can no longer proceed, allowing the GDI to maintain Rho-family proteins in a GTP-bound conformation. These structural rearrangements block intrinsic GTP-hydrolysis in the Cdc42/GDI complex. In addition, the GDI will clearly be competitive with GAP binding due to the extensive interface both proteins form with the switch regions of the Rho proteins, particularly switch II.

Structural Model for Membrane Release The structure of the Cdc42/GDI complex represents the only structural data available for a fully processed, post-translationally modified GTP-binding protein, providing insight into the ability of these proteins to associate with biological membranes and the regulation of this process by the GDI. Upon complex formation, the geranylgeranyl-binding pocket in the immunoglobulin-like domain of the GDI expands to conform to the shape of the bound geranylgeranyl group. This complimentarity in shape is likely to provide specificity for binding to the geranylgeranyl group over other hydrophobic ligands. In addition, interactions between the polybasic tail of Cdc42 and an acidic patch on the surface of the GDI further ensure the specificity of the interaction. Kinetic data on the release of Cdc42 from cell membranes by the GDI suggests that this interaction proceeds by a two-step mechanism. On the basis of this biochemical work and the structural data, we propose the model for membrane release shown in (Fig. 5). In the absence of GDI binding, the geranylgeranyl group is inserted into the bilayer and the polybasic tail of Cdc42 interacts with the acidic head groups of the phospholipids. The interaction of the GDI with Cdc42 at the membrane surface corresponds to the initial rapid phase in the kinetic mechanism. Subsequently, a slower isomerization event occurs involving transfer of the geranylgeranyl group out of the bilayer and into the hydrophobic binding pocket of the GDI. This isomerization may be driven in part by competition between an acidic patch on the GDI and the acidic phospolipid headgroups for binding to the polybasic tail of Cdc42. The importance of the GDI in regulating the cellular localization of Rho proteins makes the identification of small molecule inhibitors of the Cdc42/GDI interaction an attractive area of research. The geranylgeranyl-binding pocket is an obvious starting point for identifying such inhibitors and some progress has been made in isolating small molecules that bind in this hydrophobic pocket. Most of these analogs are geranylgeranyl cysteine analogs, which interact with relatively low (micromolar) affinity.39 The Cdc42/GDI complex has an affinity of 20 nM and, as such, the currently available small molecules will not act as efficient inhibitors of the Cdc42/GDI interaction. A recent finding highlighted the potential importance of carboxy-methylation demonstrating that carboxymethylated geranylgeranyl cysteine analogs bind to the GDI with much greater affinity than a corresponding unmethylated analog.39 The importance of methylation can be rationalized from the available structural data, which shows that the opening of the geranylgeranyl-binding pocket on the GDI is adjacent to a large acidic patch involved in interactions of the GDI with the polybasic tail of Cdc42. As described above, these interactions are thought to provide some of the driving force for extraction of Cdc42 from the membrane by competing for interactions of the polybasic tail with acidic phospholipid head groups. In an unmethylated situation, the acidic carboxy terminus would be directly adjacent to the acidic patch on the surface of the GDI, generating unfavorable electrostatic interactions between these two negatively charged regions. Methylation of the carboxyl group protects against such unfavorable electrostatic contacts and leads to a higher affinity interaction. Methylation presumably protects against similarly unfavorable interactions with the acidic head groups of phospholipids when the Rho proteins are inserted into the

Regulation of the RhoGTPases by RhoGDI

41

bilayer. The structural features of the geranylgeranyl-binding pocket seen in the Cdc42/GDI complex could provide an important starting point for designing higher affinity geranylgeranyl analogues for use as GDI inhibitors

Future Directions Regulation of the GTPase/GDI Complex In the resting cell, a large fraction of Rho proteins are found in a cytosolic pool associated with the GDI.40,41 In this role, RhoGDI is primarily considered a negative regulator of signalling through Rho GTPases, and over-expression or microinjection of the GDI can be shown to block many signalling events mediated by Rho-family proteins.42-46 In order for Rho proteins to respond to activating signals, they must first be displaced from the GDI and inserted into cellular membranes. This event has been observed most dramatically in the case of RhoA activation in neuronal cells where stimulation with LPA causes its redistribution from the cytosol to the plasma membrane.47 The actin based morphology changes induced by RhoA activation in these cells depends on its membrane localization and can be blocked by GDI over-expression, emphasizing the importance of regulating the RhoA/GDI interaction in this process. Translocation to specific membrane-bound locations can also be demonstrated upon activation of Rho GTPases in other systems.48-51 While it is possible that simple mass action by Dbl-family GEFs is sufficient to displace the GDI and drive the association of Rho proteins with cellular membranes, the high affinity and 1:1 stoichiometry of the complex argues for the existence of a specific GDI displacement factor (GDF). A GDF would be capable of displacing the GDI and provides an appealing means for regulating the GTPase/GDI interaction. A GDF activity has recently been described for the regulation of the Rab/RabGDI interaction,52 and the identification of similar activities acting on Rho-family proteins is an important area of study. The Ezrin-Radixin-Moeisin (ERM) family of proteins has been proposed to act as a GDF for the Rho proteins, regulating the Rho/GDI complex through direct interactions with the GDI.53 Binding of the amino-terminal domain of ERM proteins to RhoGDI causes the release of the GTPase from the complex allowing for its activation by GEFs (reviewed in ref. 54). The ERM proteins are actin-binding proteins involved in connecting the cytoskeleton with the plasma membrane and may play a role in the effects of Rho proteins on the actin cytoskeleton.55 In vivo, it appears that the ERM proteins are involved in Rho-mediated actin rearrangements, but many of these effects appear to be downstream of Rho activation. While the ERMs are candidates for GDI displacement factors involved in Rho signalling to the cytoskeleton, activated Rho proteins localize to a variety of cellular membranes and participate in signalling processes that do not involve ERM proteins, suggesting that there may be other GDFs acting in these pathways. The Vav proto-oncogene was recently identified as specific GDI-binding partner, and may act as a GDF in certain Rac-dependent signalling pathways. Originally identified as a Dbl-family GEF specific for Rac, Vav was recently shown to interact directly with both RhoGDI and GDI2 through a calponin homology domain at its amino-terminus, a portion of the protein that is lost upon oncogenic activation.56 While the ability of Vav to regulate the Rac/GDI interaction has not been directly demonstrated, the coupling of a DH domain responsible for GEF activity with a GDI-binding domain raises the potential for coordinated down-regulation of the GDI binding and activation of nucleotide exchange. Phosphorylation of the RhoGDI downstream of phorbol ester stimulation of PKCα has also been shown to correlate with Rho activation, providing yet another potential mechanism for control of the GTPase/GDI interaction.57 Finally, the involvement of specific lipid products in controlling GDI release has been proposed.58 While ERM proteins remain the only proteins shown biochemically to dissociate Rho GTPases from a complex with the GDI, there is emerging evidence demonstrating that

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Rho GTPases

Figure 6. Model for the GDI as a shuttle in Cdc42 signalling. The GDI protein, by virtue of its GIP activity, may act to deliver the activated form of Cdc42 to membrane locations distant from the site of activation. Similarly, the GDI can then act to recycle Cdc42 back to the original membrane compartment for another round of activation. Such a shuttling mechanism may be important in establishing the proper localization of Cdc42 and other Rho-family GTP-binding proteins.

other signalling events impact directly on the GDI to modulate its interaction with Rho-family GTPases.

Positive Signalling Roles for the GDI The cell maintains a careful balance in GDI expression, ensuring that there are nearly equal concentrations of the GDI and Rho proteins in the cell,15 and over-expression of the GDI may

Regulation of the RhoGTPases by RhoGDI

43

mask the more subtle regulatory features of its interaction with Rho GTPases. Recent work has pointed to a more complex role for GTPase/GDI complexes in directly mediating signalling events. Specifically, the Cdc42/GDI complex is able to block MAP-kinase activation downstream of Ras-GRF, suggesting a model in which Cdc42 and RhoGDI act to control signalling through the Ras/MAPK pathway.59 Disruption of the Cdc42/GDI complex and activation of Cdc42 releases the inhibition of Ras-GRF and is a prerequisite for Ras activation. In another study, the GDP-bound Rac/GDI complex can associate in the cytosol with a complex of the type I PIP-5 kinase and diacylglycerol kinase forming a preassembled signalling complex that translocates to the membrane upon GDI release,44 while the Rac/GDI complex is also able to effectively stimulate the NADPH-oxidase.25 Additional evidence for a positive signalling role for the GDI comes from the isolation of a Rac/GDI complex as a factor required for maintaining stimulated secretion in permeabilized mast cells.60 Interestingly, the GDI alone inhibited secretion, emphasizing the fact that over-expression of the GDI may mask its positive effects.

Conclusion While structural studies on the GDI provide a mechanistic understanding of the regulation of the membrane association of Rho GTPases by the GDI, the signalling pathways that modulate this interaction are only now becoming clear. As described above, the importance of membrane localization in signalling by Rho GTPases, and the ability of GDIs to regulate this activity, point to an important role for the GDI in signalling through Rho family proteins. Further, the involvement of the GTPase/GDI complex in ‘positive’ signals suggests that this complex represents an additional signalling state of the GTPase, in addition to the membrane associated GTP-bound form. The ability of the GDI to interact with both nucleotide states of the Rho GTPases suggests a mechanism in which the GDI may not only be involved in delivering the GDP-bound form of Rho proteins to the site of activation, but also in delivering the GTP-bound form to membrane compartments distant from the site of activation (Fig. 6). Interestingly, the membrane localization of certain Rho-family proteins in response to activating signals appears to be distinct from the ‘default’ membrane localization seen in over-expression studies,15 suggesting that shuttling by the GDI may be important to accessing specific membrane bound locations in response to extracellular stimuli. The structural studies described here provide a mechanistic understanding of the biochemical activities of the GDI. Even more valuable will be the tools provided by the structure, particularly the ability to specifically disrupt formation of the GTPase/GDI complex and assess the role of the GDI in the varied signalling events mediated by Rho-family GTPases.

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11. Isomura M, Kikuchi A, Ohga N et al. Regulation of binding of rhoB p20 to membranes by its specific regulatory protein, GDP dissociation inhibitor. Oncogene 1991; 6(1):119-24. 12. Nomanbhoy TK, Erickson JW, Cerione RA. Kinetics of Cdc42 membrane extraction by Rho-GDI monitored by real-time fluorescence resonance energy transfer. Biochemistry 1999; 38(6):1744-50. 13. Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem 1992; 61:355-86. 14. Casey PJ, Seabra MC. Protein prenyltransferases. J Biol Chem 1996; 271(10):5289-92. 15. Michaelson D, Silletti J, Murphy G et al. Differential localization of Rho GTPases in live cells: Regulation by hypervariable regions and RhoGDI binding. J Cell Biol 2001; 152(1):111-26. 16. Adra CN, Ko J, Leonard D et al. Identification of a novel protein with GDP dissociation inhibitor activity for the ras-like proteins CDC42Hs and rac I. Genes Chromosomes Cancer 1993; 8(4):253-61. 17. Lelias JM, Adra CN, Wulf GM et al. cDNA cloning of a human mRNA preferentially expressed in hematopoietic cells and with homology to a GDP-dissociation inhibitor for the rho GTP- binding proteins. Proc Natl Acad Sci USA 1993; 90(4):1479-83. 18. Scherle P, Behrens T, Staudt LM. Ly-GDI, a GDP-dissociation inhibitor of the RhoA GTP-binding protein, is expressed preferentially in lymphocytes. Proc Natl Acad Sci USA 1993; 90(16):7568-72. 19. Platko JV, Leonard DA, Adra CN et al. A single residue can modify target-binding affinity and activity of the functional domain of the Rho-subfamily GDP dissociation inhibitors. Proc Natl Acad Sci USA 1995; 92(7):2974-8. 20. Adra CN, Manor D, Ko JL et al. RhoGDIgamma: A GDP-dissociation inhibitor for Rho proteins with preferential expression in brain and pancreas. Proc Natl Acad Sci USA 1997; 94(9):4279-84. 21. Zalcman G, Closson V, Camonis J et al. RhoGDI-3 is a new GDP dissociation inhibitor (GDI). Identification of a noncytosolic GDI protein interacting with the small GTP-binding proteins RhoB and RhoG. J Biol Chem 1996; 271(48):30366-74. 22. Hoffman GR, Nassar N, Cerione RA. Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 2000; 100(3):345-56. 23. Scheffzek K, Stephan I, Jensen ON et al. The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI. Nat Struct Biol 2000; 7(2):122-6. 24. Grizot S, Faure J, Fieschi F et al. Crystal structure of the Rac1-RhoGDI complex involved in nadph oxidase activation. Biochemistry 2001; 40(34):10007-13. 25. Di-Poi N, Faure J, Grizot S et al. Mechanism of NADPH oxidase activation by the Rac/Rho-GDI complex. Biochemistry 2001; 40(34):10014-22. 26. Longenecker K, Read P, Derewenda U et al. How RhoGDI binds Rho. Acta Crystallogr D Biol Crystallogr 1999; 55(Pt 9):1503-15. 27. Gosser YQ, Nomanbhoy TK, Aghazadeh B et al. C-terminal binding domain of Rho GDP-dissociation inhibitor directs N- terminal inhibitory peptide to GTPases. Nature 1997; 387(6635):814-9. 28. Keep NH, Barnes M, Barsukov I et al. A modulator of rho family G proteins, rhoGDI, binds these G proteins via an immunoglobulin-like domain and a flexible N-terminal arm. Structure 1997; 5(5):623-33. 29. Lian LY, Barsukov I, Golovanov AP et al. Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1. Structure Fold Des 2000; 8(1):47-55. 30. Nomanbhoy TK, Cerione R. Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy. J Biol Chem 1996; 271(17):10004-9. 31. Golovanov AP, Chuang TH, DerMardirossian C et al. Structure activity relationships in flexible protein domains: Regulation of rho GTPases by RhoGDI and D4 GDI. J Mol Biol 2001; 305(1):121-35. 32. Golovanov AP, Hawkins D, Barsukov I et al. Structural consequences of site-directed mutagenesis in flexible protein domains: NMR characterization of the L(55,56)S mutant of RhoGDI. Eur J Biochem 2001; 268(8):2253-60. 33. Gibson RM, Wilson-Delfosse AL. RhoGDI-binding-defective mutant of Cdc42Hs targets to membranes and activates filopodia formation but does not cycle with the cytosol of mammalian cells. Biochem J 2001; 359(Pt 2):285-94. 34. Worthylake DK, Rossman KL, Sondek J. Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1. Nature 2000; 408(6813):682-8. 35. Wei Y, Zhang Y, Derewenda U et al. Crystal structure of RhoA-GDP and its functional implications. Nat Struct Biol 1997; 4(9):699-703. 36. Rittinger K, Walker PA, Eccleston JF et al. Crystal structure of a small G protein in complex with the GTPase- activating protein rhoGAP. Nature 1997; 388(6643):693-7.

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37. Nassar N, Hoffman GR, Manor D et al. Structures of Cdc42 bound to the active and catalytically compromised forms of Cdc42GAP. Nat Struct Biol 1998; 5(12):1047-52. 38. Rittinger K, Walker PA, Eccleston JF et al. Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue. Nature 1997; 389(6652):758-62. 39. Mondal MS, Wang Z, Seeds AM et al. The specific binding of small molecule isoprenoids to rhoGDP dissociation inhibitor (rhoGDI). Biochemistry 2000; 39:406-412. 40. Regazzi R, Kikuchi A, Takai Y et al. The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins. J Biol Chem 1992; 267(25):17512-9. 41. Bourmeyster N, Stasia MJ, Garin J et al. Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP- ribosylation by the C3 exoenzyme of Clostridium botulinum. Biochemistry 1992; 31(51):12863-9. 42. Miura Y, Kikuchi A, Musha T et al. Regulation of morphology by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in Swiss 3T3 cells. J Biol Chem 1993; 268(1):510-5. 43. Nishiyama T, Sasaki T, Takaishi K et al. rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13- acetate (TPA)-induced membrane ruffling in KB cells. Mol Cell Biol 1994; 14(4):2447-56. 44. Takaishi K, Kikuchi A, Kuroda S et al. Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility. Mol Cell Biol 1993; 13(1):72-9. 45. Coso OA, Chiariello M, Yu JC et al. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signalling pathway. Cell 1995; 81(7):1137-46. 46. Lu W, Mayer BJ. Mechanism of activation of Pak1 kinase by membrane localization. Oncogene 1999; 18(3):797-806. 47. Kranenburg O, Poland M, Gebbink M et al. Dissociation of LPA-induced cytoskeletal contraction from stress fiber formation by differential localization of RhoA. J Cell Sci 1997; 110(Pt 19):2417-27. 48. Takaishi K, Sasaki T, Kameyama T et al. Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows. Oncogene 1995; 11(1):39-48. 49. Dash D, Aepfelbacher M, Siess W. Integrin alpha IIb beta 3-mediated translocation of CDC42Hs to the cytoskeleton in stimulated human platelets. J Biol Chem 1995; 270(29):17321-6. 50. Mizuno T, Kaibuchi K, Ando S et al. Regulation of the superoxide-generating NADPH oxidase by a small GTP- binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins. J Biol Chem 1992; 267(15):10215-8. 51. Fleming IN, Elliott CM, Exton JH. Differential translocation of rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor. J Biol Chem 1996; 271(51): 33067-73. 52. Dirac-Svejstrup AB, Sumizawa T, Pfeffer SR. Identification of a GDI displacement factor that releases endosomal Rab GTPases from Rab-GDI. Embo J 1997; 16(3):465-72. 53. Hirao M, Sato N, Kondo T et al. Regulation mechanism of ERM (ezrin/radixin/moesin) protein/ plasma membrane association: Possible involvement of phosphatidylinositol turnover and Rho-dependent signalling pathway. J Cell Biol 1996; 135(1):37-51. 54. Sasaki T, Takai Y. The Rho small G protein family-Rho GDI system as a temporal and spatial determinant for cytoskeletal control. Biochem Biophys Res Commun 1998; 245(3):641-5. 55. Tsukita S, Yonemura S. ERM (ezrin/radixin/moesin) family: From cytoskeleton to signal transduction. Curr Opin Cell Biol 1997; 9(1):70-5. 56. Groysman M, Russek CS, Katzav S. Vav, a GDP/GTP nucleotide exchange factor, interacts with GDIs, proteins that inhibit GDP/GTP dissociation. FEBS Lett 2000; 467(1):75-80. 57. Mehta D, Rahman A, Malik AB. Protein kinase C-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function. J Biol Chem 2001; 276(25):22614-20. 58. Chuang TH, Bohl BP, Bokoch GM. Biologically active lipids are regulators of Rac.GDI complexation. J Biol Chem 1993; 268(35):26206-11. 59. Arozarena I, Matallanas D, Crespo P. Maintenance of CDC42 GDP-bound state by Rho-GDI inhibits MAP kinase activation by the exchange factor Ras-GRF. evidence for Ras-GRF function being inhibited by Cdc42-GDP but unaffected by CDC42-GTP. J Biol Chem 2001; 276(24):21878-84. 60. O’Sullivan AJ, Brown AM, Freeman HN et al. Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a rac/rhoGDI complex. Mol Biol Cell 1996; 7(3):397-408.

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Rho GTPases

CHAPTER 4

Rho Family Guanine Nucleotide Exchange Factors Ian P. Whitehead

Abstract

T

he guanine nucleotide exchange factors for the Rho GTPases (RhoGEFs) are a large family of proteins that share a dual structural motif designated the DH/PH domain. The interaction of Rho with residues within the DH domain enhances the exchange of GDP for GTP, thus converting Rho into the biologically active form. Outside of the core DH/ PH domain structure, RhoGEFs are structurally unrelated to one another suggesting that they may be involved in the regulation of a multitude of physiological processes. These sequences often contain additional structural and functional domains that provide valuable clues to the identity of these native functions. RhoGEFs are themselves subject to regulation, either through autoinhibition, or by external signals. The recent determination of the structures of RhoGEFs in complex with their substrates, coupled with the identification and analysis of RhoGEFs in model genetic systems, has provided important insights into the temporal and spatial regulation of RhoGEF function. The common theme that emerges is that RhoGEFs are often used as molecular sensors that can directly convert intra- or extra-cellular signals into localized modifications to actin-based cytoskeletal structures.

Introduction The Rho-specific guanine nucleotide exchange factors (RhoGEFs) are a large, structurally related family of enzymes that share a common ability to catalyze the exchange of GDP for GTP on members of the Rho subfamily of small GTPases (Table 1). Since Rho proteins will only bind with their effector targets when in the GTP-bound state, the RhoGEFs can be considered a family of Rho activators. Through the combined, and extensive, efforts of expression and database cloning strategies, the number of proteins that have been assigned to this family has risen at an alarming rate within the past several years. Based on the recent compilation of the complete genome sequences for a variety of organisms it has been estimated that there may be as many as 46 RhoGEFs in H. sapiens, 19 in D. melanogaster, 20 in C. elegans, 9 in S. cerevisiae, and 8 in A. thaliana.1 As an increasing number of RhoGEFs have been described and characterized, the number of cellular activities or behaviors that are known to be regulated by these proteins has also increased. Through their interactions with Rho-type GTPases, RhoGEFs have been implicated in the regulation of processes as diverse as chemotaxis, growth cone migration, bacterial invasion, cytokinesis, motility, cell polarity and oncogenic transformation. Most of these activities are presumed to occur as a consequence of the important regulatory role that RhoGEFs and their substrates play in the remodeling of the actin cytoskeleton. RhoGEFs also share a common ability to regulate signals to the nucleus, which stimulate transcriptional pathways or the cell cycle machinery. It should come as no surprise that such a large family of proteins that Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

Rho Family Guanine Nucleotide Exchange Factors

47

Table 1. Properties of RhoGEFs Organism

Designation

Specificity1

Comments

Mammals

Dbl

Cdc42, RhoA

Lbc Lfc

RhoA RhoA

Lsc/p115 -RhoGEF Dbs/Ost Tiam1

RhoA Cdc42, RhoA Rac1

Vav1

Rac1,Cdc42

Vav2

FGD1

Rac1, Cdc42, RhoA Rac1, RhoA, RhoG Cdc42

Trio

Rac1, RhoA

Abr

Tim NET1 Sos

Cdc42, RhoA, Rac1 Cdc42, RhoA, Rac1 Cdc42, RhoA, Rac1 NR RhoA Rac1

RasGRF1

Rac1

RasGRF2

Rac1

ARHGEF3 ARHGEF4

NR NR

Brx

NR

CDEP Collybist in/ hPEM-2 Ephexin

NR Cdc42

Frabin GEF337

Cdc42 RhoA

Oncogene;activated by truncation Oncogene Oncogene; binds microtubules Oncogene; RGS domain GAPs on Gα13 Oncogene Invasive phenotype in T lymphoma cells Oncogene; activated by phosphorylation Oncogene; ubiquitously expressed May regulate GTPhydrolases Disrupted in faciogenital dysplasia Encodes two DH/PH domain modules Similar to Bcr but lacks kinase domain Rearrangements associated with CML Oncogene; may regulate cytokinesis Oncogene Oncogene Contains a RasGEF domain Contains a RasGEF domain Contains a RasGEF domain Widely expressed Expression restrictcted to brain Modulates estrogen receptor Expressed in chondrocytes Induces clustering of gephryin Regulates growth cone dynamics Binds actin filaments Promotes actin stress fiber formation

Vav3

Bcr Ect-2

RhoA, Cdc42

continued on next page

Rho GTPases

48

Table 1. Continued Designation

Specificity1

Comments

GEF-H1 Intersectin

Rac1, RhoA NR

KIAA0380

RhoA

LARG

RhoA

Ngef

NR

p114-RhoGEF p116-RIP

RhoA RhoA

p190-RhoGEF PDZ-RhoGEF

RhoA NR

Pix/Cool /ARHGEF6 Kalirin

Rac1

Duet

NR

Stef

Rac1

Cdc24p/ Scd1p Rom1p, Rom2p DRG

Cdc42p

Binds microtubules Component of the endocytic machinery Contains PDZ and RGS domains Rearrangement associated with MLL Oncogene; expressed in brain Widely expressed Promotes neurite outgrowth Binds microtubules Binds Gα subunits through RGS domain Mutated in X-linked mental retardation Regulates dendritic morphogenesis Contains serine/ threonine kinase activity Closely related to Drosophila Sif Regulate cell polarity

Rac1A

MyoM

NR

DrtGEF

NR

DRhoGEF2 Pebble Sos

NR Rho1 NR

SIF

NR

C. Elegans

UNC-89 UNC-73

NR Rac1

Salmonella

SopE

Cdc42

SopE2

Cdc42

Organism

Yeast

Dictyostelium

Drosophila

Rac1

Rho1p

Implicated in bud emergence Encodes both GAP and GEF activity Unconventional myosin protein Highly expressed in morphogenic tissues Regulator of gastrulation Regulator of cytokinesis Regulates eye development Expressed in synaptic terminals Muscle protein Required for growth cone migration Bacterial toxin Contains no DH/PH domain Bacterial toxin Contains no DH/PH domain

1 Exchange specificities are for DH domain related activities only.NR = none reported.

Rho Family Guanine Nucleotide Exchange Factors

49

participates in such a diverse array of fundamental biological processes would be linked to an increasing number of clinical disorders in humans.

Structure of RhoGEFs All members of the RhoGEF family share an ≈ 300 amino acid region of similarity with Dbl, a transforming protein that was originally isolated from a diffuse B-cell lymphoma. This region can be further sub-divided into two recognizable domains, an ≈200 amino acid Dbl homology (DH) domain and an ≈100 amino acid pleckstrin homology domain. The DH domain is a unique feature of the RhoGEFs, while PH domains have been identified in numerous other signalling molecules.2 The DH and PH domains are almost always found in a tandem array in RhoGEFs with the PH domain located immediately carboxyl-terminal to the DH domain. The only exceptions to this conserved topography are DRG which contains two DH domains which flank a single PH domain,3 and a splice variant of βPIX, which contains no PH domain at all.4

DH Domains It has now been demonstrated through a number of cell-based, biochemical, and structural studies that the DH domain represents the core catalytic domain of RhoGEFs. In any instance where the biological activity of a RhoGEF is mediated via its interaction with a Rho GTPase substrate, this activity is dependent upon the structural integrity of the DH domain. For example, a subset of the RhoGEFs has been isolated based on their transforming activity in NIH 3T3 focus-formation assays.5 This activity is presumed to occur through their interaction with Rho family GTPases since mutants of Rho proteins that are constitutively activated exhibit a similar oncogenic activity. Although fragments of RhoGEFs that contain the isolated, intact DH/PH domain module retain all of their transforming activity, this activity is abrogated by deletions or point mutations that disrupt the DH domain.6-10 Biochemical studies also suggest that isolated DH domains can contain all the residues that are necessary to catalyze guanine nucleotide exchange.11-15 However, in virtually all cases, the associated PH domain is required for full biological activity in vivo. Early predictions that the DH domain structure would be all α-helical5 have been confirmed by the recent determinations of DH domain structures from Vav, Tiam1, Sos1, Trio, βPIX and Dbs.13,14,16,17 In each case the DH domain was shown to be a flat, elongated bundle of α-helices. Three conserved regions, designated CR1, CR2 and CR3,5 pack together to form the central core suggesting that other DH domains may have similar structures. In the cases of Tiam1 and Dbs, the DH domain structures have been resolved in complex with their GTPase substrates (Rac1 and Cdc42 respectively), and in both instances the interface between GTPase and GEF is extensive and is mediated primarily by residues within the CRI and CR2 domains.16 Consistent with the biochemical characterization of RhoGEFs, Tiam1 binds to the Switch I and II domains of Rac1 causing them to assume a conformation that blocks Mg++ binding and favoring the release of GDP. Interestingly, the extent of the interface between GEF and substrate precludes the possibility that the GTPase can simultaneously bind to both a GDI and GEF suggesting that both families of regulatory proteins may compete for access to the substrate. A search of the relevant databases suggests that the structure of the DH domain is unique, bearing no resemblance to other families of GEFs. Despite the fact that small GTPases are highly conserved in their structure, each subfamily is associated with a structurally distinct and highly specific family of exchange factors. For example, the RasGEF region of Sos1 and the ArfGEF domain of Gea2 are structurally unrelated to one another, and to the DH domain.18,19

PH Domains With respect to the full biological activity of RhoGEFs in vivo, the PH domain is as indispensable as the DH domain. Point mutations in conserved residues of either domain are often

50

Rho GTPases

associated with a complete loss of cellular activity.6-8 Although generally considered to be noncatalytic in nature, PH domains are thought to perform several tasks within the context of a RhoGEF. These include contributing to the correct folding of the DH domain, direct modulation of catalytic activity, and recruitment of RhoGEFs to cellular membranes. These latter two functions may be mediated by the association of PH domains with phosphoinositide second messengers and will be discussed in detail in subsequent sections of this chapter. Although there have also been numerous reports of nonRhoGEF associated PH domains mediating protein-protein interactions, such reports have been rare for RhoGEFs, one exception being the PH domain of Lfc which has been shown to bind directly to tubulin.20 The PH domain structures are known for the RhoGEF domains of Sos1, Dbs and Tiam1.16,17 Typical of other PH domains, the PH domains of RhoGEFs form antiparallel β-sandwiches composed of two β-sheets that are capped at one end by the carboxyl-terminal α-helix. Mutational analysis has identified clusters of positively charged residues contained within the highly variable loops that connect the β-strands that are the likely sites of phosphoinositide binding. In the proximity of the hinge region that connects the DH and PH domains are additional secondary structural elements that may be unique to the PH domains of RhoGEFs.

Additional Structural and Regulatory Motifs Outside of the well conserved DH/PH domain module, RhoGEFs exhibit relatively little similarity to one another. Nonetheless, a large and diverse array of motifs that are commonly found in other families of signalling molecules have also been described in RhoGEFs.5 These include consensus sites for protein phosphorylation, C2 domains that are thought to bind diacylglycerol in a calcium dependent manner, and SH2, SH3 and PDZ domains that are likely to mediate binding to other proteins. Additional catalytic domains have also been described that would allow for the coordinate regulation of multiple substrates. These include kinase domains, RhoGAP domains, RasGEF domains, additional RhoGEF domains, and domains that can bind and regulate membrane-bound heterotrimeric G proteins. The diversity of these signalling motifs suggests that the regulation of RhoGEFs will be equally diverse reflecting the multitude of biological activities in which they are known to participate.

Regulation of RhoGEFs It is generally thought that, in the absence of activating signals, RhoGEFs exist in the cell in a quiescent, catalytically inactive state. This state is maintained by autoinhibitory interactions between the DH domain and either the adjacent PH domain, or more distal regulatory sequences. In response to activating signals these constraints are relieved, exposing the catalytic interface and allowing access to the GTPase substrates. The large array of recognizable signalling motifs that have been identified within RhoGEFs provide ample opportunities for upstream signalling events to impinge upon catalytic function. The diversity of these motifs suggests that the signals that regulate RhoGEFs are likely to be equally diverse, and in some cases unique to a particular family member. Not surprisingly, there are an increasing number of distinct classes of signalling molecules that have been shown to interact directly with RhoGEFs and stimulate their catalytic activity. These include phosphoinositides, protein tyrosine or serine/ threonine kinases, heterotrimeric G protein Gα subunits and other regulators of small GTPases.

Regulation by PH Domains The invariable pairing of the DH domain with the PH domain suggests that the RhoGEF PH domains may play a specific role in regulating GEF activity. Although there is some structural and in vivo data to suggest that this may be the case, the picture that is emerging is complex, with PH domains having positive, negative or no effect at all on catalytic activity. For example, whereas the isolated DH domain of Trio is competent to catalyze guanine nucleotide exchange on its substrates in vitro, addition of the PH domain dramatically increases exchange proficiency.13 Structural studies of the DH/PH domain module of Tiam1 in complex with Rac1 provide some clues as to a potential mechanism for such positive regulation. In Tiam1 the

Rho Family Guanine Nucleotide Exchange Factors

51

carboxyl-terminus of the DH domain interacts with both Rac1 and the PH domain suggesting that the structural stabilization of Rac1 is dependent on both the DH and PH domains. In addition, the DH/PH domain of Tiam1, which is catalytically active in vitro, assumes a conformation that is favorable for Rac1 binding. However, the structural data for Tiam1 are in direct contrast to what has been observed for Sos1 and βPIX. The DH/PH domain of Sos1 is catalytically inactive in vitro, and assumes a configuration that would deny access to the substrate,17 while the DH and PH domains of β-PIX seem to act as independent units in solution.4 There is also some biological data to suggest that functional interdependence between the DH and PH domains may not be a characteristic of all RhoGEFs. For example, the DH domain of Lbc, when expressed alone, retains most of its biological activity in vivo,21 and the PH domains of Lfc and Dbs can be functionally replaced by plasma membrane targeting signals.7,15 Thus, while it is clear from the structural data that the PH domain is ideally situated and competent to directly regulate catalytic activity, there is also evidence to suggest that such regulation may not always occur.

Regulation by Distal Autoinhibitory Domains In addition to regulation by the adjacent PH domain, there is evidence that several RhoGEFs are regulated by additional autoinhibitory domains located outside of the DH/PH domain motif. Many of the RhoGEFs that were isolated in transformation assays require truncation of specific amino- or carboxyl-terminal sequences to expose their oncogenic activity. Such sequences are likely to impose intramolecular blocks on catalytic activity. For example, both Dbl and Vav contain amino-terminal sequences that can bind directly to the DH/PH domain module and block access to the binding surface of the GTPase.14,22 In the case of Vav, this autoinhibition is a consequence of an interaction between an amino-terminal α-helix and the GTPase binding site of the DH domain.14 These inhibitory contacts are relieved by phosphorylation of tyr-174 which is contained within the amino-terminal regulatory domain (discussed later in this chapter). In contrast, a structurally distinct domain within the amino-terminus of Dbl blocks access to substrates by forming contacts with the PH domain.22 Although the mechanism by which this constraint is relieved in vivo is not known, it may also involve phosphorylation on tyrosine residues. The large number of RhoGEFs that exhibit enhanced biological activity when expressed in a truncated form suggests that autoinhibition may be a common regulatory mechanism employed by RhoGEFs.

Regulation by Self-Association Recent studies suggest that oligomerization may also contribute to the regulation of at least a subset of the RhoGEFs. The crystal structure of Tiam1 in complex with Rac1 consists of heterotetramers whose formation are mediated by residues within the DH domain,16 and RasGRF1, RasGRF223 and an oncogenic derivative of Dbl24 are thought to undergo oligomerization in a DH domain-dependent manner. In the case of RasGRF1, RasGRF2 and Dbl, the association seems to be homophilic in nature since binding can be detected with closely related DH domains but not with those that are more distantly related. It is unclear what the function of oligomerization will be in vivo. The introduction of point mutations into the DH domain of Dbl that block oligomerization have no effect on GTPase binding and GEF activity in vitro, but exhibit reduced GEF activity in vivo.24 This reduction in activity is accompanied by selective loss of some biological responses that are normally associated with overexpression of oncogenic derivative of Dbl (e.g., anchorage independent growth) and an overall reduction in transforming activity. Based on these observations, it has been argued that oligomerization favors localized augmentation of Rho GTPase activation, which may be necessary for full oncogenic transformation. However, it has not been determined whether full-length Dbl also undergoes oligomerization and if so, what such an event contributes to its normal biological function. Although it will be of interest to see which other RhoGEFs undergo oligomerization, it may not be a universal phenomenon since the crystal structure of Dbs/Cdc42 does not consist of heterotetramers (John Sondek, personal communication).

Rho GTPases

52

Table 2. Binding of RhoGEFs to phosphoinositides GEF

Binds

Comments

Sos1

PtdIns(3,4,5)P3 PtdIns(4,5)P2 PtdIns(1,4,5)P3 PtdIns(4,5)P2

Stabilizes the DH/PH interface Destabilizes the DH/PH interface Function unknown Stabilizes the DH/PH interface; Inhibits exchange Destabilizes the DH/PH interface; Promotes exchange Promotes exchange Promotes exchange Weak interaction; Promotes exchange Weak interaction; No effect on exchange Function unknown Function unknown Blocks Cdc42 binding; Inhibits exchange Blocks Cdc42 binding; Inhibits exchange

Vav

PtdIns(3,4,5)P3 Tiam11

Trio1 Dbl

PtdIns(3,4)P2 PtdIns(3,4,5)P3 PtdIns(3,4)P2 PtdIns(4,5)P2 PtdIns(4,5)P2 PtdIns(4)P PtdIns(3,4,5)P3 PtdIns(4,5)P2

1 Binding properties for Tiam1 and Trio are for their amino-terminal PH domains.

Regulation by Phosphoinositides RhoGEFs are generally thought to require membrane localization in order to interact with their membrane-bound GTPase substrates. There is now substantial evidence that the association of RhoGEFs with membranes is mediated by interactions between PH domains and phosphoinositides. Not only are phosphoinositides thought to direct RhoGEFs to membranes, but it also thought that they can contribute directly to catalytic activation. Although PH domains will preferentially bind to phosphoinositides relative to other membrane lipids, they can differ in their ability to interact with specific members of the phosphoinositide family (Table 2). For example, the PH domains of Dbl, Sos1 and the amino-terminal PH domain of Tiam1 show their highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3),25-27 suggesting that they may be regulated by phosphoinositide 3-kinase (PI3K), while Dbs and Intersectin bind preferentially to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), and the carboxyl-terminal PH domain of Tiam1 binds with greatest affinity to phosphatidylinositol 3-phosphate (PtdIns(3)P) (John Sondek, personal communication). The possibility that RhoGEF PH domains may exhibit specificity with respect to phosphoinositide binding may provide a mechanism through which RhoGEFs may be targeted to specific membrane locations. Since the PH domains of RhoGEFs bind with phosphoinositides, and phosphoinositides such as PtdIns(3,4,5)P3 are involved in the recruitment of signalling proteins to the plasma membrane, it is tempting to speculate that the sole function of phosphoinositide binding with respect to RhoGEFs is to mediate membrane association. However, there is evidence that in at least one case binding to phosphoinositides is not associated with, or sufficient for membrane association. Although the amino-terminal PH domain of Tiam1 binds with high affinity to PtdIns(3,4,5)P3, this isolated PH domain is not localized to the membrane and the PI3K inhibitor Wortmannin has no effect on the subcellular localization of Tiam1.28 What then is the function of phosphoinositides with respect to the regulation of Tiam1? There is in fact

Rho Family Guanine Nucleotide Exchange Factors

53

Figure 1. A model for the sequential activation of Vav by phosphoinositides and phosphorylation. In the basal state the PH domain of Vav is bound to PtdIns(4,5)P2 which favors binding of the PH domain to the DH domain. This conformation would hinder access of tyrosine kinases to tyr-174. Vav activation is initiated by the conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 by PI3K which would disrupt the DH/PH interface, allowing access by Lck. Subsequent phosphorylation on tyr-174 causes the autoinhibitory domain to lose its structure allowing access of the DH domain to the GTPase substrate.

evidence that a number of PH domains, within the context of RhoGEFs, can directly regulate GEF activity via their interaction with phosphoinositides. This includes Tiam1 for which it has been demonstrated that two products of PI3K, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, enhance Tiam1 guanine nucleotide exchange activity in vitro.26 Interestingly, the carboxyl-terminal DH-associated PH domain of Tiam1 binds a different phosphoinositide (PtdIns(3)P), yet this binding is not associated with altered GEF activity in vitro (J. Sondek, personal communication). Therefore, the two PH domains of Tiam1 are likely to have discrete functions with respect to both membrane localization and catalytic regulation. Tiam1 is not the only RhoGEF whose catalytic activity is regulated by phosphoinositide binding. Studies on the PH domain of the Vav oncoprotein have determined that binding to the product of PI3K, PtdIns(3,4,5)P3, enhances GEF activity, whereas binding to the PI3K substrate PtdIns(4,5)P2 decreases GEF activity.29 Interestingly, the full-length Vav protein lacks GEF activity in vitro, yet Vav without the PH domain exhibits biological activity in vivo. This supports a model in which the PH domain of Vav, through its interaction with discrete pools of phosphoinositides, directly regulates exchange activity. In further support of this model, it has been demonstrated that PtdIns(4,5)P2 increases the affinity of the Vav DH domain for the PH domain, interfering with its ability to bind Rac1. In contrast, binding of the Vav PH domain to PtdIns(3,4,5)P3 reduces the affinity of the DH domain for the PH domain, and is permissive for Rac1 binding.30 Although it is still unclear what the consequences of phosphoinositide binding to RhoGEFs is in vivo, it seems clear that the tight coupling of DH domain/GTPase and PH domain/phosphoinositide complexes at the membrane should have a profound effect on catalytic activity.

Regulation by Phosphorylation Several RhoGEFs are known to become phosphorylated in response to extracellular stimuli and this event is generally accompanied by activation of an associated GTPase. Consistent with this, in virtually all cases that have been examined, RhoGEF phosphorylation has been shown to directly regulate exchange activity in vitro. For example, the activation of Rac1 by RasGRF1 is significantly enhanced following phosphorylation of RasGRF1 by the nonreceptor tyrosine kinase Src.31 Dbl and Vav also exhibit enhanced exchange activity when they are phosphorylated on tyrosines by the nonreceptor tyrosine kinases ACK1 and Lck respectively.32,33 Activation of RhoGEFs by phosphorylation is not just restricted to the tyrosine kinases. Membrane localization and enhanced nucleotide exchange toward Rac1 have also been reported for Tiam1 in response to phosphorylation on threonine by the serine/threonine kinase CamKII.34 Ect2 is phosphorylated during G2 and M phases by an unidentified kinase, and this phosphorylation is required for its exchange activity.35 Although relatively little is known about the mechanism through which phosphorylation can directly regulate the exchange activities of Tiam1, Dbl, Ect2 and RasGRF1, the mechanism through which tyrosine phosphorylation contributes to the activation of Vav has been extensively studied.

54

Rho GTPases

Figure 2. A model for RhoA activation by the Gα13 subunit of heterotrimeric G proteins. When the GPCR engages its ligand it becomes catalytically active, converting its associated Gα13 subunit to the GTP-bound state. Gα-GTP uncouples from the Gβγ dimer and interacts with the RGS domain in the amino-terminus of p115 RhoGEF, thus stimulating the GEF activity of p115 RhoGEF for RhoA.

Vav expression has been observed primarily in hematopoeitic tissues and it is known to be rapidly phosphorylated in response to antigen stimulation of the B- and T-cell receptors.36-38 Phosphorylation is mediated through the actions of Syk and Src-family nonreceptor tyrosine kinases.32,39 Phosphorylation of Vav invariably results in the stimulation of nucleotide exchange activity directed towards Rac1. In the absence of phosphorylation, exchange activity is not detectable, even in in vitro assays. The best characterized site of phosphorylation on Vav is tyr-174 which has been shown to be a substrate for both Syk and Lck.32 Phosphorylation of this residue by Lck enhances GEF activity of Vav in vitro, and amino-terminal truncations that remove this residue eliminate the catalytic requirement for phosphorylation.32 Interestingly, phosphorylation on tyr-174 of Vav is facilitated by the presence of PtdIns(3,4,5)P3 and hindered by the presence of PtdIns(4,5)P2.29 This correlates with phosphoinositide-mediated binding of the PH domain to the DH domain construct with PtdIns(4,5)P2 enhancing and PtdIns(3,4,5)P3 decreasing the binding affinity.30 This suggests that phospholipid binding to the PH domain may contribute to the structural coupling between tyr-174 and the DH domain. The structure of Vav in solution lends some support to such a mechanism. The amino-terminal inhibitory domain forms an α-helix that interacts with conserved residues in the DH domain, effectively blocking access by the substrate.14 At the center of this inhibitory complex is tyr-174 and phosphorylation of tyr-174 causes the α-helix to lose its structure and release from the catalytic interface. Based on the NMR structure of autoinhibited Vav, a model has been proposed for Vav regulation (Fig. 1). In its inactive state, the PH domain of Vav would be bound to PtdIns(4,5)P2, which would favor binding of the PH domain to the DH domain, which in turn would sterically hinder access of tyrosine kinases to tyr-174. Vav activation would be initiated by the conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 by PI3K, which would disrupt the DH/PH domain interface. This would allow access by tyrosine kinases to tyr-174. Subsequent phosphorylation would cause the autoinhibitory domain to lose its structure and expose the catalytic activity. Ect2, Dbl and Tiam1 are also regulated by amino-terminal sequences that lie outside of the DH domain. Like Vav, the regulatory sequences of Dbl have been localized and have been shown to form intramolecular contacts with the DH/PH domain module.22 Whether or not this inhibition is dependent upon phosphoinositide binding, or is released by phosphorylation, remains to be determined. The observation that Dbl GEF activity is regulated by the tyrosine kinase ACK1 suggests that Vav and Dbl may be subject to similar regulatory mechanisms.33 Although evidence for direct roles of phosphorylation in the regulation of RhoGEF activity remains scarce, it seems likely that such evidence will be forthcoming and that this will represent a relatively common mechanism of RhoGEF activation.

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Figure 3. A model for the coordinate regulation of Ras and Rac1 by Sos1. Activation of a receptor tyrosine kinase by its ligand recruits the Grb/Sos1 complex to the plasma membrane. The RasGRF domain of Sos1 activates membrane-bound Ras, which in turn can interact with and activate its effector protein PI3K. PI3K then phosphorylates PtdIns(4,5)P2, which is bound to the PH domain of Sos1, converting it into PtdIns(3,4,5)P3. This would modify the interface between the DH and PH domains allowing access to Rac1.

Regulation by Gα Subunits The heterotrimeric G proteins are a large family of membrane associated GTPases that coordinate the conversion of extracellular stimuli into intracellular signalling events. They are anchored to the intracellular surface of the plasma membrane where they are activated by an associated family of transmembrane receptors (G protein coupled receptors or GPCRs). Typically, a heterotrimeric G protein consists of a Gα subunit that contains the guanine nucleotide binding site, and a dimer that consists of tightly associated β and γ subunits (Gβγ). GPCRs contain GEF activity and can activate heterotrimeric G proteins by catalyzing the exchange of GDP for GTP on the Gα subunit. When a GPCR is engaged by its ligand it will become catalytically activated, converting Gα to the GTP-bound state. The Gα subunit will then become dissociated from the dimer allowing both components to function independently to stimulate distinct signalling events. Inactivation of heterotrimeric G proteins is regulated by a family of GAPs, which share a common catalytic domain referred to as the regulator of G protein signalling (RGS) domain. It has now been established that a distinct subset of the cellular changes that accompany the activation of heterotrimeric G proteins occur in a Rho-dependent manner.40 In cases where the heterotrimeric and the Rho GTPases are known to regulate similar cellular processes, they often appear to function within the context of the same signalling pathway. At least one mechanism has now been identified through which these two families of signalling molecules are coupled, and this mechanism involves a RhoGEF. p115 RhoGEF is a RhoA specific exchange factor that also contains an RGS domain within its amino-terminus.41,42 The RGS domain of p115 RhoGEF is specific for a subset of the Gα subunits, which includes Gα12 and Gα13.43 Importantly, binding of Gα13 to the RGS domain of p115 RhoGEF stimulates its catalytic activity for RhoA in vitro, and in vivo.44,45 Thus, a surprisingly simple model emerges (Fig. 2) in which activation of Gα13 causes the recruitment and activation of p115 RhoGEF which in turn causes activation of RhoA. Since p115 RhoGEF can also function as a GAP for Gα13, this would allow for rapid cycling of the complex. p115 is not the only RhoGEF that may be regulated by direct interactions with heterotrimeric G proteins. PDZ-RhoGEF and LARG also have RGS domains, and may also link heterotrimeric G proteins with Rho GTPases.46,47 PDZ-RhoGEF has been shown to bind to Gα subunits, but whether the RGS domains of PDZ-RhoGEF, or LARG, can function as GAPs for Gα subunits, or whether Gα binding stimulates their exchange activity, remains to be determined. Nonetheless, p115 RhoGEF, PDZ-RhoGEF and LARG are likely to represent an important subgroup of the RhoGEFs that can functionally link extracellular signalling via GPCRs to Rho-mediated effector responses.

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Regulation by Small GTPases There is also evidence that members of the RhoGEF family may have the capacity to coordinately regulate the activity of more than one small GTPase. GEFs such as Sos1 and RasGRF1 contain GEF domains for both Ras and Rho proteins and there is evidence that Sos1 can activate Rac in a Ras-dependent manner.48 Trio and DRG each contain two distinct DH domains and proteins such as BCR contain both DH and GAP domains. Although the mechanisms through which BCR, Trio and DRG may coordinately regulate multiple Rho family GTPases remain largely unknown, recent studies have suggested a mechanism through which Sos1 may coordinately regulate the activation of both Ras and Rac1.48 Sos1 is a member of the RhoGEF family that is probably better known as an activator of Ras.49 In addition to its DH domain, which contains GEF activity for Rac1, Sos1 also contains a structurally distinct domain through which it can catalyze guanine nucleotide exchange on members of the Ras subfamily. Since several studies have positioned Rac1 downstream of Ras, there has been keen interest in the role that Sos1 may play in linking these two GTPases. In a manner very similar to Vav, the binding of phosphoinositides to the adjacent PH domain regulates the DH domain of Sos1.30 As is the case with Vav, PtdIns(4,5)P2 promotes the binding of the DH and PH domains of Sos1, interfering with substrate binding, while PtdIns(3,4,5)P3 supports a conformation that favors substrate binding. Expression of the Sos1 DH domain alone in COS-7 cells can activate Rac1, while the DH/PH domain module can only activate Rac1 if it is coexpressed with a derivative of Ras that is competent to activate PI3K.48 PI3K is an effector protein for Ras that will bind and become activated when Ras is in the GTP-bound state. These observations are consistent with the phosphoinositide binding studies and suggest that one of the products of PI3K may be necessary to activate the DH domain of Sos1 in vivo. It also provides the framework for a model that would explain the coordinate regulation of Ras and Rac1 by Sos1 (Fig. 3). This model proposes that recruitment of Sos1 to the cellular membrane results initially in the activation of Ras by the Sos1 encoded RasGRF domain. Activated Ras would then interact with and activate its effector protein PI3K. PI3K could then, in turn, phosphorylate PtdIns(4,5)P2 which is bound to the PH domain of Sos1, converting it into PtdIns(3,4,5)P3. This would convert the DH domain of Sos1 into an active state and support exchange on Rac1. Although many aspects of this model must be confirmed by additional in vivo studies, it provides another mechanism through which RhoGEFs may play a critical role in converting extracellular signals into specific intracellular responses. As more is learned about the coordinate regulation of multiple GTPases by RhoGEFs the number of distinct regulatory mechanisms that they employ are likely to increase.

Physiological Roles for RhoGEFs Although RhoGEFs and their substrates have well documented roles in the regulation of cell morphology, the mechanisms that ensure that they are able to perform complex morphogenetic alterations in a spatially and temporally precise manner are not well understood. With the generation of mouse strains that are homozygous null for specific RhoGEFs, and the identification of RhoGEFs in species that are more amenable to genetic manipulation, the role that some of these RhoGEFs may play in the regulation of specific morphogenetic changes can now be examined in more detail. Since several chapters in this volume are devoted to the analysis of Rho function in model systems, we will confine the discussion here to some specific examples of biological processes in which the regulatory role of the RhoGEF has been more clearly defined. These include regulation of neuronal development, cytokinesis, bud emergence, and cellular transformation. The picture that emerges from these studies is that local activation or recruitment of RhoGEFs is an important strategy that is used by many organisms to link global changes in cell behavior to the local reorganization of the actin cytoskeleton.

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Figure 4. Activation of Pak by Trio in the growth cones of developing photoreceptors. Localized activation of Trio in the growth cone of a photoreceptor causes activation of Rac1 by the GEF1 domain. The adapter protein Dock recruits Pak to the same location and the two events converge. Rac-GTP binds to the CRIB domain of Pak, stimulating its kinase activity.

RhoGEFs and Neuronal Development During development of the nervous system intricate patterns of neuronal connections are formed as axons are directed towards their targets by guidance clues. This targeting system relies on the local reorganization of the actin cytoskeleton within the growth cone which can direct axonal growth away from, or towards, these extracellular markers. However, the mechanism by which guidance clues are able to convert positional information, via cellular receptors, into appropriate cytoskeletal changes is not fully understood. There is now mounting evidence that a subfamily of the RhoGEFs that includes proteins such as Trio, UNC-73 and Ephexin, may function within the context of growth cones to regulate these processes. Genetic studies have shown that UNC-73 is necessary for axon guidance in C. elegans,50 and a loss-of-function mutation in murine trio causes aberrant neuronal organization that would be consistent with a defect in axon pathfinding.51 However, the most compelling evidence linking RhoGEFs to the regulation of axon guidance comes Drosophila where a series of elegant genetic studies have identified a crucial role for Drosophila Trio in regulating multiple axon guidance pathways.52-54 These studies revealed that loss-of-function mutations in Trio were associated with defective guidance of photoreceptors, motor axons, and neurons of the embryonic central nervous system. Importantly, in each of these studies Trio expression was observed along the length of axons and at particularly high levels within the growth cone. With respect to photoreceptor guidance in Drosophila, Trio mutants exhibit phenotypes that are indistinguishable from those of Dock and Pak.54 Dock is an SH2-SH3 adaptor protein that binds to the amino-terminus of Pak, while Pak is a serine/threonine kinase that is an effector for Rac1. The recruitment of Pak to the plasma membrane by Dock is required for correct pathfinding by photoreceptors. What emerges from these studies is a model in which Trio is activated at the cell membrane and in turn activates Rac1 (Fig. 4). In response to a second signal, Dock recruits Pak to the cellular membrane where the two events can converge culminating in Pak activation. How extracellular guidance clues are able to cause localized activation of Trio remains an important question. Recent studies looking at the regulation of repulsive signals for axon guidance in mice may provide some answers.

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Figure 5. Ect2 may link cell cycle progression to cytokinesis in mammalian cells. During interphase Ect2 is localized primarily in the nucleus. It becomes phosphorylated during the G2-M phases of the cell cycle, which stimulates its catalytic activity. Phosphorylated Ect2 enters the cytoplasm at the beginning of mitosis where it accumulates at the midbody prior to cytokinesis. Ect2 may activate RhoA at the midbody which in turn could stimulate the assembly of actin structures. Thus, ect2 may provide an important molecular link between cell cycle progression and the initiation of cytokinesis.

The ephrins are a family of extracellular guidance molecules that exert a repulsive effect on axons during the development of the central nervous system of mice. The binding of ephrins to their receptors, the Eph receptor tyrosine kinases, results in growth cone collapse via activation of RhoA.55 Ephexin is a member of the RhoGEF family that was isolated as a binding partner for Eph receptors.56 Ephexin shows greatest activity for RhoA in vitro, and ligand engagement by the receptor stimulates the catalytic activity of Ephexin and is associated with increased GTP levels on RhoA in vivo. Expression of a dominant-inhibitory version of Ephexin blocks ephrin-induced growth cone collapse in primary neurons. It is clear that the direct association between the receptor and the RhoGEF may provide an important molecular link that couples positional clues to localized alterations in the actin cytoskeleton. It remains to be determined whether Trio is also sequestered by transmembrane receptors during photoreceptor growth.

RhoGEFs and Cytokinesis The process of cytokinesis is responsible for the accurate partitioning of cellular material between daughter cells during division. This involves the assembly and contraction of the actomyosin ring which drives the formation of a cleavage furrow. In addition to the structural components of the ring, which include actin, an assortment of actin-associated proteins, and the regulatory light chain of nonmuscle myosin, members of the Rho family have also been localized to the cleavage furrow in Xenopus embryos and mammalian cells.57,58 Not surprisingly, any cellular process that is driven by the action of Rho proteins is influenced by RhoGEFs, and cytokinesis is no exception. Recent studies in both Drosophila and mammalian systems point to an important role for RhoGEFs in this process.35,59 Pebble is a RhoGEF in Drosophila that is required for cytokinesis.59 During mitosis, Pebble accumulates at the cleavage furrow, and mutations in Pebble do not form a contractile ring and fail to undergo cytokinesis. The most likely target for Pebble is Rho1, since Rho1 mutations are also defective in cytokinesis, and the two proteins interact in a yeast 2-hybrid assay.59 What emerges from a large body of genetic data is a linear pathway that is responsible for the assembly of the contractile ring. The first step in this pathway is the activation of Rho1 by Pebble. In

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Figure 6. Cdc24p links cell cycle progression to bud emergence in Saccharomyces cerevisiae. At the beginning of G1 Cdc24p is sequestered in the nucleus by the adapter protein Far1p. Once the cell reaches a critical size, the G1-specific CDK, Cdc28p, phosphorylates Far1p. Once phosphorylated, Far1p dissociates from Cdc24p and is targeted for ubiquitin-mediated degradation. Cdc24p is then free to leave the nucleus where it is targeted to the incipient bud site via a Cdc28p-dependent mechanism. At the bud site, the Ras-like GTPase Rsr1 can directly activate Cdc24p in a GTP-dependent manner. Catalytic activation of Cdc24p could then trigger Cdc42p-dependent actin assembly. Rsr1 is activated by its own GEF, Bud5p, which is specifically localized to the budding landmark through its binding to Axl2p/Bud10p.

its GTP-bound form, Rho1 will interact with the formin-related protein Dia,60 which in turn will bind and activate profilin. Profilin is required for the polymerization of actin and is likely to be responsible for the assembly of the contractile ring. What then is the nature of the signal that coordinates cytokinesis with progression through the cell cycle, and how could such a signal direct the localized activation of Pebble? Studies on a mammalian relative of Pebble, Ect2,may provide an answer to this question. Ect2 is a RhoA specific RhoGEF that was identified as a transforming protein in NIH 3T3 focus-formation assays.10 Ect2 is closely related to Pebble, and has a pattern of expression that suggests that it may also be involved in the regulation of cytokinesis. During interphase Ect2 is localized primarily in the nucleus, but then enters the cytoplasm during the beginning of mitosis where it accumulates at the midbody prior to the onset of cytokinesis.35 Genetic inhibitors that block Ect2 function cause large multinucleate cells further supporting an important role for Ect2 in the regulation of cell division. In addition to the canonical DH/PH tandem structure, Ect2 also contains several motifs that suggest that it may be directly regulated by components of the cell cycle machinery. These include a region of homology to the yeast cyclin Clb6, two copies of the BRCA1-COOH-terminal repeat (BRCT) which is found in many checkpoint and repair proteins, and two consensus Cdc2 phosphorylation sites. Importantly, Ect2 becomes phosphorylated during the G2-M phases of the cell cycle and this phosphorylation is required for its catalytic activity.35 Thus, it is possible that Ect2 is directly phosphorylated by components of the cell cycle machinery, and that this phosphorylation may be required for its catalytic activation, as well as correct localization to the cleavage furrow. Thus, Ect2 may function as a nuclear sensor that provides a molecular link between cell cycle progression and the initiation of cytokinesis (Fig. 5). It remains to be determined whether Pebble would perform a similar function in Drosophila and whether it is also catalytically regulated by phosphorylation.

RhoGEFs and Bud Emergence in Yeast The budding yeast Saccharomyces cerevisiae exhibits polarized growth at several points during its life cycle. For example, during vegetative growth, and through a mechanism that is

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tightly linked to the cell cycle machinery, a bud will begin to emerge during late G1.61 This process is initiated by the cyclin-dependent kinase (CDK) Cdc28p which triggers a complex program of actin polarization.62 At the cell cortex, both the position and the timing of bud emergence are specified by a group of regulatory proteins that act through localized modifications to the actin cytoskeleton. Central to this regulatory complex is the Rho family GTPase Cdc42p, and its GEF Cdc24p, which become localized at the site of bud emergence.61 Mutant alleles of both CDC42 and CDC24 exhibit similar budding defects suggesting that activation of Cdc42p by Cdc24p is required for bud assembly. Although relatively little is known about the localized recruitment of Cdc42p, considerably more progress has been made in understanding the relationship between the cell cycle machinery, and the activation and recruitment of Cdc24p to the incipient bud site. At the beginning of G1 Cdc24p is sequestered in the nucleus by the adapter protein FarIp (Fig. 6).63-65 Once the cell reaches a critical size Cdc28p phosphorylates FarIp causing it to be released from Cdc24p and targeting it for ubiquitin-mediated degradation.64-66 Cdc24p is now free to exit the nucleus where it is targeted to the bud site through a Cdc28p-dependent mechanism.67 Once Cdc24p reaches the bud site it probably interacts with the Ras-related small GTPase RsrI/BudI. RsrI/BudI, which is also present at the bud site, will interact with the amino-terminal regulatory domain of Cdc24p in a GTP-dependent manner.68 Binding to GTP-bound RsrI/BudI may stimulate the catalytic activity of Cdc24p, which in turn, could trigger Cdc42-dependent actin assembly. RsrI/BudI is activated by its own GEF, Bud5p69 which colocalizes with Axl2p/Bud10p, a component of the early budding landmark.70 Thus, similar to the case with Ect2, Cdc24p may function as a nuclear sensor that can actively participate in the conversion of temporal information from the cell cycle machinery, into the assembly of a spatially regulated actin structure at a distant cellular location.

Transforming Activity of RhoGEFs Although recent studies have begun to reveal the broad assortment of normal biological activities that are regulated by members of the RhoGEF family, historically the family has attracted the most attention for its potential role in oncogenesis. A large subset of the RhoGEFs has been isolated in screens for proteins whose expression cause deregulated growth in NIH 3T3 mouse fibroblasts.5,71 Typically, NIH 3T3 cells that are transiently transfected with oncogenic RhoGEFs fail to undergo contact inhibition when confluence has been reached, forming large dense foci that are morphologically distinct from those that have been reported for oncogenes such as Ras or Src. Cell lines that stably express these oncogenic RhoGEFs also exhibit transformation as measured by a variety of parameters including growth in low serum, anchorage independence and tumorigenicity in nude mice. The transforming activity of RhoGEFs is often associated with truncated derivatives.5,71 Such truncations are presumed to relieve intramolecular structural constraints that normally mask the catalytic activity of the GEF when it is in its quiescent state. Since equivalent truncations activate both GEF and transforming activity, it is presumed that transforming activity is a consequence of the chronic stimulation of Rho GTPase targets. There are in fact several lines of evidence to suggest that this is the case. First, there is an absolute requirement for an intact DH domain for transformation.6-10,72 Second, activated derivatives of Rho GTPases, such as RhoA, exhibit a similar transforming activity to RhoGEFs when expressed in NIH 3T3 cells.73 Finally, dominant-inhibitory derivatives of Rho GTPases block RhoGEF transforming activity.15,74 Although the expression of constitutively activated derivatives of several Rho family GTPases have been shown to exhibit a transformed phenotype under a variety of conditions, the oncogenic potential is often very weak compared to the more potent RhoGEFs.75-81 There are several possible explanations for such a discrepancy. First, RhoGEFs may transform NIH 3T3 cells through activation of a common Rho GTPase target that has not yet been characterized for its transforming activity. The Rho family has grown rapidly in the past few years and many

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family members have not yet been tested in focus-formation assays. Second, mutations that lock GTPases into a constitutively activated GTP-bound form may not mimic the actions of a RhoGEF on a GTPase in vivo. For example, it may be necessary for GTPases to retain the ability to cycle, albeit at an accelerated rate, in order to mimic RhoGEF activation. However, mutants of RhoA, Rac1 and Cdc42 have been constructed that exhibit accelerated cycling, and none have transforming activity that is equivalent to the more strongly transforming RhoGEFs.82 Finally, transformation may be attributable to the promiscuous stimulation of several GTPases by a single RhoGEF. For example, it has been shown that the simultaneous activation of Cdc42, RhoA and Rac1 in NIH 3T3 cells could collectively account for the full complement of cellular effects that are associated with the overexpression of oncogenic Dbl.82 Although it is clear that RhoGEFs utilize Rho GTPases as their substrates for transformation, expression of the catalytic activity is not sufficient for full transforming activity. For example, the isolated DH domain of the Dbl protein retains GEF activity in vitro, yet has no transforming activity in vivo.12,72 Removal of the PH domains from Lfc and Dbs also eliminates their transforming activity, yet this can be restored by the addition of a prenylation site.7,15 Interestingly, the version of Dbs that lacks a PH domain is primarily found in the soluble fraction, whereas the wild-type and prenylated versions are found in membrane fractions. Since it is unlikely that an isoprenyl group can contribute to the structure of the DH domain in the same way that a PH domain does, then the PH domain-mediated membrane association must be required for full transformation. Thus, transformation by RhoGEFs seems to require both catalytic activity, and correct subcellular localization of this activity. Further support for this comes from the recent observation that Dbl mutants that are defective in phosphoinositide binding show much reduced transforming activity.25 Although these mutants are no longer able to associate with the plasma membrane, they exhibit enhanced levels of GEF activity in vivo. What then are the signalling events that are triggered when oncogenic RhoGEFs interact with membranes that contribute to cellular transformation. Oncogenic RhoGEFs can regulate many of the biological activities that are associated with their GTPase substrates including cytoskeletal rearrangements, transcriptional activation, and regulation of cell cycle progression.73 Many effector proteins have now been identified for various Rho family members and it would seem that at least some would have the potential to contribute to a phenotype of deregulated growth. In recent years particular attention has been focused on effectors for RhoA, since this is the GTPase that can most closely mimic the transforming activity of RhoGEFs in NIH 3T3 cells. There are now at least two RhoA effectors, PKN and ROCK, which have been strongly implicated in RhoA mediated transformation, and would presumably also be important for transformation by RhoA exchange factors such as Dbl, Dbs and Lfc.83-85 A detailed discussion of these effector proteins and their potential role in Rho and RhoGEF transformation can be found in Chapter 11 of this volume.

RhoGEFs and Human Disease It should come as no surprise that a family of proteins that is as large as the RhoGEFs, and which participates in such a broad spectrum of fundamental biological processes, would be linked to an increasing number of clinical disorders in humans. Chromosomal rearrangements that either disrupt or cause deregulated expression of RhoGEFs are associated with leukemias as well as human developmental disorders. It is anticipated that linkage and positional cloning studies will continue to add to the roster of human disorders that can be traced to dysfunctions within Rho-mediated signalling pathways.

Faciogenital Dysplasia (Aarskog-Scott Syndrome) Faciogenital dysplasia (FGDY) is an X-linked developmental disorder that was first described in the early 1970’s.86 Symptomology typically includes a specific pattern of skeletal defects which is generally accompanied by mental retardation. Loss-of-function mutations in

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the FGDY locus (FGD1) alter the shape and size of a number of small bones and cartilages, while leaving others unaffected. FGD1 was determined by positional cloning to be the gene responsible for FGDY and sequence analysis revealed that it is a member of the RhoGEF family.87 Both biochemical and cell-based studies have confirmed that FGD1 has catalytic, signalling and oncogenic activity that are reminiscent of Cdc42-like GTPases.88,89 A recent analysis of the expression of FGD1 during murine development revealed a pattern of expression that is consistent with the observed pattern of skeletal defects in humans.90 Murine FGD1 is expressed at a variety of sites where endochondral and intramembranous ossifications occur. Although it is still unclear what role FGD1 plays in the ossification of skeletal elements, it remains an important reminder of the critical role that members of the RhoGEF family are likely to play during normal human development.

Nonspecific X-Linked Mental Retardation Mental retardation is generally diagnosed during early childhood development and thus, by definition, is considered to be a developmental brain disorder. Almost nothing is known about the underlying biological defects that are responsible for mental retardation since informative studies are relatively scarce. However, since abnormalities in dendritic spine morphology have been observed in several developmental disabilities that are associated with mental retardation,91 this may be a common feature of the disorder. This possibility is supported by the recent cloning and biochemical characterization of a large group of proteins associated with X-linked mental retardation (MRX). Positional cloning and linkage studies have made possible the identification of nine genes that are associated with MRX.92 Of these nine genes, four encode proteins that have been previously linked to Rho-mediated signalling pathways. These include a RhoGAP,93 a member of the Pak family,94 FGD1,87 and ARHGEF6 which is a RhoGEF for Rac1.95 Interestingly, studies in mammalian cell lines had shown previously that ARHGEF6 is a binding partner for Pak, and that Pak binding may be necessary for ARHGEF6 activation.96,97 Thus both Pak and ARHGEF6 may be participants in a common signalling pathway that participates in the establishment of neuronal connections during human development. If so, this would be similar to the situation in Drosophila (discussed earlier in this chapter) in which members of the RhoGEF, Rho and Pak families form a signalling complex within the growth cones of developing photoreceptors that is required for the establishment of neural connections during Drosophila development.

Philadelphia Chromosome Associated Leukemias Virtually all cases of chronic myelogenous leukemia (CML) and a subset of the acute myelogenous leukemias (AML) are associated with a reciprocal translocation commonly referred to as the Philadelphia chromosome (Ph1). The product of the translocation is a fusion protein that contains amino-terminal sequences from the product of the breakpoint cluster region gene (Bcr) attached, in frame, to carboxyl-terminal sequences from the nonreceptor tyrosine kinase Abl. Animal models suggest that the expression of this chimeric molecule is sufficient to produce a full range of CML and AML like symptoms, and also suggest that elevated levels of Abl tyrosine kinase activity contribute to Bcr-Abl mediated leukemogenesis.98-100 However, the sequences that distinguish the chimeric protein that is commonly associated with CML (p210 Bcr-Abl) from the chimeric protein associated with AML (p185 Bcr-Abl) lie within the Bcr protein, suggesting that Bcr may also contribute to the outcome of the disorder. Bcr is a 160 KD protein that encodes three major structural domains. Contained within the amino-terminus of Bcr is a structurally unique serine/threonine kinase domain that has both trans- and auto-phosphorylation activity.101 However, the physiologically relevant substrates for this activity have not yet been identified, and its contribution to the disorder remains unclear. The carboxyl-terminus of Bcr encodes a catalytically active GAP domain that shows specificity for Rac-related GTPases in in vitro assays.102 In fact, mice that are null for the bcr locus exhibit several phenotypes that are consistent with increased Rac activity suggesting that

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Bcr can also function as a RacGAP in vivo.103 The central portion of Bcr, which is also the region that distinguishes p210 Bcr-Abl from p185 Bcr-Abl, contains a DH/PH domain module. The biochemical analysis of this domain revealed catalytic activity for Cdc42, RhoA and Rac1, suggesting that Bcr can coordinately regulate the activity of several distinct Rho family GTPases.102 Although the function of this domain in vivo, or within the context of p210 Bcr-Abl is not known, the observation that other DH/PH modules can stimulate growth regulatory pathways suggests that these Bcr encoded sequences may play an important role in the clinical outcome of Ph1 positive leukemias.

Mixed Lineage Leukemias Leukemia associated Rho guanine nucleotide exchange factor (LARG) is a second RhoGEF family member that has been implicated in the pathogenesis of human leukemia. LARG was originally identified as a fusion partner with the product of the mixed lineage leukemia gene (MLL) in a patient with acute myeloid leukemia.104 Typically, the mixed lineage leukemias are a treatment complication associated with the use of DNA topoisomerase II inhibitors as anti-cancer drugs. Although rearrangements involving MLL are quite common in these leukemias, MLL can be quite promiscuous in terms of its choice of translocation partners. Over 20 distinct partners have now been isolated from more than 30 independent chromosomal rearrangements. In virtually every case, the molecular mechanism through which these fusion products contribute to the progression of the disorder is unknown. In addition to its tandem DH/PH domain module, LARG also contains PDZ and RGS domains in the amino-terminus. Whereas the function of the PDZ domain is unknown, the RGS domain can mediate direct interactions between LARG and Gα12 and Gα13.47 However, it has not yet been determined whether LARG can function as a GAP for either Gα subunit. The DH/PH domain of LARG exhibits specificity for RhoA in both in vitro and in vivo assays, and exhibits signalling and transformation profiles in NIH 3T3 cells that are consistent with RhoA activation.105 The fusion of MLL with LARG occurs as the result of an interstitial deletion that joins amino-terminal sequences of MLL with carboxyl terminal sequences from LARG.104 Thus, both the DH/PH and RGS domains are retained, while the PDZ domain is lost. However, removal of the PDZ domain from LARG, independent of the MLL fusion, does not appear to activate its exchange or transforming activity.105 Thus, although it is intriguing that LARG harbors oncogenic potential, and is associated with a human leukemia, there is no obvious mechanism through which it may contribute to the outcome of the disorder.

Future Perspectives Tremendous progress has been made in the past few years towards an understanding of how the catalytic activity of RhoGEFs is regulated. Most are thought to exist in a basal state that is maintained by intramolecular interactions that block access to the substrate. A surprisingly diverse array of regulators have now been identified that can relieve this inhibition and it is likely that many more will be described. However, in many cases it has not yet been determined whether these regulatory mechanisms can be recapitulated in live cells. For example, although many RhoGEFs share a common ability to bind phosphoinositides in cell free systems, there is still little evidence to suggest that such binding is occurring in vivo. The identification of RhoGEFs in systems that are more amenable to genetic manipulation has provided some exciting glimpses into the dynamic and central role that many of these molecules will play in the regulation of some very fundamental cellular processes. The theme that continues to emerge is that RhoGEFs function as intracellular sensors that are able to actively convert signals coming from within or outside the cell into localized rearrangements of the actin cytoskeleton. It is anticipated that new experimental approaches that allow for the visualization of activated GTPases in live cells will contribute dramatically to our understanding of how these localized alterations in actin polymerization are initiated.106 Such techniques will be particularly useful in mammalian cells where multiple RhoGEFs with broad and overlapping specificities are often expressed simultaneously making it difficult to identify their primary substrates.

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References 1. Venter JC, Adams MD, Myers EW et al. The sequence of the human genome. Science 2001; 291:1304-51. 2. Rebecchi MJ, Scarlata S. Pleckstrin homology domains: A common fold a with diverse functions. Annu Rev Biophys Biomol Struct 1998; 27:503-28. 3. Knetsch ML, Schafers N, Horstmann H et al. The Dictyostelium Bcr/Abr-related protein DRG regulates both Rac- and Rab-dependent pathways. Embo J 2001; 20:1620-9. 4. Aghazadeh B, Zhu K, Kubiseski TJ et al. Structure and mutagenesis of the Dbl homology domain. Nat Struct Biol 1998; 5:1098-107. 5. Whitehead IP, Campbell S, Rossman KL et al. Dbl family proteins. Biochim Biophys Acta 1997; 1332:F1-23. 6. Whitehead I, Kirk H, Kay R. Retroviral transduction and oncogenic selection of a cDNA encoding Dbs, a homolog of the Dbl guanine nucleotide exchange factor. Oncogene 1995; 10:713-21. 7. Whitehead I, Kirk H, Tognon C et al. Expression cloning of lfc, a novel oncogene with structural similarities to guanine nucleotide exchange factors and to the regulatory region of protein kinase C. J Biol Chem 1995; 270:18388-95. 8. Whitehead IP, Khosravi-Far R, Kirk H et al. Expression cloning of lsc, a novel oncogene with structural similarities to the Dbl family of guanine nucleotide exchange factors. J Biol Chem 196; 271:18643-50. 9. Horii Y, Beeler JF, Sakaguchi K et al. A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links Rho and Rac signalling pathways. Embo J 1994; 13:4776-86. 10. Miki T, Smith CL, Long JE et al. Oncogene ect2 is related to regulators of small GTP-binding proteins. Nature 1993; 362:462-5. 11. Abe K, Rossman KL, Liu B et al. Vav2 is an activator of Cdc42, Rac1, and RhoA. J Biol Chem 2000; 275:10141-9. 12. Hart MJ, Eva A, Zangrilli D et al. Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product. J Biol Chem 1994; 269:62-5. 13. Liu X, Wang H, Eberstadt M et al. NMR structure and mutagenesis of the N-terminal Dbl homology domain of the nucleotide exchange factor. Trio Cell 1998; 95:269-77. 14. Aghazadeh B, Lowry WE, Huang XY et al. Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell 2000; 102:625-33. 15. Whitehead IP, Lambert QT, Glaven JA et al. Dependence of Dbl and Dbs transformation on MEK and NF-kappaB activation. Mol Cell Biol 1999; 19:7759-70. 16. Worthylake DK, Rossman KL, Sondek J. Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1. Nature 2000; 408:682-8. 17. Soisson SM, Nimnual AS, Uy M et al. Crystal structure of the Dbl and pleckstrin homology domains from the human Son of sevenless protein. Cell 1998; 95:259-68. 18. Goldberg J. Structural basis for activation of ARF GTPase: Mechanisms of guanine nucleotide exchange and GTP-myristoyl switching. Cell 1998; 95:237-48. 19. Boriack-Sjodin PA, Margarit SM, Bar-Sagi D et al. The structural basis of the activation of Ras by Sos. Nature 1998; 394:337-43. 20. Glaven JA, Whitehead I, Bagrodia S et al. The Dbl-related protein, Lfc, localizes to microtubules and mediates the activation of Rac signalling pathways in cells. J Biol Chem 1999; 274:2279-85. 21. Olson MF, Sterpetti P, Nagate et al. Distinct roles for DH and PH domains in the Lbc oncogene. Oncogene 1997; 15:2827-31. 22. Bi F, Debreceni B, Zhu K et al. Autoinhibition mechanism of proto-Dbl. Mol Cell Biol 2001; 21:1463-74. 23. Anborgh PH, Qian X, PapaGeorge AG et al. Ras-specific exchange factor GRF: Oligomerization through its Dbl homology domain and calcium-dependent activation of Raf. Mol Cell Biol 1999; 19:4611-22. 24. Zhu K, Debreceni B, Bi F et al. Oligomerization of DH domain is essential for Dbl-induced transformation. Mol Cell Biol 2001; 21:425-37. 25. Russo C, Gao Y, Mancini P et al. Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain. J Biol Chem 2001; 276:19524-31. 26. Fleming IN, Gray A, Downes CP. Regulation of the Rac1-specific exchange factor Tiam1 involves both phosphoinositide 3-kinase-dependent and -independent components. Biochem J 2000; 351:173-82. 27. Rameh LE, Arvidsson AK, Carraway KL et al. A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains. J Biol Chem 1997; 272:22059-66.

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28. Stam JC, Sander EE, Michiels F et al. Targeting of Tiam1 to the plasma membrane requires the cooperative function of the N-terminal pleckstrin homology domain and an adjacent protein interaction domain. J Biol Chem 1997; 272:28447-54. 29. Han J, Luby-Phelps K, Das B et al. Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 1998; 279:558-60. 30. Das B, Shu X, Day GJ et al. Control of intramolecular interactions between the pleckstrin homology and Dbl homology domains of Vav and Sos1 regulates Rac binding. J Biol Chem 2000; 275:15074-81. 31. Kiyono M, Kaziro Y, Satoh T et al. Induction of rac-guanine nucleotide exchange activity of RasGRF1/CDC25(Mm) following phosphorylation by the nonreceptor tyrosine kinase Src. J Biol Chem 2000; 275:5441-6. 32. Han J, Das B, Wei W et al. Lck regulates Vav activation of members of the Rho family of GTPases. Mol Cell Biol 1997; 17:1346-53. 33. Kato J, Kaziro Y, Satoh T. Activation of the guanine nucleotide exchange factor Dbl following ACK1- dependent tyrosine phosphorylation. Biochem Biophys Res Commun 2000; 268:141-7. 34. Fleming IN, Elliott CM, Exton JH. Phospholipase C-gamma, protein kinase C and Ca2+/ calmodulin-dependent protein kinase II are involved in platelet-derived growth factor- induced phosphorylation of Tiam1. FEBS Lett 1998; 429:229-33. 35. Tatsumoto T, Xie X, Blumenthal R et al. Human ECT2 is an exchange factor for Rho GTPases, phosphorylated in G2/M phases, and involved in cytokinesis. J Cell Biol 1999; 147:921-8. 36. Bustelo XR, Barbacid M. Tyrosine phosphorylation of the vav proto-oncogene product in activated B cells. Science 1992; 256:1196-9. 37. Bustelo XR, Ledbetter JA, Barbacid M. Product of vav proto-oncogene defines a new class of tyrosine protein kinase substrates. Nature 1992; 356:68-71. 38. Margolis B, Hu P, Katzav S et al. Tyrosine phosphorylation of vav proto-oncogene product containing SH2 domain and transcription factor motifs. Nature 1992; 356:71-4. 39. Deckert M, TartareDeckert S, Couture C et al. Functional and physical interactions of Syk family kinases with the Vav proto-oncogene product. Immunity 1996; 5:591-604. 40. Whitehead IP, Zohn IE, Der CJ. Rho GTPase-dependent transformation by G protein-coupled receptors. Oncogene 2001; 20:1547-55. 41. Glaven JA, Whitehead IP, Nomanbhoy T et al. Lfc and Lsc oncoproteins represent two new guanine nucleotide exchange factors for the Rho GTP-binding protein. J Biol Chem 1996; 271:27374-81. 42. Hart MJ, Sharma S, elMasry N et al. Identification of a novel guanine nucleotide exchange factor for the Rho GTPase. J Biol Chem 1996; 271:25452-8. 43. Kozasa T, Jiang X, Hart MJ et al. p115 RhoGEF, a GTPase activating protein for Gα 12 and Gα13. Science 1998; 280:2109-11. 44. Hart MJ, Jiang X, Kozasa T et al. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Gα 13. Science 1998; 280:2112-4. 45. Mao J, Yuan H, Xie W et al. Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein alpha subunit Gα13. Proc Natl Acad Sci USA 1998; 95:12973-6. 46. Fukuhara S, Murga C, Zohar M et al. A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho. J Biol Chem 1999; 274:5868-79. 47. Fukuhara S, Chikumi H, Gutkind JS. Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho. FEBS Lett 2000; 485:183-8. 48. Nimnual AS, Yatsula BA, Bar-Sagi D. Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos. Science 1998; 279:560-3. 49. Chardin P, Camonis JH, Gale NW et al. Human Sos1: A guanine nucleotide exchange factor for Ras that binds to GRB2. Science 1993; 260:1338-43. 50. Steven R, Kubiseski TJ, Zheng H et al. UNC-73 activates the Rac GTPase and is required for cell and growth cone migrations in C. elegans. Cell 1998; 92:785-95. 51. O’Brien SP, Seipel K, Medley QG et al. Skeletal muscle deformity and neuronal disorder in Trio exchange factor- deficient mouse embryos. Proc Natl Acad Sci USA 2000; 97:12074-8. 52. Awasaki T, Saito M, Sone M et al. The Drosophila trio plays an essential role in patterning of axons by regulating their directional extension. Neuron 2000; 26:119-31. 53. Bateman J, Shu H, Van Vactor D. The guanine nucleotide exchange factor trio mediates axonal development in the Drosophila embryo. Neuron 2000; 26:93-106. 54. Newsome TP, Schmidt S, Dietzl G et al. Trio combines with dock to regulate Pak activity during photoreceptor axon pathfinding in Drosophila. Cell 2000; 101:283-94.

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55. Wahl S, Barth H, Ciossek T et al. Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase. J Cell Biol 2000; 149:263-70. 56. Shamah SM, Lin MZ, Goldberg JL et al. EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin. Cell 2001; 105:233-44. 57. Takaishi K, Sasaki T, Kameyama T et al. Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows. Oncogene 1995; 11:39-48. 58. Kishi K, Sasaki T, Kuroda S et al. Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI). J Cell Biol 1993; 120:1187-95. 59. Prokopenko SN, Brumby A, O’Keefe L et al. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev 1999; 13:2301-14. 60. Wasserman S. FH proteins as cytoskeletal organizers. Trends Cell Biol 1998; 8:111-5. 61. Pruyne D, Bretscher A. Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states. J Cell Sci 2000; 113:365-75. 62. Lew DJ, Reed SI. Cell cycle control of morphogenesis in budding yeast. Curr Opin Genet Dev 1995; 5:17-23. 63. Toenjes KA, Sawyer MM, Johnson DI. The guanine-nucleotide-exchange factor Cdc24p is targeted to the nucleus and polarized growth sites. Curr Biol 1999; 9:1183-6. 64. Nern A, Arkowitz RA. Nucleocytoplasmic shuttling of the Cdc42p exchange factor Cdc24p. J Cell Biol 2000; 148:1115-22. 65. Shimada Y, Gulli MP, Peter M. Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating. Nat Cell Biol 2000; 2:117-24. 66. Henchoz S, Chi Y, Catarin B et al. Phosphorylation- and ubiquitin-dependent degradation of the cyclin- dependent kinase inhibitor Far1p in budding yeast. Genes Dev 1997; 11:3046-60. 67. Gulli M, Jaquenoud M, Shimada Y et al. Phosphorylation of the cdc42 exchange factor cdc24 by the PAK-like kinase cla4 may regulate polarized growth in yeast. Mol Cell 2000; 6:1155-67. 68. Park HO, Bi E, Pringle JR et al. Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarity. Proc Natl Acad Sci USA 1997; 94:4463-8. 69. Park HO, Sanson A, Herskowitz I. Localization of bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site. Genes Dev 1999; 13:1912-7. 70. Gulli MP, Peter M. Temporal and spatial regulation of Rho-type guanine-nucleotide exchange factors: The yeast perspective. Genes Dev 2001; 15:365-79. 71. Cerione RA, Zheng Y. The Dbl family of oncogenes. Curr Opin Cell Biol 1996; 8:216-22. 72. Ron D, Zannini M, Lewis M et al. A region of proto-dbl essential for its transforming activity shows sequence similarity to a yeast cell cycle gene, CDC24, and the human breakpoint cluster gene, bcr. New Biol 1991; 3:372-9. 73. Westwick JK, Lee RJ, Lambert QT et al. Transforming potential of Dbl family proteins correlates with transcription from the cyclin D1 promoter but not with activation of Jun NH2-terminal kinase, p38/Mpk2, serum response factor, or c-Jun. J Biol Chem 1998; 273:16739-47. 74. Khosravi-Far R, Chrzanowska-Wodnicka M, Solski PA et al. Dbl and Vav mediate transformation via mitogen-activated protein kinase pathways that are distinct from those activated by oncogenic Ras. Mol Cell Biol 1994; 14:6848-57. 75. Khosravi-Far R, Solski PA, Clark GJ et al. Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation. Mol Cell Biol 1995; 15:6443-53. 76. Lin R, Bagrodia S, Cerione R et al. A novel Cdc42Hs mutant induces cellular transformation. Curr Biol 1997; 7:794-7. 77. Murphy GA, Solski PA, Jillian SA et al. Cellular functions of TC10, a Rho family GTPase: Regulation of morphology, signal transduction and cell growth. Oncogene 1999; 18:3831-45. 78. Prendergast GC, Khosravi-Far R, Solski PA et al. Critical role of Rho in cell transformation by oncogenic Ras. Oncogene 1995; 10:2289-96. 79. Qiu RG, Chen J, McCormick F et al. A role for Rho in Ras transformation. Proc Natl Acad Sci USA 1995; 92:11781-5. 80. Qiu RG, Chen J, Kirn D et al. An essential role for Rac in Ras transformation. Nature 1995; 374:457-9. 81. Qiu RG, Abo A, McCormick F et al. Cdc42 regulates anchorage-independent growth and is necessary for Ras transformation. Mol Cell Biol 1997; 17:3449-58. 82. Lin R, Cerione RA, Manor D. Specific contributions of the small GTPases Rho, Rac, and Cdc42 to Dbl transformation. J Biol Chem 1999; 274:23633-41. 83. Marinissen MJ, Chiariello M, Gutkind JS. Regulation of gene expression by the small GTPase Rho through the ERK6 (p38 gamma) MAP kinase pathway. Genes Dev 2001; 15:535-53. 84. Sahai E, Ishizaki T, Narumiya S et al. Transformation mediated by RhoA requires activity of ROCK kinases. Curr Biol 1999; 9:136-45.

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85. Tran Quang C, Gautreau A, Arpin M et al. Ezrin function is required for ROCK-mediated fibroblast transformation by the Net and Dbl oncogenes. Embo J 2000; 19:4565-76. 86. Aarskog D. A familial syndrome of short stature associated with facial dysplasia and genital anomalies. J Pediatr 1970; 77:856-61. 87. Pasteris NG, Cadle A, Logie LJ et al. Isolation and characterization of the faciogenital dysplasia (Aarskog- Scott syndrome) gene: A putative Rho/Rac guanine nucleotide exchange factor. Cell 1994; 79:669-78. 88. Whitehead IP, Abe K, Gorski JL et al. CDC42 and FGD1 cause distinct signalling and transforming activities. Mol Cell Biol 1998; 18:4689-97. 89. Zheng Y, Fischer DJ, Santos MF et al. The faciogenital dysplasia gene product FGD1 functions as a Cdc42Hs- specific guanine-nucleotide exchange factor. J Biol Chem 1996; 271:33169-72. 90. Gorski JL, Estrada L, Hu C et al. Skeletal-specific expression of Fgd1 during bone formation and skeletal defects in faciogenital dysplasia (FGDY; Aarskog syndrome). Dev Dyn 2000; 218:573-86. 91. Williams RS, Hauser SL, Purpura DP et al. Autism and mental retardation: Neuropathologic studies performed in four retarded persons with autistic behavior. Arch Neurol 1980; 37:749-53. 92. Chelly J. Breakthroughs in molecular and cellular mechanisms underlying X-linked mental retardation. Hum Mol Genet 1999; 8:1833-8. 93. Billuart P, Bienvenu T, Ronce N et al. Oligophrenin-1 encodes a rhoGAP protein involved in X-linked mental retardation. Nature 1998; 392:923-6. 94. Allen KM, Gleeson JG, Bagrodia S et al. PAK3 mutation in nonsyndromic X-linked mental retardation. Nat Genet 1998; 20:25-30. 95. Kutsche, Yntema H, Brandt A et al. Mutations in ARHGEF6, encoding a guanine nucleotide exchange factor for Rho GTPases, in patients with X-linked mental retardation. Nat Genet 2000; 26:247-50. 96. Manser E, Loo TH, Koh CG et al. PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol Cell 1998; 1:183-92. 97. Bagrodia S, Taylor SJ, Jordon KA et al. A novel regulator of p21-activated kinases. J Biol Chem 1998; 273:23633-6. 98. Daley GQ, Van Etten RA, Baltimore D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science 1990; 247:824-30. 99. Gross AW, Zhang X, Ren R. Bcr-Abl with an SH3 deletion retains the ability To induce a myeloproliferative disease in mice, yet c-Abl activated by an SH3 deletion induces only lymphoid malignancy. Mol Cell Biol 1999; 19:6918-28. 100. Huettner CS, Zhang P, Van Etten RA et al. Reversibility of acute B-cell leukaemia induced by BCR-ABL1. Nat Genet 2000; 24:57-60. 101. Maru Y, Witte ON. The BCR gene encodes a novel serine/threonine kinase activity within a single exon. Cell 1991; 67:459-68. 102. Chuang TH, Xu X, Kaartinen V et al. Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family. Proc Natl Acad Sci USA 1995; 92:10282-6. 103. Voncken JW, Kaartinen V, Pattengale PK et al. BCR/ABL P210 and P190 cause distinct leukemia in transgenic mice. Blood 1995; 86:4603-11. 104. Kourlas PJ, Strout MP, Becknell B et al. Identification of a gene at 11q23 encoding a guanine nucleotide exchange factor: Evidence for its fusion with MLL in acute myeloid leukemia. Proc Natl Acad Sci USA 2000; 97:2145-50. 105. Reuther GW, Lambert QT, Booden MA et al. LARG, a Dbl family protein found mutated in leukemia, causes transformation by activation of RhoA. J Biol Chem 2001; 276:27145-51. 106. Kraynov VS, Chamberlain C, Bokoch GM et al. Localized Rac activation dynamics visualized in living cells. Science 2000; 290:333-7.

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CHAPTER 5

The Superfamily of Rho GTPase-Activating Proteins Sarah Jenna and Nathalie Lamarche-Vane

Abstract

G

TP hydrolysis is a key step in many intracellular processes. GTPases are the molecular machines that catalyze this reaction but their intrinsic GTPase activity is very slow in the case of the Ras superfamily of GTPases. This hydrolysis can be accelerated by orders of magnitude upon interaction with GTPase-activating proteins (GAPs), which are specific for their respective GTP-binding proteins. To date, around 42 distinct RhoGAP-containing proteins from various species have been reported in the literature. They are generally large and multifunctional. A consensus RhoGAP domain has been used to identify 220 potential RhoGAPs from the nonredundant protein database. On the basis of their multimodular structure, we have divided these proteins into 15 subfamilies. In this chapter, we will discuss the molecular basis of the RhoGAP activity and we will focus on studies that begin to address the multifunctional role of the RhoGAP subfamilies.

RhoGAPs Are Multimodular Proteins RhoGAPs appear to play a critical role in many biological functions and pathological processes. They are thought to be implicated in the regulation of cell cycle progression, cytokinesis, cell adhesion and migration, actin and microtubule cytoskeleton reorganization, membrane trafficking, host-pathogen interactions, development, and in cross-talk between the small GTPases Rho, Ras and Ral (Tables 2, 3). To date, around 42 distinct proteins containing a closely related RhoGAP domain have been reported in mammals, avian, worm, yeast and pathogenic bacteria (Fig. 1). RhoGAP domains show about 20 to 40% amino acid identity and the proteins in which they are contained are generally large and multifunctional. Based on amino acid sequence analysis of eucaryotic RhoGAPs, a consensus RhoGAP domain has been used to identify potential RhoGAPs from the nonredundant protein database using the SMART (Simple Modular Architecture Research Tool) interface.3,4 To date, around 220 potential RhoGAPs have been identified: 90 in human, 28 in mouse (Mus musculus), 24 in fruit fly (Drosophila melanogaster), 22 in Caenorabditis elegans, 8 in Arabidopsis thalia and 12 in Saccharomyces cerevisiae (supplemental data). On the basis of their multimodular structure, we have divided these proteins into 15 subfamilies (Fig. 1, Table 1 and supplemental data). In addition to their RhoGAP domain, enzymatic activities have been found in the Bcr, OCRL, Myosin-IX and p190-RhoGAP subfamilies as well as in D. discoideum DdRacGAP, the S. cerevisiae Bem2p and bacterial RhoGAPs. Bcr, GRAF and CeGAP subfamilies as well as the yeast Bem2p and Bem3p, and DdRacGAP proteins also contain the lipid-binding module, Pleckstrin homology (PH) domain. Other putative lipid-binding modules such as the protein kinase C conserved region 1 and 2 (C1 and C2, respectively) homology domains, the Sec14 and star-related lipid-transfer (START) Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Table 1. Protein modules identified within RhoGAP sequences Symbols

Names of Protein Modules

References

GTPase-activating protein for Rho GTPases (RhoGAP)

14-18,121,190

Dbl-homology (DH) domain. Catalytic domain of guanine-nucleotide exchange factor for RhoGTPases

29,191-194

GTP-binding domain of the Ras superfamily.

195-197

Ras Guanine nucleotide exchange factor domain.

190,198

Inositol polyphosphate phosphatase

199-201

Myosin ATPase domain. Actin-binding domain.

202,203

Pleckstrin homology (PH) domain. Can bind inositol phosphates or proteins.

204-207

Protein kinase C conserved region 1 (C1) (Cysteine-rich domain). Can bind phorbol esters, diacylglycerol, RasGTP or Zinc.

87,91,208,209

Protein kinase C conserved region 2 (C2). Can bind Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins

210-214

Sec14 homology domains (SEC14)Lipid binding domain 215-218 presenting homology with the S. cerevisiae phosphatidylinositol transfer protein (Sec14p). Star-related lipid-transfer (START) domain. putative lipid-binding domain in Star and phosphatidylcholine transfer protein

219,220

Src homology 2 (SH2) domain. binds phosphotyrosine-containing polypeptides.

221-227

Src homology 3 (SH3) domain. Binds generally to poly-proline sequences (PXXP)

228-230

Ras interactive binding domain. known also as RalGDS/AF-6 domains LIM domain. Zinc-binding domain family. Cdc42/Rac interactive binding (CRIB) domain. Present in Cdc42 and Rac1 effectors

231-235 236-238 239,240

PhoX homologous (PX) domain. SH3-binding motif FF domain. Contains two conserved F residues. A novel motif that often accompanies WW domains

241,242 243

Sterile alpha motif (SAM). In EPH-related tyrosine kinases, binds SH2-containing proteins to a conserved phosphorylated tyrosine.

244-249

IQ domain. calmodulin-binding motif. FCH domain. Fes/CIP4 homology domain. Non-kinase domain of the FER and Fes/Fps family of tyrosine kinases, also present in Cdc42 effector CIP4

251-255 250

Protein modules were identified using the SMART interface.3,4 Symbol corresponding to these domains refers to those used in (Fig. 1). Enzymatic domains: dashed symbols; lipid-binding modules: empty symbols; protein-binding modules: black-filled symbols; uncharacterized domains: grey-filled symbols.

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Figure 1. Organization of RhoGAPs into subfamilies based on their multi-modular structures. A) Proteins that have consensus RhoGAP domains were identified using the nonredundant database and the SMART interface in Homo sapiens, Ratus norvegicus, Mus musculus, Drosophila melanogaster and Caenorabditis elegans. Nonredundancy was verified using National Center for Biotechnology Information (NCBI, http:// www.ncbi.nlm.nih.gov) Basic Local Alignment Search Tool (BLAST®). Continued on next page.

homology domains have been found in members of the Bcr, chimaerin, Myosin-IX, p122-RhoGAP and p50-RhoGAP subfamilies. In addition, calmodulin-binding (IQ) domains have been found in the myosin-IX subfamily members. Furthermore, the majority of RhoGAP

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Figure 1 (cont’d). Continued on next page.

proteins contains various protein-binding modules. Src homology 2 (SH2) domains are found in members of the chimaerin and p85 subfamilies; Src homology 3 (SH3) domains in the GRAF, p85, C1 subfamilies and in dCdGAPr proteins and yeast Bem3p proteins; GTPase interactive binding domains in the Myosin-IX and RLIP subfamilies and in most of the A. talia RhoGAPs; LIM domains in yeast Dbm1p and Lrg1p and an actin-binding domain in the Myosin-IX subfamily. Proline-rich domains containing the SH3-binding motif (PXXP) have also been found in a large majority of RhoGAPs.5

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Figure 1 (cont’d). B) Proteins with a RhoGAP domain were identified in Saccharomyces cerevisiae, Arabidopsis thalia and Dictyostelium discoideum as described above. The protein modules identified with RhoGAP proteins are represented by symbols referring to Table 1.

To date, the precise role of these functional domains has remained elusive in most cases. It has been proposed however that these domains may serve to recruit RhoGAP-containing proteins to specific protein complexes. Then, RhoGAPs could act in a localized fashion to downregulate Rho GTPases or mediate downstream signals.5 Lipid- and protein-binding modules also have been described to regulate the RhoGAP activity of α-chimaerin, p190-RhoGAP, PSGAP and CdGAP. This regulation can be either direct by induction of a change of protein conformation or indirect through subcellular relocalization of the RhoGAP proteins.6-11 In this chapter, we will discuss the molecular basis of the RhoGAP activity and we will focus on studies that begin to address the multifunctional role of the RhoGAP subfamilies.

Molecular Basis of the GTPase-Stimulating Activity The Eucaryotic RhoGAP Domain The RhoGAP domain extends over approximately 180 amino acids. Analysis of its primary structure allowed the identification of three blocks of sequence conservation (Fig. 2A).12,13 Crystallographic analysis of the human p50-RhoGAP and of the p85-α subunit of Phosphatidylinositol-3 Kinase revealed that RhoGAP domains consist of 8 or 9 α-helices, respectively and present protein cores constituted by a four–helix bundle (Fig. 2A, filled rectangles), one face of which contains most of the conserved residues.14,15 Insight into the three-dimensional interaction between Rho GTPases and their specific GAPs came from the crystal structures of RhoA and p50-RhoGAP complexed with GDP.AlF -4 (transition state-mimicking complex) and from Cdc42 and p50-RhoGAP complexed with a nonhydrolysable GTP analogue (ground state).16,17 These studies revealed that p50-RhoGAP actively participates in the process of GTP hydrolysis by contributing an arginine residue (Arg85) to the catalytic domain of the GTPase. This residue has been shown to contact GDP.AlF4- and a critical glutamine at position 63 of RhoA (Gln63) during the transition state.16 It is thought that this “Arginine finger” acts to stabilize the transition state of the GTPase reaction. Additional residues of the RhoGAP domain also contribute to stabilize this interaction (Fig. 2A, indicated by an asterisk). The ‘Arginine finger’ model is highly supported by site-directed mutagenesis analysis of numerous RhoGAPs in which replacement of the ‘Arginine finger’ drastically impairs their

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Figure 2. Sequence alignment of a subset of RhoGAP domains. The sequences used are identified by their GenBank accession number: Human breakpoint cluster region (Bcr) (P11274); Rat p190 (A38218); Rat α1-chimaerin (Q03070); Human p85-α (P27986); human p50-RhoGAP (Z23024); Salmonella SptP (AAC44349); Pseudomonas aeruginosa ExoS (AAK39624); Yersinia YopE (P08008). Three blocks of sequence conservation are indicated by gray boxes (A). Numbering above and below the sequences respectively corresponds to the full-length human p50-RhoGAP and human p85-α (A) and to full-length SptP (B). Helical fragments are represented by open rectangles above the sequences. Helices implicated in the four-helix bundle GAP domain are represented by filled rectangles. Residues that are conserved in at least 14 sequences out of 22 aligned eucaryotic RhoGAPs14,15 and in all 3 bacterial RhoGAPs are shown in bold. Residues of p50-RhoGAP and SptP that are part of the proposed ligand-binding site during the transition state are indicated by an asterisk and the ‘arginine finger’ by an arrow.

GAP activity.12,18 Interestingly, these amino acid substitutions do not alter the ability of GAPs to bind to their specific Rho GTPases, indicating that these two functions are independent.12 This latter hypothesis is also supported by the identification and characterization of p85-α that

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Rho GTPases

has no GAP activity but can still bind its specific GTPase.5,14,15 Comparison of the p85-α sequence and structure with those of active GAPs14,15 as well as mutagenesis analysis12 has facilitated the identification of residues implicated in the GTPase-acceleration activity to those implicated in GTPase recognition and binding.

Bacterial RhoGAPs Three bacterial cytotoxins with RhoGAP activities have been isolated: the Salmonella typhimurium SptP,19 the Yersinia spp. YOPE 20,21 and the Pseudomonas aeruginosa ExoS.22 As observed for eucaryotic RhoGAPs, the crystal structures of SptP and ExoS complexed with Rac1.GDP.AlF4 showed that the cytotoxins bind Rac1 exclusively through a four-helix bundle domain (Fig. 2B, filled rectangles). This bundle inserts a conserved arginine (Fig. 2B, arrow) into the active site of Rac1, catalyzing GTP hydrolysis through stabilization of the transition state.23,25 Despite the high degree of homology between the amino acid sequences of bacterial and eucaryotic RhoGAPs, the residues shown to be implicated in GTPase-interaction during the transition-state are different from those identified in eucaryotic RhoGAPs (compare residues indicated by an asterisk in (Fig. 2A and B). Moreover, several important differences between the SptP and p50-RhoGAP secondary and ternary structures have been observed.23 Although the cytotoxins are much smaller in size, they make more extensive contacts with the GTPase and its nucleotide than observed in most eucaryotic GTPase/GAP complexes (compare the number of residues indicated by an asterisk in Fig. 2A and B). In addition, the catalytic arginine in SptP extends from a bundle helix,23 whereas in eucaryotic RhoGAPs it is distal from the bundle and contained in a loop.

RhoGAPs Are Multifunctional Proteins The Bcr Subfamily The Bcr subfamily includes the human Bcr (Breakpoint Cluster Region) which has been the first RhoGAP activity to be described,26 Abr (Active Bcr-Related), and a putative Drosophila melanogaster 80 kDa protein whose GAP activity has not been established yet (Fig. 1 and supplemental data). Bcr is a 160 kDa phosphoprotein mainly present in brain and hematopoietic cells26,27 while Abr is a 92 kDa protein predominantly expressed in brain.28,29 These two proteins display a RhoGAP domain at their C-terminus. The recombinant Bcr-GAP domain has GAP activity towards Rac1, Rac2, Cdc42 but not RhoA in vitro.26,28-30 Consistent with its Rac1-specific GAP activity, the Bcr-GAP domain has also been shown to inhibit Rac1-mediated membrane ruffling, but not RhoA-mediated stress fibre formation into Swiss 3T3 fibroblasts.31 These proteins also contain Dbl-homology (DH) domains (Fig. 1, Table 1) that has GEF activity towards Cdc42, RhoA, Rac1 and Rac2 but not Rap1 or Ha-Ras in vitro.30 DH domains are usually found together with pleckstrin-homology (PH) domains thought to be involved in the regulation of the GEF activity.32-35 However, such a regulatory role in Bcr and Abr has not been established yet. Interestingly, GAP and GEF domains of both proteins bind in vitro to the GTPases in a noncompetitive manner suggesting that these two domains may interact simultaneously with two members of the Rho GTPase family of proteins30 and may act in a coordinated manner to regulate Rho GTPases. Bcr, but not Abr, also contains an amino-terminal Ser/Thr kinase domain that displays in vitro activity towards Bcr itself27 and towards members of the 14-3-3 protein subfamily: 14-3-3β and BAP-1 (Bcr-associated protein 1).36,37 The role of the Ser/Thr kinase activity of Bcr, in vivo, remains to be determined. Bcr was first identified as part of the Bcr-Abl fusion oncogene, which is generated by a translocation event between the coding sequence of the Abl tyrosine kinase on chromosome 9 and the Bcr sequence on chromosome 22 in human leukemias positive for the philadelphia chromosome.38-40 Two distinct translocations result in the generation of chimeric proteins p210Bcr-Abl and p185Bcr-Abl, which are characteristic of chronic myelogenous leukaemia (CML) and acute lymphocytic leukemia (ALL), respectively. Interestingly, both proteins lack

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the Bcr-GAP domain and p185Bcr-Abl lacks the DH domain. Moreover, neutrophils from Bcr-null mice show a pronounced increase in reactive oxygen metabolite production upon activation as well as a significant increase in Rac2 membrane translocation compared to control mice.41 Taken together, these data suggest that Bcr may act to regulate Rac-mediated superoxide production in leukocytes, pointing to a role for the Bcr-GAP activity in Bcr-Abl-mediated leukemia development. However, misregulation of Rac is not the only pathological mechanism responsible for leukemia development. Constitutive activation of Bcr-Abl tyrosine kinase activity results in deregulated activation of signalling pathways mediated by Ras, p120-RasGAP and p62(dok)42-47 and is thought to play a key role in the development of this pathology. In addition, association of Bcr and Bcr-Abl with 14-3-3β and tyrosine phosphorylation of BAP-1 by Bcr-Abl36 have been proposed to participate into mitogenic signalling by Bcr and Bcr-Abl.36,37 c-Fes, a nonreceptor tyrosine kinase involved in myeloid cell growth and differentiation has also been shown to phosphorylate Bcr on tyrosine residues48 leading to its association with multiple signalling molecules,48,49 and to abolish its serine/threonine kinase activity.49-50 Interestingly, c-FES has been shown to suppress cytokine-independent outgrowth of myeloid leukemia cells induced by Bcr-Abl.51 Together, these data suggest a link between Bcr and the regulation of cell cycle progression in hematopoietic cells and suggest a regulatory role for Bcr sequences in Bcr-Abl oncogenicity. In addition to hematopoietic cells, Bcr is strongly expressed in brain. Moreover, mice lacking the Bcr product show an abnormal stress response and increased fighting behavior, suggesting a role of Bcr in brain functions.52

The p190-RhoGAP Subfamily The p190-RhoGAP subfamily consists of two isoforms, p190-A (originally named p190) and p190-B. These two proteins have been identified in rat,53,54 mouse55 and human (Fig. 1).56,57 Both p190-A and -B contain a RhoGAP domain at their C-terminus and a GTP-binding domain at their N-terminus. The central domain of these proteins also contains a repetition of four FF domains (Table 1) whose role remains unknown. In vitro, both rat p190-A and human p190-B display GAP activity towards Rac1, Cdc42 and RhoA, with a preference for RhoA.31,57,59 When expressed in Swiss 3T3 cells however, p190-A exclusively acts on RhoA.31,60 p190-A was first isolated from mitogenically stimulated and tyrosine kinase-transformed cells as a p120-RasGAP-associated protein.53,61,62 It is phosphorylated on tyrosine and to a larger extent on serine residues.61 It is the first RhoGAP whose activity has been shown to be regulated. The molecular mechanisms implicated in this regulation are not fully understood yet, but may involve both the GTP-binding domain, association with p120-RasGAP and tyrosine phosphorylation. The guanine nucleotide binding domain of the p190 proteins is similar to those of monomeric small GTPases.53 This domain has GTPase activity and appears to play a role in the regulation of p190-RhoGAP activity in vivo.63,64 p190-A interacts with p120-RasGAP in both a phosphotyrosine-dependent and independent manner.65 Phosphorylation of Tyr1105 appears to be mainly responsible for the phosphotyrosine-dependent association of these proteins.65 p190-A appears to interact with the two SH2 domains of p120-RasGAP, 62,66 resulting in a conformational change of p120-RasGAP.67 In addition to regulating the potential scaffolding activity of p120-RasGAP, this association may control the p190-A RhoGAP activity towards RhoA. The N-terminus of p120-RasGAP (GAP-N), containing the SH2 and SH3 domains but lacking the RasGAP domain, when overexpressed in Rat-2 fibroblasts, constitutively interacts with p190-A9 and induces changes in the actin cytoskeleton and cell adhesion that are consistent with the stimulation of p190-A activity towards RhoA.9 This stimulation however is likely to be indirect, since the GAP activity of p190-A is not increased by GAP-N in vitro.9,60 Phosphorylation on tyrosine residues has been shown to play a major regulatory role on p190-RhoGAP and GTP-binding activities. p190-A is a preferred substrate of the non recep-

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Rho GTPases

tor tyrosine kinase c-Src.68,69 Roof and coworkers have shown that phosphorylation of p190-A by Src in vitro results in a dramatic loss of p190 GTP-binding activity,69 suggesting a first level of regulation of p190 RhoGAP activity by Src. c-Src has been shown to phosphorylate p190-A on Tyr1105 both in vivo and in vitro, resulting in the association of p190-A with p120-RasGAP.70 Since this association regulates p190-RhoGAP activity, these data suggest a second level of regulation of p190-A activity by Src. Moreover, p120-RasGAP has been proposed to modulate the tyrosine phosphorylation of p190-A since this phosphorylation is abnormal in p120-RasGAP null fibroblasts.71 Regulation of p190-A tyrosine phosphorylation and GAP activity has been recently proposed to involve novel p190-A protein partners. In breast cancer cells, p190-A tyrosine phosphorylation by Src upon heregulin (HRG) stimulation seems to be regulated by the related adhesion focal tyrosine kinase (RAFTK), a kinase that associates in these conditions with both p190-A and Src.72 In NIH 3T3 cells, p190-A tyrosine phosphorylation as well as its GAP activity are regulated by a low molecular weight protein-tyrosine phosphatase (LMW-PTP) upon integrin engagement or platelet derived growth factor (PDGF) stimulation.73 It should be pointed out however that the regulatory role of p190-A tyrosine phosphorylation on its RhoGAP activity may be cell type specific, since p190-A activation correlates with its tyrosine phosphorylation in some cell types and conditions72-75 but not others.60,76 A role for p190-A has also been proposed in fibroblast migration74,77 as well as in melanoma and breast cancer cell invasion.72,75 It has been shown that RhoA is transiently inhibited prior to its activation during integrin-mediated adhesion to fibronectin and that integrin stimulates p190-A RhoGAP activity as well as its c-Src-dependent-tyrosine-phosphorylation.74,77 Thus, it has been postulated that initial localized suppression of RhoA activity by integrin via c-Src and p190-A may be a critical step to allow cells to migrate and may also contribute to focal adhesion disassembly at the rear of the motile cell.74 Recently, a role for p190-A in neuronal development has been proposed.78,80 p190-A is expressed widely throughout the nervous system at all stages of development and is highly phosphorylated in all regions of the brain.78,79 p190-A is coenriched with F-actin at the distal tip of axons and its overexpression promotes Src-dependent, integrin-mediated neurite outgrowth in neuroblastoma cells.79 Moreover, p190-A null mice exhibit several defects in neuronal development that are reminiscent of those described in mice lacking certain mediators of neuronal cell adhesion.78

The Chimaerin Subfamily Members of the chimaerin subfamily present both a RhoGAP domain located at their C-terminus and a cysteine-rich (C1) domain in their amino-terminal extremity (Fig. 1, Table 1). This subfamily consists of α1- ( or n-), α2-, β1- and β2-chimaerin variants, rotund-like, and PARG1-like proteins. α1-, α2, β1 and β2-chimaerin are 34 to 46 kDa proteins and are alternative spliced variants from the α- and β-chimaerin genes.81-84 α2 and β2-chimaerin variants contain an additional Src homology 2 (SH2) domain at their N-terminus whose role remains undefined.81,83 The expression of these four isozymes appears to be spatially and temporally regulated in vivo and some of the molecular mechanisms of their tissue-specific expression have been described.85,86 For instance, the brain-specific α1-chimaerin expression is detected at day 15 and appears to increase postnatally from birth to 20 days, a time of extensive cellular differentiation and synaptogenesis.84 It has been also proposed that α1-chimaerin expression might be regulated by neuronal/synaptic activity.88 The cerebellar β2-chimaerin is expressed predominantly in granule cells and exhibits post-natal developmental increase.83 Moreover, human β2-chimaerin is differently expressed in normal brain and low-grade astrocytoma compared to malignant gliomas where its expression is drastically reduced.89 Thus, it was proposed that down regulation of β2-chimaerin expression might be implicated in the development of malignant glioma.89

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Both α- and β-chimaerin variants show in vitro GAP activity towards Rac1 and to a lesser extent Cdc42, but not RhoA.26,82 This activity has been also confirmed in vivo, since the expression of the α1-chimaerin GAP domain inhibits Rac1-induced lamellipodia formation in Swiss 3T3 cells.90 Surprisingly, the expression of the full-length protein seems to cooperate with Rac1 and Cdc42 to induce the formation of lamellipodia and filopodia in a GAP activity-independent but GTPase-binding dependent manner.90 This suggests a putative role for α1-chimaerin as a Rac1 and/or Cdc42 effector. In addition to their GAP domain, chimaerins contain a cysteine-rich (C1) domain homologous to the protein Kinase C (PKC) regulatory domain. The C1 domain of PKC is responsible for phospholipid–dependent phorbol ester binding.91 Similar to PKC, α1- and β2-chimaerins bind phorbol ester in a phospholipid- and zinc-dependent manner.6,7 Moreover, it has been shown that the α1-chimaerin GAP activity towards Rac1 is stimulated in vitro through its C1 domain by phosphatidylserine and phosphatidic acid in a synergistic manner with phorbol esters, whereas lysophosphatidic acid (LPA) inhibits this GAP activity.8 Therefore, this suggests a potential regulation of chimaerin GAP activity by lipid binding that may orchestrate the dual function of chimaerin as negative regulators or effectors of Rac and/or Cdc42. Rotund proteins constitute a second subgroup within the chimaerin subfamily. Drosophila rotund, a 41 kDa protein, has been isolated as a male sterility mutant.94,95 Rotund transcripts have been localized in germ line cells of testis and primary spermatocytes.94,95 Very homologous to rotund, the human MgcRacGAP and the C. elegans CYK-4, are predominantly expressed in testis and in spermatocytes or in gonads and oocytes respectively.96,258 Both proteins have GAP activity towards Rac1 and Cdc42 but not RhoA in vitro.96,258 In contrast to other RhoGAPs that are mainly cytosolic, MgcRacGAP and CYK-4 are localized to the nucleus during interphase, accumulates at the mitotic spindle during metaphase, and are concentrated in the midbody during cytokinesis.97,258 Experiments using a GAP-inactive mutant or embryonic stem cells defective in MgcRacGAP expression and RNA interference for CYK-4 support the idea that these proteins are required for normal mitosis and cytokinesis.98,258 MgcRacGAP has also been shown to interact with a sulfate transporter specifically expressed in male germ cell line, suggesting that MgcRacGAP may link sulfate transport to Rho GTPases in male germ cells.99 Additional roles for MgcRacGAP have been also proposed in the control of growth and differentiation of hematopoietic cells100 and in neuronal development.101 Finally, a 150 kDa protein PARG1 (PTPL1-associated RhoGAP1) has been isolated from its ability to bind to the five PDZ domains present in the protein-tyrosine phosphatase PTPL1.102 PARG1 has GAP activity in vitro towards both Rac1, Cdc42 and RhoA with a clear preference for RhoA.102 To date, the in vivo function of PARG1 is still unknown.

The GRAF Subfamily The GRAF subfamily contains the human proteins GRAF (GAP for Rho associated with focal adhesion kinase), oligophrenin-1, the murine PSGAP-S and PSGAP-M. Most of them are constituted from their N- to C-terminus of a PH domain, a RhoGAP domain and a SH3 domain (Fig. 1, Table 1). Chicken GRAF was the first protein of this subfamily to be identified as a focal adhesion tyrosine kinase (FAK)-interacting protein through its SH3 domain and was subsequently shown also to interact with cell adhesion kinase β (CAKβ), a tyrosine kinase that is closely related to FAK.103,104 GRAF has GAP activity towards RhoA and Cdc42 in vitro.103 However, when expressed in Swiss 3T3 cells, it inhibits RhoA-induced stress fibers formation but not Cdc42-induced filopodia.106 In chicken embryo cells, GRAF colocalizes with actin stress fibers, cortical actin and focal adhesion complexes.103 Human GRAF localizes to the gene locus 5q31,107 which is disrupted in some cases of myelodysplastic syndrome and acute myeloid leukemia, suggesting that deletions and mutations of the GRAF gene may be instrumental in the development and progression of hematopoietic disorders.107 Mutations resulting in oligophrenin-1 loss of function are related to nonspecific X-linked mental retardation (MRX), a pathology affecting 0.15 to 0.3% of men.108 Oligophrenin-1

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Rho GTPases

mRNA is present in various adult and fetal tissues and appears largely expressed in fetal brain. Oligophrenin-1 displays in vitro GAP activity towards both RhoA, Cdc42 and to a lower extent Rac1.108 To date, the specificity of this GAP activity in vivo and the molecular mechanisms linking mental retardation to loss of oligophrenin-1 activity are still unknown. There is two splice variants of PSGAP: PSGAP-S, a 683 amino acid protein (85 kDa) and PSGAP-M, a 786 amino acid protein (95 kDa). Three mRNAs have been detected in a variety of mouse tissues.10 Generation of specific anti-PSGAP antibodies revealed that these two protein variants were expressed in different cell types: PSGAP-M was expressed in fibroblastic cells whereas PSGAP-S appeared to be expressed ubiquitously. PSGAP has GAP activity towards RhoA and Cdc42 both in vitro and in vivo10 and interacts through its SH3 domain with PYK2, a FAK homologous tyrosine kinase. PSGAP colocalizes with PYK2 to focal adhesion complexes through its PH domain and is also phosphorylated by PYK2. In addition, PYK2 has been shown to inhibit PSGAP activity towards Cdc42 in vivo.10 These results suggest that PSGAP may be an important mediator for PYK2 to regulate cytoskeletal dynamics via Cdc42.

The Myosin-IX Subfamily Members of the unconventional myosin-IX subfamily are large multimodular proteins. They are constituted from their N- to C-terminus of a Ras interactive binding domain, whose biological relevance has not been reported, a myosin ATPase domain, four to six short calmodulin binding motifs (IQ), a cysteine-rich domain (C1), and a RhoGAP domain (Fig. 1, Table 1). Rat Myr 5 and its human ortholog myosin-IXB are detected as 225-229 kDa proteins in several rat and human tissues.109,110 Human myosin-IXB is more abundant in cells of myeloid origin.111 Its expression increases upon differentiation of human leukocytes into macrophage-like cells,111 concomitantly with relocation of myosin-IXB from actin-rich structures at the cell periphery to a more cytosolic and perinuclear location.111 Isolated from rat brain, Myr 7 is expressed at high levels in developing and adult brain, while Myr 5 is expressed at moderate levels in this tissue at all developmental stages.112 These proteins display specific GAP activities towards RhoA but not Rac1 or Cdc42 in vitro.110,112,113 Cloning of the mouse ortholog of the rat myr 5 and the human myosin-IXB genes has allowed the identification of two alternatively spliced exons that have still no equivalent in the human or Rat genes.115 Mouse myosin-IXB coding sequence has been mapped to chromosome 8 and is a candidate target for the mouse mutation myodystrophy and quinky.115 Recently, myosin-IXA has been identified in human as a candidate gene involved in the Bardet-Bield syndrome (BBS), an heterogenous autosomal recessive disorder partially characterized by mental retardation, obesity, short stature and hypogenitalism.116 The biological role of these unconventional myosins remains to be determined. The presence of the actin-binding myosin domain suggests that they may act as effectors of RhoA by regulating the organization of actin filaments. However, no evidence for such an effector role of unconventional myosins was obtained. In the case of myr 5, it seems plausible that the myosin domain participates in controlling the subcellular localisation of myr 5, thereby contributing to the spatial and temporal regulation of RhoA.113

The RLIP Subfamily The RLIP proteins consist of a central RhoGAP domain and a C-terminal Ral- binding domain. The human Ral-interacting protein of 76 kDa (RLIP76), the mouse Ral-interacting protein 1 (RLIP1) and the rat Ral-binding protein 1 (RalBP1) bind to activated GTP-bound RalA. RLIP76 and RLIP1 show GAP activity towards Cdc42 and Rac1 but not RhoA in vitro,117,118 while RalBP1 GAP activity seems specific towards Cdc42. The GAP activities and Ral-effector properties of the rat cytocentrin and the D. melanogaster member of this subfamily (supplemental data) are so far unknown, although the high degree of identity (98.4%) between cytocentrin and RalBP1 amino acid sequences suggest that they have similar properties.119 The RhoGAP domain appears not to be required for RLIP1 binding to RalA-GTP117 and the

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79

RalA-binding domain does not affect RLIP1 GAP activity in vitro,118 suggesting that these two domains may function independently. The biological relevance of these activities in vivo has not been established yet. In mouse and rat, RLIP1 and cytocentrin appear to be ubiquitously expressed.118,119 Interestingly, the localization of cytocentrin can cycle between the cytosol and the mitotic poles.120 Moreover, overexpression of this protein inhibits the assembly of mitotic spindles whereas loss of cytocentrin expression leads to the formation of monopolar spindle, suggesting that cytocentrin may play a regulatory role in mitosis.119

The p85 Subfamily p85-α and -β are the regulatory subunits of the phosphatidylinositol-3 kinase (PI-3K). They are constituted from their N- to C-terminus by a SH3 domain, a RhoGAP domain, and two SH2 domains that mediate interaction with activated protein tyrosine kinases121-123 (Fig. 1, Table 1). The RhoGAP domain of p85 is similar to p50-RhoGAP (45% homology) but has no GAP activity.124 However, p85-α is able through its RhoGAP domain to bind specifically to Cdc42 and Rac1 but not RhoA and this interaction has been shown to modulate PI-3 Kinase activity in vitro.124,125 The association between PI-3K and Rac1 induced upon PDGF stimulation of Swiss 3T3 cells suggests a potential role of this interaction in PDGF-mediated actin reorganization.125,126 However, more recently, Rac1 has been shown to inhibit apoptosis in epithelial cells in anchorage-dependent conditions and PI-3K appears to mediate the effects of Rac1 on cell survival.257 Therefore, PI-3K may act both upstream and downstream of Rac1, suggesting the possibility of a positive feedback loop.257

The p50-RhoGAP Subfamily p50-RhoGAP isoforms have been found in human, mouse, D. melanogaster, and C. elegans. They present a RhoGAP domain at their C-terminus and a putative lipid-binding domain (Sec14 domain) whose function has not yet been established (Fig. 1, Table 1 and supplemental data). p50-RhoGAP (named also Cdc42Hs-GAP) displays GAP activity towards Cdc42, RhoA and Rac1 with notable preference for Cdc42 in vitro.31,127-129 Although p50-RhoGAP activity has not been tested in vivo towards Cdc42, it has been shown to inhibit RhoA- but not Rac1-induced cytoskeleton reorganization when expressed in Swiss 3T3 cells.31 p50-RhoGAP has also been shown to interact in vitro with the SH3 domains of p85-α and of c-Src through a proline-rich domain.127 To date, the biological function of these interactions remains to be established.

The OCRL Subfamily The Lowe syndrome, also known as oculocerebrorenal syndrome, is caused by mutations in the X chromosome-encoded OCRL gene.130-132 The OCRL protein is 51% identical to inositol polyphosphate 5-phosphatase II (5-phosphatase II) from human platelets and contains a RhoGAP domain at its C-terminus. OCRL is expressed in a number of tissues and functions as a phosphatidylinositol 4,5-bisphosphate 5-phosphatase both in vitro and in vivo133 but does not present any GAP activity towards Cdc42, Rac1 or RhoA (Lamarche-Vane, unpublished data). OCRL is associated with lysosomal membranes suggesting that OCRL may regulate a specific pool of phosphatidylinositol 4,5-bisphosphate that is associated with lysosomes.134

The p122-RhoGAP Subfamily In addition to their RhoGAP domain, members of this subfamily display a star-related lipid transfer (START) domain (Fig. 1, Table 1 and supplemental data). p122-RhoGAP displays GAP activity in vitro and in vivo towards RhoA but not Rac1. In addition, it interacts with phospholipase C-δ1 and can activate its phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysing activity.135,136 PIP2 modulates the function of numerous actin-binding protein, including gelsolin and profilin, and p122-RhoGAP therefore may be involved in remodeling the actin cytoskeleton through the control of the intracellular levels of PIP2.

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Rho GTPases

The C1 Subfamily Members of the C1 subfamily are constituted by a N-terminal Fes/CIP4 homology domain, a RhoGAP domain, and a C-terminal SH3 domain (Fig. 1, Table 1 and supplemental data). To date, the function of these RhoGAPs is poorly described. Human C1 protein, also named p115, is synthesized predominantly in cells of hematopoietic origin (spleen, thymus and leukocytes) and localized within the cell just below the plasma membrane.137 Overexpression of this protein into fibroblasts disrupts stress fibers suggesting that this protein may have GAP activity towards RhoA in vivo.137 Whether this protein has GAP activity towards Rac1 and Cdc42 has not been investigated.

The CeGAP Subfamily Members of C. elegans GAP (CeGAP) subfamily contain a PH and a RhoGAP domain (Fig. 1, Table 1 and supplemental data). CeGAP presents GAP activity towards human and C. elegans Rac1, Cdc42 and RhoA as well as the C. elegans and human Ras protein (let-60 and Ras) and Rab3A.138 The biological relevance of this cross reactivity in vivo has not been established yet.

The 3BP-1 Subfamily 3BP-1 contains a RhoGAP and a SH3-binding domain. It is mainly expressed in brain and spleen and displays GAP activity towards Rac1 and Cdc42 but not RhoA in vitro.139 Its Rac1-specific GAP activity has been also confirmed in vivo.139 Moreover, this protein is able to bind to the SH3 domain of the protooncogene c-Abl.140 It will therefore be interesting to see whether this interaction can regulate the transforming potential of c-Abl. Nadrin is another member of the 3BP-1 subfamily that has been first identified in rat. Nadrin is a neuronal protein whose expression correlates well with differentiation of neurons. It has GAP activity towards RhoA, Rac1 and Cdc42 in vitro and its expression in NIH 3T3 cells disrupts stress fibers and membrane ruffles formation.141 Since overexpression of a GAP-inactive mutant inhibited Ca2+-dependent exocytosis in PC12 cells, it has been postulated that nadrin may play a regulatory role in this process.141

The CdGAP Subfamily Mouse CdGAP consists of a N-terminal RhoGAP domain and five potential SH3-binding motifs localized within a large C-terminal proline rich domain. CdGAP mRNA is detected in a number of mouse tissues but seems more abundant in brain, heart and lung. CdGAP has GAP activity towards Rac1 and Cdc42 but not RhoA, both in vitro and in vivo.142 Moreover, this activity has been shown to be directly regulated through its interaction with the scaffolding protein intersectin both in vitro and in vivo.11 Since intersectin has been implicated in clathrin-dependent endocytosis and mitogenic signalling, the CdGAP-intersectin interaction may play a role in the regulation of these cellular processes. However, to date such a function has not been established. d-CdGAPr is a D. melanogaster CdGAP related protein that contains an additional SH3 domain at the N-terminus of the GAP domain. This protein has not been characterized functionally yet, but its mRNA has been detected throughout development as well as in adult and presents a highly dynamic spatio-temporal pattern.143

The ARHGAP6 Subfamily

ARHGAP6 proteins display GAP activity towards RhoA both in vivo and in vitro.144,145 ARHGAP6 localizes to the X chromosome in a critical region deleted in patients suffering of microphthalmia with linear skin defects (MLS),144,145 but whether alteration of this gene contributes to MLS remains to be shown.

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The Saccharomyces Cerevisiae GAPs Bem2p consists of a RasGEF, a PH, and a RhoGAP domain (Fig. 1 and Table 1). Bem2 mutations cause cells to become large and multinucleate, suggesting that Bem2p is involved in bud emergence.146,147 Bem2p has GAP activity towards Rho1p in vitro.148 In addition, increasing the gene dosage of rho1 or rho2 can partially suppress bem2 mutant phenotypes. Thus, it appears that Rho1p and Rho2p GTPases might be the targets of Bem2p in vivo.149 Since bem2 mutations have abnormal microtubule arrays, Bem2p has been also proposed to be involved directly or indirectly in regulating microtubule function.150 Bem3p is a suppressor of the temperature-sensitive lethality caused by mutations in bem2.151 In addition to its C-terminal RhoGAP domain, it has PX and PH domains (Fig. 1, Table 1). The recombinant GAP domain of Bem3p has GAP activity towards human Cdc42 in vitro.13,151 Dbm1p (named also Rga1p) has two LIM domains at its N-terminus and a RhoGAP domain at its C-terminus (Fig. 1, Table 1). It has been identified as a negative regulator of the pheromone response pathway152 and has been implicated in bud site selection.153 Mutations within the LIM domains result in mutant forms of Dbm1p that can no longer function in bud site selection.153 Using the yeast two-hybrid system, this protein has been shown to interact with Cdc42p, suggesting that the latter protein may be a specific target for Dbm1p GAP activity.152 Like Dbm1p, Lrg1p consists of three LIM domains and a RhoGAP domain at its C-terminus. Lrg1p has been shown to be expressed during sporulation and is thought to play a role during mating.154 To date, nothing is known about its GAP activity. The sac7 gene was identified as a genomic suppressor of actin mutations.155 Its protein product has GAP activity towards Rho1p in vitro.156 Signal transduction mediated by the mitogen-activated protein kinase (MAPK) Slt2 pathway is essential to maintain the cell wall integrity in Saccharomyces cerevisiae. Disruption of the Rho1-GTPase-activating protein genes sac7 and bem2 leads to constitutive Slt2 activation, indicating their involvement as negative regulators of the MAPK pathway and a putative role in the maintenance of cell wall.157,158

The Arabidopsis Thalia GAPs Recently, RhoGAP-like proteins have been identified in plants: the RopGAPs. They all present a consensus RhoGAP domain and most of them contain a Cdc42/Rac-interactive binding (CRIB) motif as well (Fig. 1, Table 1, supplemental data). Interestingly, the CRIB domain has been shown to regulate the in vitro GAP activity of the RopGAPs towards a Rho GTPase closely related to the mammalian Rac protein.159

The Dictyostelium Discoidum GAP Dictyostelium discoideum DdRacGap1 (DRG) contains from its N- to C-terminus a RhoGAP domain, a SH3 domain, and a PH domain surrounded by two DH domains (Fig. 1, Table 1, see also Chapter 14). Its GAP domain is active towards DdRac1A, DdRacC, DdRacE, HsRhoA and HsRac1 in vitro, but not HsN-Ras.160,161 Disruption of DRG stimulates membrane ruffling, similar to overexpression of a constitutively active DdRac1 mutant, suggesting that DdRacGAP is a Rac1-specific GAP in vivo.161,162 DRG also has GEF activity towards Rac1, but not DdRacE. Interestingly, DRG has a high GAP but not GEF activity towards DdRabd, the dictyostelium homolog of Rab4. Moreover, DRG-GAP activity appears to be required for RabD-dependent regulation of the contractile vacuole, suggesting that the RabD-specific GAP activity of DRG may have some biological relevance in vivo.161

RhoGAPs in Host-Pathogen Interaction Multiple studies indicate that Rho GTPases are the preferred eucaryotic substrates of various bacterial protein toxins and exoenzymes.163 ExoS, YopE and SptP are cytotoxins secreted via a type III secretion pathway by the opportunistic bacteria Pseudomonas aeruginosa, Yersinia spp. and Salmonella typhimurium respectively.164-168 All proteins present a RhoGAP domain.

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Table 2. Specific GAP activities, tissue distributions and putative cellular and pathological functions of characterized RhoGAPs Subfamily

Name

GAP Activity

Tissue Distribution

Role In

References

Bcr

Bcr Abr

Rac1, Rac2, Cdc42 Rac1, Cdc42

Brain, hematopoietic cells Brain

•cell cycle progression •mediation of glucocorticoid effects

26-31, 52

p190-RhoGAP

p190-A

Rac1,Cdc42, RhoA*

Most tissues

•Ras-RhoA cross-talk

31, 57, 59, 60, 72, 74, 75, 77-79, 188, 189

p190-B

Rac1,Cdc42, RhoA*

Most tissues

•fibroblast migration •melanoma and breast cancer cell invasion •neurogenesis •Shigella flexneri entry

Alpha 1 Alpha 2 Beta 1 Beta 2 MgcRac-GAP

Rac1, Cdc42 Rac1, Cdc42 Rac1, Cdc42 Rac1, Cdc42 Rac1,Cdc42

Brain Brain, testis Testis, germs cells Brain, pancreas Testis,Thymus, CNS

•Rac1, Cdc42 effector?

Rotund CYK-4 PARG1

ND Rac1,Cdc42 Rac1, Cdc42, RhoA*

testis gonad, oocytes Most tissues

Chimaerin

26, 81-83, 87, 90, 94-97, 100-102, 258

continued on next page

Rho GTPases

•malignant glioma? •mitosis, cytokinesis •hematopoiesis spermatogenesis? •neurogenesis? •spermatogenesis •cytokinesis

Subfamily

Name

GAP Activity

Tissue Distribution

Role In

References

GRAF

GRAF Oligophrenin1 PSGAP

RhoA,Cdc42 RhoA,Cdc42, Rac1 RhoA,Cdc42

Most tissues Most tissues, fetal brain* Most tissues

•hematopoietic disorders? •X-linked mental retardation

10, 103, 106-108

Myosin-IX

Myosin-IXB

RhoA

Most tissues, cells of myeloid origin*

Myosin-IXA Myr 5 Myr 7

ND RhoA, RhoB, RhoC RhoA

Most tissues Most tissues, brain*

RLIP

RLIP76 RIP1 RalBP1 Cytocentrin

Cdc42,Rac1 Cdc42,Rac1 Cdc42 ND

Most tissues Most tissues Most tissues Most tissues

Ral-Rho crosstalk Ral-Rho crosstalk Ral-Rho crosstalk Ral-Rho crosstalk •mitosis

117-119

p85

P85-alpha P85-beta p50-RhoGAP (Cdc42Hs-GAP)

(-)

Most tissues Most tissues

•actin reorganization

124, 125

p50-RhoGAP

109-111, 112, 113, 114,116 •Bardet-Bield sydrome?

Rac1,Cdc42*,RhoA

OCRL

OCRL1

(-)

p122-RhoGAP

P122-RhoGAP

RhoA

The Superfamily of Rho GTPase-Activating Proteins

Table 2. Cont.

31, 127-129

Most tissues

•Lowe syndrome

130-133

RhoA effector? activates PLC-δ1

135,136,256

83

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84

Table 2. Cont. Subfamily

Name

GAP Activity

Tissue Distribution

C1

Hs C1

RhoA

spleen,thymus, leukocytes

CeGAP

CeGAP

Rac1,Cdc42, RhoA,Ras, let60,Rab3A

3BP1

3BP1 Nadrin

Rac1, Cdc42 Rac1, Cdc42,RhoA

Brain,spleen neurons

•Abl signalling? •exocytosis in PC12

139, 141

CdGAP

CdGAP

Rac1, Cdc42

Most tissues

•endocytosis? •mitogenic-signalling?

142

ARHGAP6

ARHGAP6

RhoA

Most tissues

•Microphthalmia with Linear Skin defects?

144, 145

Role In

References

137 138

Rho GTPases experimentally identified as in vitro and/or in vivo RhoGAP substrates are indicated respectively in italic and/or bold. Organs in which the RhoGAP transcript or protein has been detected and the proposed cellular and/or pathological roles are indicated. Asterisks indicate the preferred substrates or tissues. ND indicates noncharacterized activity.(-) indicates that the protein does not present any GAP activity in vitro.

Rho GTPases

The Superfamily of Rho GTPase-Activating Proteins

85

Table 3. Specific GAP activity and potential cellular function of S. cerevisiae, D. discoideum and pathogen RhoGAPs Species

Name

GAP Activity

Role In

References

S. cerevisiae

BEM2

Rho1p,Rho2p

13, 146, 147 149, 151, 152, 154, 156-158

BEM3 DBM1 LRG1

HsCdc42 Cdc42p ND

SAC7

Rho1p

-bud emergence - microtubule regulation? -cell wall -bud emergence -pheromone response -sporulation, mating -cell wall

D. discoideum

Yersinia spp.

P. aeruginosa S. typhimurium

DdRacGAP DdRac1A, DdRacC,DdRacE, HsRhoA, HsRac1, DdRabd YopE Rac1,Cdc42, RhoA ExoS SptP

Rac1,Cdc42, RhoA, Rac1,Cdc42

160-162

-inhibition of phagocytosis -cell rounding -antagonizes SOPE, SOPB

175, 180-183

Rho GTPases experimentally identified as in vitro and/or in vivo RhoGAP substrates are indicated respectively in italic and/or bold. Cellular roles proposed for these proteins are indicated. ND indicates noncharacterized activity.

In addition to its RhoGAP domain, ExoS has a C-terminal ADP-ribosyltransferase domain. Intracellular delivery of ExoS is cytotoxic for eucaryotic cells and has been shown to ADP-ribosylate Ras in vivo and uncouple Ras-mediated signal transduction pathways.169-174 ExoS displays a GAP activity towards RhoA, Rac1, and Cdc42 in vitro.175 In addition, intracellular expression of the RhoGAP domain of ExoS elicits the rounding of Chinese hamster ovary (CHO) cells and the disruption of actin filaments in a dose-dependent manner.176 The functional role of ExoS GAP activity in microbial pathogenesis is not fully understood, however it may play a role in microbial entry since both Rac1, RhoA and Cdc42 have been implicated in phagocytosis.178,179 Similar to ExoS, YopE exhibits in vitro GAP activity towards RhoA, Rac1 and Cdc42.180,181 Infection assays carried out with a Yersinia pseudotuberculosis strain producing a YopE GAP-deficient mutant (R144A) demonstrated that the GAP function is essential for the disruption of actin filaments, cell rounding and inhibition of phagocytosis by YopE.180-183 SptP consists of a RhoGAP domain and a protein tyrosine phosphatase domain.184 Microinjection of purified GST-SptP into cultured cells results in the disruption of the actin cytoskeleton.185 SptP has GAP activity towards Rac1 and Cdc42 but not RhoA in vitro and antagonizes the Rac1/Cdc42 GEF activity of SopE in vivo.19,186,187

Conclusion Analysis of the various subfamilies of RhoGAP proteins points out the high degree of redundancy of RhoGAPs within higher eucaryotic organisms such as human and mouse in com-

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parison with C. elegans that in most cases has only one member of each subfamily (supplemental data). This analysis also highlights that within a same family of proteins, RhoGAPs are not clearly homogenous when considering their Rho GTPase targets and tissue distribution (Table 2). The hypothesis considering RhoGAPs as spatio-temporal regulators of Rho GTPases gives a reasonable explanation to such an abundance of RhoGAP proteins. This hypothesis is nicely illustrated in the case of p190-A thought to be implicated in the temporally and spatially restricted inactivation of RhoA in fibroblasts upon integrin engagement allowing cell movement.74 This also implies that RhoGAP activities may be submitted to a spatio-temporal regulation that could be at the level of their subcellular localization and/or GAP activity. In this study, 24 and 22 potential RhoGAPs have been identified in D. melanogaster and C. elegans, respectively. However, little is known about the in vivo characterization of most of these putative GAPs encoded genes. Clearly, disruption of the GAP genes in fruit fly and worm will be of great interest and will provide novel insights into the biological functions of RhoGAPs. Future studies of RhoGAP proteins and their cellular partners will help to elucidate how the regulation of GAP activities and the integration of inputs from a number of different signalling pathways are translated into a specific pattern of Rho GTPase activities.

Acknowledgments Nathalie Lamarche-Vane and Sarah Jenna hold a junior scholarship and a fellowship, respectively, from the Fonds de la Recherche en Sante du Quebec and the Canadian Institute of Health Research

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CHAPTER 6

Rnd Proteins: Intriguing Members of the Rho Family Pierre Chardin

Abstract

T

he three Rnd proteins form a distinct sub-branch of the Rho family. They appear to differ from most other Rho family proteins in their basic functional cycle (see Preface). Rnd1 and Rnd3 exchange GTP rapidly, have a reduced affinity for GDP and have no GTPase activity suggesting that they might be constitutively in the GTP-bound form. However, compared to active Rho, they have opposite effects on the actin cytoskeleton and cell adhesion. Rnd1 or Rnd3 expression in fibroblasts inhibits the formation of actin stress fibers and focal adhesions. Rnd1 is expressed mostly in brain and liver, the highest level of Rnd2 expression is in testis and Rnd3 is present at very low levels in most tissues, but can be induced by a variety of signals.

General Features Rnd proteins share 54-63% identity pairwise, 45-49% identity with RhoA, B or C and about 40% identity with Rac or Cdc42 (ref. 1, for a detailed phylogenic analysis, see Guasch et al2). The first member of this group, RhoE, was discovered by Foster et al3 in 1996, mouse RhoE was also cloned independently.2 RhoE is identical to Rnd3, except that it is missing 15 N-terminal residues. The first cloned RhoE cDNAs had shorter 5’ends, but there are now at least 4 mouse cDNAs (for instance GB: BC009002) and 1 pig cDNA (GB:AF078839) encoding full length proteins. Rnd proteins display striking differences from other members of the Rho family in their size, charge and biochemical properties.1 Their expected molecular weights are higher due to N-terminal extensions of 10-20 a.a. for Rnd1 and Rnd3/RhoE, and C-terminal extensions of about 30 a.a. for all three. Calculated molecular weights predict that Rnd1 is a 26 kD protein, Rnd2 is 25 kD and Rnd3 is 27 kD. Their apparent molecular mass on SDS-PAGE is about 3032 kD, whereas other Rho proteins have apparent mobilities of about 24 kD. They also have very different isoelectric points: whereas most Rho family proteins are negatively charged at neutral pH (pI 5.5 to 6.8, ref. 4), Rnd proteins are positively charged with pIs of 8.1-8.7.

Prenylation As other Rho family members, Rnd proteins end with a “CAAX box” that serves as a recognition signal for prenylation (see Chapter 2). However, unlike Rho, Rac or Cdc42, which have a C-terminal leucine specifying geranyl-geranylation, Rnd proteins end with a methionine residue, suggesting that they are farnesylated. Indeed, it has been shown that in-vivo, RhoE/ Rnd3 is farnesylated.3 Interestingly, the farnesyltransferase inhibitor L-739,749 developed to inhibit Ras farnesylation, induces cell flattening, actin stress fiber formation and inhibition of anchorage-independant growth after only 18 hours of treatment,5 too fast to be explained by Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Figure 1. Alignment of Human Rnd1, 2 and 3 with RhoA, RhoD, Rac1 and H-Ras. Top, Stars over the sequence indicate the positions corresponding to Ras activating mutations, “effector”: region considered as the main target binding site including the asparagine (FENY) ADP-ribosylated by the C3 transferase in Rho. Bottom, P1-3: regions involved in binding phosphate, G1-3: regions binding the guanine moiety of GTP, below, α-helices and β-sheets (based on Ras, Rac and Rho structures). The “Rho insert” (α3b) is a 14 amino acids helical insertion found only in Rho family proteins. The C-terminal parts are poorly conserved, except for the presence of basic residues. The cysteines of the CAAX sequences for prenylation are aligned. Accession numbers for human nucleotide and protein sequences: Rnd1: Y07923, Q92730 Rnd2: X95456, P52198, Rnd3: X95282, P52199. Xenopus laevis Rnd1: AF151014, AAD40670. Pig Rnd3: AF078839, O77683.

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the loss of farnesylated Ras which is stable (half-life of 24 h). Since Rnd1 or 3, that have shorter half-lives, induce a loss of stress fibers and cell rounding, (see next paragraphs) the inhibition of Rnd farnesylation would presumably induce opposite effects, namely stress fiber formation and cell spreading, as seen with L737-749. Rnd proteins are thus interesting candidate targets for this class of drugs.

Chromosomal Localization Rnd1: 12q13, Rnd2: 17q21, Rnd3: 2q22. Sequencing of the BRCA1 region and now the human genome sequence indicate that the Rnd2 gene is one of the closest to BRCA1 (ref. 6). Rnd2 maps to a region frequently rearranged in breast and ovarian tumors. Breast cancer is one of the most frequent malignancies in women and rearrangements of chromosome 17 are the most frequent genetic alterations observed in breast and ovarian tumors. Mutations in BRCA1 increase susceptibility to breast and ovarian cancers, but other genetic rearrangements of this region are also frequent. Although Rnd2 is located very close to BRCA1, no implication of Rnd2 in breast cancer could be detected at present.

Biochemical Properties of Rnds Although Rnds are related to Rho in terms of sequence, they possess strikingly different biochemical properties.

GTP Binding The three motifs for guanine binding (noted G1-3 on Fig. 1) are conserved in Rnd proteins, the two loops and a conserved threonine involved in phosphate binding (noted P1-3 on Fig. 1) can also be recognized. The three major residues that coordinate magnesium in the GTP bound form of Ras: T17, T35 (noted P2 on Fig. 1) and D57 (ref. 6) are strictly conserved. Compared to most other small GTP-binding proteins they have a weaker affinity for GTP: in a physiological buffer (with 1 mM magnesium) it takes only 1 or 2 minutes to dissociate half of the GTP (for Rnd1 t1/2=1.4 min at 37°C), but most strikingly they have a 100 fold weaker affinity for GDP, suggesting that they are constitutively in a GTP-bound form.

GTP Hydrolysis Three residues of the phosphate binding site that are important for the intrinsic GTPase activity differ in Rnd. The equivalent of Ras glycine 12 is replaced by valine, alanine or serine in Rnd proteins, Ras glycine 13 is replaced by glutamine or glutamic acid and Ras alanine 59 and glutamine 61 are both replaced by serine. Any of these substitutions in Ras decreases the GTPase rate and leads to constitutively active, oncogenic proteins. Based on the effects of these substitutions on Ras, the prediction was that Rnd proteins will have little or no intrinsic GTPase activity. Indeed, Rnd1 has no detectable GTPase activity, even in the presence of RhoGAP.1 Foster et al 3 also reported that p190 and another RhoGAP do not stimulate GTP-hydrolysis on RhoE, but if the three residues residues of the phosphate binding site that differ in RhoE are mutated to those found in RhoA, then the intrinsic GTPase activity is restored. The activity is even higher than in RhoA, indicating that the presence of a proline immediately after Glutamine 61 (ras numbering) does not impair the proper positioning of this glutamine relative to the γPhosphate. In addition, Foster et al3 showed that a tagged version of RhoE expressed in COS cells, was constitutively GTP-bound. Taken together these results strongly suggest that in-vivo, Rnd proteins are constitutively GTP bound. This would be the first example of a GTPase that does not switch between GDP- and GTP-bound forms. This raises to question as to why do they bind nucleotide? In fact, it seems that these proteins have appeared rather recently in evolution. Rnd1 is present in Xenopus, but there are no obvious homologs in the fly or nematode. One possible explanation is that Rnds may have retained GTP binding mostly for reasons of stability, since it is well known that nucleotide-free small G proteins are very unstable.

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Do Rnd Proteins Need GEFs for Activation The observation that Rnd1 exchanges GTP rapidly, binds GDP with a very low affinity and has no GTPase activity already suggests that it is constitutively GTP bound in the cell. A feature of the primary structure of Rnds further supports this idea. Glutamate 62 from Ras (DTAGQE) is conserved in almost all proteins of the Ras family (Ras, Rho, Rab, Ran), although examination of the structure of isolated proteins either bound to GDP or GTP does not suggest any obvious reason for the conservation of this residue. However, in the nucleotide free complex of Ras or Rac, with their GEFs, this glutamate contacts the strictly conserved Lysine 16. In the GDP or GTP forms, Lys16 contacts the phosphates, but when GDP leaves, the positive charge of Lys16 needs to interact with a negative charge, provided by Glu62. The fact that this glutamate is not conserved in Rnd proteins suggests that they may never form a nucleotide free complex with GEFs. Their biochemical properties suggest that they may not need GEFs to accelerate GTP binding and therefore, they may never see GEFs. Another intriguing possibility would be that Rnd-GTP forms a complex with GEFs and inhibits them, behaving as endogenous “dominant negatives”. Rnds in their GTP form would mimic the nucleotide free form of Rho to bind its GEFs and inhibit endogenous Rho activation. At present there is no experimental data to support this hypothesis. It is also possible that GTP binding plays a regulatory role through a completely new mechanism that we currently do not understand.

Rnds As Targets of Bacterial Toxins The Clostridium botulinum C3 transferase is an exo-enzyme that specifically ADP-ribosylates Rho proteins on asparagine 41 (ref. 7,8). Rnd proteins are almost identical to Rho proteins in the region surrounding the ADP-ribosylated Asn41, but surprisingly Rnd1, Rnd2 and Rnd3 proteins, either expressed and purified from bacteria, or in intact tissues, are not ADP-ribosylated by C3 (ref. 1). Positions Val43 and Glu54 of Rho are involved in C3 interaction (P. Chardin, unpublished). These positions, as well as Arg5, Lys6, and Glu47 were recently shown to participate in C3 binding.9 Lys 6 and V43 are not conserved in Rnd proteins, explaining why they are not C3 substrates. Recently, a novel C3-like ADP-ribosyltransferase from Staphylococcus aureus was shown to ADP-ribosylate Rnd3, although less efficiently than RhoA.10 At present the functional relevance of this observation is not clear since this novel C3-like ADPribosyltransferase from Staphylococcus aureus causes the same cytoskeletal changes on NIH/ 3T3 cells as C3 from Clostridium botulinum. It is possible however, that in other cell types expressing high levels of Rnd3 and less Rho, this novel C3-like toxin might have different effects on the cytoskeleton.

Expression in Human Tissues Northern blots show that Rnd1 is expressed as a major 1.9 kb mRNA, mostly in brain and liver. In the brain, Rnd1 is mostly expressed in different types of neurons (see Nobes et al,1 for a detailed description of the brain regions expressing Rnd1). The Rnd2 mRNA of 1.5 kb is abundant in testis, and is expressed at lower levels in brain and liver. Rnd3/RhoE is expressed at low levels in all tissues studied, as a 3.1 kb mRNA. Rnd3/RhoE expression is specially low in brain, pancreas, thymus, testis, and peripheral bloods leukocytes.1 The expression of Rnd1 in rat brain and liver could also be detected at the protein level, with antibodies raised against the bacterially expressed Rnd1. In post-nuclear supernatants Rnd proteins are mostly found in the pellet (P100) fraction, whereas most Rho appears in the supernatant (S100) fraction. In rat liver, Rnd1 expression is mostly found in hepatocytes. Nishi et al,11 have cloned the rat and mouse homologs of Rnd2 and named them RhoN. In rats, they found expression of RhoN in neurons from the brain and spinal cord (see Nishi et al,11 for a detailed description of the brain regions expressing RhoN/Rnd2) and in stellate cells of the liver. Surprisingly, they find only a faint expression in rat testis, whereas in human this was the place where Rnd2 was expressed at the highest levels and expression of the Rnd2 protein has been observed in rat testis.1

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Figure 2. Rnd1 or Rnd3 expression in fibroblasts inhibit actin stress fibers formation. Serum-starved confluent Swiss 3T3 fibroblasts micro-injected with expression vectors for: w.t. Rnd1 (A, B), T27N Rnd1 (C, D), N-terminal truncated Rnd1 (E, F) or Myc-tagged Rnd3 (G, H) and then stimulated with LPA for 30 minutes, 2 hours after injection. Cells were fixed, permeabilised, and stained with Anti-Rnd1 antibodies (A, C, E) or Anti-Myc-tag antibodies (G) and phalloidin to decorate actin filaments (right: B, D, F, H).

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Localization of Rnd1 in Swiss 3T3 Fibroblasts and Microinjected MDCK Cells In Swiss 3T3 fibroblasts, the anti-Rnd1 antibody gives a dotted staining of the plasma membrane at points of cell-cell contacts, but no staining at places where there is no neighboring cell. The peripheral staining co-localizes with an anti-pan-cadherin antibody, indicating that Rnd1 is localized in adherens junctions. In MDCK epithelial cells, microinjection of wild type Rnd1 does not cause cell rounding and Rnd1 localises at the cell periphery. The staining pattern closely resembles E cadherin staining in these cells. Interestingly, a truncated form, in which the N-terminal 6 amino acids are deleted, no longer localizes to adherens junctions, instead the expressed protein is present in the cytoplasm and cell nucleus. Deletion of the CAAX motif for prenylation leads to a predominant nuclear localization. Thus, the integrity of the N-terminus and the CAAX box are required for normal localization of Rnd1 to adherens junctions.

Rnd1 or Rnd3 Expression Inhibit the Formation of Actin Stress Fibers Microinjection of Rnd1 expression vectors in quiescent serum-starved Swiss 3T3 fibroblasts does not induce the formation of actin stress fibers. In contrast, Rnd1 expression inhibits actin stress fibers formation in cells stimulated with LPA (Fig. 2A,B). After longer times, Rnd 1 expressing cells round up and detach from the glass coverslips. In cells expressing T27NRnd1 (equivalent of a dominant negative S17N mutation in Ras) (Fig. 2C and D) or T45ARnd1 (equivalent of a T35A, effector site mutation in Ras), LPA-induced stress fiber formation was not inhibited. Thus the binding of GTP and a functional effector domain are both required for Rnd1 to block stress fiber formation.1 In Ras the substitution of glutamine 61 by leucine gives a higher transforming potential because it impairs GAP stimulation of GTP hydrolysis. Rap proteins that have a threonine at this position do possess an intrinsic GTPase activity, that is stimulated by RapGAP. However, Rnd1 with a leucine instead of serine at position 71 (equivalent to codon 61 in Ras) has the same activity as wild-type Rnd1 in blocking LPA-induced stress fiber formation, suggesting that fibroblasts do not contain a RndGAP. Interestingly, a mutant Rnd1 deleted of its first 6 amino-acids was unable to block stress fiber formation (Fig. 2E and F). Thus, the six N-terminal amino-acids of Rnd1 (strictly conserved in Rnd3/RhoE) are also required. This could explain the lack of effects on the cytoskeleton reported for RhoE, a N-terminal truncated version of Rnd3 (ref.3). Expression of fulllength Rnd3/RhoE also blocks LPA induced stress fibers formation (Fig. 2G, H), similar to wild-type Rnd1 (Fig. 2B). This N-terminal sequence is highly polar (MKERRA) and mostly basic, there are no other proteins in the database with a very similar N-terminus, it might be that the positive charges interact with the negative charges of phospholipid head groups and stabilize membrane binding. At high levels of expression a Rnd1 mutant deleted of its CAAX box also blocked stress fiber formation, but was less potent than wild type Rnd1. Thus, it seems that the N-terminal motif is even more important than the prenylation for the effects of Rnd1 or Rnd3 on actin structures. In this respect, it is interesting to note that expression of Rnd2 has no obvious effects on the cytoskeleton, and that Rnd2 does not possess this N-terminal motif. Furthermore, N or Cterminal truncations that impair the proper localization of Rnd1 also abolish or decrease the effects of Rnd1 on actin stress fibers, indicating that both GTP binding and localization are essential for these biological effects.

Rnds Inhibit Smooth Muscle Contraction Smooth muscle contraction is regulated by the level of phosphorylation of Myosin Light Chain (MLC). Calcium increase activates a calcium/calmodulin dependent MLC kinase.

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Smooth muscle contraction can also occur at constant calcium (this is called ”calcium sensitization”) when Rho is activated.12,13 One of the Rho-stimulated kinases (p160ROKα, or ROCK) can phosphorylate a regulatory subunit of myosin light chain phosphatase, leading to its inhibition.14,15 In physiological conditions, calcium increase and Rho activation are both required and cooperate in MLC phosphorylation, calcium activating the kinase and Rho inhibiting the phosphatase. Consistently, the Rho-kinase inhibitor Y27632 inhibits agonist induced contraction.14 Thus, activation of Rho increases the phosphorylation of MLC and leads to the bundling of actin into stress fibers.15 Loirand et al16 found that Rnd1 inhibits agonist induced calcium sensitization, by acting on the Rho-dependent pathway. Furthermore, treatment of rats with sex hormone steroids, that are known to alter smooth muscle contractivity, induced the expression of Rnd proteins in intestinal and aortic smooth muscle and decreased agonistinduced contraction.16 Interestingly, RhoA induced calcium sensitization was completely inhibited when Rnd1 was added together with RhoA, but Rnd1 could not reverse the tension induced by addition of RhoA alone for 15 minutes. Thus, on this short time scale, when RhoA has been activated and engaged in the interaction with its partners, Rnd1 is no longer able to inhibit, suggesting that Rnd1 directly inhibits RhoA activation, or competes for the interaction with a subset of RhoA partners. Although these functional studies in smooth muscle were performed with Rnd1, it is possible that the protein involved in-vivo is Rnd3, that has similar biological effects. Interestingly, Rnd3 expression increases in myometrium during pregnancy, when smooth muscle contraction needs to be repressed, and decreases close to delivery, before contraction can occur.17 These results confirm that Rnd proteins can inhibit Rho pathways in several physiological situations.

Proteins Interacting with Rnds The effects of Rnds suggest that they might compete for the interaction with some Rho effectors, however, using the two-hybrid system, no interaction of Rnd1 with citron, rhotekin, the Rho binding domain of ROK, or PAK could be detected. Interestingly, the interactions with RhoGDI-3 and LyGDI were also negative. Positive controls with known interacting proteins were included in all cases (Gerard Zalcman and Jacques Camonis, unpublished). Surprisingly, a fragment of Rnd1 (61-144) was found in a two-hybrid screen with the Grb7 adaptor protein as bait. Full length Rnd1 also interacts with Grb7 in the two-hybrid system. Further mapping indicated that the SH2 domain of Grb7 is sufficient for the interaction with Rnd1, no interaction could be detected with a large variety of other SH2 domains. Direct binding was demonstrated in-vitro: Recombinant Rnd1 interacts with GST-Grb7, suggesting that tyrosine phosphorylation of Rnd1 is not required for its interaction with the SH2 domain of Grb7. Rnd1 expressed in mammalian cells is also able to bind Grb7, but no tyrosine phosphorylation of Rnd1 could be detected.18 Rnd1 and Grb7 are both expressed in liver and Grb7 appears to be involved in cell migration.19 A role for Grb7 interaction with phospho-caveolin1, near focal adhesions, in EGF-stimulated cell migration has recently been proposed.20 Since Rnd1 overexpression leads to the loss of focal adhesions, one could imagine that Rnd1 traps Grb7 away from these sites. Several groups have reported that the Rho antagonist effects of Rnds are due to the stimulation of RhoGAPs and inhibition of a Rho effector, these recent results are discussed in last paragraph.

Effects of Rnd1 in Neurons Rnd1 is expressed in specialized neurons and has major effects on the actin cytoskeleton in fibroblasts. Since Rho proteins have been involved in neurite retraction whereas Cdc42 and Rac promote neurite extension in PC12 cells it was tempting to look at Rnd1 effects in these cells. Aoki et al,21 found that Rnd1 expressed in PC12 cells localizes to the plasma membrane and induces the formation of long branched neuritic processes, as a control the inactive T27N mutant of Rnd1 had no effect. NGF induces long neurites having thick bundles of microtu-

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bules and neurofilaments, and F-actin at their tips, in contast Rnd1 expression induced shorter neuritic processes containing microtubules but little neurofilaments and F-actin. Treatment of PC12 with cytochalasin D, that inhibits actin polymerization, induced processes similar to those seen with Rnd1, suggesting that the morphological effects of Rnd1 are mostly due to the collapse of peripheral actin filaments, with no active role of Rnd1 in the elongation of dendritic processes. Although Rnd1 induced processes seem to contain little F-actin, co-transfection of dominant negative Rac with Rnd1 inhibited the formation of these long processes and led to the formation of multiple thin and short processes.21 In the macrophage cell line Bac1.2F5 RhoE /Rnd3 also induces filopodial or pseudopodial extensions, but in this case they are actincontaining structures.2 Interestingly, Rnd1 was found to interact with PlexinA1, a trans-membrane protein with an intracellular domain showing some homology with R-RasGAP. Plexins are the signal transducing subunits associated with Neuropilin-1, the receptor for the axon guidance signal semaphorin 3A that induces growth cone collapse. A double mutant of PlexinA1 at two arginines likely to be involved in GAP activity no longer induces collapse in a model system, but still binds Rnd1, indicating that other properties than Rnd1 binding, possibly GAP activity, are required for the collapse induced by PlexinA1 (ref. 22). It is suggested that PlexinA1 could be a Rnd1 GAP, but this has not been tested experimentally. Rnd3/RhoE has first been isolated as a p190RhoGAP binding protein3 but is not sensitive to the GAP activity of p190. Alternatively, PlexinA1 could act simply by sequestering Rnd1.

Induced Expression of Rnd3 Participates in the Transformation of Epithelial Cells by Raf Rho family proteins play an important role in cell proliferation and their exchange factors are also involved in transformation and invasiveness26 (reviewed by Symons,27 see also Chapter 11). Cells microinjected with activated Ras rapidly spread, with increased ruffling and actin stress fibers,28,29,30 but display a completely different morphology at later times, with cell rounding and a fusiform appearance as seen in foci of Ras-transformed cells.27 Several events contribute to cell rounding: for instance activation of the Ras/raf/MAP Kinase pathway leads to the suppression of integrin activation, in the absence of de-novo protein synthesis.31 At later times, transformation by Ras also induces the expression and secretion of cathepsins D and L, matrix proteases involved in the acquisition of invasiveness. Decreases in α-actinin, vinculin, or tropomyosins, have also been proposed to explain the loss of stress fibers in transformed cells and more recently, oncogenic Ras has been shown to downregulate Rho-kinase.32 Activation of the Raf–MEK-ERK pathway in MDCK cells induces transformation, with characteristic morphological changes: loss of basal actin stress fibers, increased cortical actin and multilayering, with a loss of cell polarity.33 The increase in cortical actin correlates with Rac activation, but no change in active Rho could be detected. However, Raf activation induced a strong increase of Rnd3 protein expression that could be blocked by MEK inhibitors. This increased Rnd3 expression correlated with the decrease in actin stress fibers. Moreover, in MDCK cells expressing Rnd3 under the control of tetracyclin (Tet-off system), induction of Rnd3 expression results in a loss of actin stress fibers. To confirm the central role of the Rho pathway, it was shown that Raf induced multilayering could be abrogated by constitutive expression of activated RhoA, but surprisingly most stress fibers were lost, even in the activated RhoA expressing cells, indicating that the loss of basal actin stress fibers is not sufficient to induce multilayering.33 Since Rho-GTP levels do not seem to decrease when Rnd3 is induced, this suggests that Rnd does not interfere with Rho activation, but rather with its stimulation of downstream targets. One could imagine that Rnd3 traps some Rho effectors in membrane domains where they can not be active. Increased Rnd3 expression has also been found in pancreatic cancer.34 Furthermore, in keratinocytes, Rnd3 expression also increases 1-2 hours after the binding of activated Factor VIIa to Tissue Factor, a transmembrane glycoprotein.35 In this case, increased Rnd3 expression

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together with transcription factors, growth factors, cytokines, and other proteins involved in cell migration, appears to be part of a wound-healing response. Interestingly, increased expression of Rnd3/RhoE has also been found after treatment of colon cancer cell lines by the nonsteroidal anti-inflammatory drug Sulindac that has an anti-proliferative effect. Although this last result does not seem to fit with the observed increase in Rnd3 expression in proliferating cells or wound-healing, it should be noted that Rnd3 expression was already quite high in the colon cancer cell lines before treatment, whereas expression of Rnd3 in normal colon cells is very low. Thus, overall, a higher expression of Rnd3 in cancer cells is frequently observed and it is clear that Rnd3 gene expression is modulated in response to a variety of different signals.

Rnds in Development Several proteins of the Rho family, as well as their regulators, are involved in developmental process such as dorsal closure or gastrulation and have also been involved in the establishment of cell polarity. Wünnenberg-Stapleton et al37 (1999) have studied the role of RhoA and Rnd1 in Xenopus development. XRnd1 was originally isolated from a cDNA library enriched in ventral mRNAs, and screened for inhibitors of secondary axis formation induced by a dominant negative receptor for the ventralizing factor BMP. However, XRnd1 does not seem to be involved in BMP signalling since it does not ventralize embryos when injected into the dorsal side. XRnd1 is expressed predominantly in tissues undergoing extensive morphogenic movements and reduces cell adhesion, whereas activated XRhoA has an opposite effect, increasing cell adhesion. In addition, XRnd1 is also mostly associated with the plasma membrane.37 Guasch et al,2 have also observed that Rnd3 increases MDCK cell migration in response to HGF (Scatter factor). In contrast, activated RhoA expression in MDCK cells leads to an increase in basal stress fibers that impairs locomotion, thus in epithelial cells too Rnd3 and RhoA have antagonistic effects. In testis, the differentiation of germ cells and maturation of spermatids is tightly coupled to their migration between the sertoli cells epithelium in seminiferous tubules. At places of germ cell/sertoli cell contacts, the rearrangements of cell junctions and associated cytoskeletal elements in sertoli cells controls germ cells differentiation. The expression of Rnd2 could contribute to the control of these rearrangements of cellular junctions. Rnd2 interacts and co-localizes with MgcRacGAP in male germ cells38 (spermatocytes and early spermatids) where it most likely controls the RhoGAP activity of MgcRacGAP.

Perspectives Expression of Rnd1, which is probably constitutively in the GTP form, inhibits the formation of actin stress fibers. These dramatic effects of Rnd1 on stress fiber formation could be due to the high level of expression, whereas at physiological levels the effects of Rnd1 on the actin cytoskeleton might be localized to more specific intracellular regions. Several groups have reported that the Rho antagonist effects of Rnds are due to the stimulation of RhoGAPs and inhibition of the Rho effector Rock. The group of Anne Ridley found an interaction of Rnd3/ RhoE with Rock, an observation that seemed to contradict ours, that Rnd3 does not bind the Rho Binding Domain (RBD) of Rock in the two-hybrid assay. In fact, it appears that Rnd3 does bind Rock, not in the RBD, but close to the kinase domain, inhibiting kinase activity.39 In addition, Steen Hansen’s group has shown that Rnd3 interacts with p190 RhoGAP, not in the GAP domain itself, as first assumed, but in the central domain of previously unknown structure and function. Membrane-associated Rnd3 most likely recruits p190-RhoGAP to the membrane where it can act on its substrate: membrane-associated Rho-GTP. At high levels of expression this leads to a massive inhibition of stress fiber formation, but in more physiological conditions it is likely that Rnds act at discrete places, such as adherens junctions, where other actin structures are formed. A protein named Socius is also proposed to participate in Rnd effects on the actin cytoskeleton,41 but at present it is not clear how it fits in the general picture. Since Socius is mostly expressed in testis, as Rnd2, they may interact to affect the cytoskeleton

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in male germ cells. It has recently been shown that Cadherins, the main components of adherens junctions, recruit p190-RhoGAP to inactivate Rho.42 Since Rnd1 and 3 co-localize with cadherins,1 and interact with p190-RhoGAP,40 it is tempting to think that they are involved in p190-RhoGAP recruitment on cadherins, and local Rho inhibition. Until now, little was known on RhoGAPs regulation. These recent discoveries emphasize their importance and open new perspectives, suggesting other complex cross-talks between different small G proteins. Several recent results indicate that the membrane micro-domains where small GTP binding proteins get activated might be essential for their functions. Cdc42, Rac and Rho control dynamic rearrangements of the cytoskeleton that can occur in the time-scale of minutes. They can be rapidly activated by exchange factors and inactivated by GAPs, both being precisely localized and tightly coupled to regulatory signals. We suggest that Rnd proteins, which may constitutively be in the GTP-bound form, are regulated on a longer time-scale (hours). Their expression appears to be restricted to a few specialized cells in the adult. In these cells, Rnd proteins could be used to induce a local inhibition of actin stress fiber formation, at places of cell-cell contacts, for axon guidance and growth cone extension in neurons, for cell migration, or during cytokinesis.

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20. Lee H, Woodman SE, Engelman JA et al. Palmitoylation of caveolin-1 at a single site (Cys-156) controls its coupling to the c-Src tyrosine kinase. J Biol Chem 2001. in press. 21. Aoki J, Katoh H, Mori K et al. Rnd1, a novel rho family GTPase, induces the formation of neuritic processes in PC12 cells. Biochem Biophys Res Commun 2000; 278:604-8. 22. Rohm B, Rahim B, Kleiber B et al. The semaphorin 3A receptor may directly regulate the activity of small GTPases. FEBS Lett 2000; 486:68-72. 23. Qiu R-G, Chen J, Kirn D et al. An essential role for rac in Ras transformation. Nature 1995; 374:457-459. 24. Qiu R-G, Chen J, McCormick F et al. A role for Rho in Ras transformation. P N A S 1995; 92:11781-11785. 25. Olson MF, Ashworth A, Hall A. An essential role for Rho, rac, and Cdc42 GTPases in cell cycle progression through G1. Science 1995; 269:1270-1272. 26. Habets GG, Scholtes EH, Zuydgeest D et al. Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins. Cell 1994; 77:537-49. 27. Symons, M. Rho family GTPases: The cytoskeleton and beyond. T I B S 1996; 21:178-181. 28. Bar-Sagi D, Feramisco JR. Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by Ras proteins. Science 1986; 233:1061-1068. 29. Ridley AJ, Hall A. The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992; 70:389-399. 30. Ridley AJ, Patterson HF, Johnston CL et al. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992; 70:401-410. 31. Hughes PE et al. Suppression of integrin activation: A novel function of a Ras/raf-initiated MAP Kinase pathway. Cell 1997; 88:521-530. 32. Sahai E, Olson MF, Marshall CJ. Cross-talk between Ras and Rho signalling pathways in transformation favours proliferation and increased motility. EMBO J 2001; 20:755-66. 33. Hansen SH, Zegers MM, Woodrow M et al. Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway. Mol Cell Biol 2000; 20:9364-75. 34. Gress TM, Muller-Pillasch F, Geng M et al. A pancreatic cancer-specific expression profile. Oncogene 1996; 13:1819-30. 35. Camerer E, Gjernes E, Wiiger M et al. Binding of factor VIIa to tissue factor on keratinocytes induces gene expression. J Biol Chem 2000; 275:6580-5. 36. Akashi H, Han HJ, Iizaka M et al. Growth-suppressive effect of non-steroidal anti-inflammatory drugs on 11 colon-cancer cell lines and fluorescence differential display of genes whose expression is influenced by sulindac. Int J Cancer 2000; 88:873-80. 37. Wunnenberg-Stapleton K, Blitz IL, Hashimoto C et al. Involvement of the small GTPases XRhoA and XRnd1 in cell adhesion and head formation in early Xenopus development. Development 1999; 126:5339-51. 38. Naud N, Toure A, Liu J et al. Rho family GTPase Rnd2 interacts and co-localizes with MgcRacGAP in male germ cells. Biochem J 2003; 372(Pt 1):105-112. 39. Riento K, Guasch RM, Garg R et al. RhoE binds to ROCK I and inhibits downstream signalling. Mol Cell Biol 2003; 23(12):4219-4229. 40. Wennerberg K, Forget M-A, Ellerbroek S et al. Rnd proteins function as RhoA antagonists by activating p190-RhoGAP. Current Biol 2003; 13:1106-1115. 41. Katoh H, Harada A, Mori K et al. Socius is a novel Rnd GTPase-interacting protein involved in disassembly of actin stress fibers. Mol Cell Biol 2002; 22(9):2952-2964. 42. Noren NK, Arthur WT, Burridge K. Cadherin engagement inhibits RhoA via p190RhoGAP. J Biol Chem 2003; 278(16):13615-13618.

CHAPTER 7

Rho GTPases in the Organization of the Actin Cytoskeleton Ed Manser

Abstract

C

ells change their shape by altering their internal cytoskeleton. It has become apparent that in all eukaryotes Rho-family GTPases orchestrate changes to the actin cytoskeleton. In the last few years we have gained important insights into how these GTPases carry such processes via a variety of downstream ‘effectors’. These target proteins have a range of functions which can be under the direct control of the GTPase.

Introduction All organisms have cellular machinery dedicated to modulating cell shape, which is important in many biological processes, particularly for development, in the immune system and for plasticity in the central nervous system. Actin is an ancient protein that is able to form filaments that can be organized into a variety of structures, providing shape and allowing force generation within the cell. This abundant eukaryotic proteins, is a cytoskeletal component that assembles into paired filaments, and is conserved from yeast to humans.1 The ~43 kDa protein has an ATP binding domain with intrinsic ATPase activity and assembles primarily at the ‘barbed’ or ‘plus’ end of filaments when charged with ATP. This subsequently undergoes hydrolysis within the filament producing a phenomenon known as treadmilling, whereby monomers apparently move through the filament at steady-state. Proteins such as profilin and cofilin bind to monomeric or globular (G) actin, while many others actin binding proteins interact preferentially with filamentous (F) actin. Just recently proteins distantly related to actin have been discovered in prokaryotes such as Bacillus subtilis2 where they also regulate cell shape. Crystallization of the one such bacterial protein (MreB) indeed revealed a tertiary structure highly related to that of mammalian actin.3 Many dynamic actin structures are organized by a Rho family GTP-binding proteins. It is ten years since Alan Hall and colleagues first described4 how active versions of two such proteins, Rac1 and RhoA, can assemble distinct actin arrays in mammalian cells.5 Significantly, these studies found that external signals acting on transmembrane cell-surface receptors use these GTPases to initiate such changes in the actin cytoskeleton. Specifically, Rac1, acting downstream of receptor tyrosine kinases (RTKs), generates peripheral extensions known as membrane ruffles or lamellipodia while RhoA assembles more internally located actin stress fibres and associated adhesion complexes. The related GTPase called Cdc42, first described in budding yeast, was found to organize peripheral actin structures known as filopodia in mammalian cells.6,7 Work from many laboratories has since established that the Rho-GTPases family including Rho, Rac and Cdc42, alter the cytoskeleton by recruiting protein effectors. These, in turn, act in cascades that are responsible for changes to the actin cytoskeleton providing the basis for Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Table 1. Effector proteins involved in actin organization Effector

GTPase Selectivity

Protein Type

Target

mDia1/2

Rho

adaptor

MRCK (α,β)

Cdc42

S/T kinase

MBS

PAK (α,β,γ)

Cdc42 Rac

S/T kinase

PAK4

Cdc42

S/T kinase

LIM-K MLCK LIM-K

PIP5kinase

Rac Rho

Lipid kinase

PIP2

ROK (α,β)

Rho

S/T kinase

N-WASP IRS-p53/58

Cdc42 Cdc42 Rac

adaptor adaptor

MBS vimentin Arp2/3 WAVE

Cytoskeletal Effect actin polymerization/ microtubule organization actin/myosin II/ focal adhesions actin polymerization/ inhibits actin/myosin II actin polymerization actin polymerization/ focal adhesions actin/myosin II IFs disassembly actin polymerization actin polymerization

both cell shape and adhesion. Thus, many signals that impinge on cells act via a common set of Rho switches. Here I discuss the established mechanisms by which three well-characterized GTPases — Rac, Cdc42 and Rho — use a variety of ‘effectors’ to alter the actin cytoskeleton. Lessons learned about these pathways tell us how cells coordinate complex internal reorganisation, as required for processes such as cell movement.

Different Actin Networks: Membrane Ruffles, Filopodia and Stress Fibres Reorganization of the actin cytoskeleton is most easily visualized in cultured cells responding to soluble factors or extracellular matrices, undergoing progression through the cell-cycle, or during invasion by pathogens. These systems have provided models which are the basis of most of our understanding of actin dynamics. Many studies have pointed to Rac playing a critical role in cell migration,8 a property of many cultured cells that can be directly observed and quantified. In migrating cells, the forward ‘leading’ edge consists of protruding flat lamellipodia in which there exists a dense array of actin. These filaments are orientated with their plus ends pointing towards the plasma membrane.9 The actin elements provide a ‘pushing’ force to expand the plasma membrane at the leading edge of the cell or, in combination with myosin, provide contractile ‘pulling’ forces between different cell attachment sites. Recent real-time imaging techniques have allowed the production of active Rac.GTP by growth factors to be visualized directly to the leading edge,10 confirming that the GTPase is activated in a polarized fashion. Cdc42 drives formation of filopodia and is important for cells to orientate correctly towards a chemotactic gradient11 or for fibroblasts to align themselves during wound healing.12 Recent evidence suggests that this involves organization of the microtubule network.13 Rho-dependent actin stress fibres are responsible for the contractile forces generated in cultured cells, both within leading-edge structures14 and between integrin-containing focal-adhesion complexes (FCs), particularly in the retracting ‘tail’ of migrating cells.15 It is in these central and rear areas that Rho promotes the formation of actin stress fibres. Rho, acting through its effector Rho kinase (ROK), plays a key role in producing these structures by organizing myosin II16 in combination with other Rho effectors (see next section). Several types of protein are needed to organize and regulate the actin cytoskeleton. Key among these are pro-

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Figure 1. Pathways linking Rho GTPase effectors to actin/myosin II assembly. Phosphorylation events that have inhibitory effects are marked (-). Rho regulation of actin organization occurs through multiple effectors. The Rho-associated kinase (ROK) blocks the protein phosphatase delta (PP1δ), by phosphorylating the myosin binding subunit (MBS) p85 or p130 subunits. This prevents inactivation of phosphorylated MLC~P assembled into actin stress fibres. Myosin II driven assembly of stress fibres favours the formation of focal adhesion complexes. Myosin light chain kinase (MLCK) is a key enzyme for maintaining the myosin heavy–light chain complex in an active state, but is negatively regulated by Rac via the p21-activated kinase (PAK). Cdc42 regulates myotonin-related Cdc42-binding kinase (MRCK) and PAKs, which act via LIM-kinases (LIMKs) to inactivate cofilin. This stabilizes actin filaments because cofilin serves to accelerate actin dissociation.

teins that promote actin-filament assembly, treadmilling, crosslinking, branching, severing and linkage of actin filaments to the cell membrane. We now have some understanding of how a number of Rho effectors couple to these actin-related processes. The importance of Rho GTPases is exemplified by the way in which microbial pathogens have developed specific proteins that interact with Rho GTPases to turn them on or off, thus allowing pathogens to subvert actin function. This fascinating subject has recently been reviewed in some detail.17

Actin–Myosin II Contractility and Stress Fibre Formation In nonmuscle cells, actin is organized into functional motor units analogous to those found in muscle. This involves the assembly of actin filaments into higher-order structures bridged by myosin II. Such structures are readily observable as the actin stress fibres, which are often seen

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Figure 2. Pathways linking Rho GTPase effectors to Arp2/3 mediated actin assembly. At peripheral locations Cdc42 interaction with N-WASP, or Rac1 interaction with IRS-p53/WAVE can recruit and activate the Arp2/3 complex. This protein complex thereby increases the number of actin filaments through branching onto existing filaments. The N-WASP can also be activated by PIP2, or through interactions of SH3 domain containing proteins such as WIP and Nck. PtdIns(4,5)P2 also binds to and activates N-WASP. It is suggested from dominant interfering experiments that N-WASP is required for actin assembly underlying filopodia formation: data using N-WASP knockout cells is required to confirm these findings. The IRS-p53/WAVE complex can also recruit the Arp2/3 complex to drive net actin polymerization - Arp2/3 is a key component of lamellipodia.

in cultured cells stained with phalloidin, and assemble under the control of Rho.4 Myosin II is primarily under the control of the calcium-dependent myosin light chain kinase (MLCK), whose activity is absolutely required to maintain stress fibres, however the kinase also functions at the leading edge of cells. For example in motile eosinophils, photolysis of caged inhibitory peptides directed to either calmodulin or MLCK results in a rapid block of forward motion of the leading lamellipod.18 The serine/threonine kinases ROK and the myotonin-related Cdc42-binding kinase (MRCK) cause MLC phosphorylation, but not by affecting MLCK. Rather these promote maintenance of the phosphorylated state of myosin light chain (P~MLC) (Fig. 1) by blocking the action of the key phosphatase in the cycle.4 Both ROK and MRCK target a single inhibitory threonine phosphorylation site on the p8519 or p13020 myosin-binding subunit (MBS) of protein phosphatase 1δ. This MBS phosphorylation directly blocks the activity of the phosphatase and causes acccumulation of P~MLC. Such activity occurs in the context of other Rho effectors, for example the mammalian Diaphanous (mDia) a formin which can generate F-actin in combination with the action of ROK.21

Controlling Actin Treadmilling The maximal forward movement of the leading edge of mammalian cells is of the order of 5–10 µm per minute. In vitro studies of the properties of actin have shown that disassembly is rate limiting for actin to cycle quickly enough to drive such rates of cell movement, therefore in vivo proteins must accelerate the intrinsic rate. ADF/cofilin fulfils this role (Fig. 1), by stimulating ‘treadmilling’ of actin.22 This increases net actin turnover in the cell and net loss of F- actin. However, cofilin is under tight negative regulation by LIM kinases phophorylation. While expressing a cofilin mutant that cannot be inactivated (S3A) does not block formation of filopodia, lamellipodia or actin stress fibres,23 cofilin inactivation facilitates the dynamics of assembly/disassembly. LIM-kinase is regulated by ROK16,24 and this is in part why Rho promotes net actin assembly (Fig. 1). LIM-kinase 1 is

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also reported to be activated by the serine/threonine p21-activated kinase PAK1/αPAK,25 one of the first Cdc42/Rac effectors to be identified.26 It has recently been shown27 that the Cdc42 effector PAK4 is a potent in vivo activator of LIM-kinase 1.

Arp2/3-Mediated Actin Nucleation and Branching One of the key findings of the last few years is that Wiscott-Aldrich syndrome protein (WASP) and its ubiquitously expressed homologue N-WASP are important actin organizers downstream of Cdc42.28,29 Use of dominant-inhibitory constructs of N-WASP suggest it is required for the formation of filopodia, although Arp2/3 is not actually present in these structures.30 Structural studies have revealed how binding of Cdc42 releases the auto-inhibitory region from the WASP carboxy-terminal region31 (referred to as the VCA domain), allowing it to activate a multi-protein component called the Arp2/3 complex (Fig. 2). The VCA region of WASP contains a sequence homologous to the ActA protein of the pathogenic bacterium Listeria monocytogenes, which also recruits Arp2/3 to drive actin-based bacterial movement. The Arp2/3 complex is capable of branching actin filaments and thereby rapidly increasing the number of ends. N-WASP can apparently be recruited for this purpose by proteins other than Cdc42. For example, the enteropathogenic E. coli Tir protein binds the adaptor Nck, which in turn recruits N-WASP to assemble a pedestal below the bacterium.32 In lamellipodia, electron-microscopic analysis of the actin network has revealed an extensive branched network of actin filaments, where the barbed ends face the leading edge.9 Here, 70 Y junctions between actin filaments contain Arp2 and Arp3 in addition to five other subunits: this complex is enriched at the leading edge of crawling cells.33 Many studies have indicated the nucleation and crosslinking of actin filaments provides a force to push against the leading-edge membrane. But what organizes this behaviour downstream of Rac? Truncated versions of a WASP-related protein (WASP family Verprolin homologous), WAVE, can block formation of Rac-induced lamellipodia but not Cdc42-induced filopodia.34 WAVE has the same type of VCA domains as WASP, but does not seem to be regulated by an analogous interaction with Rac.35 Instead it might be brought into play through additional signalling proteins, such as the binding partner insulin receptor substrate protein IRSp53/58 (Fig. 2). IRSp53/58 is reported to bind Rac.GTP via an amino-terminal domain,36 and this adaptor clearly contains a distinct central Cdc42.GTP binding site resembling that in PAK and WASP.37 Thus Rac is which potently assembles leading edge actin, is biochemically linked to actin assembly—however why both Rac1 and Cdc42 bind the IRSp53 protein remains to be resolved

Stimulating Actin Assembly The polymerization of actin is controlled by actin-binding proteins that either promote new filament nucleation or stimulate elongation of existing structures. The role of Arps in creating new ends has been discussed. A different player is the actin-binding protein profilin, which can stimulate the rate of actin addition from sequestered pools to the barbed end of the filament.38 Profilin’s name derives from its affinity for proline-rich sequences, which are a feature of profilin-binding proteins such as vasodilator-stimulated phosphoprotein (VASP) and formins. Profilin can be recruited by bacterial pathogens that use actin polymerization for movement, indicating its central role in actin-based motility.29 Rho effectors are indeed linked to profilin: profilin is recruited by the Cdc42 effector WASP. Significantly, mutants of profilin that do not bind actin, block filopodia formation downstream of Cdc4239 and can suppresses N-WASP-induced actin reorganization in cells.34 The WAVE similarly requires profilin to promote the formation of lamellipodia.40 The ability of profilin to stimulate actin assembly is augmented by the binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) to profilin to release actin from the complex. Levels of cellular PtdIns(4,5)P2 are elevated by PtdIns 4-phosphate 5-kinases (PIPKs), which associate with both Rho and Rac41 and are stimulated by ROK.42 Thus, elevating the levels of PtdIns(4,5)P2 causes extensive actin polymerisation.43

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Tethering Assembled Actin to Membranes In most cell types the plasma membrane is intimately associated with the underlying actin cytoskeleton. Rho GTPases, which coordinate changes in the plasma-membrane shape, are ideally placed to modulate this attachment - via ERM proteins. This acronym derives from the first three members to be identified - ezrin, radaxin and moesin, which all have an amino-terminal membrane-associated domain and a carboxy-terminal actin-binding region. Such proteins are known to be associated with plasma membrane structures such as microvilli, filopodia and membrane ruffles,44 but require phosphorylation in the C-terminal domain to become active.45 Activation of RhoA leads to phosphorylation of ERMs on a conserved regulatory threonine residue.45 Activated Cdc42 also drives such phosphorylation of ERMs but at the tips of filopodia, via the ROK-like kinase MRCK.46 Although ROK has been implicated in this process,47,48 other in vivo experiments showed that phosphorylation of ERMs is not suppressed by the ROK inhibitor Y-27632. Rather overexpression of the lipid kinase effector PIPKα, is capable of increasing levels of phosphorylated ERM sixfold and concomitant formation of microvilli.49 A lipid regulated protein kinase might mediate such ERM phosphorylation. Interestingly a nonphosphorylatable mutant of ezrin has been reported to block actin contractility, focal-adhesion formation and transformation downstream of Rho.50

The Focal Adhesion Complex-Actin Connection Introduction of active Cdc42, Rac and Rho drives the formation of integrin-containing focal complexes (FCs) in serum-starved fibroblasts.6 Formation of FCs is reinforced by intracellular tension between the integrins and underlying actin cytoskeleton, and is very sensitive to myosin II inhibitors.51 To sustain the structure of FCs in turn requires components that link actin filaments to the integrin complex, including vinculin, talin and α-actinin. Vinculin is directly regulated by PtdIns(4,5)P2, which disrupts intramolecular interactions to allow binding to talin and α-actinin52—key interactions in the assembly of FCs. Thus, injection of antibodies to PtdIns(4,5)P2 blocks the formation of FCs.52 Rho, ROK53 and Rac42 are known to increase cellular levels of PtdIns(4,5)P2 through the action of PIPKs. This promotes protein components to assemble with integrins into bona fide FCs. In spite of the close connection between Rho GTPases and FCs, the only identified effector that has been localized to FCs is PAK.54 Because assembly of new FCs leads to activation of Rac and Cdc42, PAK is probably locally activated in these fledgling structures.55 Much work is yet required to resolve the various connections between integrin containing complexes and Rho GTPases. This will continue to be a key area of focus because of the importance of integrin signalling for development, organogenesis and metastasis.

Conclusions and Future Directions We are beginning to build a more detailed picture of how Rho GTPases regulate the actin cytoskeleton, but much of our knowledge is derived from work using cultured cells. It will be a challenge to understand how these processes are coordinated in whole organisms. As we have seen, the pathways that control the actin cytoskeleton downstream of Rho GTPases are mainly controlled by phosphorylation but identification of their targets in vivo continues to remain a challenge. New methodologies such as sensitive mass spectrometry are anticipated to speed up the identification of protein modifications resulting from Rho signalling.

Acknowledgement The author is supported by the Glaxo-IMCB Research Fund.

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29. Cameron LA, Giardini PA, Soo FS et al. Secrets of actin-based motility revealed by a bacterial pathogen. Nature Reviews Mol Cell Biol 2001; 1:110-120. 30. Castellano F, Montcourrier P, Guillemot JC et al. Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation. Curr Biol 1999; 9:351-360. 31. Kim AS, Kakalis LT, Abdul-Manan N et al. Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein. Nature 2000; 404:151-8. 32. Gruenheid S, DeVinney R, Bladt F et al. Enteropathogenic E. coli Tir binds Nck to initiate actin pedestal formation in host cells. Nat Cell Biol 2001; 3:856-859. 33. Machesky LM, Gould KL. The Arp2/3 complex: A multifunctional actin organizer. Curr Opin Cell Biol 1999; 11:117-121. 34. Miki H, Sasaki T, Takai Y et al. Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP. Nature 1998; 391:93-96. 35. Rohatgi R, Ma L, Miki H et al. The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 1999; 97:221-231. 36. Miki H, Yamaguchi H, Suetsugu S et al. IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature 2000; 408:732-735. 37. Govind S, Kozma R, Monfries C et al. Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin. J Cell Biol 2001; 152:579-594. 38. Didry D, Carlier MF, Pantaloni D. Synergy between actin depolymerizing factor/cofilin and profilin in increasing actin filament turnover. J Biol Chem 1998; 273:25602-25611. 39. Suetsugu S, Miki H, Takenawa T. Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex. Biochem Biophys Res Commun 1999; 260:296-302. 40. Sasaki N, Miki H, Takenawa T. Arp2/3 complex-independent actin regulatory function of WAVE. Biochem Biophys Res Commun 2000; 272:386-390. 41. Ren XD, Bokoch GM, Traynor-Kaplan A et al. Physical association of the small GTPase rho with a 68-kDa phosphatidylinositol 4-phosphate 5-kinase in Swiss 3T3 cells. Mol Biol Cell 1996; 7:435-442. 42. Tolias KF, Hartwig JH, Ishihara H et al. Type Ialpha phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly. Curr Biol 2000; 10:153-156. 43. Shibasaki Y, Ishihara H, Kizuki N et al. Massive actin polymerization induced by phosphatidylinositol4-phosphate 5-kinase in vivo. J Biol Chem 1997; 272:7578-7581. 44. Bretscher A. Regulation of cortical structure by the ezrin-radixin-moesin protein family. Curr Opin Cell Biol 1999; 11:109-116. 45. Hirao M, Sato N, Kondo T et al. Regulation mechanism of ERM (ezrin/radixin/moesin) protein/ plasma membrane association: Possible involvement of phosphatidylinositol turnover and Rho-dependent signalling pathway. J Cell Biol 1996; 135:37-51. 46. Nakamura N, Oshiro N, Fukata Y et al. Phosphorylation of ERM proteins at filopodia induced by Cdc42. Genes Cells 2000; 5:571-581. 47. Matsui T, Maeda M, Doi Y et al. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/ radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol 1998; 140:647-657. 48. Shaw RJ, Henry M, Solomon F et al. RhoA-dependent phosphorylation and relocalization of ERM proteins into apical membrane/actin protrusions in fibroblasts. Mol Biol Cell 1998; 9:403-419. 49. Matsui T, Yonemura S, Tsukita S et al. Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases. Curr Biol 1999; 9:1259-1262. 50. Tran Quang C, Gautreau A, Arpin M et al. Ezrin function is required for ROCK-mediated fibroblast transformation by the net and dbl oncogenes. EMBO J 2000; 19:4565-4576. 51. Small JV, Rottner K, Kaverina I. Functional design in the actin cytoskeleton. Curr Opin Cell Biol 1999; 11:54-60. 52. Gilmore AP, Burridge K. Regulation of vinculin binding to talin and actin by phosphatidyl-inositol-4 -5-bisphosphate. Nature 1996; 381:531-535. 53. Oude Weernink PA, Schulte P, Guo Y et al. Stimulation of phosphatidylinositol-4-phosphate 5-kinase by Rho-kinase. J Biol Chem 2000; 275:10168-10174. 54. Manser E, Huang HY, Loo TH et al. Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes. Mol Cell Biol 1997; 17:1129-1143. 55. Zhao ZS, Manser E, Loo TH et al. Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complex disassembly. Mol Cell Biol 2000; 20:6354-6363.

CHAPTER 8

Cell-Cell Adhesion Vania M.M. Braga and Martha Betson

Introduction

C

ell-cell adhesion is an essential process for tissue architecture. During development and differentiation, intercellular adhesion provides a selective way for choosing like-neighbours and spatial cues for cell positioning within a tissue. In addition, by modulating the type and the surface levels of cell-cell adhesion receptors, cells can change not only their morphology but also their phenotypic properties (differentiation status, gene expression profile, proliferation and migration rates). As the above cellular processes are perturbed during tumorigenesis, much effort has been put into understanding how cell-cell contacts are regulated and how they can affect so many diverse signalling events. The role of cell-cell adhesion is particularly relevant for epithelium function. An epithelium is formed by a sheet of cells tightly attached to each other. The major functions are in secretion/ absorption and as a protective barrier. Although cell-cell adhesion is essential for the differentiation of epithelia, distinct epithelial types (simple or stratified) differ somewhat in the adhesive structures present and in the distribution of proteins at junctions (see below). In this chapter, we discuss the major cell-cell adhesive system found in epithelia (adherens junctions), its interaction with the cytoskeleton and the signalling pathways that have been implicated on its functional regulation.

Characteristics of Epithelial Cells Adhesive Structures The epithelial junction is a complex cellular structure cosisting of different adhesive components: adherens junctions, desmosomes and tight junctions. Adherens junctions and desmosomes contain members of the cadherin superfamily of transmembrane proteins, which mediate calcium-dependent cell-cell adhesion. Classical cadherins form the adherens junctions and are indirectly connected to the actin cytoskeleton. Other cadherin-family members, desmogleins and desmocollins, form the desmosomes and interact with the intermediate cytoskeleton (keratins) via a cytosolic plaque containing different proteins.1 The tight junction transmembrane proteins are claudins and occludins that, similarly to cadherins in adherens junctions, are indirectly connected to actin filaments. Adherens junctions and desmosomes give strength to the epithelial junction and their association with the cytoskeleton provides resistance to tension and friction. Tight junction formation yields impermeability to ions and solutes across the epithelial sheet, virtually sealing the space in between adjacent cells (paracellular permeability).

Epithelial Phenotype and Cytoskeleton Epithelial cells are characterised by a cuboidal morphology and differential distribution of surface and cytosolic proteins into specific domains (Fig. 1). This is known as the polarised Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Figure 1. Cell shape and cytoskeletal organization of epithelial and fibroblast cells. Actin bundles are shown as red lines; cadherin receptors are represented as green dots at the membrane. The main differences between these epithelia and fibroblasts are on the cell shape (flat versus tall; irregular versus cobblestone, respectively) and cytoskeletal organization (stress fibers versus circumferential bundles of actin). Distinct organization of the cytoskeleton is found in simple epithelial cell such as MDCK. Polarised MDCK cells have stress fibers at the base; adherens junctions and the circumferential actin ring are present as a single spot at the apex of the lateral domain.

epithelial phenotype and a cooperation between cell-cell adhesion and remodelling of the cytoskeleton is necessary to achieve it. Adherens junctions provide points of attachment for the actin cytoskeleton at the lateral domain. From these points, the actin cytoskeleton can generate contraction/tension to create the cuboidal cell morphology. This rearrangement programmes the sorting machinery to redirect membrane proteins to different domains (apical, basal or lateral), promoting the recruitment to the junctions of cytosolic and signalling proteins. The presence of tight junctions in simple epithelia prevents the mixing of proteins between apical and baso-lateral domains. The end point is the formation of dynamic structures across the whole length of the adhesive lateral domain, from which external clues are transmitted via the signalling proteins nearby and the cytoskeleton. The association with the cytoskeletal network is essential for the assembly and function of the junctional adhesive structures. Mutation or deletion of the junctional proteins that mediate the interaction with the actin cytoskeleton or intermediate filaments completely abolishes the appropriate assembly of adherens junctions, tight junctions and desmosomes, respectively (reviewed by ref. 2). The cytoskeleton organization in polarised epithelial cells is distinct from the one found in fibroblasts (Fig. 1),3,4 with differences in the composition and organization of actin bundles between these two cell types. In fibroblasts, filamentous actin is organised into thick bundles known as stress fibers. Stress fibers originate from points of attachment to the extracellular matrix and can traverse the whole length of a fibroblast cell. Epithelial cell lines grown in vitro show stress fibers at the base of the cell (i.e., MDCK cells derived from kidney epithelia). In

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contrast, in primary/normal epithelia, stress fibers are not prominent. Instead, thin actin cables can traverse the epithelial cell body, but the majority of actin cables are found in circumferential bundles adjacent to cell-cell junctions.5 Because of the functional interaction between adhesive receptors and the cytoskeletal network, understanding how this connection is established and maintained may provide insights into how cell-cell adhesion is regulated.

Cadherin Receptors Cadherins and Catenins Classical cadherin receptors found in epithelial tissues are E- and P-cadherin. Additional family members may be found to be expressed in epithelial cells, as new cadherin types are investigated in epithelia. However, E-cadherin seems to be the most important receptor for epithelia differentiation. First, while P-cadherin knockout mice are viable and fertile, embryos depleted of E-cadherin gene die very early (embryonic day E6.5), as the first epithelium, the trophectoderm, is not properly formed.6,7 Second, expression of E-cadherin gene in fibroblasts and tumour cell lines is sufficient to restore the cuboidal morphology and asymmetrical distribution of proteins typical of epithelial cells.8 Cadherin receptors interact with similar type of molecules from adjacent cells (homophilic binding). At the cytoplasmic side, the cadherin tail associates directly with proteins of the armadillo family (β-catenin, plakoglobin, δ-catenin, p120ctn, reviewed by ref. 9). Some of these proteins (β-catenin and plakoglobin) serve as a docking site for α-catenin, which can bind to and bundle actin filaments.10 These three components (cadherin, α-catenin and β-catenin) provide the core and essential unit of the cadherin complexes. Similar to the E-cadherin knockout phenotype, depletion of β-catenin in vivo is lethal due to impairment in epithelial differentiation.11 The catenins δ-catenin, ARVCF and p120ctn differ from the other armadillo family members, as they bind to a distinct region in the cadherin cytoplasmic tail.12-14 The role of δ-catenin in cadherin adhesion is less clear than for β-catenin as it seems to destabilise adhesive complexes.13 In addition, reports have described either a positive or negative role for p120ctn on cadherin-dependent adhesion.12,15-17 Most probably these discrepancies reflect the cellular context, the presence of multiple p120ctn isoforms and the modulation of p120ctn by phosphorylation (reviewed by ref. 18). Thus, while the roles that α-catenin, β-catenin and plakoglobin play on cadherin function are established, it has been more challenging to pin point the functional contribution of the other catenins.

Proposed Model for Cadherin-Dependent Adhesion Formation of cell-cell contacts is a dynamic process that results in complex changes both locally at the site of adhesion as well as in the overall cell morphology. Cadherin-mediated cell-cell adhesion is formed by sequential steps of increasing levels of complexity. While there is a predicted temporal sequence of events, in a given cell the different steps can overlap with each other considerably, as cell-cell contact formation does not occur synchronously along the lateral domain. Although the key steps in the cadherin adhesion have been known for the past fifteen years, the mechanisms via which they take place are still not completely understood. These steps are discussed below.

Homophilic Binding Cadherin receptors are found at the surface as lateral or cis dimers, which form the structural unit of the adhesive complexes (Fig. 2).19-24 Lateral dimerization is mediated by the extracellular domain (containing 5 calcium-binding repeats), and its stability is independent of the association with calcium ions.21,25 In the presence of calcium ions, the extracellular domain is stabilised as an elongated, rigid conformation, allowing the interaction with the same type of molecules on opposing cells (homophilic binding; Fig. 2).22,24 Although the formation of lat-

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Figure 2. Homophilic binding, clustering and puncta formation after induction of cell-cell adhesion. (a) Homophilic binding. Cadherin molecules have 5 extracellular calcium-binding repeats and are found as lateral dimers at the surface. Association with β- and α-catenin form the cadherin complex. As calcium ions interact with the extracellular domain, it is rearranged in a rod-like structure and receptors become adhesive. These dimers interact with similar dimers from neighbouring cells, in a process known as homophilic binding. Two models have been proposed for how homophilic binding takes place. Model 1 predicts that only the first repeat of cadherin receptors interact with each the apposing dimers. Model 2 argues for a more extensive overlap of all 5 extracellular repeats. (b) Apposing membranes from two neighbour cells are shown with cadherin dimers represented in green. After homophilic binding, cadherins progressively cluster at sites of cell-cell contacts. These clusters are seen at the microscopic level as distinct punctum. As clustering progresses, each punctum is enlarged until a complete line of adhesive cadherin dimers is found at cell-cell contacts.

eral dimers is important for the adhesive interaction of cadherin receptors, it is not known whether these dimers can form before reaching the cell surface or if they are regulated in any way. Two models have been proposed for how the homophilic binding occurs (Fig. 2a). The two models differ in the amount of overlap between the dimers engaged in adhesion, and there is supportive experimental evidence for both models. One model predicts that only the first extracellular repeat of each dimer overlap with each other.20,22 This model is consistent with the identification of cadherin specificity domain at the first repeat.26 and the mapping of epitopes for inhibitory antibodies to this region.26,27 The second model proposes that a more extensive overlap may occur, with the participation of 3 to 5 extracellular repeats.28 The later model suggests that cadherin adhesiveness results from multivalent low affinity interactions between cadherin dimers. It is supported by (a) mutations of E-cadherin gene known to affect adhesion that do not lie in the first N-terminal repeat;29,30 (b) inhibitory antibodies binding to the repeat 5;30 (c) biophysical measurements of force between the adhesive dimers;31 and (d) direct in vitro binding of different combinations of the cadherin repeats.28

Clustering After the homophilic binding, the interacting receptors cluster at sites of cell-cell contacts, originating what are known as “puncta”.32,33 These structures are visible by immunofluorescence very quickly after induction of cell-cell contacts.33,34 Scattered along the lateral domain,

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multiple puncta enlarge as more receptors are engaged in adhesion and they then coalesce to form a contiguous line of E-cadherin staining at the lateral domain to form the junctions (Fig. 2B). It is not clear how the clustering events take place nor how pucta are formed and enlarged. Lateral mobility of the receptors in the plane of the membrane as well as recruitment of intracellular pools is thought to play a role. Because these structures are not stable if the actin cytoskeleton is disrupted, actin filament reorganization/polymerisation may participate in the process.33,35 In addition, myosin-dependent contractility also may play a role in the clustering process, as different myosin isoforms do localise at the junctions in epithelial cells.36-38 Yet, a functional link between the myosin localization and cadherin adhesion has not been addressed experimentally. It has been proposed that cells make transient contacts prior to stable cell-cell attachment using “finger-like” projections at their membrane.39-41 Upon cadherin homophilic binding, these projections are then stabilised and embedded into the neighbour’s cell body. Actin cytoskeletal rearrangement results in the formation of a cadherin zipper, which seals the lateral domains of adjacent cells from bottom to top. During junction formation of primary keratinocytes, Drosophila and C.elegans epidermis these finger-like projections are evident.39-41 However, in keratinocytes, the extent of interdigitation of the adhesive membranes is variable across the monolayer. Moreover, these structures are not observed in other epithelia such as MDCK cells.32,33 This may reflect a cell-type specific event and suggests that these “finger-like” projections are not essential for junction formation.

Stabilization via Association with the Actin Cytoskeleton The association of cadherin complexes with the actin cytoskeleton results in the stabilization of the clustered receptors. One characteristic of the puncta is that these are sites for de novo actin polymerisation.33,34 A couple of minutes after induction of cell-cell adhesion, actin is already colocalised with cadherin receptors,34 suggesting that actin recruitment occurs concomitantly or shortly after homophilic binding. The mechanism via which clustered cadherin receptors can induce actin polymerisation is not known. Proteins such as VASP and Mena, which regulate actin polymerization, colocalise with cadherin receptors at junctions40 and are good candidates for participating in actin recruitment. Interestingly, around 50% of the cadherin molecules not engaged in adhesion already associate with actin filaments, and this interaction is important for the mobility of receptors in the plane of the membrane.42,43 However, as there are no reports of detectable actin incorporation at nonadhesive cadherin complexes, the latter association is qualitatively different from the interaction observed at junctions. Another characteristic of the puncta is that a proportion of its cadherin receptor content is detergent-insoluble.33,34 The detergent insolubility of cadherin receptors is interpreted as their immobilisation at the puncta via an interaction with the actin cytoskeleton.44,45 However, lipid association may also render proteins insoluble in detergents.

Remodelling of the Cytoskeleton After cell-cell contact formation, a combination of new polymerisation and remodelling of existing filaments may play a role in the reorganization of the epithelial cytoskeleton. However, it is not know how actin dynamics are altered to produce the cuboidal cell shape typical of epithelia (reviewed by ref. 46). Nor is it clear how the filaments are reorganised to form the circumferential ring of actin, which is connected to the puncta and provides support and contractility.5,33,47 Generation of tension and contraction is then coordinated to produce compaction between adjacent cells that ultimately leads to the cuboidal morphology. Concomitant with the cytoskeleton remodelling, other cytoskeletal and signalling molecules are recruited to sites of cadherin-dependent cell-cell contacts (see below). Among them are spectrin, ZO-1, vinculin and α-actinin that bind directly to α-catenin.48-53 Although these

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interactions are thought to reinforce the binding of cadherin complexes to the actin cytoskeleton, this has not been formally proven. Alternatively, these proteins may help to cross-link and bundle actin filaments at the subcortical cytoskeleton, contributing to the reorganization of cell shape.

Polarised Epithelial Phenotype In addition to cytoskeletal proteins, many other transmembrane and cytosolic molecules are relocalised to junctions. Among the transmembrane proteins, some are involved in cell-cell adhesion, but others are clearly not, such as growth factor receptors, ion pumps, etc. (reviewed ref. 9). Signalling proteins are also localised at cell-cell contacts, presumably by assembly of multi-molecular complexes at the tail of transmembrane receptors. It is not clear how cadherin-dependent adhesion triggers the differential distribution of proteins to the baso-lateral and apical domains of epithelia. The formation of other adhesive structures also contributes to the stabilization of junctions, remodelling of the cytoskeleton and the recruitment of additional signalling complexes.

Maturation of Junctions The maturation of junctions is a poorly characterised process. It derives from the observation that with time after junction formation and after confluence, the biochemical properties of cadherin complexes change. There is an enhanced stability of the complexes at junctions that can be evaluated as an increased detergent-insolubility, inability of mature contacts to be disassembled by cytochalasin D (a drug interfering with actin polymerization), and insensitivity of desmosomes to withdrawal of calcium.33,44,45,54 The molecular mechanisms underlying these processes are not known.

Experimental Models to Investigate Cell-Cell Adhesion Regulation Different models for studying the regulation of cadherin adhesion are available. They allow investigation of junction regulation from different angles: the establishment of new cell-cell contacts or the stability of previously formed junctions (mature junctions). The formation of cell-cell contacts assay investigates which elements are important for the initial events during the establishment of cadherin-dependent adhesion. The mature junction assay studies the relevant events for the maintenance of cadherin complexes at junctions. This can be done by analysing the stability of the complexes under steady state conditions or after stimuli that interfere with cadherin-dependent adhesion such as oncogene expression and treatment with growth factors or HGF/scatter factor.55-59 Interpretation of the published results should take into account the experimental model system, cell type and methodology used. These different settings can account for some of the discrepancies reported in the literature. For instance, it is not known whether the same signalling pathways participate in the formation of new cell-cell contacts and in the maintenance of mature junctions (steady state). It is possible that they are somewhat different, as the stability of cadherin complexes at junctions is clearly increased during maturation.60 Similarly, it is not yet clear whether the same regulatory mechanisms operate after a destabilising stimulus is applied to epithelial cells containing mature junctions. Growth factors and oncogenes are known to activate multiple signalling pathways. Thus, under these conditions, regulation of cadherin adhesion occurs in a different setting, in which additional signalling events and increased phosphorylation levels are triggered by growth factor treatment/oncogene expression. The same applies for the use of established transformed epithelial cell lines. Another cautionary note applies to results obtained with fibroblasts expressing different cadherin constructs. First, although an epithelial morphology is achieved after full length cadherin expression in fibroblasts, the junctions formed are different as no other epithelial adhesive

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Figure 3. Working model of how Rho small GTPases participate on the stabilization of cadherin-dependent cell-cell contacts. Homophilic binding is represented as model 1. After clustering of cadherin dimers at the cell surface, Rho proteins are activated. Rho and Rac are independently activated; it is not known whether Cdc42 activation is required for Rac activation. Each GTPase interact and activate their specific targets. These targets may play a role in cellular processes that may be distinct or overlap (see Fig. 4). The cooperation between these multiple activities leads to the development of the epithelial phenotype: tight cell-cell adhesion, cytoskeletal rearrangement to produce a cuboidal cell shape and the asymmetric distribution of membrane proteins. Some of these activated pathways form a feed back loop to stabilise clustered cadherin receptors (dashed curved arrows).

structures are present (i.e., desmosomes). Second, as mentioned above, the cytoskeletal network of these two cell types is distinct. This concerns both the actin filaments organization and intermediate filament composition (vimentin instead of keratin expression). Finally, and importantly, the same stimuli can activate different signalling pathways in fibroblasts and epithelial cells.61,62 In particular, the regulation of cadherin-adhesion by small GTPases is affected by the cell type in which the receptors are expressed.60

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Regulation of Cadherin Function Small GTPases The Rho family of small GTPases plays a role in cytoskeletal reorganization in a variety of cellular processes. As many of these processes are covered in other chapters, only a detailed discussion on the role of small GTPases in the regulation of cadherin adhesion is found below. Small GTPases are activated when associated with GTP (guanine nucleotide triphosphate). This is the form competent to interact with downstream targets and activate distinct signalling pathways. Inactivation occurs by hydrolysis and elimination of the phosphate, a step that is facilitated by GAPs (GTPase Activating Proteins). When a new activation cycle starts, GDP is released and replaced by GTP. This step is regulated by molecules that promote the GDP-GTP exchange (GEF or Guanine nucleotide Exchange Factors). Thus, signalling pathways that impinge on the small GTPase activity can operate at two different levels, by regulating the function of either GAP or GEF proteins (inactivation and activation of small GTPases, respectively). There are many members in the Rho family, but in epithelia the function of 3 members has been investigated in detail: RhoA, Rac1 and Cdc42 (thereafter referred to as Rho, Rac and Cdc42). In fibroblasts, activated Rho induces bundling of actin filaments in the cytoplasm known as stress fibers.63 Following stimulation by different growth factors, Rac form large protusions rich in polymerised actin at the cell periphery (lamellipodia) or on top of the cell (ruffles).64 Cdc42 can be activated by a distinct set of growth factors and can induce the formation of finger-like projections containing bundles of actin filaments (filopodia).65,66

Working Model for the Participation of Small GTPases in Junction Regulation Studies in vivo all reveal the important role of Rho proteins in junction regulation and epithelial differentiation.41,67-71 Similar to what has been described in fibroblasts, expression of activated forms of Rho, Rac and Cdc42 in epithelial cells in vitro leads to the formation of stress fibers, lamellipodia and filopodia, respectively. However, it is not clear how these specific cytoskeletal rearrangements play a role in cadherin function or the development of epithelial cell shape. The working model of how the small GTPases may participate in the regulation of cadherin adhesion is shown in Figure 3; specific steps are discussed below. Upon induction of new cell-cell contacts, activation of the small GTPases occurs. This has been demonstrated for Rho, Rac and Cdc42 (see below).72-74 Experimental data also suggest that, following cell-cell contact formation, Rho and Rac pathways are activated independently.75,76 This is in contrast to migration in fibroblasts, where the activation levels of Rho and Rac are interrelated.77-80 The activated small GTPases then bind to different effectors and trigger distinct signalling pathways (Fig. 3). These pathways collaborate with each other to produce the cytoskeletal rearrangements and originate the epithelial phenotype. Some of these pathways provide a feedback loop for the maintenance of stable cadherin-dependent cell-cell contacts. The ability of small GTPases to regulate cadherin adhesion depends on the maturation status of the junctions and the cell type.60 For example, in endothelial cells blocking Rho or Rac function does not affect VE-cadherin localization at junctions. However, in CHO cells expressing VE-cadherin, this receptor is readily removed by Rho or Rac inhibition. Thus, the current evidence indicates that Rho and Rac activities are important for cadherin-dependent adhesion, while Cdc42 function is relevant for the development of the polarized phenotype. An attractive possibility is that, in addition to the morphological changes, the small GTPases may participate in other signalling activities important for epithelia differentiation (see below).

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Activation of Small GTPases Recently, it was demonstrated that the activation of small GTPases can be induced by cadherin-dependent adhesion in epithelial cells.72-74 When expressed in MDCK cells, wild type, activated and dominant negative forms of Rac and Cdc42 localise at the junctions.73,76,81-83 It is not clear why all these mutants localise at the same place and no reports on the localization of endogenous proteins at junctions have been published. In contrast, no such localization has been reported for Rho.82 The functional significance of the distinct localization of each Rho family member is not known. Nevertheless, the localization of exogenous Rho molecules seems specific and dependent on cadherin adhesion: clustering of cadherin receptors with antibodies results in the recruitment of Rac (activated and dominant negative).73

Rac and Cdc42 The pattern observed for Rac and Cdc42 activation following cell-cell contact formation is similar in many cell types.72-74 First, activation of Rac and Cdc42 occurs very quickly, although depending on the study the time when activation is first detected varied (5 to 30 minutes). Second, this process requires cadherin adhesion, as inhibitory antibodies against cadherins can block activation of both GTPases. Third, PI3 kinase appears to be involved in Rac and Cdc42 activation following junction assembly.72,73 Regarding the role of PI3 kinase in increasing levels of Rac.GTP and Cdc42.GTP, it is noteworthy that PI3 kinases are themselves activated by induction of cell-cell adhesion.72,84 However, in MDCK cells, PI3 kinase inhibition can not interfere with junction assembly nor with Rac localization at these sites.73 One possible explanation is that PI 3-kinase activity is not essential for cadherin function and Rac recruitment to junctions, but may participate in subsequent steps (i.e., Rac activation; junction maintenance).73 Nevertheless, as during junction assembly PI3 kinase inhibition did not abolish completely the levels of active Rac in MDCK,73 it is conceivable that, in addition to PI3 kinase, alternative mechanisms exist to promote Rac activation. Although activation of Rac and Cdc42 during junction formation in epithelia has many similarities, it has not yet been demonstrated whether their activation is linked as described in fibroblasts (i.e., Cdc42 activates Rac; Fig. 3).66 While the above studies demonstrated that E-cadherin function is necessary for the activation of Rac and Cdc42 small GTPases, the question of whether cadherin adhesion is sufficient to drive their activation has not yet been addressed.

Rho Regarding Rho activation, conflicting results using MDCK cells are available. In one study, no upregulation of Rho activity was detected after new cell-cell contacts formation.73 In another study, Rho is activated following 5 minutes of induction of cell-cell adhesion, but measurement of Rho activity in other cells lines also gave conflicting data.74 A possible explanation for these discrepancies is that different levels of endogenous Rho in the distinct MDCK strains that would make it more difficult to detect variations in the activity levels. The above results led the authors to propose that Rho activity was due to calcium signalling, and not cadherin-mediated adhesion.74 However, no controls to pin point the contribution of calcium signalling versus cadherin adhesion for the process are provided (i.e., using anti-cadherin blocking antibodies). In addition, the use of different cell lines such as CHO and fibroblasts poses additional complications, due to the distinct cellular context in which Rho operates.60 These cell lines also express different members of the cadherin family, and at the moment, it is not known whether all the family members are regulated in a similar way by the small GTPases. Thus, Rho activation following junction formation is controversial and requires additional experiments to demonstrate its specificity by cadherin-mediated adhesion. Nevertheless, irrespective of how Rho is activated, its function is clearly necessary for the stability of the cadherin complexes at junctions (see below).60,75,82

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How Are Small GTPases Activated Following Cell-Cell Adhesion? It is also not known how the GTPases activation takes place, but presumably it involves the recruitment of GEFs to these sites. Alternatively, formation of cell-cell contacts may inactivate specific GAPs, allowing increased levels of activated Rho proteins. At the moment, no candidate GEF or GAP has been put forward to fulfill this task. Vav-2, an exchange factor for Rac and Cdc42, is not activated by phosphorylation following induction of cell-cell adhesion in MDCK cells.74 Thus, Vav-2 has been excluded to participate on the cadherin-dependent activation of the small GTPases.74 Interestingly, Vav-2 is able to interact with the catenin p120ctn, but this association occurs mostly with the p120ctn cytoplasmic pool (see section Regulation of Cadherin Function/Cross-Talk between Small GTPase, Cadherin- and Integrin-Dependent Adhesion).74 Future studies will investigate the mechanisms via which cadherin receptors can signal to activate the small GTPases.

Regulation by Rho Many of the reported cellular functions of Rho have a component of contractility and generation of tension. These include the Rho functions in the formation of stress fibers, phagocytosis and cytokinesis (reviewed by ref. 85). However, it is not clear whether generation of tension plays a role in other Rho activities such as gene transcription, growth control and in cadherin adhesion. Inhibition of Rho by C3 transferase destabilises cadherin receptors from both newly formed and mature junctions.60,75,82 Rho activity play also a role in tight junction function.86 In two studies, transfection of the N19RhoA mutant to block endogenous Rho function was unable to disturb the presence of cadherins or actin at the junctions.76,87 One possibility is that expression of the N19Rho mutant was not high enough to completely block Rho function. A complete inhibition of Rho function by transfection is not achievable, as the cells would round up and detach during the selection procedure. On the other hand, in addition to RhoA, C3 is also able to inhibit RhoB and RhoC, and it is feasible that these other members may play a role on cadherin adhesion. However, a contribution of RhoB- and RhoC-specific signalling pathways seems unlikely: coexpression of a RhoA molecule insensitive to C3 inhibition can completely rescue the inhibitory phenotype induced by C3 alone.88 One current model argues that Rho activities are relevant for the cytoskeletal organization of epithelia, and that this effect indirectly stabilises cadherin receptors at junctions.89 Another model predicts that Rho function is important for both cell shape changes and events more proximal to the cadherin receptors.88 Both models agree on the participation of Rho in cytoskeletal organization. Because there are profound cell shape changes as the epithelial morphology develops, Rho-dependent contractility may be important for this process. The main difference between the two models is whether Rho can participate in events more proximal to the receptors, such as cadherin clustering. In support of the second model, when Rho is inhibited, cadherins are removed from junctions before other adhesion receptors, implying a certain degree of specificity in this process.75 Cells are still touching each other when cadherins are removed, suggesting that there is not a major disorganization of the cytoskeleton at this time. In addition, Rho is known to modulate myosin function, and different myosin molecules are found at the subcortical actin cytoskeleton and junctions.36-38 In endothelial cells, Rho mediates clustering of adhesion receptors such as ICAM-1 and VCAM-1.90,91 Based on the above evidence, it is possible that pathways activated by Rho may impinge on the cadherin clustering process in the following steps (see below): (a) lateral mobility of the receptors; (b) clustering to form the punctum; or (c) coalescence of the different puncta to form the adherens junctions. However, direct experimental evidence for an involvement in these processes are not available at the moment.

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Rho Targets One of Rho targets that has been investigated in epithelial cell-cell adhesion is ROCK (also known as Rho kinase or Rok).92,93 ROCK is a key target in the induction of stress fibers and generation of contractility.93-95 Transfection of a dominant negative version of ROCK (ROCK-KD-IA) can partially perturb the localization of cadherins at the MDCK junctions.89 However, expression of the same construct in breast epithelial cells do not disturb the recruitment of cadherins or actin to cell-cell contacts.87 In addition, inhibition of ROCK function by treatment with the compound Y27632 in keratinocytes cannot prevent establishment of new cell-cell contacts (ref. 88 and data not shown). These discrepancies may reflect different methodology and cellular context used in these studies. ROCK is able to regulate myosin II function and induce contraction.95-97 As blocking ROCK activity in certain epithelial types does not prevent cadherin clustering, these results imply that ROCK is not necessary for this process. One possible role for ROCK is in the development of epithelial cell shape. Indeed, by using the compound Y27632 during induction of cell-cell contacts in keratinocytes, the cells were unable to shape up and become tall and polarised (ref. 88 and data not shown). As the compound is not effective in cells containing mature junctions, it is possible that the ROCK inhibitory effects require the remodelling of the cytoskeleton. Thus, these results can dissociate between cadherin adhesiveness and cell compaction, and suggest that ROCK operates downstream of cadherin receptor clustering.

Regulation by Rac In different cell types, Rac has an important role in the regulation of lamellipodia formation, cell spreading and actin polymerisation. Recent evidence suggests that induction of lamellipodia is not relevant for cadherin-mediated adhesion.88 Indeed in epithelial cells, there is an inverse correlation between the presence of junctions and lamellipodia, suggesting an antagonism between these structures.47,88 Interestingly, Rac regulates the clustering of acetylcholine receptors in myotubes and integrins in T-lymphocytes and fibroblasts.64,91,98 Thus, it is conceivable that Rac may also play a role in cadherin clustering. In epithelial cells, Rac plays a role on the structural and functional organization of tight junctions.86 During cell-cell contacts formation, the subcortical cytoskeleton needs to be relaxed locally to allow formation of protusions which probe neighbouring cells for adhesion and polarization. Similar relaxation of the cell cortex is seen during spreading, a process regulated by Rac.77,91,99,100 Based on these results, Rac may contribute to cadherin adhesion on three fronts: clustering of the receptors, relaxation of cell cortex and receptor stabilization via actin recruitment and remodelling (Fig. 4b). However, the hypothesis that Rac participates in the clustering of cadherin receptors and in cell cortex relaxation has not yet been tested during junction assembly in epithelia. Experimental evidence suggests that Rac-dependent actin polymerisation plays a role in cadherin adhesion. Inhibition of endogenous Rac function in keratinocytes and MDCK cells removes cadherin receptors from both newly formed and mature junctions.60,75,82 Actin recruitment to junctions and to clustered cadherin receptors is also inhibited by blocking endogenous Rac function.75,82 On the other hand, expression of activated Rac results in (a) enhanced staining of cadherins and actin at the junctions and (b) more intermingled cell-cell contacts.38,82,83 As mentioned earlier, it is not know how actin dynamic changes and cytoskeletal reorganization takes place following junction assembly. Actin polymerization at cell-cell contacts strictly requires the presence of adhesive cadherin receptors. Interestingly, activation of Rac in the absence of cell-cell adhesion in keratinocytes does not lead to actin polymerisation at the cell borders.60 The results suggest the Rac activation per se cannot bypass the requirement for cadherin adhesion. It is possible that clustering of cadherin receptors provides spatial clues for a localised, Rac-dependent actin polymerisation in epithelia. Taken together, the data indicate that a tri-partite process takes place, in which cadherin-dependent adhesion, Rac activation and actin recruitment cooperate with and depend on each other for the formation of stable cell-cell contacts.

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Figure 4. Possible cellular processes triggered by the small GTPases Rho (a) and Rac (b) during establishment of cadherin-dependent cell-cell adhesion. Dashed arrows and question marks denote possible activities based on data from other cell types; no direct evidence from epithelia and cadherin-based adhesion. Possible cellular processes triggered by the small GTPase Cdc42 (c) during establishment of cadherin-dependent cell-cell adhesion. Dashed arrows and question marks denote possible activities based on data from other cell types; no direct evidence from epithelia and cadherin-based adhesion. See text for details.

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Regulation by Cdc42 In addition to formation of filopodia, Cdc42 has also been implicated in actin polymerisation.101-103 In other cell types, Cdc42 regulates polarity during cell migration and bud formation in yeast, as well as chemotactic responses.79,104-106 Similarly in epithelial cells, Cdc42 contributes to the development of polarity, as it regulates the fidelity of the transport of secretory vesicles, and thus promotes the asymmetric distribution of proteins at the membrane (see section Regulation of Cadherin Function/Rac and Cdc42 Binding Proteins/Cell Polarity).107,108 In Drosophila, after blocking Cdc42 function two main effects are observed: (a) inhibition of the apicobasal elongation of epithelial cells,67,109 and (b) perturbation of dorsal closure (embryonic ectodermal epithelium; Jacinto et al, 2000). However, in the latter case it is difficult to distinguish between defects in migration of the leading edges and a failure in junction formation per se when the wound is closed. Nevertheless, the staining for junctional components is not substantially perturbed in these studies.41,67,109 While the above evidence suggests that Cdc42 is important for the development of the epithelial phenotype, the specific role of Cdc42 in cadherin-mediated adhesion per se is not clear (Fig. 4c). Inhibition of Cdc42 has no effect on the levels of cadherin staining at junctions in MDCK cells.83,107,110 However, upon expression of dominant negative Cdc42, a reduction in cadherin-mediated adhesion (aggregation assays) is observed in E-cadherin transfected fibroblasts.111 These data might reflect an inefficient inhibition of endogenous Cdc42 by the dominant negative approach. Further experiments using alternative tools to inhibit Cdc42 are necessary to confirm this phenotype. In epithelial cells, activated Cdc42 produces similar effects to activated Rac: it can promote more intermingled cell-cell contacts and accumulation of F-actin at junctions38,83 These results suggest that Cdc42 may cooperate with Rac in actin recruitment at junctions. However, there are many transmembrane proteins at junctions in addition to cadherin receptors that may act as recipients for recruited actin. As opposed to Rac,75 direct recruitment of actin to clustered cadherin receptors has not yet been show for Cdc42. Another step in which Cdc42 may pay a role is on the induction of the finger-like structures observed during new cell-cell contacts formation in keratinocytes, Drosophila or C. elegans.39-41 These projections appear similar to filopodia, and, after establishment of cell-cell adhesion in keratinocytes, bundles of actin filaments are seen inside.40 However, it is not clear whether before cell-cell adhesion these projections already contain actin bundles. During dorsal and ventral closure, typical filopodia are clearly seen, but the epithelial sheets are actively migrating towards each other.39,41 While these morphological considerations needs further investigation, the question is whether these finger-like projections are dependent on Cdc42 activity. Direct evidence for a role for Cdc42 on this process using a confluent epithelial monolayer (where no migration is involved) is still lacking. The fact that at present Cdc42 inhibition cannot abolish the localization of cadherins at junctions would argue against a participation of these small GTPase in this process. Alternatively, these finger-like structures are not essential for junction assembly as discussed previously.

Rac and Cdc42 Binding Proteins In different cell types, Rac has a clear effect on actin organization via activation of different targets such as PI4,5P kinase and LIM kinase.112-114 In epithelial cells, no Rac target has yet been identified that mediates Rac-dependent actin recruitment at junctions. IQGAP, a Rac and Cdc42 target, is detected at cell-cell contacts in epithelial cells.110 This molecule has homology with RasGAP, but no detectable GAP activity.110,115,116 IQGAP1 interacts with the cadherin tail and β-catenin. The latter interaction occurs in the same region as α-catenin, and therefore IQGAP competes with α-catenin for binding to β-catenin.117 The displacement of α-catenin from cadherin complexes is supposed to weaken the interaction with the actin cytoskeleton. However, IQGAP is also able to bind and bundle actin filaments,116 and may partially substitute for α-catenin in the cytoskeletal interactions. Nevertheless, the

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presence of “less adhesive” cadherin receptors at the membrane may contribute to the dynamics of cell-cell adhesion in different cellular processes. Expression of IQGAP in fibroblasts expressing E-cadherin can reduce their aggregation levels in a quantitative assay for cell-cell adhesion.117 Consistent with that, activated Cdc42 and Rac bind to IQGAP and prevent it from associating with β-catenin, thereby inhibiting disassembly of cell-cell contacts.111,117 Nevertheless, IQGAP expression does not abolish E-cadherin staining at junctions.117 Another level of regulation is via calmodulin, which interacts with IQGAP1 and competes with E-cadherin for IQGAP1 binding. Inhibition of calmodulin function increases junctional localization of IQGAP1 and negatively modulates E-cadherin adhesion in MCF-7 cells.118 The physiological role of IQGAP in junction regulation is unclear at present. Similarly to α-catenin, IQGAP can bind to actin bundles, and it could therefore mediate attachment of cadherin complexes to the actin cytoskeleton.116,119 A putative dominant negative truncation of IQGAP1 prevents HGF- and TPA-induced disruption of cell-cell contacts in MDCK cells,120 suggesting that IQGAP1 may play a role on cell scattering. Mice with a targeted disruption of the IQGAP1 gene show no defects in cadherin-dependent adhesion and develop normally, although there is an increased incidence of late-onset gastric hyperplasia.118 IQGAP2 is a close homologue of IQGAP1 (62% identical) and it may functionally compensate for the lack of this protein.119 Arguing against this, IQGAP2 has a much more restricted expression than IQGAP1 and there is no obvious increase in IQGAP2 expression upon loss of IQGAP1.118,119 Tiam-1 is an exchange factor for Rac that localises at cell-cell contacts in epithelia and has been connected to components of cadherin complexes. When expressed in fibroblasts and transformed MDCK cells, Tiam-1 induces an “epithelial-like” phenotype with an increased localization of cadherin and actin at the cell-cell borders.80,121 The enhanced actin staining at junctions may result from an increased localization of Rac at these sites, a process that is mediated by Tiam-1. Although in transformed MDCK Tiam-1 activity appears to promote cell-cell adhesion, Tiam-1 can induce invasion and migration in T-lymphocytes and breast tumour cells.122-124 Thus, it seems that Tiam-1 may play a role in migration as well as cell-cell adhesion, depending on the cell type in which it is expressed. Nevertheless, it is not know whether Tiam-1 function is essential for cadherin-mediated adhesion as no dominant negative experiments have yet been reported. The Cdc42 effector MRCKα localises at cell-cell contact sites, but its role in the regulation of junctions has not yet been determined.125 A new family of Cdc42 effector proteins, CEPs has been implicated in regulation of cell-cell contacts.126 However, since the CRIB domain (Cdc42/Rac Binding Domain) of CEP is necessary for this response, it is possible that CEP perturb junctions by titrating out endogenous Cdc42.

Cell Polarity Cdc42 plays a role in epithelial polarity (asymmetric distribution of proteins in distinct domains) as well as in the polarity of cell migration. A Cdc42 target that may play a role on these processes is PAR-6.127-129 PAR-6 forms a complex with activated Rac/Cdc42, PAR-3 and PKCζ.127-129 In Drosophila and C.elegans, this complex participates in the establishment of epithelial polarity.130 In mammalian cells, PAR-6/PAR-3/PKCζ complex localizes at tight junctions via a direct interaction of PAR-3 with JAM, a member of the immunoglobulin family.131,132 This association is important, as overexpression of PAR-6 or the N-terminal domain of PAR-3 disrupts tight junctions.129 In contrast, localization of cadherin at adherens junctions is not perturbed. As JAM localises at tight junctions through an interaction with ZO-1, it is likely that JAM recruits the complex to tight junctions. Thus, Cdc42 and the PAR-6/PAR-3/ PKCζ complex appear to be involved in the establishment of epithelial polarity, but not specifically on the localization of cadherin receptors at junctions. Moreover, this Cdc42 function has been conserved in evolution from worm to man.

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Regulation by Other Small GTPases Additional members of small GTPase superfamily may also play a role on the organization of cadherin-based junctions, in particular, members of the ARF and Rab subfamilies that play a role in vesicular transport. The recycling of endocytosed cadherin receptors is clearly important for the establishment of new cell-cell contacts in MDCK cells.133 Dominant negative versions of Arf6 and Rab5 can block the HGF-induced disruption of cell-cell contacts.134,135 Consistent with these results, Arf6 activation can induce disassembly of cell-cell adhesion and cell scattering in MDCK cells.135,136 The mechanisms via which cadherin-based adhesion is disrupted are not clear, but it may involve activation of both Rac and phospolipase D.136 Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) may exist in a constitutively activated form, as they show very low intrinsic levels of GTP hydrolysis.137 Endogenous Rnd 1 colocalizes with cadherin receptors at cell-cell contacts in epithelia and fibroblasts. Expression of wild type Rnd1 or Rnd3 leads to disassembly of actin filaments and loss of cell-matrix adhesion.137-139 However, no major effect is seen on the organization of actin at cell-cell adhesion sites when Rnd1 is expressed in MDCK cells.137,138 Thus, it appears that even though Rnd1 localises at junctions, so far its biological effect has been primarily described at sites of adhesion to substratum and stress fibers. Further experiments are necessary to define a putative function for Rnd proteins on cadherin-dependent cell-cell contacts. Another example is the Ras target, AF-6 or Afadin, which has been shown to localise at adherens and/or tight junctions.140-142 While a direct interaction between AF-6 and ZO-1 and AF-6 and JAM have been demonstrated, no functional implication for these interactions has been described.140,143 Interestingly, deficiency of AF-6/afadin causes embryonic lethality and both adherens and tight junctions are not properly organised in the ectoderm.141 However, these results need to be reconciled with the fact that Ras activity is not apparently necessary for formation of cadherin dependent cell-cell contacts.144

Cross-Talk between Small GTPases Cadherin and Integrin-Dependent Adhesion For many years, different reports have described a cross-talk between cadherin and integrin adhesion. This cross-talk is observed in different systems such as keratinocyte and muscle differentiation,145,146 migration of neural crest cells,147 MDCK polarization,148,149 regulation of receptor expression levels150 and determination of malignant phenotype.151,152 This cross-talk is apparently a two-way street, in which a reciprocal relationship is observed between the adhesiveness of both types of adhesion receptors. This phenomenon is interpreted as the need to coordinate cell-cell and cell-extracellular matrix adhesion in different cellular processes, and ultimately determine a sessile or migratory phenotype. However, the mechanism via which such coordination is achieved is poorly understood. Rho small GTPases regulate both cadherin- and integrin-mediated adhesion, and they are likely candidates to participate in a cross-talk between the two receptors. Recent results suggest that adhesion to different extracellular matrices can influence the manner in which Rac interferes with cadherin-dependent cell-cell contacts.80 Depending on the substratum to which the cells are attached, either a positive or negative effect is observed on junctions following Rac activation. These results suggest that the engagement of different types of integrin receptors can have a profound effect on the signalling pathways activated by Rho small GTPases. Interestingly, expression of β1 integrin in epithelial GE11 null cells (β1-/-) leads to a disruption of cadherin-based adhesion, decrease in cadherin and α-catenin levels and rearrangement of focal adhesions.78 Expression of β1 integrin in these null cells activates Rac and Rho, but not Cdc42. The morphological and scattering phenotype can be reversed by expression of dominant negative versions of the small GTPases.78 In contrast, reports in other cell types point out to a balance between the activity levels for Rho and Rac to determine cell-cell adhesion or migratory behaviour.99,153

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Similarly, the presence of cadherin-dependent adhesion can modify the migratory behaviour of cells, by reducing the speed and motility rate. This balance between motility and cell-cell adhesion is perturbed by treatment with specific growth factors/scatter factors. HGF/scatter factor, TGF-β and Ras can all induce disruption of cell-cell adhesion, scattering and enhanced motility via activation of Rho small GTPases.87,144,154,155 However, these effects are dependent on the cell type, substratum and the levels of growth factor receptors expressed.80,156 In cells containing normal levels of growth factor receptors, cadherin-dependent cell-cell contacts are not easily disrupted: addition of inhibitory antibodies against cadherins is necessary to induce the migratory phenotype.157,158 A candidate player for the coordination between cell adhesion and migration is the nonreceptor tyrosine kinase Fer.159 This molecule associates with the juxta-membrane domain of N-cadherin, and has been shown to participate in the cross-talk between N-cadherin and integrins.160,161 Indeed, the juxta-membrane region of E-cadherin cytoplasmic tail is also involved in the regulation of cell adhesion and cell motility.162 Another possible point of cross-talk between cadherins, integrins and small GTPases is the catenin p120ctn. Recent results suggest that p120ctn can modulate the activation status of Rho proteins as overexpression of this catenin induces branching protusions.163-165 This phenotype is dependent on Rho inhibition and upregulation of Rac and Cdc42. Only p120ctn molecules not associated with the cadherin receptors (cytosolic pools) regulate the activity of small GTPases. Expression of E-cadherin can titrate out p120ctn and inhibit these effects.163-165 It is predicted that disruption of cadherin-based junctions would free up p120ctn in the cytoplasm, which would then be able to modulate the activity of RHO small GTPases to an appropriate setting for migration (Rho inhibition and activation of Rac/Cdc42). Although p120ctn can interact with Vav-2, an exchange factor for Rho, Rac and Cdc42,166 overexpression of Vav-2 cannot reproduce the same phenotype as p120ctn overexpression.164 On the other hand, using purified proteins, it has been shown that p120ctn can inhibit the intrinsic GDP/GTP exchange activity of Rho.163 However, a direct interaction between these two proteins has been unsuccessful. Thus, the mechanisms via which p120ctn may regulate the activity of Rho small GTPases is an open issue. Taken together, these results reflect the ability of cells to coordinate when is necessary to move and when to stop and adhere to each other. The coordination between these two decisions observed in vitro represent glimpses of a biological process that occurs often during tissue morphogenesis. During developmental processes such as migration of neural crest cells, kidney epithelial tubular formation and muscle differentiation, the precise spatial and temporal control of migration to specific regions in the embryo is essential. Here cells become sessile, adhere to their neighbours and differentiate. It will be important to confirm whether the molecular players identified in the cross-talk between cadherins and integrins in vitro may also play a role on the differentiation processes in vivo.

Signalling from Cadherin Receptors Because addition of extracellular calcium ions is required for cadherin function, there is a concern about the role of calcium signalling in the cellular processes that are triggered following cell-cell contact formation. An important role for calcium signalling seems unlikely because: (a) an increase in intracellular calcium ions is readily buffered; (b) the time course for calcium signalling is usually very short, and does not match the time course for cytoskeletal rearrangement triggered by cell-cell adhesion (1 – 2 hours); (c) inhibition of cadherin function has so far blocked the relevant cellular processes (formation of other adhesive structures and cell shape change).73-75,145,167,168 Thus, calcium signalling may contribute to some cellular processes after cadherin-dependent adhesion.169 However, this pathway is by no means essential or sufficient for the development of the epithelial phenotype. Although it was originally thought that cadherin receptors play a purely mechanical role in mediating attachment of adjacent cells, it is now becoming apparent that cadherin-mediated adhesion can also activate intracellular signalling pathways. The pathways activated in a

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cadherin-dependent manner participate in different cellular processes such as gene expression, differentiation, growth control, apoptosis and phosphorylation/activation of specific signalling cascades. The small GTPases of the Rho family have also been implicated in these processes in different cell types.85 One interesting question is to whether in epithelial cells small GTPases can participate in these processes in a cadherin-dependent manner. Some studies give insights particularly when the specificity of the signalling output depends on a particular type of cadherin expressed. For example, in keratinoctytes, cell-cell contact formation induces expression of differentiation markers in an E-cadherin- and P-cadherin-dependent manner.170,171 Reexpression of E-cadherin in embryonic stem cells derived form E-cadherin -/- mice triggers epithelial differentiation in vitro (Larue et al, 1996). In contrast, expression of N-cadherin induces chondrogenic, skeletal and cardiac muscle differentiation.6,172-174 However, E-cadherin can functionally substitute for N-cadherin when expressed in cardiomyocytes null for N-cadherin expression.175 These results imply that some functions of different cadherin receptors are interchangeable, but not all of them. A role for cadherins in the contact-inhibition of growth of normal cells has long been speculated. This involvement has now been shown for both E- and N-cadherin, although the mechanisms are somewhat different.110,176 In addition, there is some evidence suggesting that E-cadherin may function as a tumor suppressor, as it can slow the proliferation rate of some cell lines.176-179 A participation of E-cadherin-, N-cadherin- and VE-cadherin-mediated adhesion in suppression of apoptosis has also been demonstrated.175,180,181 Moreover, cell-cell adhesion mediated by cadherin receptors can trigger the transcription of specific genes.146,182-184 Cell-cell contacts mediated by cadherins in fibroblasts can activate stretch-sensitive calcium permeable channels, which induces a transient actin polymerisation at contact sites.169 Finally, in addition to the activation of Rho proteins, cell-cell contact formation in epithelial cells can activate PI3 kinase, Akt/PKB and MAPK.84,185 Establishment of junctions induces phosphorylation of the docking protein Gab1 in an E-cadherin-dependent manner.186 As phosphorylation of Gab1 can negatively regulate HGF receptor signalling,187 it will be interesting to determine if these phosphorylated residues are similar to the residues modified after cell-cell adhesion formation. In many of the studies described above, particularly the long-term differentiation studies, it is not clear whether the activation of signalling pathways is a direct consequence of cadherin association or results from the assembly of other junctional complexes or recruitment of signalling molecules to the junction. This issue has been addressed in some instances, where clustering of cadherin receptors using specific antibodies or antibody-coated beads can mimic the ability of cadherin-mediated adhesion to activate distinct pathways.171,173,188 However, in two of these studies, cell-cell contact assembly is also enhanced by incubation with antibody-coated beads. Therefore, this increase in junction formation, rather than the bead-treatment per se, may be responsible for the activation of signalling cascades.173,188

Possible Mechanisms for Signalling Following Cell-Cell Contact Formation Since the cadherins have no intrinsic catalytic activity, the manner in which they may activate signalling cascades is unclear at present. One possibility is by the association with scaffolding proteins or proteins with enzymatic activity. Indeed, an association between a number of growth factor receptors (NGFR, EGFR, VEGFR) and cadherin-adhesion complexes has been demonstrated in different cell types.180,185,189-191 Shc has also been shown to coprecipitate with cadherin receptors.192 However, it is conceivable that Shc was present in the cadherin precipitates via association with a growth factor receptor, an established partner for Shc. A current hypothesis is that clustering of cadherins and growth factors receptors at sites of cell-cell contact may be sufficient to activate the receptors and thus downstream signalling pathways. Alternatively, cadherin receptors may associate with kinases and phosphatases that can then mediate some of the reported effects. These interactions have been demonstrated for N-cadherin and the tyrosine phosphatases PTPµ, PTP1B and LAR193-195 and nonreceptor tyrosine kinase Fer.159

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It has long been demonstrated that the formation of desmosomes, gap junctions and tight junctions require the prior assembly of cadherin-dependent cell-cell contacts.8,167,168,196,197 Two possibilities have been suggested to explain these events: first, cadherin receptors form stronger cell-cell contacts and, by holding the two opposing membranes from neighbouring cells together, it would facilitate the formation of these other adhesive structures. Alternatively, cadherin-dependent adhesion would trigger signalling events that facilitate the assembly of desmosomes and tight junctions (see section Signalling from Cadherin Receptors). Some recent experiments indicate that PKC isoforms may play a role in these processes.198-202 The mechanism via which PKC can induce the localized assembly of tight junctions and desmosomes at cell borders in the absence of cadherin function is far from clear. Nevertheless, this data imply that a possible consequence of cadherin adhesion is the activation of PKC isoforms, which then participate in the assembly of tight junctions. One possible connection that has not yet been explored is via association of PKCζ with Par3/Par6 complexes. This complex can directly interact with JAM receptors present at tight junctions.131,132 Another example is the involvement of Src kinases (Src, Fyn and Yes). In keratinocytes, inhibition of Src activity in vitro leads to the stabilization of transient cadherin-dependent cell-cell contacts, even in the absence of calcium ions.203 This suggests that after cell-cell contact formation, cadherins might inactivate Src to enable stable junction formation. These results are consistent with early experiments in which expression of the oncogenic form v-Src can perturb cadherin-mediated cell-cell adhesion.55,57,204 In contrast, a recent report showed that fyn-/- keratinocytes are deficient in the formation of cell-cell junctions in vitro.205 Disruption of cell-cell contacts in the epidermis in vivo requires ablation of both src and fyn genes.205 These contradictions might reflect that, in addition to the catalytic domain, additional adaptor functions (i.e., SH2, SH3 domains) of the Src molecule are necessary for its effects on junctions.

Conclusions Exciting results in the past few years have highlighted the potential mechanisms for regulation of cell-cell contacts in epithelial cells. Cadherin receptors emerge as a key component for the activation of distinct signalling pathways during formation of new cell-cell adhesion. These pathways have been shown to participate in many different cellular processes relevant to epithelia function: assembly of adhesive structures, cell shape change, polarisation, differentiation, survival and growth control. Some of the activated pathways, particularly related to Rho and Rac small GTPases, participate in a feed back loop as they may be an important contribution to the stability of the cadherin receptors at junctions. The challenge now is to understand the signalling components and the molecular interactions that leads to the activation of these diverse signalling pathways following clustering of cadherin receptors.

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CHAPTER 9

Rho GTPases and Cell Motility Alexander Beeser and Jonathan Chernoff

Abstract

T

he ability to regulate movement is a fundamental property of most eukaryotic cells. All phases of cell motility; the establishment and maintenance of polarity, the formation of actin-rich protrusions, the remodeling of adhesive contacts and the generation of force, are regulated by various members of the Rho GTPase family. The method by which this regulation is achieved is beginning to be understood at the molecular level. In this chapter, we review the signalling mechanisms used by Rho GTPases to regulate cell motility. A cell that has received a signal to move must undertake a profound reorganization of its cytoskeleton. This reorganization includes changes in polarity, in the amount and location of actin polymerization, in the strength and duration of integrin attachments, and in the activity of myosin motors, all choreographed in the proper time and place. The signalling machinery that is required to bring about these events is complex and far from being understood in detail, but our understanding of how cell locomotion is regulated is rapidly advancing. Cell movement is a complex phenomenon, involving Rho GTPases and a large number of effector proteins that act upon a large number of cellular targets. A list of critical effectors and their contributions to motility is presented in Table 1. For simplicity, cell motility can be subdivided conceptually into four components: a) the polarization of the cytoskeleton, b) the protrusion of the plasma membrane, in the form of lamellipodia, at the leading edge; c) the establishment of transient contacts with new substratum, accompanied by the release of preexisting contacts, and d) the production of a contractile force to allow for the rear of the cell to retract in the direction of movement (Fig. 1). These four processes are inextricably linked and must be properly coordinated to allow normal cell movement. As the molecular mechanisms of mammalian cell motility are perhaps best understood in fibroblasts, we have chosen to focus primarily on the motility of this cell type, although we have on occasion considered other cell types or other motile organisms where exceptionally informative. Although a universal model of motility likely does not exist, the influence of the Rho family GTPases cannot be overestimated. Humans contain more than 20 Rho family GTPases, by convention divided into three subfamilies, Rho, Rac and Cdc42. All three subfamilies are known to affect one or more components of motility, including promoting cytoskeletal rearrangement, localized actin polymerization, establishment and dissolution of focal adhesions, and effects on myosin contractility. In addition to regulating the fundamental aspects of motility, the Rho family GTPases are also critical in ensuring that movement is not random but appropriately directed in response to extracellular signals. That the Rho family GTPases are involved in every aspect of cell movement and that this influence is evolutionarily conserved from the most primitive social eukaryotes, suggests that these proteins are a fundamental component of the motility apparatus, and that understanding their action is a prerequisite to comprehending cell locomotion.

Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Table 1. Contributions of the individual GTPases and their effectors to various aspects of motility Effect

Rho-GTPase

Effectors

Actin polymerization Focal complex assembly Polarity Membrane Protrusion Contractility

Rac, Cdc42, Rho Rho, Rac Cdc42 Cdc42, Rac Rho

WASP, WAVE PI3K Par6, BORG, Pak? WASP, WAVE, Pak ROCK, mDia

This list is not comprehensive nor is the order of the GTPases listed reflective of their absolute importance to the process under consideration. For descriptions of the effectors, please refer to the appropriate sections in the text.

Rho GTPases and Cell Polarity In response to various growth factors, quiescent fibroblasts rapidly direct membrane protrusions, in the form of lamellipodia, towards the direction of movement. This establishment of a single, dominant lamellipodium defines the leading edge of the cell. The ability to restrict the directed protrusion to a single direction effectively polarizes the cell and establishes the direction of movement in the event of prolonged haptotactic or chemotactic signalling. Following these initial events, motile cells reorient their microtubules and Golgi apparatus in the direction of membrane protrusion. These later events are not required for the initial establishment of polarity, but contribute to its maintenance. The ability to establish and maintain cellular polarity is not restricted to motile fibroblasts, as many different cell types require polarization in order to function effectively in a biological context, suggesting that the regulation of motility by Rho family GTPases is merely a specialized component of their role in polarization. For example, in Saccharomyces cerevisiae, bud site selection is under defined genetic constraints and Rho family GTPases are known to regulate this process.1 Of the three Rho GTPase subfamilies, Cdc42 is the one most often associated with regulating cell polarity. In budding yeast, Drosophila, and C. elegans, loss of Cdc42 function is accompanied by profound polarity defects. Cdc42-null mice die at an early stage of embryogenesis, and ES derived from these embryos proliferate, but display aberrant actin cytoskeletal morphologies and fail to support phosphatidylinositol 4,5-bisphosphate (PIP2)-induced actin polymerization in vitro.2 These genetic studies, and many other studies using dominant negative forms of Cdc42 (e.g., see ref. 3), have firmly established a role for Cdc42 in cell polarity that is conserved throughout eukaryotic evolution. How does Cdc42 regulate cell polarity? The clearest data are from budding yeast, in which it has been shown that Cdc24p, a specific guanine nucleotide exchange factor (GEF) for Cdc42, is anchored in the nucleus by Far1p until the G1 phase of the cell cycle, at which time Cdc28p-Cln-dependent destruction of Far1p allows Cdc24p to exit the nucleus and localize to the nascent bud, where it is tethered to the adaptor Bem1p.3 The polarized distribution of Cdc24p provides a mechanism by which Cdc42p can become activated in an asymmetric manner to a location that is in some ways analogous to the leading edge of a mammalian cell. Interestingly, in yeast, Cdc42p activates Cla4p (a protein kinase of the p21-activated kinase (Pak) family), which then phosphorylates and inactivates Cdc24p, releasing it from Bem1p.4 Thus, the polarized distribution of Cdc42p is tightly linked to the cell cycle and is achieved by shuttling of a specific GEF from the nucleus to a cytoplasmic adaptor protein that is positioned at the site of bud emergence. The location of Cdc42p activation in mammalian cells is uncertain, though indirect tests using antibodies that specifically recognize an activated form of Pak1 are consistent with activation of the GTPase at leading edge filopodia.5 More direct studies,

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Figure 1. Generalized model for cellular motility. The aspects of motility can be divided into four stages. Here a resting cell has established contacts with the extracellular matrix are represented in stage one. Initiation of motility results from lamellopodial extension followed by the transient formation of focal complexes at the lading edge of the cell, as demonstrated in stage two. Dissolution of preexisting focal complexes at the cells trailing edge occurs at stage three followed by the generation of contractive forces to promote the motility of the trailing edge as demonstrated in stage four. As these distinctions are static representations of a dynamic process, they are not meant to imply that each stage requires the completion of previous stages, for example the dissolution of focal adhesions at the cells trailing edge and the generation of contractile forces may occur simultaneously rather than sequentially. In all stages, green circles represent polymerized actin.

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Figure 2. Establishment and maintenance of polarity. Upon the addition of growth factors this idealized cell imitates polarization by promoting actin proliferation and membrane protrusion at the cells leading edge. This process occurs rapidly and establishes cellular polarity. The maintenance of polarity requires the reorganization of the MTOC towards the initial site of polarization. This polarization is thought to provide continued deliverance of both signalling and structural components required for membrane protrusion in the form of intracellular vesicles.

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using fluorescence resonance transfer techniques, should be useful in determining the location of activated Cdc42 in mammalian cells.6,7 The connections that link Cdc42 activation to changes in cell polarity are currently a topic of intense interest. Cdc42 triggers reorientation of the microtubule organizing center (MTOC) in fibroblasts8 and in astrocytes9 and Cdc42 function has been shown to be required for such reorientation in these cells and in T cells.10 MTOC reorientation does not depend on changes in actin organization, but does require dynein function.8 The link from Cdc42 to dynein may be provided by a cytoplasmic mPar6/PKCζ complex9 though the precise details of this mechanism remain to be established. Cdc42 effector domain mutants that are defective in recruiting Cdc42/Rac interactive binding (CRIB)-containing proteins are still able to stimulate MTOC reorientation, suggesting that effectors such as Wiskott Aldrich Syndrome Protein (WASP) and Pak are not involved in this process, whereas over expression of mPAR6 completely blocked MTOC reorganization. However, the interpretation of these results need to be viewed with caution, as particular isoforms of WASP and Pak (i.e., N-WASP and Pak4) are still activated by the Cdc42 effector domain mutants that were used in these studies.11 It should also be noted that, in budding yeast, both the WASP homolog Bee1p (via effects on the Arp2/3 complex), and the Pak homolog Cla4p (via phosphorylation of myosin I), play a definite role in Cdc42-regulated cell polarity.12 Based on data from wound healing models for cell motility, it has been suggested that Cdc42 is required for two independent processes, one which involves the reorientation of the MTOC towards the wound, and the other is to activate Rac which then promotes localized actin polymerization that drives membrane protrusion.9 This latter process is thought to be aided by the delivery of membrane components along microtubules towards the leading edge, a process that requires the recycling of membrane components and microfilament motors. A role for Rho family GTPases in these processes has been identified in S. cerevisiae budding yeast. As S. cerevisiae buds in a polarized fashion, it requires the ability to direct exocytic vesicles to the sites of active membrane protrusion. It is thought that post Golgi vesicles migrate along microfilaments driven my myosin motors where they dock at the appropriate site of the plasma membrane by association with a multi-subunit complex known as the exocyst. Once localized to the appropriate region the vesicles fuse to the plasma membrane. Although members of the Rab GTPase family have long been associated with defects in secretion (reviewed in ref. 13) recent evidence suggests that both Rho1p and Cdc42p, are required for proper exocyst localization13,14 in yeast and that a component of the exocyst, Sec3p, is capable of binding to either GTP loaded Cdc42p or Rhop. It is interesting to note that, in mammalian cells, Cdc42 is present in membranes of the Golgi apparatus, which is known to reorient towards the leading edge following wounding. Perhaps, by analogy to the yeast system, membrane vesicles in mammalian cells are directed by Cdc42 to the sites of membrane protrusion. The mechanism by which Cdc42 might drive such a process is not known, but one attractive target are the BORG proteins, which have recently been shown to bind Cdc42 and also septins.15 Septins are known to regulate cytokinesis, cell polarity, and vesicular transport and, as such make attractive targets for future studies. A model for the initiation and maintenance of polarity is presented in (Fig. 2).

Actin Polymerization at the Leading Edge Once polarity has been established, the next requirement for motility is plasma membrane protrusion. In Swiss 3T3 cells injection of activated Rac or platelet-derived growth factor (PDGF) treatment initiates cell protrusion in the form of lamellipodia at the cell’s leading edge.16,17 Examination of these structures demonstrates that they are regions of extensive actin polymerization and it is believed that polymerization is the driving force for membrane protrusion at these sites (reviewed in ref. 19). For this reason, the effectors that link the Rac to actin polymerization and assembly have been intensely studied. To date, three major pathways have been uncovered: the regulation of Arp2/3 activity by WASP family verprolin-homologous protein

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(WAVE); the regulation of cofilin and myosin light chain kinase (MLCK) by Pak; and the regulation of actin capping proteins by PIP2 (reviewed in ref. 20).

WASP/WAVE and Arp2/3 The seven-protein complex known as the Arp2/3 complex is the major regulator of de novo actin assembly in eukaryotes. Arp2/3 dependent polymerization of actin is strongly stimulated by proteins of the WASP and WAVE (a.k.a. SCAR) family.18 WASPs contain a conserved Cdc42/Rac1 interactive binding (CRIB) domain that is specific for Cdc42, establishing these proteins as downstream effectors of this GTPase19 (reviewed in ref. 23). Although the related WAVE proteins lack a CRIB domain, they are linked to Rac. Here, the connection to the GTPase is indirect, mediated by the docking protein IRSp53.20 The activation of WASP and WAVE proteins by Cdc42 and Rac, respectively, provides a major link between these small GTPases and actin polymerization, and hence to cell motility. As WASP and WAVE are major regulators of Arp2/3 activity, a brief look at how Cdc42 activates WASP is warranted. In the absence of GTP loaded Cdc42, WASP adopts a conformation in which its CRIB domain forms an intermolecular association with the VA domain, which, when unmasked, is responsible for activation of Arp2/3.21 Upon binding of GTP loaded Cdc42, the C-terminal portion of WASP, including the VA domain, is relieved of the intermolecular inhibition, and is free to bind actin and to activate Arp2/3. The dynamics of WAVE activation are likely to differ from WASP in some respects, as its interaction with Rac is mediated by a third partner, but the structure of WAVE makes it likely that it operates along similar principles as WASP. It is intriguing that this method of transducing signals from GTPases through the relief of autoinhibition of downstream effectors is common to some of the best characterized small GTPase effectors, including WASP, Pak, and the protein kinase Raf that acts downstream of Ras. The importance of WASP/WAVE proteins to cell motility is underscored by in vitro reconstitution experiments using purified components. These experiments show that WASP, Arp2/ 3, cofilin, and capping protein, are sufficient to drive actin ‘rocketing.22 Other factors that modulate actin assembly, such as profilin, VASP and α-actinin, are not essential for in vitro motility, but either increase the speed of rocketing or its persistence.22 Based on these observations, the authors proposed a model for expansion of the lamellipodia in which active Rac binds to and activates WAVE which promotes the formation of actin nucleation via Arp2/3. This nucleation restricted to the lamellipodial edge through a conserved region of WAVE called the SHD for Scar Homology Domain, as this region is sufficient to target GFP to the same region of the lamellipod as full length SCAR.23 Following the initial stimulation by Rac, continued stimulation of Arp2/3 might be provided by the ENA/VASP family proteins that are known to affect actin polymerization.24,25

The Role of Pak Paks are a highly conserved family of protein kinases that are activated by Cdc42 and Rac. Overexpression of Paks affects cell morphology26 and motility5,27 and experiments using activation specific antibodies demonstrate that Pak1 is activated at the leading edge of motile cells.5 The ability of Paks to act as GTPase effectors for actin organization is reasonably well established, but its mechanisms of action are not well defined. In part, this stems from the observation that Pak has both kinase-dependent and kinase independent activities.26,28,29 In the last three years, a great deal of progress has been made in explaining these effects, through identification of Pak substrates and binding partners that are relevant to the cytoskeleton. Both Pak1 and Pak4 have been shown to phosphorylate and subsequently activate LIM kinase.30,31 As a consequence of phosphorylation by LIM kinase, cofilin is inactivated and as cofilin severs preexisting actin filaments and promotes actin disassembly. In this way, Pak activation might play a key role in the formation and/or stabilization of actin-based protrusions. Pak1 has also been shown to phosphorylate, and inactivate, MLCK.32 This action in turn leads

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Figure 3. Regulation of actin polymerzation by Rac. Rac is though to affect actin polymerzation through three pathways. In conjunction with IRSp53, Rac activates WAVE that directly activates the Arp2/3 complex required for de novo actin polymerization. Additionally Rac activates the protein kinase Pak that phosphorylates two substrates, LIM kinase and myosin light chain kinase. Activation of LIMK by Pak inhibits the ability of cofilin to promote severing and or depolymerization of actin filaments. Inhibition of MLCK by Pak leads to decreased levels of phosphorylated myosin and inhibits the formation of contractile filaments known as stress fibers. A third pathway is through the activation of PI5 kinase which leads to the production of the second messenger PIP2. PIP2 is known to inhibit the function of capping proteins, which results in the generation of free ends required for prolonged actin polymerization.

to decreased MLC activity. Presumably, this occurs in a spatially restricted manner at the leading edge, as myosin activity is required at the trailing edge of the cell to generate tractile force in the later stages of movement (see below). A third substrate for Pak has recently been described by Daub et al, who showed that this kinase phosphorylates and inhibits stathmin.33

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Stathmin, in its unphosphorylated state, destabilizes microtubules, and the inhibition of this effect by Pak suggests that, in addition to its effects on actin, Pak might also regulate microtubule dynamics. Pak may also play a second, a kinase-independent role, through the recruitment of signalling molecules to sites of membrane protrusion. A kinase-dead mutant of Pak1 is sufficient to promote large lamellipodia in Swiss 3T3 cells5 and Pak associates with a number of important signalling molecules, including the adaptor protein Nck (which binds receptor tyrosine kinases via its SH2 domain and to WASP via one of its three SH3 domains) and also the GEF PIX. Binding of Pak to Nck could put Pak in proximity to receptor tyrosine kinases and to WASP at the cell surface and recruit both the GTPase as well as the exchange factors that activate GTPase to result in sustained activation of downstream effectors. Finally, Rac also influences actin protrusion via stimulating the production of PIP2. Rac has been reported to associate with PI-4,5 kinase (PI5K), an enzyme which produces PIP2 from precursor phosphatidylinositides.34,35 As discussed in more detail below, the production of PIP2 has a profound effect on actin polymerization due to uncapping of actin filaments (reviewed in ref. 36). Thus, the stimulation of polymerization by Rac via PI5K provides a third link between this GTPase and the formation of actin-based protrusions (Fig. 3).

Remodeling Focal Contacts In order to promote motility the cell must reorganize its relationship to the extracellular matrix, or in the case of multi-cellular tissues, to other cells. The predominant link between a cell and the ECM are the focal adhesions; complexes of membrane spanning integrins and a variety of associated proteins such as vinculin, paxillin, talin, and several signalling molecules such as focal adhesion kinase. The establishment of focal adhesions directly links the extracellular matrix to the actin cytoskeleton, as vinculin is known to bind to both talin as well as filamentous actin. Of the small GTPases known to regulate focal adhesion formation, Rho is of primary importance. Constitutively active alleles of Rho increase the appearance of focal adhesions and inhibit motility. It is thought that Rho promotes focal adhesions mainly through stimulation of PI5K;37 the resulting increase in PIP2 is thought to promote a conformational change in vinculin that allows it to bind to both talin and actin,38 enabling the linking of the cytoskeleton to the extracellular matrix. Although Rho is known to be the predominate GTPase regulating the assembly of focal adhesions, it is not the only one. When cells extend filopodia or lamellipodia during movement, these new processes must also make transient contacts with the extracellular matrix in order to generate a traction force to allow the rest of the cell body to follow (see Fig. 1). A description of these forces is presented in the recent work of Beningo et al.39 This process is very much akin to a treadmill, where protrusions at the leading edge form transient contacts that eventually mature into focal adhesions, whereas at the trailing edge of the cell, the contacts to the ECM are dissolved. In this manner, the focal contacts established remain immotile with respect to the ECM, but the cells ability to form new contacts and dissolve old contacts allows it to move with respect to the substratum. As noted previously, treatment of confluent starved Swiss 3T3 cells with PDGF induces the formation of lamellipodia.16 Such protrusions are rich in vinculin-containing complexes that are reminiscent of focal adhesions. These PDGF-induced complexes however, do not have the classical rhomboid staining associated with focal adhesions and are formed independently of Rho. These complexes do require Rac, however, suggesting that both Rac and Rho can independently induce vinculin clustering.16 If both Rho and Rac can induce a similar phenotype, what is difference between these complexes? Rac dependent focal complexes, like Rho-dependent focal adhesions, contain both paxillin and focal adhesion kinase, suggesting that these two complexes are related.16 While molecular differences, if any, between these two structures have not been convincingly demonstrated, the distinction has been useful to describe the different morphologies and to relate them to their dependence on Rac or Rho activity. The most rel-

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evant effector in this instance may be PI5K, which is recruited by both Rac and Rho, and which induces (via PIP2) the conformational changes in vinculin required for focal adhesion assembly. Supporting this model is the fact that Cdc42 is not known to stimulate PI5K, and is not known to promote focal complex or adhesion assembly in the absence of Rac.16 In addition to the formation of new focal complexes by activated Rac, the cell must also dissolve preexisting focal adhesions at the trailing edge of the cell in order to regulate movement. Clustering of focal adhesion components might allow cells to create temporary sites of attachment to provide traction to motile cells.17 Experiments using time-lapse microscopy have demonstrated that assembly of focal contacts at the leading edge contain integrins. Although down regulation of focal adhesions is required for cell motility, this is not simply a dissolution of the preexisting complexes, as complexes formed at the leading edge of cells continue to contain integrins attached to the ECM after the cells have bypassed the position with respect to the substratum. This suggests that integrin release from the substratum is not required for motility of some cell types.40 In other cell lines, it has been shown that a functional microtubule network is required for focal contact dissociation. In fibroblasts, the focal complexes that form at the lamellipodia are generally recycled as they approach the perinuclear region.41 When the microtubule network is depolymerized by the addition of nocodazole, these complexes are far more stable and can persist even at the very trailing edge of the cell.42 These results are consistent with the idea that proper microtubule organization is required for focal complex turnover. Even more surprising was the observation that directional motility in cells with disrupted microtubules could be restored by simultaneously locally inhibiting MLCK, a component of the acto-myosin motor. Disruption of microtubules has long been known to prevent polarization in cells and to have concomitant effects on the actin cytoskeleton. However, cells treated locally with an MLCK inhibitor (ML-7) displayed a profound polarization opposite to the site of addition, and the lamellipodia that formed appeared normal. These results suggest that not only is a microtubule network required for focal adhesion disassembly, but that microtubules are not required for the establishment of polarity and that local actin dynamics are unperturbed and responsible for lamellipodial protrusion.42 These sets of experiments clearly demonstrate a relationship between the protrusive activity of the leading edge and the retractile forces at the cells trailing edge. Rho function is critical to the relationship between the creation of new adhesions and the dissolution of preexisting ones. Rho is known to mediate focal adhesion assembly, in part due to its stimulation of PI5K and its resulting effect on vinculin. However a role for Rho in the dissolution of focal adhesions has recently been reported.43 Using time-lapse microscopy and transduced proteins to modulate leukocyte migration, the authors demonstrated that in addition to being required for focal adhesions, Rho, and its downstream effector kinase Rho-kinase (ROK or ROCK) is also required for the dissolution of preexisting focal adhesions. Leukocytes that retained the ability to promote leading edge migration but were unable to promote dissociation of the preexisting focal adhesions at the rear of the cell, displayed defects in motility.43 That this effect was mediated by the Rho was discerned by three lines of evidence; 1) inhibition of Rho by C3 exoenzyme or inhibition of ROK by the specific antagonist Y27632 prevented uropod release, 2) Transduction of cells with constitutively active forms of Rho allowed for uropod release in the absence of serum and transduction of dominant negative alleles of Rho (RhoN19) prevented uropod release that is normally seen by the addition of serum.43 It appears therefore that it is both the activation level of Rho and its cellular location that determines the cytoskeletal effects: at the leading edge activation of Rho leads to the formation of focal complexes and focal adhesions, and in the uropod activation of Rho can lead to the dissolution of preexisting focal adhesions and stress fiber formation. These combined activities allow for the contractile forces required for motility. Whether this represents a difference between fibroblasts and leukocytes or represents a more generalized mechanism remains to be elucidated.

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The Generation of Contractile Force Of the three common GTPases, Rho is most often associated with the production of stress fibers. Stress fibers are highly bundled actin filaments associated with myosin and actin cross-linking proteins such as α-actinin and are believed to mediate the predominant contractile force in cells. In order to exert contractile force both ends of stress fibers must be fixed to different cellular components. Stress fibers are known to terminate at focal adhesions through binding to one, or many, components of focal adhesions (reviewed in ref. 44), thus providing a link between the actin cytoskeleton and the ECM.45-47 As Rho is known to regulate focal adhesion assembly, it is not surprising that it also coordinately promotes the formation of stress fibers. Delineating the effects of Rho on stress fiber formation has relied greatly on the use of the bacterial C3 toxin that specifically inhibits Rho through ribosylation,48 but does not affect either Cdc42 or Rac. As a result of this specificity, it is likely that Rho is the best characterized of these GTPases, and unlike either Rac or Cdc42, it appears that the majority of the effects on stress fiber formation and a focal adhesion formation depends almost entirely on one of three effectors, ROKα, mDia, and to a lesser extent, PI5K. ROK and mDia, a member of the evolutionarily conserved diaphanous-related formins,49-52 associate with GTP loaded Rho.53-55 The combination of constitutively active ROK and mDia is sufficient to restore stress fiber formation in cells whose Rho function had been ablated by C3 treatment.56 It is thought that these effectors regulate cytoskeleton reorganization as follows. Activated mDia binds profilin, a known actin binding protein that affects actin polymerization in vitro and which is thought to control the levels of free monomeric actin available for polymerization in vivo. The ability of ROK to modulate cytoskeletal structures is thought to result from its ability to phosphorylate two downstream effectors, myosin phosphatase and LIM kinase. Phosphorylation of myosin phosphatase is known to inhibit its ability to dephosphorylate myosin.52,57-61 As MLC phosphorylation is required for interaction between actin and myosin fibers in non muscle cells61 and for maximal actin-activated ATPase activity of myosin II,62 a preliminary model for how Rho affects contractility can be generated; Rho activates ROK which then phosphorylates myosin phosphatase and inactivates it. As a result of inactivating myosin phosphatase, the relative levels of the phosphorylated regulatory MLC increases, promoting contractility and the assembly of stress fibers. That contractility regulates the formation of stress fibers is shown by MLCK inhibitors such as KT5976, that inhibit contractility and dissolve preexisting stress fibers and focal adhesions.63 In addition to regulating myosin phosphorylation, ROK activates LIM kinase, leading to phosphorylation of the actin depolymerizing protein cofilin. This phosphorylation prevents cofilin from binding to actin.64 As the major role of cofilin in cells is to promote cleavage of polymerized actin structures and to promote selective depolymerization of existing actin structures,65 activation of ROK can therefore promote the assembly of actin filaments into contractile fibers as well as stabilize these fibers by preventing internal cleavage. The second major effector of Rho, mDia, is though to promote stress fibers in a different manner. mDia is known to bind to the actin-binding protein profilin, and is thought to regulate local actin polymerization in vivo through regulating the activity of profilin.53 Recently a novel mDia interacting protein called DIP (diaphanous-interacting protein) was isolated as a two-hybrid partner of mDia, was subsequently shown to be involved in mDia stress fiber formation, and in the activation of v-Src at focal contacts.66 The region of mDia known to be involved in profilin binding, the FH1 region, is also the same region that is known to promote binding o both v-Src and DIP, prompting the question as to whether the mDia binding partners compete for binding. Although not as well elucidated as the pathways downstream of ROK, and to a lesser extent mDia, another Rho effector known to promote actin fiber bundling is PI5K and its second messenger product PIP2. As mentioned previously in this chapter, it has been suggested that Rho activates PI5K to generate PIP2,67 although whether this effect is direct or through ROK remains an unanswered question.68 Irrespective of the mechanism, activation of Rho increases

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Figure 4. Generation of contractile force and focal adhesions by activation of Rho. Activation of Rho leads to three specific effects. The activation of the diaphanous related protein mDia that is known to activate profilin and also has a role in microtubule dynamics. Regulating the effects of profilin promotes actin polymerization. In the second pathway, Rho activates ROCK that simultaneously activates LIMK and inhibits myosin light chain phosphatase. The net effect of activation of ROCK is to decrease the ability of cofilin to sever and or depolymerize preexisting acting filaments and decrease contractility by increasing the extent of myosin phosphorylation. A third mechanism in which Rho activates focal adhesion formation is through the stimulation of PI5K, which leads to increased levels of the second messenger PIP2, which is known to promote conformational changes in vinculin as well as affecting actin polymerization. This model does not take into account the subcellular localization of these activities, which is likely of great importance in determining the spatio-temporal activation of each of these pathways.

PIP2 levels. This increase in PIP2 decreases the affinity of actin-destabilizing proteins for actin, while increasing the affinity of ezrin; a protein required for reconstitution of Rho dependent stress fibers in permeabilized cells.69 Independently of affecting proteins that regulate preexist-

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ing actin filaments, PIP2 also stimulates N-WASP, which, via the Arp2/3 complex, results in increased de novo synthesis of actin fibers (reviewed in ref. 70). A model describing how Rho modulates stress fiber formation and contractility is presented in (Fig. 4).

Summary While much remains to be learned, considerable headway has been made in the last few years regarding the signalling machinery that governs cell movement. All four phases of movement, generation of polarity, actin protrusion, changes in adhesion, and generation of force, are strongly influenced by Rho GTPases, and, in many cases, the outlines of the effector pathways have been delineated. These pathways are strongly conserved in evolution, and impact proteins that control both the actin and tubulin cytoskeleton. The challenge in the coming years will be to integrate these findings into a cohesive picture that explains the elegant choreography of the motile cell.

References 1. Chant J. Cell polarity in yeast. Annu Rev Cell Dev Biol 1999; 15:365-391. 2. Chen F, Ma L, Parrini MC et al. Cdc42 is required for PIP(2)-induced actin polymerization and early development but not for cell viability. Curr Biol 2000; 10(13):758-765. 3. Shimada Y, Gulli MP, Peter M. Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating. Nat Cell Biol 2000; 2(2):117-124. 4. Gulli M, Jaquenoud M, Shimada Y et al. Phosphorylation of the cdc42 exchange factor cdc24 by the PAK-like kinase cla4 may regulate polarized growth in yeast. Mol Cell 2000; 6(5):1155-1167. 5. Sells MA, Pfaff A, Chernoff J. Temporal and spatial distribution of activated Pak1 in fibroblasts. J Cell Biol 2000; 151(7):1449-1458. 6. Mochizuki N, Yamashita S, Kurokawa K et al. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 2001; 411(6841):1065-1068. 7. Graham DL, Lowe PN, Chalk PA. A method to measure the interaction of Rac/Cdc42 with their binding partners using fluorescence resonance energy transfer between mutants of green fluorescent protein. Anal Biochem 2001; 296(2):208-217. 8. Palazzo AF, Joseph HL, Chen Y et al. Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization. Curr Biol 2001; 11(19):1536-1541. 9. Etienne-Manneville S, Hall A. Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell 2001; 106(4):489-498. 10. Stowers L, Yelon D, Berg LJ et al. Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42. Proc Natl Acad Sci USA 1995; 92(11):5027-5031. 11. Abo A, Qu J, Cammarano MS et al. PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia. Embo J 1998; 17(22):6527-6540. 12. Lechler T, Jonsdottir GA, Klee SK et al. A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast. J Cell Biol 2001; 155(2):261-270. 13. Guo W, Tamanoi F, Novick P. Spatial regulation of the exocyst complex by Rho1 GTPase. Nat Cell Biol 2001; 3(4):353-360. 14. Zhang X, Bi E, Novick P et al. Cdc42 interacts with the exocyst and regulates polarized secretion. J Biol Chem 2001; 10:10. 15. Joberty G, Perlungher RR, Sheffield PJ et al. Borg proteins control septin organization and are negatively regulated by Cdc42. Nat Cell Biol 2001; 3(10):861-866. 16. Nobes CD, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995; 81(1):53-62. 17. Rottner K, Hall A, Small JV. Interplay between Rac and Rho in the control of substrate contact dynamics. Curr Biol 1999; 9(12):640-648. 18. Miki H, Suetsugu S, Takenawa T. WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac. Embo J 1998; 17(23):6932-6941. 19. Miki H, Sasaki T, Takai Y et al. Induction of filopodium formation by a WASP-related actindepolymerizing protein N-WASP. Nature 1998; 391(6662):93-96. 20. Miki H, Yamaguchi H, Suetsugu S et al. IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature 2000; 408(6813):732-735.

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21. Kim AS, Kakalis LT, Abdul-Manan N et al. Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein. Nature 2000; 404(6774):151-158. 22. Loisel TP, Boujemaa R, Pantaloni D et al. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 1999; 401(6753):613-616. 23. Nakagawa H, Miki H, Ito M et al. N-WASP, WAVE and Mena play different roles in the organization of actin cytoskeleton in lamellipodia. J Cell Sci 2001; 114(Pt 8):1555-1565. 24. Laurent V, Loisel TP, Harbeck B et al. Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes. J Cell Biol 1999; 144(6):1245-1258. 25. Bear JE, Krause M, Gertler FB. Regulating cellular actin assembly. Curr Opin Cell Biol 2001; 13(2):158-166. 26. Sells MA, Knaus UG, Bagrodia S et al. Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr Biol 1997; 7(3):202-210. 27. Kiosses WB, Daniels RH, Otey C et al. A role for p21-activated kinase in endothelial cell migration. J Cell Biol 1999; 147(4):831-844. 28. Daniels RH, Hall PS, Bokoch GM. Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells. Embo J 1998; 17(3):754-764. 29. Wang J, Frost JA, Cobb MH et al. Reciprocal signalling between heterotrimeric G proteins and the p21- stimulated protein kinase. J Biol Chem 1999; 274(44):31641-31647. 30. Dan C, Kelly A, Bernard O et al. Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin. J Biol Chem 2001; 276(34):32115-32121. 31. Edwards DC, Sanders LC, Bokoch GM et al. Activation of LIM-kinase by Pak1 couples Rac/ Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol 1999; 1(5):253-259. 32. Sanders LC, Matsumura F, Bokoch GM et al. Inhibition of myosin light chain kinase by p21-activated kinase. Science 1999; 283(5410):2083-2085. 33. Daub H, Gevaert K, Vandekerckhove J et al. Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16. J Biol Chem 2001; 276(3):1677-1680. 34. Anderson RA, Boronenkov IV, Doughman SD et al. Phosphatidylinositol phosphate kinases, a multifaceted family of signalling enzymes. J Biol Chem 1999; 274(15):9907-9910. 35. Toker A. The synthesis and cellular roles of phosphatidylinositol 4,5- bisphosphate. Curr Opin Cell Biol 1998; 10(2):254-261. 36. Pollard TD, Blanchoin L, Mullins RD. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct 2000; 29:545-576. 37. Schoenwaelder SM, Burridge K. Bidirectional signalling between the cytoskeleton and integrins. Curr Opin Cell Biol 1999; 11(2):274-286. 38. Critchley DR. Focal adhesions - the cytoskeletal connection. Curr Opin Cell Biol 2000; 12(1):133-139. 39. Beningo KA, Dembo M, Kaverina I et al. Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts. J Cell Biol 2001; 153(4):881-888. 40. Regen CM, Horwitz AF. Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts. J Cell Biol 1992; 119(5):1347-1359. 41. Izzard CS, Lochner LR. Formation of cell-to-substrate contacts during fibroblast motility: An interference-reflexion study. J Cell Sci 1980; 42:81-116. 42. Kaverina I, Krylyshkina O, Gimona M et al. Enforced polarisation and locomotion of fibroblasts lacking microtubules. Curr Biol 2000; 10(12):739-742. 43. Alblas J, Ulfman L, Hordijk P et al. Activation of rhoa and rock are essential for detachment of migrating leukocytes. Mol Biol Cell 2001; 12(7):2137-2145. 44. Burridge K, Chrzanowska-Wodnicka M. Focal adhesions, contractility, and signalling. Annu Rev Cell Dev Biol 1996; 12:463-518. 45. Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992; 70(3):389-399. 46. Nusrat A, Giry M, Turner JR et al. Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia. Proc Natl Acad Sci USA 1995; 92(23):10629-10633. 47. Yamamoto M, Morii N, Ikai K et al. Effect of botulinum C3 exoenzyme on cell growth and cytoskeleton organization in transformed human epidermal cells in culture: A possible role for rho protein in epidermal cells. J Dermatol Sci 1994; 8(2):103-109. 48. Morii N, Teru-uchi T, Tominaga T et al. A rho gene product in human blood platelets. II. Effects of the ADP- ribosylation by botulinum C3 ADP-ribosyltransferase on platelet aggregation. J Biol Chem 1992; 267(29):20921-20926. 49. Ishizaki T, Naito M, Fujisawa K et al. p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett 1997; 404(2-3):118-124.

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50. Narumiya S, Ishizaki T, Watanabe N. Rho effectors and reorganization of actin cytoskeleton. FEBS Lett 1997; 410(1):68-72. 51. Sahai E, Ishizaki T, Narumiya S et al. Transformation mediated by RhoA requires activity of ROCK kinases. Curr Biol 1999; 9(3):136-145. 52. Maekawa M, Ishizaki T, Boku S et al. Signalling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 1999; 285(5429):895-898. 53. Watanabe N, Madaule P, Reid T et al. p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. Embo J 1997; 16(11):3044-3056. 54. Leung T, Chen XQ, Manser E et al. The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol 1996; 16(10):5313-5327. 55. Leung T, Manser E, Tan L et al. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem 1995; 270(49):29051-29054. 56. Watanabe N, Kato T, Fujita A et al. Cooperation between mDia1 and ROCK in Rho-induced actin reorganization. Nat Cell Biol 1999; 1(3):136-143. 57. Ohashi K, Nagata K, Maekawa M et al. Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop. J Biol Chem 2000; 275(5):3577-3582. 58. Kosako H, Yoshida T, Matsumura F et al. Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow. Oncogene 2000; 19(52):6059-6064. 59. Takaishi K, Matozaki T, Nakano K et al. Multiple downstream signalling pathways from ROCK, a target molecule of Rho small G protein, in reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells. Genes Cells 2000; 5(11):929-936. 60. Shimizu Y, Dobashi K, Iizuka K et al. Contribution of small GTPase Rho and its target protein rock in a murine model of lung fibrosis. Am J Respir Crit Care Med 2001; 163(1):210-217. 61. Kimura K, Ito M, Amano M et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho- kinase). Science 1996; 273(5272):245-248. 62. Adelstein RS, Conti MA, Daniel JL et al. The interaction of platelet actin, myosin and myosin light chain kinase. Ciba Found Symp 1975; 35:101-109. 63. Chrzanowska-Wodnicka M, Burridge K. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol 1996; 133(6):1403-1415. 64. Moriyama K, Iida K, Yahara I. Phosphorylation of Ser-3 of cofilin regulates its essential function on actin. Genes Cells 1996; 1(1):73-86. 65. Moriyama K, Yahara I. Two activities of cofilin, severing and accelerating directional depolymerization of actin filaments, are affected differentially by mutations around the actin-binding helix. Embo J 1999; 18(23):6752-6761. 66. Satoh S, Tominaga T. mDia-interacting protein acts downstream of rho-mdia and modifies src activation and stress fiber formation. J Biol Chem 2001; 276(42):39290-39294. 67. Chong LD, Traynor-Kaplan A, Bokoch GM et al. The small GTP-binding protein Rho regulates a phosphatidylinositol 4- phosphate 5-kinase in mammalian cells. Cell 1994; 79(3):507-513. 68. Yamamoto M, Hilgemann DH, Feng S et al. Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells. J Cell Biol 2001; 152(5):867-876. 69. Mackay DJ, Esch F, Furthmayr H et al. Rho- and rac-dependent assembly of focal adhesion complexes and actin filaments in permeabilized fibroblasts: An essential role for ezrin/radixin/moesin proteins. J Cell Biol 1997; 138(4):927-938. 70. Higgs HN, Pollard TD. Regulation of actin filament network formation through Arp2/3 complex: Activation by a diverse array of proteins. Annu Rev Biochem 2001; 70:649-676.

CHAPTER 10

The RhoGTPase in Tumor Invasion Arthur M. Mercurio

Abstract

T

he progression from benign disease to invasive carcinoma is a complex and dynamic process that involves profound changes in the adhesive interactions and cytoskeletal organization of premalignant cells. Such changes culminate in the acquisition of cell motility and the ability of carcinoma cells to penetrate tissue compartments. Given the importance of the Rho family GTPases in the regulation of the cytoskeleton and cell migration, it is not surprising that their involvement in tumor cell motility and invasion has received considerable attention. Although most studies have focused on Rac in this context because of its established role in lamellipodia formation and migration, recent studies have suggested an important role for Rho itself in tumor invasion and progression. The contribution of Rho to tumor progression is substantiated by several reports indicating that the expression of specific Rho isoforms is elevated in invasive and mestastatic tumors. Studies that attempt to define a mechanistic role for Rho in migration and invasion, however, are fragmented and somewhat contradictory. This review attempts to synthesize these studies into a comprehensive model and highlight important areas for future work.

Introduction Invasion, or the penetration of tumor cells into adjacent tissues, is one of the hallmarks of malignant tumors.1 In contrast to benign tumors that are encapsulated, invasive tumors have the potential to metastasize because of their access to lymphatics and the vasculature. The fact that most human cancers are carcinomas and arise from epithelia, highlights the importance of epithelial cell biology in understanding cancer progression. In this context, a key difference between benign and invasive carcinomas is that invasive carcinoma cells often lose contact with each other and exhibit a mesenchymal or migratory phenotype that is distinct from normal epithelial structure.2-4 This realization led to the hypothesis that an epithelial to mesenchymal transition is a major component of the invasive process. Subsequent studies on the loss of cadherin-mediated cell-cell adhesion in invasive carcinomas established a mechanistic basis for this epithelial to mesenchymal transition.5-7 More recent data indicate that cadherin-mediated signalling and not cadherin-mediated adhesion is important for suppressing the invasive phenotype.8 Cell migration is an important component of invasion because numerous experimental observations indicate that the translocation of tumor cells from one tissue compartment to another (e.g., epithelium to stroma) involves active cell movement.9-12 For this reason, a detailed understanding of the mechanisms that underlie the migration and invasion of tumor cells is essential. Given the importance of the Rho family GTPases Rho, Rac and cdc42 in the regulation of the cytoskeleton and cell migration,13-14 it is not surprising that their involvement in tumor invasion and metastasis has received considerable attention.

Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Many studies on the contribution of Rho family GTPases to invasion have highlighted Rac because of its established role in lamellipodial formation and cell migration. Indeed, the contribution of Rac to migration and invasion has been a focus of several excellent reviews15-18 and will not be emphasized here. A more intriguing GTPase, however, is Rho itself. Data derived largely from studies on fibroblasts have established a function for Rho in stress fiber formation and focal adhesion assembly and a more tangential role in cell migration.13 As will be discussed below, recent studies have provided evidence that the expression of specific Rho isoforms is elevated in invasive and metastatic tumors. These findings are substantiated by other work that has provided insight into possible mechanisms by which Rho may be linked to invasion. Although the functions of Rho family GTPases are inter-connected, studies on the involvement of Rho itself in tumor migration and invasion are fragmented and somewhat contradictory. This review attempts to synthesize these studies into a comprehensive model and highlight important areas for future work.

RHO Involvement in Tumor Progression Evidence from the Analysis of Solid Tumors to Implicate Rho in Progression Initial evidence to support the involvement of specific genes and proteins in tumor progression is often obtained by comparing their expression in tumor tissue relative to either benign lesions or normal tissue. This approach has yielded sufficient data to implicate specific Rho isoforms in progression and to justify more mechanistic studies. For example, an analysis of RhoA, Rac and cdc42 proteins in colon, lung and breast tumors revealed an increase in their expression in comparison to corresponding normal tissue.19 Also, a study of genes expressed in highly metastatic rat osteosarcomas relative to low metastatic tumors identified the rat homologue of the Drosophila melanogaster diaphanous gene (Dia).20 This finding is of interest because Dia is an effector of Rho involved in actin polymerization and microtubule stability.21-22 Several studies have noted enhanced expression of RhoC in a range of tumors. Suwa et al reported that increased expression of the RhoC gene correlates with the progression of ductal adenocarcinoma of the pancreas.23 Moreover, the report that RhoC expression is elevated in inflammatory breast carcinomas is particularly interesting because this type of breast cancer is highly aggressive.24-25 Perhaps, the most definitive and unbiased study to date involved the use of a microarray-based screen for genes that are expressed preferentially in metastatic melanomas.26 This screen identified RhoC as one such gene. These studies not only reveal a potentially important role for RhoC in tumor progression but they also highlight the need to define possible functional differences between RhoA and RhoC. There is also a need to define at which steps of the metastatic cascade Rho activity is most critical. The melanoma study, for example, implicated RhoC in migration and invasion.

Evidence from in Vitro Studies to Implicate Rho in Tumor Migration and Invasion Given the central importance of RhoGTPases to cell function and the availability of reagents to study these molecules, it is not surprising that papers abound on the role of RhoGTPases in processes that are linked to tumor progression, especially migration and invasion. My comments here will emphasize those studies that have provided the most compelling evidence to date to support the involvement of Rho itself and its effectors in these processes. A role for Rho in invasion has been established primarily by the work of Akedo, Itoh and their colleagues.27-34 Initially, they reported that the serum-stimulated invasion of rat MM1 hepatoma cells could be inhibited by C3 transferase and that expression of activated Rho (Val 14) promoted invasion in the absence of serum. The involvement of RhoA in invasion was attributed to its ability to stimulate actomyosin contraction. Subsequent work by this group identified Rho-associated kinase (ROCK), an effector of Rho, as being essential for invasion.28

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Importantly, they demonstrated that expression of activated ROCK in MM1 cells stimulated invasion in the absence of serum and Rho. A ROCK inhibitor (Y-27632) not only blocked the invasion of MM1 cells in vitro but it also prevented the dissemination of these cells in mice.28,32 These findings provided a foundation for the use of ROCK inhibitors as therapeutic agents for reducing tumor spread and clinical trials for this purpose are underway in Japan (Itoh, personal communication). Two other lines of evidence provide strong support for the involvement of Rho in tumor migration and invasion. Our group has shown that the migration of invasive colon carcinoma cells requires RhoA activity and, more interestingly, that RhoA is needed for the formation and stabilization of lamellae in these cells (see below).35 Studies on lysophosphatidic acid (LPA) also provide compelling evidence to implicate Rho in tumor migration and invasion. A potentially important role for LPA in tumor progression, has been noted, especially for ovarian carcinomas, which produce relatively high levels of LPA.36 Interestingly, LPA is a potent activator of Rho by a mechanism that involves its binding to specific G protein coupled receptors,37 and a direct connection between LPA and RhoA-dependent tumor migration and invasion has been shown.27,30,35,38-40

Mechanisms of Rho Involvement in Invasion The conclusion that Rho is important for tumor migration and invasion is not surprising. The more timely and challenging problem is to define specific aspects of the invasive process that require Rho and to elucidate its mechanistic contribution to these processes. In this section, the possible involvement of Rho in the epithelial-mesenchymal transition and the acquisition of an invasive phenotype will be discussed. In addition, the existing data on the involvement of Rho in migration and invasion will be summarized and a hypothesis that unifies these data will be derived.

RhoA and the Epithelial-Mesenchymal Transition The involvement of Rho family GTPases in the formation and maintenance of adherens junctions in epithelia has been demonstrated.41-43 The issue of interest here is the potential contribution of RhoA to the epithelial-mesenchymal transition and, more specifically, the possibility that an increase in RhoA expression or activity facilitates this transition. This possibility is supported by several findings. The mesenchymal transition of mammary epithelial cells that is induced by TGF-β requires RhoA and ROCK activity and these activities promote the acquisition of a fibroblastic, motile phenotype.44 Other studies have shown that Ras-induced epithelial-mesenchymal transition and cell scattering correlate with increased Rho activity and a decrease in Rac activity.45 As argued by Collard and colleagues, the shift between an epithelial and mesenchymal phenotype is influenced by a dynamic balance between Rac and Rho activity, as well as the specific matrix to which cells are anchored.15,45 A key problem that needs to be addressed is whether increased RhoA (or Rho C) expression in a well-differentiated tumor promotes an epithelial-mesenchymal transition. In support of this possibility, Jou and Nelson42-43 reported that the regulated expression of RhoAV14 in polarized MDCK cells resulted in membrane ruffling and lamellipodia formation and they noted that ‘gaps’ formed between cells. A relevant conclusion from their study is that RhoA appears to regulate actin stress fibers at the basal membrane that are characteristic of single, motile cells. Collectively, the existing data suggest that increased RhoA activity and a possible decrease in Rac activity facilitate an epithelial-mesenchymal transition.

Rho A and Migration With respect to the contribution of Rho to the migration of invasive carcinoma cells, it is most fruitful to focus on those cells that have undergone an EMT and exhibit a fibroblastic morphology. Such cells do not form cell-cell adhesive junctions so that the contribution of Rho to the formation of these junctions and epithelial morphology is not a factor. Importantly,

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studies on these cells are clinically relevant because they are usually much more invasive and aggressive than are more differentiated carcinoma cells, which retain epithelial features.5-7 The fact that highly invasive carcinoma cells display a fibroblast-like morphology and are motile suggests that insight into the contribution of RhoA to their migration can be obtained from studies on fibroblast migration. Although RhoA has been implicated in stress fiber and focal adhesion assembly in fibroblasts,13 recent studies have examined its contribution to migration more directly. A consensus is that some RhoA activity is needed for migration presumably to maintain some adhesion to the substrate but that high RhoA activity impedes migration.16,18 More specific insight into this problem has been provided by the work of Burridge and colleagues. Their studies have demonstrated that integrin-mediated activation of p190RhoGAP, which is mediated by src, inhibits RhoA function and is necessary for fibroblast spreading and migration.46-47 Presumably, transient suppression of RhoA activity is needed to alleviate RhoA-mediated contractile forces that would otherwise impede protrusion at the leading edge of migrating cells. Interestingly, however, studies by the same group on the migration of leukocytes revealed that the activity of RhoA and its effector ROCK are required for the release of integrin-mediated attachments at the trailing edge of migrating cells.48-49 Collectively, these studies highlight a spatial regulation of RhoA function (inactivation at the leading edge and activation at the trailing edge) that is necessary for efficient migration. In contrast to the fibroblast studies described above, our group noted that RhoA activity is needed for the formation and stabilization of lamellae at the leading edge of invasive colon carcinoma cells. This conclusion was based on the expression of either a dominant inhibitory RhoA construct35 or C3 transferase (O’Connor and Mercurio, unpublished). Interestingly, the inhibition of RhoA activity by these reagents did not influence cell attachment and spreading. Additional evidence to implicate RhoA in leading edge dynamics was provided by our finding that an increase in the [cAMP]i, which suppresses RhoA activation, also prevents lamellae formation and stabilization in migrating colon and breast carcinoma cells.35,40 Lamellae formation and migration in some invasive colon carcinoma cells appears to be independent of Rac activity based on the use of a dominant inhibitory Rac construct.35 As suggested by Ridley,16 this observation raises the possibility of a link between RhoA and actin polymerization in these cells, perhaps analogous to the finding that RhoA and Arp2/3 are required for integrin-mediated phagocytosis.50 However, in most carcinoma cell lines that we have examined, lamellae formation and migration require the activities of both RhoA and Rac, and the contribution of each of these GTPase to these processes needs to be defined. One attractive hypothesis is that the contractile events that are needed for cell body translocation during migration require RhoA and ROCK activity and that this contraction occurs at the base of lamellae in migrating carcinoma cells (see below). Rac in this scenario probably contributes to actin polymerization at the leading edge, similar to its role in other cell types. In this direction, recent work from our group raised the possibility of a spatial and temporal regulation of RhoA and Rac activity during the migration of carcinoma cells. This possibility is based on our finding that PKA impedes migration because it inhibits RhoA activity but that it also required for migration because it is needed for Rac activation.51 A comparison of the contribution of RhoA to the migration of fibroblasts and carcinoma cells must also consider important differences between these cell types. Fibroblasts in culture form large stress-fiber, associated focal adhesions and the formation of such structures is RhoA-dependent.13 Thus, the need to suppress RhoA activity to enable migration is much greater in fibroblasts than in carcinoma cells, which lack focal adhesions. Other points need to be considered as well. Most notably, most, if not all, invasive carcinoma cells harbor mutations in key signalling molecules such as Ras and PTEN and they over-express other signalling molecules such as Rho itself and Akt/PKB. These alterations are likely to have profound effects on signalling pathways involved in migration. Another important factor that distinguishes fibroblasts from carcinoma cells is that carcinoma cells express the integrin α6β4.52 We and others have provided compelling evidence that

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this integrin plays a dominant role in the migration and invasion of carcinoma cells.40,53-57 More specifically, this integrin is linked to activation of both PI3-kinase54,57 and RhoA35 and it stimulates both lamellae formation and migration in many carcinoma cells. An important venue for future work is to elucidate differences in the nature of migration that occurs in the presence or absence of α6β4 expression. In this direction, we observed that the expression of α6β4 enables cells to respond to chemotactic factors such as LPA and form large, fan-shaped lamellae, and stimulate RhoA activation, processes that appear to be linked.35,40 Based on these observations, the hypothesis can be derived that expression of α6β4 regulates either the expression or function of LPA receptors, and this mechanism may account for the distinct ability of this integrin to stimulate RhoA activation.

RhoA and Invasion Although migration is an important component of invasion, invasion also requires the ability of tumor cells to penetrate basement membranes.58 Localized proteolysis of basement membranes is considered to be a major mechanism for tumor cell penetration.58 Recent studies by our group,59 however, have revealed a potentially novel mechanism of invasion involving the α6β4 integrin that may explain why RhoA has been implicated in invasion in previous studies. In epithelial cells, the α6β4 integrin is a laminin receptor that stabilizes epithelial cell adhesion to the basement membrane through its ability to associate with cytokeratins and form hemidesmosomes.60 In invasive carcinoma cells, hemidesmosomes are disrupted and α6β4 is localized in actin-rich protrusions.56 In fact, this integrin actually stimulates the formation and stabilization of such protrusions.55 More specifically, α6β4 colocalizes with actin filament bundles that are present at the base of lamellae in invasive carcinoma cells, and function-blocking antibodies specific for α6β4 disrupt these actin bundles.55 These observations are of considerable interest in light of our recent finding that carcinoma cells are able to remodel basement membranes through α6β4-mediated compression forces by a process that involves the packing of BM material under the cells and the mechanical removal of BM from adjacent areas.59 Moreover, we demonstrated that this ‘compression machine’ is localized to the base of lamellae, and that compression requires ROCK activity. Given that α6β4 and actin filament bundles colocalize at the base of lamellae and that these actin bundles are probably responsible for basement membrane compression, it is likely that α6β4 activation of RhoA is needed for the formation of these bundles and their myosin-mediated contraction that occur as a part of the compression process. In fact, compression is blocked by either cytochalasin B or the myosin inhibitor BDM.59 Our findings reveal a potentially novel mechanism of basement membrane penetration that involves the ability of tumor cells to compress and remodel the basement membrane, thus creating ‘gaps’ for tumor cell egress. An essential component of this model is that the activity of RhoA and its effector ROCK are required for compression and remodeling. Thus, these data may provide a mechanism to explain the studies of Itoh and colleagues that have implicated RhoA, ROCK and actomyosin contraction in invasion.28

Conclusions The available data implicate an important role for the RhoGTPase in tumor invasion. One hypothesis that unifies much of these data is that RhoA activity is needed for actomyosin contraction and that this contraction is necessary not only for efficient migration, but more importantly, for the ability of tumor cells to compress and remodel basement membranes.59 A critical effector of RhoA in this regard is ROCK because of its ability to regulate myosin light chain phosphorylation.61 In addition, the involvement of RhoA in releasing adhesive contacts at the trailing edge of migrating cells, as indicated from studies on fibroblasts,48 could have important implications for invasion that need to be elucidated. Despite recent advances in our understanding of the contribution of Rho to invasion, much remains to be learned. The possibility that RhoA and RhoC differ in function is a problem of

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major importance because of the reports demonstrating enhanced RhoC expression in a number of tumors.23-26 Another challenge is to elucidate the mechanisms that contribute to Rho activation in invasive cells and to link Rho signalling and function to signalling pathways that have been implicated in invasion such as PI3-K54,62 and the PKCs.63 Moreover, it is apparent that extensive ‘cross-talk’ exists among Rho family GTPases and that the balance of these activities is critical.15 Studies on Rho in invasion, therefore, must consider the contribution of other Rho family GTPases, especially Rac, and the possibility that a spatial and temporal regulation of Rho and Rac activation underlies tumor migration and invasion.51 In this regard, a detailed understanding of the differential regulation of these GTPases by cAMP and PKA could be very insightful. Another area that is ripe for investigation is the contribution of Rho effectors to invasion. Studies to date have highlighted an important role for ROCK but other effectors may play important roles. Dia, for example, has been implicated in actin polymerization and microtubule stability,21-22 processes that are critical for migration and invasion. Finally, the involvement of specific Rho GEFs and GAPs in invasion needs to be investigated. In this direction, studies on p190RhoGAP could be particularly fruitful for several reasons. This molecule has been implicated in fibroblast migration46 and its activity is regulated by src, a kinase that has been implicated in invasion.64 Also, several studies have suggested a potential role for p190RhoGAP in invasion.65-67 Clearly, future studies on Rho and other GTPases will add considerably to our understanding of tumor invasion.

Acknowledgement NIH Grant CA80789 supported the work from the author’s laboratory discussed in this review.

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17. Wittmann T, Waterman-Storer CM. Cell motility: Can Rho GTPases and microtubules point the way? J Cell Sci 2001; 114:3795-3803. 18. Schmitz AA, Govek EE, Bottner B et al. Rho GTPases: Signalling, migration, and invasion. Exp Cell Res 2000; 261:1-12. 19. Fritz G, Just I, Kaina B. Rho GTPases are over-expressed in human tumors. Int J Cancer 1999; 81:682-687. 20. Fukuda T, Kido A, Kajino K et al. Cloning of differentially expressed genes in highly and low metastatic rat osteosarcomas by a modified cDNA-AFLP method. Biochem Biophys Res Commun 1999; 261:35-40. 21. Satoh S, Tominaga T. mDia-interacting protein acts downstream of rho-mdia and modifies src activation and stress fiber formation. J Biol Chem 2001; 276:39290-39294. 22. Palazzo AF, Cook TA, Alberts AS et al. mDia mediates Rho-regulated formation and orientation of stable microtubules. Nat Cell Biol 2001; 3:723-729. 23. Suwa H, Ohshio G, Imamura T et al. Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas. Br J Cancer 1998; 77:147-152. 24. van Golen KL, Davies S, Wu ZF et al. A novel putative low-affinity insulin-like growth factor-binding protein, LIBC (lost in inflammatory breast cancer), and RhoC GTPase correlate with the inflammatory breast cancer phenotype. Clin Cancer Res 1999; 5:2511-2519. 25. van Golen KL, Wu ZF, Qiao XT et al. RhoC GTPase, a novel transforming oncogene for human mammary epithelial cells that partially recapitulates the inflammatory breast cancer phenotype. Cancer Res 2000; 60:5832-5838. 26. Clark EA, Golub TR, Lander ES et al. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000; 406:532-535. 27. Yoshioka K, Matsumura F, Akedo H et al. Small GTP-binding protein Rho stimulates the actomyosin system, leading to invasion of tumor cells. J Biol Chem 1998; 273:5146-5154. 28. Itoh K, Yoshioka K, Akedo H et al. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat Med 1999; 5:221-225. 29. Yoshioka K, Imamura F, Shinkai K et al. Participation of rhop21 in serum-dependent invasion by rat ascites hepatoma cells. FEBS Lett 1995; 372:25-28. 30. Imamura F, Shinkai K, Mukai M et al. rho-Mediated protein tyrosine phosphorylation in lysophosphatidic-acid- induced tumor-cell invasion. Int J Cancer 1996; 65:627-632. 31. Imamura F, Mukai M, Ayaki M et al. Involvement of small GTPases Rho and Rac in the invasion of rat ascites hepatoma cells. Clin Exp Metastasis 1999; 17:141-148. 32. Imamura F, Mukai M, Ayaki et al. Y-27632, an inhibitor of rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase. Jpn J Cancer Res 2000; 91:811-816. 33. Mukai M, Nakamura H, Tatsuta M et al. Hepatoma cell migration through a mesothelial cell monolayer is inhibited by cyclic AMP-elevating agents via a Rho-dependent pathway. FEBS Lett 2000; 484:69-73. 34. Kusama T, Mukai M, Iwasaki T et al. Inhibition of epidermal growth factor-induced RhoA translocation and invasion of human pancreatic cancer cells by 3-hydroxy-3-methylglutaryl- coenzyme a reductase inhibitors. Cancer Res 2001; 61:4885-4891. 35. O’Connor KL, Nguyen BK, Mercurio AM et al. RhoA function in lamellae formation and migration is regulated by the α6β4 integrin and cAMP metabolism. J Cell Biol 2000; 148:253-258. 36. Goetzl EJ, Dolezalova H, Kong Y et al. Distinctive expression and functions of the type 4 endothelial differentiation gene-encoded G protein-coupled receptor for lysophosphatidic acid in ovarian cancer. Cancer Res 1999; 59:5370-5375. 37. Fukushima N, Chun J. The LPA receptors. Prostaglandins 2001; 64:21-32. 38. Yanai N, Matsui N, Furusawa T et al. Sphingosine-1-phosphate and lysophosphatidic acid trigger invasion of primitive hematopoietic cells into stromal cell layers. Blood 2000; 96:139-144. 39. Stam JC, Michiels F, van der Kammen RA et al. Invasion of T-lymphoma cells: Cooperation between Rho family GTPases and lysophospholipid receptor signalling. Embo J 1998; 17:4066-4074. 40. O’Connor KL, Shaw LM, Mercurio AM. Release of cAMP gating by the α6β4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells. J Cell Biol 1998; 143:1749-1760. 41. Sander EE, Collard JG. Rho-like GTPases: Their role in epithelial cell-cell adhesion and invasion. Eur J Cancer 1999; 35:1905-1911. 42. Jou TS, Schneeberger EE, Nelson WJ. Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases. J Cell Biol 1998; 142:101-115. 43. Jou TS, Nelson WJ. Effects of regulated expression of mutant RhoA and Rac1 small GTPases on the development of epithelial (MDCK) cell polarity. J Cell Biol 1998; 142:85-100.

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44. Bhowmick NA, Ghiassi M, Bakin A et al. Transforming growth factor-beta1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism. Mol Biol Cell 2001; 12:27-36. 45. Zondag GC, Evers EE, ten Klooster JP et al. Oncogenic Ras downregulates Rac activity, which leads to increased Rho activity and epithelial-mesenchymal transition. J Cell Biol 2000; 149:775-782. 46. Arthur WT, Petch LA, Burridge K. Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism. Curr Biol 2000; 10:719-722. 47. Arthur WT, Burridge K. RhoA Inactivation by p190RhoGAP Regulates Cell Spreading and Migration by Promoting Membrane Protrusion and Polarity. Mol Biol Cell 2001; 12:2711-2720. 48. Worthylake RA, Lemoine S, Watson JM et al. RhoA is required for monocyte tail retraction during transendothelial migration. J Cell Biol 2001; 154:147-160. 49. Worthylake RA, Burridge K. Leukocyte transendothelial migration: Orchestrating the underlying molecular machinery. Curr Opin Cell Biol 2001; 13:569-577. 50. May RC, Caron E, Hall A et al. Involvement of the Arp2/3 complex in phagocytosis mediated by FcgammaR or CR3. Nat Cell Biol 2000; 2:246-248. 51. O’Connor KL, Mercurio AM. Protein kinase A regulates rac and is required for the growth factorstimulated migration of carcinoma cells. J Biol Chem 2001; 276:47895-47900. 52. Mercurio AM, Rabinovitz I. Towards a mechanistic understanding of tumor invasion-lessons from the α6β4 integrin. Semin Cancer Biol 2001; 11:129-141. 53. Chao C, Lotz MM, Clarke AC et al. A function for the integrin α6β4 in the invasive properties of colorectal carcinoma cells. Cancer Res 1996; 56:4811-4819. 54. Shaw LM, Rabinovitz I, Wang HH et al. Activation of phosphoinositide 3-OH kinase by the α6β4 integrin promotes carcinoma invasion. Cell 1997; 91:949-960. 55. Rabinovitz I, Mercurio AM. The integrin α6β4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin- containing motility structures. J Cell Biol 1997; 39:1873-1884. 56. Rabinovitz I, Toker A, Mercurio AM. Protein kinase C-dependent mobilization of the α6β4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells. J Cell Biol 1999; 46:1147-1160. 57. Gambaletta D, Marchetti A, Benedetti L et al. Cooperative signalling between α6β4 integrin and ErbB-2 receptor is required to promote phosphatidylinositol 3-kinase-dependent invasion. J Biol Chem 2000; 75:10604-10610. 58. Stetler-Stevenson WG, Aznavoorian S, Liotta LA. Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu Rev Cell Biol 1993; 9:541-573. 59. Rabinovitz I, Gipson IK, Mercurio AM et al. Traction forces mediated by α6β4 integrin: Implications for basement membrane organization and tumor invasion. Mol Biol Cell 2001; 12:4030-4043. 60. Green KJ, Jones JCR. Desmosomes and hemidesmosomes - structure and function of molecular components[Review]. FASEB Journal 1996; 10:871-881. 61. Amano M, Fukata Y, Kaibuchi K. Regulation and functions of Rho-associated kinase. Exp Cell Res 2000; 261:44-51. 62. Keely PJ, Westwick JK, Whitehead IP et al. Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K. Nature 1997; 390:632-636. 63. Gomez DE, Skilton G, Alonso DF et al. The role of protein kinase C and novel phorbol ester receptors in tumor cell invasion and metastasis (Review). Oncol Rep 1999; 6:1363-1370. 64. Atabey N, Gao Y, Yao ZJ et al. Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions. J Biol Chem 2001; 276:14308-14314. 65. Chakravarty G, Roy D, Gonzales M et al. P190-B, a Rho-GTPase-activating protein, is differentially expressed in terminal end buds and breast cancer. Cell Growth Differ 2000; 11:343-354. 66. Zrihan-Licht S, Fu Y, Settleman J et al. RAFTK/Pyk2 tyrosine kinase mediates the association of p190 RhoGAP with RasGAP and is involved in breast cancer cell invasion. Oncogene 2000; 19:1318-1328. 67. Nakahara H, Mueller SC, Nomizu M et al. Activation of beta1 integrin signalling stimulates tyrosine phosphorylation of p190RhoGAP and membrane-protrusive activities at invadopodia. J Biol Chem 1998; 273:9-12.

CHAPTER 11

Rho Family GTPases and Cellular Transformation Antoine E. Karnoub and Channing J. Der

Abstract

M

embers of the Ras-related Rho family of GTPases act as key signalling nodes that regulate a variety of crucial cellular activities including morphology, motility, and proliferation. Their aberrant expression causes uncontrolled growth and transformation, and they are required for the transforming actions of Ras and other oncoproteins. However, whereas the identification of mutated Ras proteins in human cancers prompted immediate and intense interest in investigating their role in tumor formation, the evidence for the involvement of Rho GTPases in oncogenesis has come mainly from experimental cell culture and animal studies. Nevertheless, novel evidence for a direct link between Rho proteins and cancer development is beginning to accumulate, rendering them potential targets for drug discovery and cancer intervention.

Introduction Malignant transformation is a complex multifaceted process involving systematic alterations in a variety of cellular properties. Cancer cells are characterized by deregulated cell cycle control, reduced contact inhibition, loss of matrix-dependent growth regulation, increased cell survival, morphologic alteration, increased motility, and acquisition of invasive and metastatic properties. Rho family GTPases act as key regulators of all of these cellular processes and mounting evidence implicates aberrant Rho GTPase function in human oncogenesis. Like the Ras oncoproteins, Rho GTPases function as regulated GDP/GTP switches that relay extracellular cues to cytoplasmic signalling cascades. In contrast to Ras proteins however, where direct mutational activation of Ras is prevalent in human cancers, much of the evidence linking Rho GTPases to oncogenesis is indirect, and implicates Rho proteins as critical downstream signalling components of a diverse array of oncoproteins. Recent reviews have provided excellent overviews on various facets of normal Rho GTPase function.1-5 Therefore, in this review we have especially focused on a summary of the evidence linking Rho GTPases to oncogenesis.

The Rho Family of GTPases Is a Major Branch of the Ras Superfamily The Rho family constitutes one of the major branches of the Ras superfamily of small GTPases. Presently, 18 mammalian members have been described that share significant sequence identity ranging from 50 to 90%, with an overall 25% identity with Ras. This family is further subdivided into several groups and include the Rho (isoforms A, B, and C), Rac (isoforms 1, 2, 3), Cdc42 (and Wrch-1), TC10 (and TCL), Rnd (isoforms 1, 2, and 3), RhoD, RhoG, and TTF subfamilies. The most heavily studied and best characterized members are Rac1, Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Figure 1. Rho GTPases are regulated molecular switches. Similar to Ras, Rho family proteins shuttle between the inactive GDP-bound and the active GTP-bound states. Activation is supported by guanine nucleotide exchange factors (GEFs) which promote exchange of GDP for GTP. Rho GTPases are downregulated by GTPase activating proteins (GAP) and guanine nucleotide dissociation inhibitors (GDI). GAPs promote the hydrolysis of GTP to GDP, while GDIs prevent nucleotide exchange and sequester Rho from membranes. Activated GTP-bound Rho binds to effectors (E) which mediate downstream signal propagation. RhoA, Cdc42, and RhoA interact with many effectors, some unique to each GTPase, many shared by multiple Rho GTPases. These include protein kinases such as PAK and ROCK. Active Rho family proteins transmit extracellular signals by activation of effector proteins.

RhoA, and Cdc42.6-8 Rho GTPase function is conserved in evolution, with members identified in yeast, plants, slime mold, worms, and flies.

Rho GTPases Function As Membrane-Associated GDP/ GTP-Regulated Molecular Switches In addition to sharing strong primary amino acid sequence identity, Ras and Rho GTPases share substantial structural, biochemical, and functional similarities. In the sections below, we have summarized these similarities (as well as differences) with regards to their function as regulated GDP/GTP signal transducers.

The Rho GDP/GTP Cycle Is Regulated by GEFs and GAPs Like Ras, Rho GTPases act as binary switches that cycle between active GTP-bound and inactive GDP-bound states (Fig. 1). The GTP-bound forms complex with and activate multiple downstream effector target molecules to stimulate cytoplasmic signalling cascades. Subsequently, an intrinsic GTP hydrolysis activity regenerates the GDP-bound form terminating the active state. The intrinsic GTP hydrolysis and the GDP/GTP nucleotide exchange reactions of Rho and other Ras superfamily GTPases are both very slow, and require the enzymatic assistance of two families of regulators: GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) (Fig. 1). GAPs enhance the intrinsic GTP hydrolysis rate and therefore attenu-

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Figure 2. Regulation of Rho proteins by membrane association. Rho proteins undergo posttranslational modification by farnesyltransferases (FTase) (e.g., RhoE/Rnd3), geranylgeranylases (e.g., Rac1), and carboxymethyltransferase. FTase and GGTaseI recognize carboxyl terminal CAAX tetrapeptide sequences and cause covalent addition of the isoprenoid moieties to the cysteine residue. Endoprotease activity cleaves the AAX residues and carboxymethyltransferase adds a methyl group to the now terminal prenylated cysteine. These activities are essential for anchoring Rho proteins to membranes, where they are active. GDIs mask the isoprenoid group and sequester Rho proteins from membranes to the cytoplasm, and result in their inactivation. FTase and GGTase inhibitors (FTI and GGTI) inactivate Rho proteins by inhibition of prenylation, and mimic GDI action.

ate Rho protein activity by promoting the GDP-bound state.9 More than 20 Rho GAPs have been identified to date.10 Among these is BCR, which was identified originally as the translocation partner for the Abl tyrosine kinase in the Philadelphia Chromosome translocation found in a majority of human chronic myelogenous and acute lymphocytic leukemias.11 GEFs act as positive modulators by catalyzing the exchange of GDP for GTP, thereby increasing the GTP-bound protein levels in the cell (Fig. 1). Rho GEFs, like Ras GEFs, eject the bound nucleotide (GDP or GTP) to promote the formation of the nucleotide-free form of the GTPase. However, since there is about 15-fold excess of GTP versus GDP in the cell, the net result of GEF-mediated exchange is to enhance formation of the GTP-bound protein.12 Rho GEFs are also referred to as Dbl family proteins and are named after the founding member characterized originally as a transforming protein from a human diffuse B-cell lymphoma (Dbl) cell line.13,14 To date, over 60 Dbl family proteins have been identified, and like Dbl, many were identified initially in screens for novel transforming oncoproteins. All Dbl family proteins contain a Dbl homology (DH) domain that catalyses GDP/GTP exchange, as well as a pleckstrin homology (PH) domain that regulates DH domain function.15,16 The tandem DH/PH domains are typically flanked by different modules (SH2, SH3, calponin homology (CH) domains, kinase domains, etc.) that contribute to the DH domain activation through diverse signalling activities. Unlike Ras, Rho GTPases are under the control of a third functional class of regulators called guanine nucleotide dissociation inhibitors (GDIs) comprising three members: GDI1

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Figure 3. Activated versus dominant negative Rho GTPases. The GAP-insensitive (G12V and Q61L) mutants are constitutively GTP-bound, and have therefore been used to characterize the different cellular functions of a variety of GTPases. By inhibiting Rho GEF function, dominant negative S17N versions have been key tools in studies assessing the contribution of Rho proteins to various extracellular signalling pathways. However, since they target multiple GEFs, the activities of S17N may have broader inhibitory actions on several GEFs, which then may block other Rho GTPases. Peptide fragments consisting of the GTP-dependent Rho binding regions (RBDs) of specific effectors can also act as select dominant negative versions of specific Rho proteins and have been used similarly to S17N.

(RhoGDI or GDIα), GDI2 (D4/Ly-GDI or GDIβ), and GDI3 (GDIγ).17 GDIs inhibit Rho GTPase signalling by exerting three concerted functions (Fig. 1 and Fig. 2). First, they inhibit the nucleotide dissociation and block the GDP/GTP exchange, therefore antagonizing GEF activity.18-20 Second, GDIs interfere with GTP hydrolysis and hinder GAP activity.21 Finally, perhaps the most important function of GDIs involves their stimulation of the release of Rho GTPases from cellular membranes (Fig. 2). The net effect is the transfer of the Rho protein from membrane compartments, where it is active, to the cytosol, shutting down the GTPase activation altogether.

Membrane Asscociation Is Critical for Rho GTPase Function Like Ras oncoproteins, Rho proteins also undergo COOH-terminal posttranslational modification by isoprenoid lipids, a process critical for their biological activity. Both Ras and Rho GTPases terminate in a carboxyl-terminal CAAX tetrapeptide motif (where C = cysteine, A = aliphatic amino acid, and X = terminal amino acid) that is necessary and sufficient for posttranslational modification. In the first step, a farnesyltransferase (FTase; when X = serine, methionine) or the related geranylgeranyltransferaseI (GGTaseI; when X = leucine), catalyze the covalent addition of a C15 farnesyl or a C20 geranylgeranyl isoprenoid moiety, respectively (Fig. 2). Although the majority of Rho GTPases are modified by GGTaseI (e.g., RhoA, Rac1, and Cdc42), others are processed solely by FTase (e.g., RhoD and RhoE/Rnd3), or by both enzymes (such as RhoB). Prenylation is followed by a proteolytic activity that cleaves the terminal AAX residues prior to carboxymethylation of the terminal prenylated cysteine. The CAAX-signaled modifications, together with an additional membrane-targeting motif directly upstream of the CAAX motif, anchor Rho GTPases to membranes, a necessary step for GTPase function.22

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Figure 4. Deregulated Rho GTPase mutants are tools for functional studies. Dominant, gain-of-function (G12V, F28L, and Q61L), or dominant-negative (S17N) mutants of Rho GTPases have been developed based on Ras mutants. The majority of studies have evaluated the contribution of residues in the effector domain using site-directed mutagenesis. Chimeric studies also involved swapping effector domains or carboxyl terminal sequences among GTPases to assess function. Mutations in the CAAX motif (e.g., C186S) abolish the ability of Rho proteins to be prenylated and hence result in inactive, cytosolic proteins. Mutations in the Rho insert domain have also implicated this region in effector activation.

In light of the critical importance of prenylation for the function of Ras and Rho GTPases, pharmacologic inhibitors of FTase (FTIs) or GGTaseI (GGTIs) have been developed as novel anti-cancer therapeutics (Fig. 2). Initially developed as anti-Ras drugs, the anti-tumor activity of FTIs is speculated to be mediated, in part, by targeting other GTPases such as RhoB.23 Nevertheless, FTIs have demonstrated potent anti-oncogenic activity in cell culture and animal models, and are now under evaluation in phase III clinical trials.24 Similarly, inhibitors of GGTaseI (GGTIs) have been developed as inhibitors of Rho GTPases and have also shown anti-tumor activity in experimental models and tumor xenograft studies.25

Rho GTPase Mutants Are Molecular Tools for Dissection of Function Alterations of specific residues in Rho GTPases can genetically modify their ability to interact with their regulators, effectors, or with membranes.26 Depending on the residue, these mutations can either generate dominant activated, inactive, or dominant negative GTPase. In particular, two classes of mutants have provided powerful reagents to evaluate the signalling and biological functions of Rho GTPases (Fig. 3). Mutations analogous to those found in tumor-associated mutants of Ras (e.g., G12V and Q61L) interfere with the intrinsic GTP hydrolysis reaction, and de-sensitize the G-protein to GAP regulation, resulting in constitutively active GTP-bound proteins that are activated in a ligand-independent fashion27-29 (Fig. 4). A second type of activated mutants is based on the Ras(F28L) variant, which shows decreased affinity for guanine nucleotides and results in a “fast-cycling” protein.30 Their increased intrinsic nucleotide exchange rate, coupled with excess in cellular GTP, favor the formation of GTP-bound proteins. Like the GAP-deficient mutants, F28 versions of Ras, Cdc42, RhoA, and Rac1 are constitutively activated and transforming.31

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Figure 5. Extracellular signal activation of Rho family GTPases. Rho GTPases function downstream of a variety of extracellular stimuli. For example, Rho proteins are activated by ligand-stimulated G protein-coupled serpentine receptors (GPCR), receptor tyrosine kinases (RTKs), nonreceptor tyrosine kinase-associated cytokine receptors, and matrix-interacting integrins.

Another important class of GTPase mutants is those that display a dominant-negative phenotype.32 These mutants have been essential reagents that implicated Rho GTPases as mediators of oncogenesis.33 In particular, the S17N mutant of Ras has been the prototype for analogous versions in Rho proteins. Introducing an aspargine at position 17 (17N) favors formation of nucleotide-free GTPases that bind GEFs with higher affinity than their wild type counterparts. Hence, they prevent GTPase activation via inhibition of GEF function. When introduced in cells, Rac1(17N) for instance, would compete with the endogenous Rac for GEF binding, forming tight complexes unable to relay effector-mediated downstream signalling. As described in the following sections, both dominant activating and dominant negative mutants of Rho GTPases have been instrumental in linking Rho GTPases to oncogenesis. A third functional class of mutants are those that are impaired in downstream effector interaction26 (Fig. 4). As described below, Rho GTPase function is mediated through multiple downstream effector targets. Although all effector interactions are dependent on a core effector domain (corresponding to Ras residues 32-40), select missense mutations in the Rho protein core effector domain can result in differential impairment in effector binding (e.g., see refs. 34 and 35). Such mutants have provided powerful reagents to assess the contribution of a particular effector to a specific GTPase signalling and/or function. In contrast to the Ras superfamily of proteins, Rho GTPases possess an additional ~13 amino acid “insert region” positioned between amino acids corresponding to Ras residues 122 and 123.36 Although the insert region is distant some 18 Å from the switch regions and the

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effector domain, it still participates in effector-mediated functions, such as cytoskeletal remodeling.37 The sequence and structural divergence of the insert domain between different Rho GTPases suggests it may serve very distinct roles, particularly in transformation.37-39

Rho Family GTPases Are Critical Components of Diverse Signalling Pathways A large and diverse variety of cell surface agonists utilize Rho GTPases to modify cellular behavior.40 An expanding body of evidence has implicated Rho proteins in growth factor, cytokine, cell-cell as well as cell-extracellular matrix signalling. The ligand-activated receptors are thought to regulate Rho GTPase activation mainly via activation of Rho GEFs, although in some situations regulation of Rho GAP activity has been described. Once activated, the GTP-bound Rho GTPase interacts with a wide spectrum of downstream effectors, initiating signalling cascades that regulate events in the cytoplasm and the nucleus (Fig. 5). In the following sections, we have described the cellular responses regulated by signalling cascades that may contribute to the aberrant growth properties of cancer cells.

Rho GTPases Regulate Actin Cytoskeletal Organization The best-characterized cytoplasmic function of Rho family proteins is their role as regulators of actin cytoskeletal organization.3,6,8 For example, Rac1 promotes the reorganization of filamentous actin into membrane structures called lamellipodia which serve as new adhesive contacts typically found at the leading edge of migrating fibroblasts.41 On the other hand, RhoA promotes the formation of actin stress fibers and focal adhesions, promoting cell attachment, whereas RhoE causes a disruption of these structures and causes cell rounding.42-44 Other GTPases such as Cdc42 promote the protrusion of finger-like actin-rich projections that act as sensors of the extracellular environment.45 Additional processes regulated by Rho proteins and requiring cytoskeletal changes extend to membrane transport, organization of tight junctions, chemotaxis, cytokinesis, and cell polarity.46-54 Collectively, these dynamic RhoGTPase-controlled cytoskeletal rearrangements promote shape adjustment and regulate cell-cell and cell-matrix contacts, processes that may influence invasion and metastasis.

Rho GTPases Regulate Transcription Factor Function and Gene Expression A second major function of Rho family GTPases involves their regulation of the activity of a variety of transcription factors.55 For instance, the three GTPases Rac1, RhoA, and Cdc42 activate the serum response factor (SRF).56 SRF is a transcription factor that cooperates with ternary complex factors (e.g., Elk-1) to modulate the activity of serum response element (SRE)-containing promoters of many early response genes such as c-fos.57 Rho proteins also stimulate the activation of various other transcription factors such as NF-κB, E2F-1, c-Jun, ATF-2, Elk-1, AP-1, and STAT3.4,58-67 The diverse multitude of transcription factors that are regulated by Rho GTPases emphasizes that changes in gene expression form an important outcome and may be a critical mediator of Rho GTPase-induced transformation.

Rho GTPases Regulate Cell Cycle Progression and Cell Proliferation In light of their ability to promote uncontrolled growth, it is not surprising that Rho GTPases, like Ras, also regulate cell cycle progression.5 Dominant negative Rac1, RhoA, and Cdc42 block, whereas activated forms of these GTPases stimulate, progression through the G1 phase of the cell cycle.68,69 Rho GTPases promote cell cycle progression by modulating the activity of both positive as well as negative regulators of the Rb tumor suppressor, which functions in regulating G1 progression. For example, RhoA and Rac1 up-regulate cyclin D1 gene expression34 and the transforming activity of various Dbl family oncoproteins correlated with their ability in promote cyclin D1 expression.70 Conversely, RhoA has been found to antagonize the expression of two negative regulators of G1 progression, the cyclin-dependent kinase inhibitors

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p21CIP1 and p27KIP1.71 For example, RhoA inhibition of p21CIP1 has been described as one mechanism by which Rho GTPases may facilitate the growth promoting actions of Ras.72

Rho GTPases and Regulation of Apoptosis Rho GTPases have also been shown to regulate cell survival by either promoting or antagonizing apoptosis depending on the cell type and signalling contexts. Several reports have indicated that RhoA, RhoC, Rac1, Rac2 and Cdc42 proteins were involved in apoptotic stimulation in cells such as fibroblasts, thymocytes, neurons, and epithelial cells. NIH 3T3 cells expressing Rac1 seem to be sensitized to serum withdrawal through a mechanism involving up-regulation of Fas ligand transcription.73 Overexpression of RhoA also correlates with an increased ceramide production, which is a second messenger for apoptosis.74,75 In addition, RhoG is thought to mediate the toxic effects of FTI treatment and Taxol.77,76 On the other hand, there is also considerable evidence for the anti-apoptotic activities of Rho GTPases. For example, Rac1 appears to protect Rat-1 fibroblasts from serum deprivation-induced apoptosis, or MDCK epithelial cells from matrix deprivation-induced apoptosis.78,79 Inhibition of RhoA, but not of Rac1, caused apoptosis of RET/PTC1-expressing thyroid cells.80 Similarly, Cdc42 inhibition through caspase-mediated degradation of Cdc42 was suggested to facilitate Fas ligand-induced apoptosis in T47D breast cancer cell line.81 While oncogenesis clearly involves increased cell survival, how Rho GTPase regulation of apoptosis factors into Rho GTPase-mediated oncogenesis is presently not clear.

Rho GTPases Promote Oncogenic Transformation Several lines of evidence have implicated Rho GTPases in cellular transformation. First, Rho GTPases are required for Ras-mediated oncogenesis.29,82-88 Rho proteins are also required for the functions of other oncoproteins such as polyoma virus middle T antigen, tyrosine kinases such as Abl, Met, Fps, BCR-Abl, RET, epidermal growth factor receptor, insulin-like growth factor receptor, G protein-coupled receptors (Mas, G2A, M1-muscarinic receptor, and Par-1), and Dbl family oncoproteins (see below).80,89-101 Finally, Rho GTPases are associated with tumor suppressor function. For example, the NF2 tumor suppressor merlin may mediate its actions, in part, by inhibiting the functions of Rac.102 Moreover, the hematopoietic-specific Rho GTPase Rac2 was shown to cooperate with Ras to promote the proliferation of mast cells heterozygous for the tumor suppressor neurofibromin (NF1).103 Second, ectopic expression of dominant active forms of Rho GTPases, analogous to the oncogenic mutants of Ras (e.g., G12V and Q61L), cause growth and morphologic transformation, and tumorigenic and metastatic growth of rodent fibroblasts.29,82,83,85,104,105 Similarly, activated Rac1 was shown to induce anchorage-independent growth29,82,83 and to promote motility, invasion, and metastasis in a variety of cell types, including fibroblasts, T lymphoma, epithelial, and melanoma cells.106-109 In the following sections below, we provide a more detailed discussion of the evidence that implicates Rho GTPases in the transforming functions of Ras, Dbl family oncoproteins, and GPCRs, as well as the emerging evidence that Rho GTPases themselves are aberrantly expressed in human cancers (see below).

Rho GTPases Are Critical for Ras-Mediated Oncogenesis The evidence implicating Rho GTPases as critical mediators of oncogenic Ras function has come mainly from studies of NIH 3T3 and Rat-1 rodent fibroblast cell lines. First, it was found that dominant negative versions of Rac1, RhoA, RhoB, RhoG, Cdc42, and TC10 blocked Ras-induced transformation of rodent fibroblasts.29,82-87,110 Second, constitutively activated mutants of Rho GTPases were shown to cooperate with activated Raf to cause synergistic transformation of rodent fibroblasts. Similarly, coordinate expression of activated Rac1 and MEK1 caused synergistic growth transformation of FRTL-5 rat thyroid epithelial cells.111 Finally, like Ras, Rac and Raf cooperated with E1A to cause transformation of primary BRK rat epithelial cells.112 These observations suggested a model where Ras activation of Rho GTPases

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via a Raf-independent effector(s) pathway can promote various aspects of Ras transformation of both fibroblasts and epithelial cells. Studies with activated or dominant negative Rho GTPase mutants have delineated the contribution of specific Rho family GTPases to distinct aspects of the transformed phenotype caused by oncogenic Ras. Although activated Rho GTPases are not sufficient to reproduce the drastic morphologic transformation caused by activated Ras or Raf, NIH 3T3 cells stably-expressing activated RhoA or Rac1, or Rat-1 cells expressing activated Cdc42, grow in anchorage-independent conditions and form tumors in nude mice, indicating that each GTPase alone can contribute to the growth-promoting actions of oncogenic Ras.29,31,84 Furthermore, analyses of Ras transformation of Rat-1 fibroblasts indicated that while RhoA, Rac1 and Cdc42 all contribute to Ras induction of anchorage-independent growth, Rac1 contributed to the reduced growth factor requirements, whereas Cdc42 and RhoA were required for morphologic transformation.82-84 Interestingly, there is evidence that Rac1 may also contribute to Ras-mediated transformation by blocking apoptosis.113 Similarly, Symons and colleagues determined that Rac1 protected MDCK epithelial cells79 and Rat1 cells78 against apoptosis induced by matrix deprivation. Finally, activated Rac1 can promote the invasion and metastasis of a variety of cell types, suggesting that the invasive properties of Ras may be mediated, in part, by activation of Rac1.107,114 Whether Rho GTPases are found to be constitutively activated in Ras-transformed rodent fibroblasts, and whether Rho GTPases are important for Ras transformation of epithelial cells, are issues that have been addressed with conflicting results, and are yet to be fully resolved. Earlier microinjection analyses showed that transient expression of oncogenic Ras caused activation of Rac, which in turn caused activation of RhoA.41,42 However, Ras-transformed rodent fibroblasts lack well-developed actin stress fibers, an observation that is inconsistent with an up-regulated RhoA activity. In fact, activated RhoA restored stress fibers to Ras-transformed cells, arguing for a down-regulated Rho function.115 Similarly, inhibition of RhoA/B/C function in mouse T cells by the C3 toxin promoted the development of malignant thymic lymphoblastic lymphomas, consistent with the model that the loss of Rho function supports oncogenesis.116 On the other hand, Rho activation in Ras-transformed MDCK cells was found to contribute to their transformation from an epithelial to a mesenchymal morphology, an observation consistent with RhoA up-regulation in Ras-transformed cells.117 Finally, as described below, observations from RhoA-specific GEF studies as well as others using pharmacologic inhibition of RhoA signalling, provide further evidence that up-regulation, rather than down-regulation, of RhoA activity is consistent with promoting oncogenesis. Similarly, Ras-transformed cells show increased membrane ruffling, which corresponds to an up-regulated Rac1 activity by oncogenic Ras.118 Furthermore, transient expression of oncogenic Ras was shown to increase Rac1-GTP levels in COS-1 cells.119 In contrast, Collard and colleagues determined that sustained activation of Ras down-regulated Rac activity in MDCK epithelial cells.117 Interestingly, activated Rac inhibited cell migration in these cells, suggesting an invasion-suppressing, rather than promoting, function for Rac.120 It was argued that while transient Ras activation can cause Rac activation, sustained Ras stimulation leads instead to down-regulation of Rac activity. Finally, Nur-E-Kamal and colleagues showed that Cdc42-GTP levels were indeed up-regulated in Ras-transformed NIH 3T3 cells and that stimulated receptor tyrosine kinases that activate Ras also activate Cdc42.121 In accordance, inhibition of Cdc42 by the Cdc42-specific GTPase binding domain of ACK-1 blocked Ras transforming activity. Thus, similar to Ras, Rho GTPases may have positive or negative roles in growth regulation that will be influenced by cell type differences and/or genetic context. The signalling pathways that link Ras with Rho family GTPases are currently incompletely understood.122-124 Several possible links between Ras and Rac have been suggested (Fig. 6). First, both Ras and Rac1 associate with the two subunits of phosphatidylinositol 3-kinase (PI3K) p110 and p85, respectively. Ras binding to p110 catalytic subunit may regulate the

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Figure 6. Linking Ras to Rho. Three main pathways relate Ras signalling events to Rho. Growth factor mediated activation of Sos leads to Ras activation which can (1) activate RalGEF/Ral pathway, leading to activation of a RhoGAP and inhibition of Rho activation. (2) Ras activation of PI3K may lead to Rho activation by lipid second messenger-mediated Dbl stimulation. (3) The Ras exchanger Sos1 associates with the newly described E3b1/Eps8 proteins, which have been shown to bind the Rho GTPase Rac1, and may modulate its activity.

association of p85 with Rac1, which may determine Rac1 function.125,126 Additionally, Ras activation of PI3K leads to the production of lipid products such as phosphatidylinositol 3,4,5-trisphosphate (PIP3), which in turn may interact with the PH domain of Dbl family proteins to promote GEF activity. For example, PI3K activation has been described to cause activation of Vav and Tiam1.127,128 Second, the fact that the Ras exchange factor Son of sevenless (Sos), which in addition to the Cdc25 Ras GEF domain also contains a functional DH/PH unit that exhibits some activity towards Rac1 suggests that Sos-dependent Ras activation may potentially lead to Sos-dependent Rac1 activation.129 Another mechanism has been proposed where the activation of Grb2/Sos1 leads to the formation of a complex (Eps8-E3b1-Sos1) able to bind and possibly modulate Rac1 activity.130 Third, Ras activation of its effector RalGDS (for Ral guanine nucleotide dissociation stimulator) leads to the activation of the Ral small GTPase, which in turn binds a Rac/Cdc42 GAP (RalBP1/RIP1/RLIP76), and consequently, may modulate its activity towards Rac1.131,134 Several reports have emphasized the importance of the RalGDS pathway in Ras transformation of mouse and human cells.135,136 Depending on the cell type and the mechanisms used, these observations place Rho GTPases at the point of convergence of a multitude of membrane-triggered events ultimately determining cellular fate.

Rho GTPases are Involved in Tyrosine Kinase Oncoprotein Transformation The implication of Ras in transformation caused by tyrosine kinase oncoproteins has also associated Rho GTPases with this process. For example, the BCR/Abl oncoprotein renders 32D mouse myeloid cells resistant to IL-3 deprivation-induced growth cessation and apoptosis, in part, by activation of Ras. Coexpression of dominant negative Rac1 rendered Ras-transformed 32D cells sensitive to IL-3 withdrawal growth inhibition and impaired invasion in vitro and

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Table 1. Aberrant Rho GTPase activity in human cancers Protein Rho GTPase Rac1 Rac2 Rac3 Rac1b

Alteration

Increased protein expression Increased protein expression Increased GTP-binding Increased transcript and protein expression of a splice variant of Rac1; increased intrinsic guanine nucleotide exchange activity RhoA Increased protein expression Increased mRNA expression RhoC Increased mRNA expression Cdc42 Increased protein expression TTF/RhoH Fusion with LAZ3/BCL6 Rho GTPase Regulator BCR Fusion protein with Abl LARG Fusion protein with MLL Tiam1 Point mutations RhoGDI Protein overexpression RhoGDI2 Transcript suppression

Tissue

Reference

Breast, colon, and lung Head and neck Breast Colon, breast

155 157 158 167,168

Breast, colon, and lung, head and neck; testes Pancreas, breast, melanocyte Breast, colon, and lung Hematopoietic cells

155,156,157 159,160,162 155 164,166

Myeloid cells Myeloid cells Kidney Ovary Bladder

142 144 141 148 147

leukemogenesis in vivo.94 Along the same line, tyrosine oncoproteins rely on multiple Rho GTPases to cause transformation via Ras. For example, Fps/Fes transformation of Rat-2 fibroblasts was impaired not only by dominant negative Ras, but also by Rac1(N17) and Cdc42(N17).93 A Ras-independent requirement of Rho GTPases in tyrosine kinase-mediated transformation has also been documented. For example, although Ras was not found to be required for the transforming activity of an avian viral mutant of EGFR, dominant negative RhoA, but not Rac1 nor Cdc42, was found to block its transforming activity.137 Thus, while tyrosine kinases can cause activation of Ras, the signalling pathways that link tyrosine kinase oncoproteins with Rho GTPases may not always involve Ras activation. This possibility is supported by earlier observations that oncogenic but not normal Ras triggers the activation of Rho GTPase cascades.

Dbl Family Oncoproteins Are Activators of Rho GTPases Further evidence that up-regulated Rho GTPase activity promotes oncogenesis comes from the observations that many Dbl family proteins were identified initially as oncoproteins before they were characterized as Rho GEFs.15,16,138 Many Dbl family proteins were identified initially in biological screens for novel transforming (e.g., Dbl, Vav, Ect2, Tim, Net1, Lfc, Lsc) or invasion-inducing (Tiam1) oncogenes106,114,139,140 and in most cases, structural alterations were identified as the mechanism of activation of their transforming activities. For example, amino terminal truncation of negative regulatory sequences accounts for the formation of constitutively activated and transforming mutants of Dbl, Vav, Ect2, and Tiam1. However, despite their identification in screens of tumor cell-derived DNA or RNA, these genetic alterations occurred during the transfection procedure and did not originate from the patient tumor sample. Despite the tantalizing links between Dbl family proteins and oncogenesis, evidence that Dbl family proteins are aberrantly activated in human cancers has been limited to three ex-

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Figure 7. Heterotrimeric G protein activation of Rho. The lysophosphatidylcholine-stimulated G2A GPCR causes activation of RhoA by activation of the p115RhoGEF/Lsc Dbl family protein. The G2A-activated Gα13 subunit binds the regulator of G protein signalling (RGS) box of p115RhoGEF/Lsc resulting in DH domain-dependent activation of RhoA.

amples (Table 1). Recently, mutations in Tiam1 have been reported in human renal carcinomas leading to constitutive activation of the protein.141 The two other Dbl family members were identified initially as proteins whose genes are rearranged in human leukemias. First, the product of the breakpoint cluster region (BCR) gene is truncated and forms a fusion protein with the Abl tyrosine kinase caused by the Philadelphia chromosome translocation, an aberration associated with about 95% of chronic myelogenous leukemias.142 Although some versions of the BCR-Abl fusion protein do contain the Rho GEF catalytic domain, these versions are less transforming than those that lack this domain. While BCR-Abl has been found to activate Rac, this has been attributed to its association with and activation of the Vav Dbl family protein.143 Second, the leukemia associated RhoGEF (LARG) was also identified as a gene rearranged in mixed lineage leukemia (MLL) and encoded a chimeric fusion protein with MLL.144 LARG has been subsequently shown to cause growth transformation of rodent fibroblasts via activation of its target GTPase RhoA.145,146 While these rearrangements mimic those that render other Dbl family proteins constitutively activated, there is no current evidence that these BCR or LARG fusion proteins promote oncogenesis by causing persistent activation of Rho GTPases. Finally, there are preliminary observations that altered expression of RhoGDI may be involved in oncogenesis (Table 1). Decreased mRNA expression of RhoGDI2 was associated with a more invasive variant of the T24 bladder cancer cell line147 suggesting a role for Rho activation in metastasis and invasion. In contrast, proteomic analyses identified RhoGDI protein overexpression in invasive ovarian cancers, suggesting that loss of Rho activity may promote tumor progression.148 However, whether Rho GTPase activity was altered by changes in RhoGDI activity were not determined in these studies.

Transforming G Protein-Coupled Receptors Are Linked to Rho GTPases Investigations into the mechanisms of receptor signalling have uncovered a pivotal role for Rho GTPases in heterotrimeric G protein-coupled receptor (GPCR) oncogenesis. The orphan GPCR Mas, initially identified in a genetic screen for transforming proteins, provided the first evidence for the transforming potential of GPCRs.149 Mas was later shown to cause lamellipodia formation as well as Rac-dependent transformation of NIH 3T3 cells.99 However, the fact that

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Mas is normally expressed in nonmitotic neural cells raises the possibility that it may not play an important role in oncogenesis. The link between GPCRs and Rho GTPases has been best delineated for the p115 RhoGEF/ Lsc, where association of activated Gα13 with the NH2-terminal RGS (regulator of G protein signalling) domain up-regulates the DH catalytic activity towards RhoA124,150 (Fig. 7). In agreement, constitutively activated variants of Gα13 and Lsc have also been shown to cause RhoA-dependent transformation of NIH 3T3 cells.151 Other GPCRs coupled to the Gα13 heterotrimeric subunit are G2A and Par-1. Like Mas, G2A and Par-1 were also identified in NIH 3T3 expression library screens for novel transforming proteins. Both G2A and Par-1 activate RhoA and their transforming activities were shown to require Rho GTPase activation.100,152 Interestingly, G2A expression is up-regulated by BCR-Abl, and Par-1 overexpression has been associated with tumor cell invasion.153,154

Aberrant Expression of Rho GTPases in Oncogenesis Evidence for the direct involvement of Rho GTPases in human cancer development, although less well documented, is rapidly accumulating (Table 1). For example, human breast, colon, and lung tumors exhibit high protein expression levels of Rac1, Cdc42, and in particular RhoA protein, when compared to normal tissue from the same patient.155 Increased levels of RhoA mRNA was detected in testicular germ cell tumor tissue and correlated with tumor progression.156 Similarly, a recent study also indicated that RhoA and Rac2 were largely overexpressed in head and neck cancer tissues and proposed their use as markers for squamous cell carcinoma of the head and neck.157 In addition, Rac3 was found to be hyperactive in human breast tumor cell lines and appears to be required for breast cancer cell proliferation.158 A role for another Rho family member, RhoC, in human cancer metastasis and invasion has been uncovered in melanoma cell lines.159 RhoC overexpression was found in 90% of inflammatory breast cancer and may promote tumor angiogenesis.160,161 RhoC was also found to be overexpressed in adenocarcinomas of the pancreas and its high levels of expression correlated with poor prognosis.162 A recent addition to the Rho family, a Cdc42-homolog termed Wrch-1, was found to be regulated by Wnt-1 oncogenic signalling in human mammary tumors.163 Overexpression of Wrch-1, like Wnt-1, promoted morphological transformation of mouse mammary epithelial cells. Taken together, Wrch-1 could mediate the transforming effects of Wnt-1 signalling in the regulation of cell morphology, cytoskeletal organization, and cell proliferation. The identification of mutant Rho proteins in human cancer specimens has so far been limited to two examples. The first represents a fusion of the Rho family member RhoH/TTF to the lymphoma-associated LAZ3/BCL6 gene, a mutation identified in nonHodgkin’s lymphoma (NHL) cell lines164,165 and was later found in patients with NHL and multiple myelomas.166 The second example is that of Rac1b, a novel splice variant of Rac1, found to be aberrantly expressed in human colorectal167 and breast168 carcinomas, establishing a direct link between Rho GTPase activation in general, and Rac in particular, and tumor development in humans. Rac1b contains an additional 19 amino acid insert just before the switch II region of Rac1, and in vitro analyses indicated that this variant exhibits a fast-cycling guanine nucleotide exchange activity, suggesting that it may be an activated variant of Rac1.

Rho GTPases Mediate Transformation through Multiple Effectors The conformations adopted by switch 1 and switch 2 in the active GTP-bound state enable high affinity interactions with multiple downstream molecules termed effectors which transmit their signals downstream.1,6,7,169 For example, over 25 known or candidate effectors of Rac1 have been identified to date. In addition to the complexity provided by multiple effectors, the fact that effector utilization can be shared by different Rho GTPases adds another level of regulatory complication to Rho GTPase function. For instance, despite their distinct functions, Rac1 and Cdc42 share a wide spectrum of effectors. 1 The majority of these

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effector-mediated signalling pathways remain to be fully delineated, but enough evidence points to the involvement of few key effectors in Rho GTPase transformation. In the following sections, we summarize what is known regarding the effectors and signalling activities important for the transforming actions of RhoA, Rac1, and Cdc42.

RhoA Transformation and ROCK Effector domain mutants have provided critical clues regarding what effector signalling pathway is important for RhoA transformation. These analyses have dissociated transformation from a variety of effector-mediated signalling pathways such as stress fiber formation and SRE activation.170,171 Interestingly, these studies have also shown strong correlations between the ability of RhoA to bind ROCK, and its ability to cooperate with Raf to cause transformation of NIH 3T3 cells.170 Indeed, treatment with the Y-27632 pharmacologic inhibitor of ROCK impaired RhoA transforming activity, as well as the transforming activity of oncoproteins (Ras, Dbl, and Net) that activated RhoA.170 In addition, a role for ROCK in facilitating RhoA-mediated invasion has been documented. Itoh and colleagues showed that dominant active ROCK promoted invasion of MM1 hepatoma cells, whereas inhibition of ROCK activity by Y-27632 reduced the ability of these cells to invade in mice.172 On the other hand, the fact that activated ROCK does not reproduce the same transforming activity seen with activated RhoA suggests that additional effectors are likely to contribute to RhoA transformation. This may involve, for example, an effector(s) that interacts with the unique insert domain of RhoA, a region required for transformation but not for ROCK binding.173

Role of Reactive Oxygen Species (ROS) NF-κB and Par-6 in Rac1 Transformation Effector domain mutants have also been used to address the importance of specific effector signalling functions in Rac1 transformation. PAK binding, as well as induction of lamellipodia, activation of the JNK and p38 MAPKs, SRF, the MEKK1 and p70 S6 kinases, were all shown to be dispensable for Rac1-induced transformation of NIH 3T3 cells.34,174,175 PAK and JNK activation were also dissociated from the ability of Rac1 to induce cell cycle progression.176 Since a role in growth induction as well as apoptosis has been established for several of these pathways, it is likely that multiple signalling pathways may also contribute to Rac1-induced cellular transformation. Rac1 is distinguished from RhoA and Cdc42 in its ability to activate the phagocyte NADPH oxidase system177,178 by interacting with and modulating the activity of the p67 subunit of the complex.179 Rac also activates a related oxidase system in nonphagocytic cells by an as yet to be identified effector. The reactive oxygen species (ROS) by-products of this reaction induce NF-κB activation, a process inhibited by ROS inhibitors such as N-acetylcystine (NAC).58 ROS have been shown to be important second messengers for Ras-mediated transformation180 and Rac-mediated cell cycle progression.181 Deletion of the Rho insert region of Rac1 impaired its mitogenic activity and ROS production in certain cell types. This finding, together with the observation that inhibitors of ROS production inhibit Rac1 mitogenic activity, support an important contribution of ROS in Rac transformation, possibly by its ability to activate the NF-κB-mediated cell survival pathway. NF-κB was found to be required for the transforming activity of two Dbl oncoproteins, Dbl and Dbs (GEFs for RhoA and Cdc42), providing further evidence for the importance of this signalling pathway in Rho GTPase transformation.182 A novel effector for Rac1 and Cdc42 has been identified as the human homologue of the cell polarity determinant in Caenorhabditis elegans, hPar-6, and has also been linked to cellular transformation.183,184 Rac1 and Par-6 associate with an atypical protein kinase C isoform, PKCζ, an interaction that releases the kinase activity of PKCζ leading to transformation. Additionally, Par-6 has been suggested to play an important role in the polarization of mammalian cells by functioning as an adaptor protein that links activated Rac and Cdc42 to PKCζ

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signalling.185,186 While it was found that PKCζ potentiated Rac1 transformation, whether PKCζ is a critical effector of Rac and Cdc42 transformation remains to be clarified.183

Cross-Talk between Vesicular Transport and Cdc42 Transformation Cerione and colleagues identified the γ-subunit (γ-COP) of COPI, a cytoplasmic ‘coatomer’ protein complex as an effector important for Cdc42 transformation.187 The coatomer coats membrane regions that bud and form vesicles, a process controlled by the Arf small GTPase. Interactions between Arf and COPI direct the formation of vesicles that are involved in the selective transport of proteins from the Golgi complex back to the endoplasmic reticulum (ER) and in protein and lipid recycling from the plasma membrane through endosomes.3 Interestingly, Cdc42 mutants that no longer bound to γ-COP or affect coatomer interaction with the Golgi (or perhaps endosomal compartments) were also unable to cause growth transformation. However, the nontransforming insert mutant of Cdc42188 retained the ability to bind γ-COP and promote vesicle trafficking, suggesting that γ-COP may not be sufficient to mediate transformation by Cdc42.

Conclusions and Future Directions While establishing the importance of Rho GTPases in human carcinogenesis will require further experimental support and validation, the vital role these GTPases play in diverse cellular functions establish them as key components of malignant tumor progression. As mediators of a variety of oncoproteins, Rho proteins represent attractive targets for intervention and inhibition of oncogene function. GGTIs represent one possible approach for blocking the action of Rho GTPases. However, in light of the many other cellular proteins that are also substrates for GGTaseI, it remains to be determined whether more precise approaches will be needed to block Rho GTPase function. Information regarding the effectors and downstream signalling pathways that promote transformation by Rho GTPases remains limited. This task is certain to be complicated by the expanding ensemble of effectors and the plethora of cellular functions attributed to Rho GTPases. Regulation of gene expression is an important consequence of Rho GTPase function and the critical gene targets that mediate Rho GTPase activities will need to be identified. Further delineation of these pathways may characterize more precise points of therapeutic intervention. Finally, while most of the attention has been focused on Rac1, RhoA, and Cdc42, the emergence of other GTPases such as RhoC and Rac3 as important GTPases in cancer suggest that the involvement of other “nonclassical” members of the family deserves more attention.

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CHAPTER 12

Rho GTPases in Lipid Metabolism Maikel P. Peppelenbosch

Abstract

I

n view of the fundamental importance of both the activation of Rho-like GTPases as well as the stimulation of phospholipid metabolism for cellular signal transduction, it should come as no surprise that the two processes are highly interconnected. For instance PI3-kinase-generated phosphorylated derivatives of phosphatidylinositol biphosphates act upstream of Cdc42 and Rac proteins, whereas Rac proteins in turn mediate arachidonic acid release. Additionally phospholipase D acts as an important effector of Rho proteins in this chapter it will be attempted to give an impression as to both the nature and the function of these interconnections between Rho family proteins and phospholipid metabolism and to review the consequences of this interaction for cellular signal transduction.

Introduction Among the myriad of events that involved the origin of life, more than 4 billion years ago, one of the most crucial developments must have been the development of an outer membrane capable of containing self-replicating assemblies of biological catalysts. Although the exact nature of the processes that led to the formation of the first cell have long been lost in the mists of time, the spontaneous aggregation of amphipathic phospholipids into vesicles easily fulfilled the need for an outer membrane. Accordingly all living cells remain enclosed by a phospholipid bilayer. Phospholipid structure has remained a constant factor for cellular physiology during evolution and thus phospholipid-derived metabolites, for which the substrate availability is virtually unlimited, could become important molecules for signal transduction. Three major groups of phospholipases exist (Fig. 1), phospholipases A2, phospholipases C, and phospholipase D, and the enzymatic products of all three groups are highly relevant for intracellular signal transduction and in the case of phospholipase A2 also for intercellular signalling. In view of the fundamental importance of both the activation of Rho-like GTPases as well as the stimulation of phospholipid metabolism for cellular signal transduction, it should come as no surprise that the two processes are highly interconnected. In the present chapter, it will be attempted to give an impression as to both the nature and the function of these interconnections. To this end, four important different aspects of the interaction between lipid metabolism and Rho family GTPases will be dealt with: i) Rho family GTPases and phosphatidylinositol phosphates ii) Rho family GTPases and arachidonic acid metabolism, iii) Rho family GTPases and inositol-3-phosphate generation and iv) Phospholipase D as en effector of Rho family GTPases.

Rho Family GTPases and Phosphatidylintositol-3-Phosphates Membrane phospholipids containing a phosphate-linked inositol moiety on the sn-3 position of the glycerol backbone are a substrate for phosphatidylinositol 3-kinases (PI3-kinase). The most biologically relevant product of the resulting enzymatic reaction is phosphatidylinositol-3,4,5-triphosphate (PIP3), as this lipid is thought to fulfill crucial funcRho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Figure 1. Action of phospholipases in generating phospholipid-derived second messenger molecules. Membrane phospholipids constitute an important source for the production of second messengers. Various enzymes are involved in the production of second messengers from phospholipids, hydrolysing esterbonds or phosphorylating inositol sugars. Phospholipase A2 action produces arachidonic acid, phospholipase C diacylglycerol and inositol-1,4,5-triphosphate, phospholipase D produces phosphatidic acid and PI3-kinase phosphatidyl inositol 3,4,5-triphosphate (X represents the various possible headgroups, e.g., choline, serine, ethanolamine, and inositol for phosholipase C and PI3-kinase)

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Figure 2. A connection map for the activation of Rho GTPases by PI3-kinase.

tions in cellular signalling (for a review,1). Enhanced production of PIP3 is noted in response to a variety of different stimuli, in particular growth factors and cytokines, and has now been implicated in various different signal transduction events, in particular those associated with preventing apoptosis, either via protein kinase B-dependent2 or independent pathways.3 In addition, enhanced PI3-kinase activity is implicated in cytoskeletal reorganization (e.g.,refs. 4,5) Interfering with PI3-kinase enzymatic activity inhibits cytoskeletal reorganization upstream of Rac activation (e.g., in insulin or platelet derived growth factor (PDGF)-stimulated Swiss 3T3 cells and CHO cells6,7 or after activation of the chemoattractant formyl peptide receptor in Cos cells8). Also the increase in Rac-GTP to PDGF is sensitive to inhibition of PI3-kinase in various experimental systems (e.g., ref. 9). Rac activation by, for instance tissue factor/factor VIIa interaction10 and Kit receptor activation11 seems to be solely mediated via Src-dependent PI3-kinase activity. Most often, however, there is not an absolute requirement for stimulation of the lipid kinase in receptor-dependent Rac activation whereas in human neutrophils the coupling between PI3-kinase and Rac does not even seem to exist.12,13 Thus, the physiological importance of PI3-kinase as an upstream regulator of Rac appears to be restricted to a subset of cell types. Although there is little evidence that Rho is a direct target for PI3-kinase, inhibition of PI3-kinase abolishes Cdc42 activation in A14 fibroblasts in response to factor VIIa.10 In agreement, injection of PI-3kinase blocking antibodies, especially those directed against p110δ subunit, into Bac1.2F5 macrophages inhibited chemotaxis and reduced filopodia formation14 which are both well-established to be Cdc42-dependent in this cell type.15 Also Rac-dependent effects were inhibited by these antibodies, suggesting that the coupling between PI3-kinases and Rac has relevance for cells of monocytic lineage. The coupling between PI3-kinase and Cdc42 may even be of greater general importance. The mechanistic understanding as to how intracellular PIP3 accumulation may result in GTPase activation remains obscure. The best understood direct effector of PIP 3 is PIP3-dependent protein kinase-1 (PDK1)16 but there is no evidence that this kinase is somehow implicated in the activation of Rac or Cdc42 by PI3-kinase. More plausible candidates are exchange factors for Rac like Vav and Tiam1, of which the exchange activity may be enhanced in vitro by PIP3.17,18 It seems likely that this effect is responsible for enhanced GTP loading of Rac and Cdc42 following stimulation of PI3-kinase enzymatic activity (Fig. 2). Additional links between Rho and Rac and phosphoinositide metabolism are found in the direct association between these small GTPases and PIP kinases. Type-I phosphatidylinositol 4-phosphate 5-kinases are activated by this association resulting in the production phosphatidylinositol 4,5-bisphosphate. This lipid appears to mediate some of the effects of Rho and Rac on the

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actin cytoskeleton.19-21 Thus phosphoinositide metabolism is both an upstream and downstream mediator of Rho-like GTPases.

Rac-Mediated Arachidonic Acid Release Where as PIP3 may act mainly upstream of Rac, arachidonic acid metabolism is a downstream effector of Rac activation. Expression of dominant negative Rac proteins, using either a vaccinia virus or stable transfected cells abrogates arachidonic acid release or peptidoleukotriene formation in response to receptor tyrosine kinase activation.22 Also TNFα-dependent LTB4 synthesis is sensitive to dominant negative Rac proteins.23 Likewise, the calcium ionophoredependent translocation of 5-lipoxygenase (the enzyme converting arachidonic acid to leukotriene A4) is not observed in cells expressing Rac N17.24 Conversely, overexpression of wild type Rac or the constitutively active Rac V12 results in enhanced production of arachidonic acid and its metabolites.22,23,25 Thus Rac is both sufficient and necessary for arachidonic release in a variety of experimental systems. The molecular details underlying Rac-dependent arachidonic acid release remain largely obscure. Generally speaking arachidonic acid metabolism can be initiated either indirectly, via the action of phospholipase C to produce diacylglycerol from membrane phospholipids followed by diacylglycerol lipase-mediated release of arachidonic acid, or directly from membrane phospholipids via hydrolysis of the ester bond at the sn-2 position by phospholipase A2. In turn, the phospholipase A2 family is fairly large group of enzymes, most often subdivided in the smaller 14 kD isoforms and the larger 85 kD cytosolic phospholipase A2s. The smaller 14 kD isoforms are further subdivided in type I and type II, based on their primary structures (mainly the presence or absence of a cysteine at position 11). Hence, multiple routes by which Rac activation could lead to the release of arachidonic acid exist and it is unclear what the relative contribution of these different isoforms to Rac-dependent arachidonic acid production is. A role for diacylglycerol lipase in Rac-dependent arachidonic acid metabolism may be inferred from experiments involving fibroblast growth factor. Both the arachidonic acid release, the Ca2+ influx as well as the neurite outgrowth in response to fibroblast growth factor are sensitive to inhibitors of diacylglycerol lipase.26-28 The findings that growth factor-induced calcium influx and actin reorganisation29-31 are mediated by Rac, suggest a function for diacylglycerol lipase in Rac-dependent arachidonic acid release. This is further supported by observations indicating that Rac is capable of activating phospholipase C activity;32,33 see also below) and thus it is possible that Rac-dependent enhanced cellular levels of phospholipase C-produced diacylglycerol lead to increased substrate availability for diacylglycerol lipase, resulting in increased arachidonic acid release. Experiments assessing the effect of dominant negative Rac proteins on diacylglycerol lipase-mediated arachidonic acid release could help assessing the possible importance of this pathway. One of the major sources of epidermal growth factor-induced production of arachidonic acid seems to be the 85 kD cytosolic phospholipase A2. 34 As the epidermal growth factor-dependent release of arachidonic acid is highly sensitive to the expression of Rac N17,30 it is likely that this phospholipase is at least partially responsible for Rac-mediated arachidonic acid production. In agreement with this, antisense studies show that Ha-Ras V12-induced Rac-mediated arachidonic acid release requires the 85 kD cytosolic phospholipase A2 35. Furthermore, TNF-induced leukotriene B4 generation is sensitive to both Rac N17 expression and the 85 kD cytosolic phospholipase A2 inhibitor AACOCF3. In this experimental system also TNF-dependent transactivation of the serum response element (SRE) was inhibited by both Rac N17 as well as by antisense oligonucleotides of the 85 kD cytosolic phospholipase A2.36 In addition, expression of the constitutively active Rac V12 is sufficient for 85 kD cytosolic phospholipase A2 translocation to the membrane25 and Rac V12-induced generation of reactive oxygen species is not observed in the presence of AACOCF3.25 Thus the existing literature data are difficult to explain without al least some role for the 85 kD cytosolic phospholipase A2 in mediating Rac dependent arachidonic acid release.

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Figure 3. Rac-dependent pathways leading to arachidonic acid release. Rac activates the 85 kD cytoplasmic phospholipase A2 (cPLA2) via at least to pathways, one being dependent on p38 MAP kinase, the other leading to increased cellular calcium levels via the activation of a 10 pS calcium channel. Rac-dependent phospholipase C activation may also increase cytoplasmic calcium levels and, in addition provide substrate for diacylglycerol (DAG) lipase. IP3 is inositol-1,4,5-triphosphate. See text for details

Activation of the 85 kD cytosolic phospholipase A2 involves translocation to intracellular membranes after Ca2+-binding to the C2 domain37 and phosphorylation of serine 505.38 Serine 505 lies in a MAP kinase phosphorylation consensus motif and in vitro p42/p44 MAP kinase is capable of phosphorylating this site.39 In addition, phosphorylation of serine 437, serine 454 and especially serine 727 have emerged as an important regulatory events.40,41 Finally, tyrosine phosphorylation of the 85 kD cytosolic phospholipase A2 seems important for its regulation (Marcel Spaargaren, Academic Medical Centre, Amsterdam, Personal Communication). Thus regulation of the 85 kD cytosolic phospholipase A2 is highly complex and may intersect at different points with Rac-dependent signalling. Interestingly, p38 MAP kinase seems important for controlling the 85 kD cytosolic phospholipase A2. The p38 MAP kinase activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate in vitro the lipase uniquely on serine 727.42 In agreement, pharmacological inhibition of p38 MAP kinase inhibits phosphorylation of the 85 kD cytosolic phospholipase A2 in general43 and of serine 727 in particular.41,42 Furthermore, arachidonic metabolism also is sensitive to pharmacological p38 MAP kinase inhibition in a variety of in vitro systems (e.g., ref. 44), as well as in LPS-challenged volunteers in vivo (Bernt van den Blink and Maikel P. Peppelenbosch, unpublished data). As the action of p38 MAP kinase downstream of Rac is now well recognised (e.g., refs. 10,45,46) it seems likely that Rac-dependent p38 MAP kinase activation contributes to the activation of the 85 kD cytosolic phospholipase A2. In addition, Ca2+ signalling may be involved. Arachidonic acid release mediates growth factor-induced Ca2+

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influx47,48 and accordingly Rac N17 inhibits growth factor-induced Ca2+ influx through the plasma membrane.30 Ca2+ influx, however, facilitates arachidonic acid release37 and thus a positive feed back loop exists. It possible that Rac-dependent phospholipase C32,33 activity and subsequent Ca2+ release is involved in Rac-dependent arachidonic acid metabolism by stimulating this positive feed back loop (Fig. 3). Further complications are added by the observation that 4-bromophenacylbromide seems capable of inhibiting Rac-dependent arachidonic acid release and metabolism (e.g., refs. 49,50). This inhibitor is more potent on the 14 kD phospholipase A2 when compared to the 85 kD cytosolic phospholipase A2. It is possible, therefore, that also this group of small phospholipases A2 is involved in arachidonic acid release downstream of Rac, but this question remains to be addressed.

Rho A As an Effector of Arachidonic Acid Metabolism Stress fibers formation often involves the sequential activation of Rac and Rho (e.g., refs. 29,51) as well as metabolism of arachidonic acid (e.g., refs. 52-54). Interestingly, arachidonic acid metabolism-dependent stress fibre formation requires functional Rho proteins.22 Also transactivation of the SRE by arachidonic acid is sensitive to either Rho N19 expression or to botulinum C3 transferase.55 It appears, therefore, that arachidonic acid metabolism is capable of activating Rho. It should be noted, however, that evidence exists showing that arachidonic acid may directly activate Rho kinase.56,57 Also the Rho effector PKN is directly activated by arachidonic acid58 and it is therefore possible that Rac-dependent arachidonic acid metabolism acts in concert with Rho to produce cytoskeletal effects rather than acting upstream of Rho. Hence, now that pull down techniques exist that allow direct visualization of the Rho activation state, it may be important to reevaluate the action of arachidonic acid metabolism on Rho activation. The molecular details by which arachidonic acid metabolism could effect Rho GTP loading are still unresolved. Interestingly, a large number of studies have shown that arachidonic acid metabolites modulate the activity of GAPs in vitro. For instance, RasGAP is inhibited by arachidonic acid and lipoxygenase products, whereas it is stimulated by prostaglandins.59 Importantly, RhoGAP and other interesting GAPs are regulated by arachidonic acid,60-62 probably via a physical association.63 Thus direct inhibition of RhoGAP is a possible mechanism for arachidonic acid metabolism to increase Rho GTP loading. Alternatively, members of the Rho family of GTP-binding proteins are localized in the cytosol of cells by complexation with a protein known as (Rho) GDP-dissociation inhibitor, preventing their activation. This interaction can be disrupted in the presence of various lipids and arachidonic acid is particularly effective in this respect.64 Finally, arachidonic acid metabolites also act via extracellular G protein-linked receptors, and activation of these receptors may cause secondary signal transduction and Rho activation.65,66 As stated above, Rho activation by arachidonic acid metabolites has not been investigated directly and thus the relative contribution of these possible mechanisms to Rho activation is unknown.

Rho Family GTPases and Inositol-1,4,5-Triphosphate Generation In addition to phospholipase A2, phospholipase Cγ also has emerged as a possible connection between lipid metabolism and Rho family proteins. Phospholipase C hydrolyses phosphatidyl 4,5-biphosphate (PIP2), resulting in the release of the second messengers diacylglycerol and inositol-1,4,5-triphosphate, the former activating protein kinase C and acting as substrate for diacylglycerol lipase, whereas the latter releases Ca2+ from the intracellular stores.67 After growth factor stimulation, phospholipase Cγ binds to phosphotyrosine residues of the receptor or an associated signalling molecule, followed by tyrosine phosphorylation of the enzyme itself. This latter phosphorylation allows the enzyme to overcome the inhibitory influence of profilin binding to PIP2 on the hydrolysis of this lipid.68 Recent data however, indicate an alternative activation route, involving Rho family proteins.

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In RBL-2H3 mast cells, expression of either constitutively active Rac or Cdc42 increased the antigen-induced production of inositol-triphosphate and Ca2+ mobilization, whereas the dominant negative Rac N17 proteins partially inhibited calcium responses.69 Also, in a mutant RBL-2H3 mast cell that is defective in Ca2+ mobilization, expression of activated Rac and Cdc42 proteins restored Ca2+ signalling.33 The observation that these constitutively active Rac and Cdc42 proteins stimulated Ca2+ responses, but were not active on phosphoinositide metabolism per se, suggest that Rho family proteins are not directly responsible for the activation of phospholipase Cγ, but rather provide an important accessory signal. The nature of this signal is unknown, but is likely to involve the direct interaction of Cdc42 with phospholipase Cγ detected by these authors.69 An inhibitory effect of dominant negative Rac proteins on T cell receptor-dependent PIP2 hydrolysis was also observed by Arrieukerlou et al.32 These authors show that Rac acts in T cell receptor signal transduction to orchestrate the formation of a multiprotein complex, including ZAP-10, LAT, and phospholipase Cγ. This complex then mediates phosphorylation and activation of phospholipase Cγ as evident by the decreased phospholipase Cγ phosphorylation and PIP2 hydrolysis in Rac N17 expressing T cells. Although a similar, Rac or Cdc42 containing complex may mediate phospholipase Cγ activation in mast cells, effects of dominant negative Cdc42 expression on phospholipase Cγ phosphorylation were explicitly not noted in mast cells.69 Hence, further research is needed to clarify the molecular mechanisms explaining the effects of Rac proteins on phospholipase Cγ activity.

Phospholipase D As an Effector of Rho Family GTPases Phospholipase D functions as a direct downstream effector of Rho (reference). The function of this ubiquitously expressed enzyme however is still ill-defined. The enzyme releases phosphatidic acid from membrane phospholipids. Addition of phosphatidic acid or purified phospholipase D to fibroblasts causes stress fiber formation,70 a hallmark of Rho signal transduction.51 Furthermore Rho-activating stimuli, like LPA, activate phospholipase D and increase phosphatidic acid production.71,72 Importantly, reducing phospholipase D enzymatic activity, using either butan-2-ol or expression of inactivated phospholipase D inhibits LPA-induced stress fiber formation, but does not interfere with Rho GTP loading or the activation of different Rho effectors by LPA.73,72 As LPA-induced stress fiber formation is mediated by Rho,51 phospholipase D seems to fulfil an important function downstream of Rho, mediating extracellular signals to the cytoskeleton. In agreement, the C3 exoenzyme from Clostridium botulinum and dominant negative Rho proteins substantially reduce phospholipase D activation.74,75 Phospholipase D directly interacts with GTP-Rho but does not bind to GDP-Rho. Furthermore, only forms of PLD that can bind to Rho are activated by the GTPase.76 Many other Rho effectors have been implicated in Rho-mediated stress fibre formation77 and the relative contribution of different Rho effectors to cytoskeletal reorganisation remains only very partially resolved. The function of phospholipase D in the spectrum of events needed for stress fibre formation seems to lie in the cross-linking of actin filaments mediated by α-actinin, maybe by producing a local increase in PIP2,73 although as acknowledged by these authors themselves, much work has still to be done to corroborate this suggestion. Interestingly, also Cdc42 is capable of directly binding phospholipase D and this physical interaction only occurs when Cdc42 is GTP-loaded.78 Furthermore, this interaction is capable of activating phospholipase D enzymatic activity, but only if Cdc42 is geranylgeranylated –and thus functionally active. Finally, mutants of Cdc42 that are unable to activate phospholipase D inhibit stimulation of this enzyme by Arf and protein kinase C.78 Thus, phospholipase D is not only an effector of Rho, but also of Cdc42. Hence, it is tempting to speculate that phospholipase D has a general facilitating role downstream of Rho GTPases, participating in cross-linking of actin filaments by α-actinin, a process that is important for actin reorganization in general and not only for stress fiber formation.79,80 It will be particularly interesting to investigate whether also Rac is capable of activating phospholipase D and some circumstantial evidence for this exists.81

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Figure 4. Activation of Rho by arachidonic acid. Arachidonic acid may release Rho proteins from GDIs, may inhibit RhoGAP, or arachidonic metabolites may activate extracellular serpentine receptors, in turn activating Rho.

Final Considerations Rho family GTPases exert a myriad of effects on the activity of phospholipases and conversely, lipid metabolism modulates the function of Rho family GTPases in a critical fashion. Thus it is clear that the long coexistence of lipid biochemistry and Rho family signal transduction during evolution has resulted in an intertwining of these two classes of biological processes. As a crude rule of the thumb, it could be said that PIP3 generation is important for the activation of Rho family GTPases, and that phospholipase C and phospholipase D may be important downstream effectors of Rho family proteins. In addition, phospholipase A2 may be important for signal transduction between GTPases. The important question that now needs to be addressed is to assess the importance of these interactions for pathophysiology in a living being. With advent of techniques capable of measuring in vivo signal transduction, answering this question may soon turn in to reality.

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64. Chuang TH, Bohl BP, Bokoch GM. Biologically active lipids are regulators of Rac.GDI complexation. J Biol Chem 1993; 268:26206-26211. 65. Gronroos E, Andersson T, Schippert A et al. Leukotriene D4-induced mobilization of intracellular Ca2+ in epithelial cells is critically dependent on activation of the small GTP-binding protein Rho. Biochem J 1996; 316:239-245. 66. Setoguchi H, Nishimura J, Hirano K et al. Leukotriene C(4) enhances the contraction of porcine tracheal smooth muscle through the activation of Y-27632, a rho kinase inhibitor, sensitive pathway. Br J Pharmacol 2001; 132:111-118. 67. Van Den Brink GR, Bloemers SM, Van Den Blink B et al. Study of calcium signalling in nonexcitable cells. Microscopy Research & Technique 1999; 46:418-433. 68. Nishibe S, Wahl MI, Hernandez-Sotomayor SM et al. Increase of the catalytic activity of phospholipase C-gamma 1 by tyrosine phosphorylation. Science 1990; 250:1253-1256. 69. Hong-Geller E, Cerione RA. Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells. J Cell Biol 2000; 148:481-494. 70. Ha KS, Exton JH. Activation of actin polymerization by phosphatidic acid derived from phosphatidylcholine in IIC9 fibroblasts. J Cell Biol 1993; 123:1789-1796. 71. Ha KS, Yeo EJ, Exton JH. Lysophosphatidic acid activation of phosphatidylcholine-hydrolysing phospholipase D and actin polymerization by a pertussis toxin-sensitive mechanism. Biochem J 1994; 303:55-59. 72. Cross MJ, Roberts S, Ridley AJ et al. Stimulation of actin stress fibre formation mediated by activation of phospholipase D. Curr Biol 1996; 6:588-597. 73. Kam Y, Exton JH. Phospholipase D activity is required for actin stress fiber formation in fibroblasts. Mol Cell Biol 2001; 21:4055-4066. 74. Frankel P, Ramos M, Flom J et al. Ral and Rho-dependent activation of phospholipase D in v-Raf-transformed cells. Biochem Biophys Res Commun 1999; 255:502-507. 75. Guillemain I, Exton JH. Role of rho proteins in agonist regulation of phospholipase D in HL-60 cells. Biochim Biophys Acta 1998; 1405:161-170. 76. Cai S, Exton JH. Determination of interaction sites of phospholipase D1 for RhoA. Biochem J 2001; 355:779-785. 77. Ridley AJ. Stress fibres take shape. Nat Cell Biol 1999; 1:E64-66. 78. Walker SJ, Wu WJ, Cerione RA et al. Activation of phospholipase D1 by Cdc42 requires the Rho insert region. J Biol Chem 2000; 275:15665-15668. 79. Malcolm KC, Ross AH, Qiu RG et al. Activation of rat liver phospholipase D by the small GTP-binding protein RhoA. J Biol Chem 1994; 269:25951-25954. 80. Hess JA, Ross AH, Qiu RG et al. Role of Rho family proteins in phospholipase D activation by growth factors. J Biol Chem 1997; 272:1615-1620. 81. Banno Y, Takuwa Y, Akao Y et al. Involvement of phospholipase D in sphingosine 1-phosphate-induced activation of phosphatidylinositol 3-kinase and Akt in Chinese hamster ovary cells overexpressing EDG3. J Biol Chem 2001; 23:23.

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CHAPTER 13

The Role of Rho GTPases in Vesicle Trafficking Nicole Rusk and Marc Symons

Summary

M

embers of the Rho family of GTPases play important roles in the regulation of many stages of vesicular trafficking, including vesicle internalization from the plasma membrane, vesicle recycling and exocytosis. Rho-mediated regulation of vesicular trafficking has an impact on diverse biological functions, including cell proliferation and motility. Here we discuss the role of the Rho GTPases and their salient effectors in various aspects of vesicular trafficking and focus on the biological implications of these functions.

Introduction The plasma membrane of eukaryotic cells is in a constant flux that is determined by various modes of endocytic and exocytic trafficking. This dynamic is important for communication across the plasma membrane and allows for sampling of the external environment in addition to nutrient uptake. Vesicular trafficking is controlled by members of several subfamilies of the Ras superfamily of small GTPases. Members of the Arf family control the formation of transport vesicles, whereas Rab proteins function in directing transport of vesicles to their appropriate target compartments. There also is increasing evidence that GTPases of the Rho and Ras subfamilies are important modulators of membrane trafficking.1-4 In this chapter, we will review recent developments in our understanding of the role of Rho GTPases in vesicle trafficking. We will also attempt to put some of these findings in the context of other functions that are regulated by Rho GTPases, including cell proliferation and migration.

Role of Rho GTPases in Clathrin-Mediated Endocytosis Rac The first evidence for the involvement of Rac1 in clathrin mediated endocytosis came from studies in HeLa cells showing that overexpression of constitutively active Rac1 causes inhibition of EGF and transferrin receptor internalization.5 A possible mechanism that could mediate this effect is that reorganization of the actin cytoskeleton by Rac somehow modulates the formation of clathrin-coated pits or subsequent vesicle internalization. Interestingly however, destabilization of the actin cytoskeleton by cytochalasin D does not change the inhibitory effect of Rac, indicating that Rac does not inhibit endocytosis via altering the actin cytoskeleton.5 A subsequent study addressing the possible role of actin dynamics on clathrin-mediated endocytosis indicated that an intact actin cytoskeleton is required for internalization only in certain cell types. Whether cells are attached to extracellular matrix or are in suspension further modulates this requirement.6 Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Figure 1. Rho GTPases on the crossroad between membrane trafficking and the actin cytoskeleton. The Rac1 effectors synaptojanin 2 (SJ2) and type I PIPkinase directly modulate PI(4,5)P2 levels at the plasma membrane. PI(4,5)P2 is critical for the assembly of the endocytic coat, e.g., binding of the adaptor protein AP2 to the plasma membrane, assembly of the clathrin lattice and recruitment of dynamin to the coated pit. The interaction of huntingtin interacting protein 1 related (Hip1R) with clathrin is also PI(4,5)P2-dependent, as is the Arp2/3 activation by N-WASP. Amphiphysin, another endocytic scaffold protein binds to SJ2 and dynamin.102 In addition to its role in the fission of clathrin-coated pits, dynamin plays a role in actin dynamics. The latter function may be mediated by actin binding protein 1 (mAbp1), profilin and cortactin, all of which are direct binding partners of dynamin. The Cdc42 effector N-WASP binds syndapin103 as well as the endocytic scaffold protein intersectin. In neuronal cells, N-WASP can enhance the GEF activity of intersectin on Cdc42, suggesting the existence of a positive feedback loop. Rho GTPase effectors are framed, proteins dependent on PI(4,5)P2 for their membrane localization or binding activity are circled.

The identification of synaptojanin2, a phosphatidylinositol lipid phosphatase, as a Rac1 effector has suggested another mechanism for the role of Rac1 in modulating receptor internalization.7 PI(4,5)P2 is a substrate of synaptojanin 2 and an essential binding partner for many components of the endocytic machinery.8 A model was proposed in which activated Rac1 promotes translocation of synaptojanin 2 to the plasma membrane, where it hydrolyzes PI(4,5)P2, thereby inhibiting clathrin coated pit formation. Interestingly, and complicating this picture, Rac1 also binds to PI 3'-kinase and members of the phosphatidylinositol4-phosphate5'-kinase family (also termed type I PIPkinases),9,10 all of which modulate PI(4,5)P2 levels. Thus, Rac may be a key player in the control of PI metabolism. Even though the precise role of the actin cytoskeleton in mammalian cell endocytosis is still unclear, more and more actin binding proteins with PI(4,5)P2-dependent roles in endocytosis are being identified: Huntingtin interacting protein1-related (Hip1R) binds both F-actin and clathrin,11 the latter in a PI(4,5)P2-dependent fashion. EGF receptor internalization is blocked with a Hip1R ENTH-domain mutant defective in PI(4,5)P2 binding.12 Dynamin, the GTPase necessary for scission of endocytic vesicles, depends on PI(4,5)P2 for its recruitment to the plasma membrane.13 Recently, dynamin has been implicated in the regulation of actin filament nucleation and has been shown to be a component of actin comet tails on intracellular vesicles.14,15 This, together with the finding that dynamin also binds profiling,16 cortactin16 and mammalian actin binding protein1,17 proteins involved in F-actin assembly, shows that the actin cytoskeleton and endocytosis share many of the same players (see also Fig. 1).

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Rac1 could control Hip1R’s role in clathrin assembly and dynamin’s recruitment to the plasma membrane by modulating PI(4,5)P2 levels via its effectors SJ2 and type 1 PIPkinase. It will be important however to elucidate the spatio-temporal regulation of these Rac effectors for a full understanding of the role of Rac in the crosstalk between clathrin-mediated endocytosis and the cytoskeleton. Rac is important for cell transformation induced by Ras and other oncogenes18,19 (see also Chapter 11). There is increasing evidence that retardation of the internalization and degradation of growth factor receptors can stimulate cell proliferation (reviewed in refs. 20-21). Thus the inhibitory effect of Rac on clathrin-mediated endocytosis could be one of the means by which this GTPase contributes to cell proliferation and transformation.

Rho Different members of the Rho subfamily appear to regulate receptor-mediated endocytosis at distinct steps along the endocytic pathway. In HeLa cells, constitutively active RhoA-V14 blocks internalization of the EGF and transferrin receptors, similar to the effect of constitutively active Rac1.21 Overexpression of wild type RhoA in HEK 293 cells also leads to a marked reduction of muscarinic acetylcholine receptor internalization.22 On the other hand, wild type RhoB or constitutively active RhoB have no effect on EGF receptor internalization, but prevent intracellular trafficking of the receptor from early to late endosomes.23 A Rho effector that could mediate the effect of RhoB on receptor trafficking is protein kinase C related kinase 1 (PRK1). Rho B targets PRK1 to endosomal membranes24 and overexpression of a kinase dead version of PRK1 releases the inhibition caused by RhoB.23 Even though the exact substrates of PRK1 remain to be identified, it is conceivable that the formation and/or maintenance of endocytic complexes are altered by protein phosphorylation. Instances of this type of regulation of endocytic proteins have been documented. Phosphorylation of dynamin1 and synaptojanin1 for example inhibits their binding to amphiphysin, and phosphorylation of amphiphysin inhibits its binding to AP-2 and clathrin.25 It should be noted that the exact intracellular localization of RhoB remains controversial. Using epitope-tagged versions of RhoB, Adamson et al showed a partial localization of RhoB in early endosomes26 and in multivesicular bodies.27 In a recent study however, RhoB was shown to colocalize with Golgi markers, rather than with endosomes.28 In summary, it appears that both RhoA and B may cause prolonged growth factor receptor signalling. As is the case for Rac1, it will be important to determine the contribution of these effects to cell transformation. Elucidation of the precise mechanisms that mediate the roles of these GTPases in clathrin-mediated endocytosis may lead to the identification of novel drug targets for cancer therapy.

Cdc42 Two recent reports showed that clathrin heavy chain is a binding partner of the Cdc42 effector activated Cdc42-associated kinase (Ack).29,30 Interestingly, low expression levels of Ack1 enhance transferrin receptor internalization in a kinase-independent fashion, whereas high levels of Ack1 inhibit transferrin uptake.29 A stimulatory role for Ack in receptor internalization is further supported by findings in C. elegans showing that ARK-1, a tyrosine kinase that is related to Ack, is a negative regulator of epidermal growth factor (EGF) receptor signalling.31 The inhibitory effect of overexpressed Ack on receptor internalization might be a consequence of preventing AP-2 from binding to clathrin.30 AP-2 is an essential adapter protein of the endocytic complex and failure to associate with clathrin would lead to inhibition of coated pit formation. Interestingly, ARK-1 binds to the Grb2 homolog SEM-5, establishing a direct link to the EGF-like receptor LET-23, suggesting that the effect of ARK-1 on endocytosis may be independent of Cdc42. Ack1 also contains multiple proline-rich domains that provide docking sites for SH3-containing proteins such as Nck.29 Like Rac1, Cdc42 may also be involved in linking the actin cytoskeleton to the control of endocytosis. The Cdc42 effector N-WASP, an

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activator of Arp2/3,32 binds intersectin, a key scaffolding protein for the endocytic complex.33 It is noteworthy that intersectin may be involved in a positive feedback loop, regulating the activity of the GTPases by acting as a GEF for Cdc42 in neurons34 and by inactivating the GAP activity in cdGAP toward Rac1.35

Macropinocytosis Macropinosomes are formed from plasma membrane protrusions which fuse at the point of membrane-membrane contact and bring their fluid content into the cytoplasm of the cell. Macropinocytosis is dependent on actin polymerization and a number of recent studies set out to elucidate the importance of Rho GTPases in this process (reviewed in ref. 36). In fibroblasts, activated Rac strongly enhances pinocytosis.37 Recently, the Rac/Cdc42 effector PAK has been implicated in this activity. PAK colocalizes to membrane ruffles as well as pinocytic vesicles and the autoinhibitory domain of PAK inhibits Rac-induced pinocytosis, indicating that the PAK kinase activity is important for pinocytosis.38 Possible mediators of this function include Lim-domain-containing protein kinase (LIMK), myosin light chain kinase (MLCK) and myosin light chain (MLC). PAK may directly phosphorylate LIMK,39 that in turn phosphorylates and inactivates cofilin,40,41 a protein involved in actin depolymerization. MLCK and MLC itself are additional potential substrates of PAK which could provide a relay to the actomyosin contractile machinery.42,43 For a detailed discussion on the signal transduction machinery that controls the actin cytoskeleton downstream of Rho GTPases, see Chapters 7 and 9. The physiological significance of pinocytosis in fibroblasts remains to be determined. An intriguing possibility is that PAK-regulated plasma membrane recycling may contribute to directed cell migration. Fibroblasts overexpressing either wild type or constitutively active PAK have large lamellipodia at the leading edge and are more motile than control cells. Cells expressing a kinase-dead PAK are also highly motile, but show defects in directed motility.43 In endothelial cells, a model has been suggested where PAK coordinates the formation of new adhesion sites at the leading edge of the cell, while inducing contraction and detachment at the trailing edge.44 It remains to be seen how directed cell motility is influenced by the cell’s ability to pinocytose and whether inhibition of pinocytosis can affect motility. An example where the connection between pinocytosis and cell function has been characterized in more detail is presented by dendritic cells (DC), antigen presenting cells. In their immature form they are highly pinocytic, efficiently capturing antigen. As they mature due to an inflammatory stimulus, they cease to pinocytose and instead process and present the captured antigen on their cell surface to T cells (reviewed in ref. 45). Garrett et al showed that immature DCs have increased levels of activated Cdc42 and that macropinocytosis can be blocked by dominant negative Cdc42. In addition, microinjection of constitutively active Cdc42 into mature DCs restores their ability to pinocytose. The authors suggest that maturation stimuli could control Cdc42 at the level of nucleotide exchange, i.e., by regulation the activity and/or expression level of a Cdc42-GEF.45 Rac activity is also essential for macropinocytosis in DCs, although activated Rac1 does not restore the endocytic ability of mature DCs, indicating that, in contrast to Cdc42, it is not the control point in the downregulation of pinocytosis that occurs during DC maturation.45,46 It is interesting to note that the activity of Rac does not appear to be essential for membrane ruffling in DCs, suggesting that Rac may be required for pinocytosis at a point downstream of lamellipodial formation.46,47 It will therefore be important to determine which of the other Rho family members controls membrane ruffling in these cells.

Phagocytosis The uptake of bacterial pathogens or apoptotic cells is essential for host defense and tissue repair. Phagocytosis is routinely carried out by highly specialized cells like macrophages and neutrophils, although a number of additional cell types are able to phagocytose. Complement

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Figure 2. Model for the control of degranulation by Rho GTPases. IgE stimulates PLC in a Rac- and Cdc42-dependent fashion, leading to an increase in IP3 production, calcium release and degranulation. After EGF stimulation, Rac activates PLD, leading to phosphatidic acid (PA) production, which in turn contributes to degranulation in a PLC-independent fashion. PA also is a cofactor for type I PIPkinases, which directly bind to Rac, leading to PI(4,5)P2 production, a substrate for PLD.

(CR) and immunoglobulin receptors (FcR) are the major phagocytic receptors. Their engagement triggers reorganization of the actin cytoskeleton. In the case of FcR, this consists of the extension of pseudopods which engulf the particle to be taken up and so form a phagosome.48 The phagosome gets taken up by the cell and fuses with endo-lysosomal vesicles to digest the content of the phagososme.49 CR activation requires additional stimuli like chemokines and integrins to form the F-actin cup. Here no pseudopod formation is necessary, particles sink into the phagocytic cup and get taken up. (reviewed in ref. 47, 50) Since actin plays a pivotal role in phagocytosis, it comes as no surprise that Rho GTPases also have been implicated as regulators of this process. Rac and Cdc42 have been described as essential for FcR-mediated phagocytosis, leading to a respiratory burst and subsequent inflammatory response.50 Expression of dominant negative Rac or Cdc42 abolishes particle uptake via the Fc receptor.51,50 The role of Rho in FcR-mediated phagocytosis is still somewhat controversial. Caron and Hall showed that expression of dominant negative Rho has no effect,50 whereas Hackam and colleagues observed that inactivation of Rho by C3 exotoxin causes inhibition of FcR-mediated phagocytosis, both in macrophages and COS cells.52 The use of C3 exotoxin causes a more complete inhibition of Rho activity than dominant negative Rho. It is therefore possible that the different findings could be explained by differences in the extent of Rho inhibition. Use of the new technique of RNA interference (RNAi) 53 should help to clarify unambiguously whether or not a particular GTPase is involved in a certain process. For complement receptor-mediated phagocytosis, the dependence on Rho has been clearly established; here only Rho colocalizes with F-actin and the phagosome. A Rho inhibitor blocks phagosome formation, while inhibiting Rac or Cdc42 has no effect.50 Phagocytic clearance of apoptotic cells also requires actin rearrangement as well as integrin engagement. αvβ5 receptor mediates internalization of apoptotic cells via recruitment of the adapter complex p130Cas-CrkII and the Rac GEF Dock180. This complex leads to Rac activation and phagosome formation.54,55 Cdc42 also appears to be an essential player in apoptotic cell phagocytosis. Inhibition of either GTPase leads to impaired uptake of apoptotic cells, possibly by reducing the formation of F-actin at the site of the nascent phagosome.56 The crucial role of GTPase-controlled actin dynamics in phagocytosis is underscored by the finding that the Cdc42 effector WASP together with the Arp2/3 complex is recruited to the phagocytic cup. Absence of WASP leads to a delay in phagocytosis of apoptotic cells.57 PAK also localizes

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to the phagosome and may regulate the initial extension of the pseudopod about to engulf the particle to be taken up.58 Exocytosis of endosomal vesicles has been shown to be of importance for pseudopod formation in FcR mediated phagocytosis (ref. 59 and references therein). One of the regulators of endosomal vesicle trafficking is the coatomer complex COPI. It participates in the budding and trafficking of vesicles originating in the Golgi and has been shown by Hackam et al to be important for pseudopod extension. Loss of COPI leads to inhibition of phagocytosis by inhibiting pseudopod formation.59 Interestingly, the γ-subunit of COPI has recently been demonstrated to be a binding partner for Cdc42 and this binding may be essential for Cdc42-induced transformation.60 It will be interesting to see if Cdc42 is also involved in COPI-controlled vesicle trafficking and thereby in the formation of pseudopods and control of phagocytosis. While the importance of phagocytosis for host defense is obvious, there are other biological aspects for which uptake of extracellular particles may be important. In Rat1 fibroblasts a correlation has been observed between Rac1-induced increase in cell motility and increase in the uptake of extracellular matrix particles into vesicles enriched in cathepsin B.61,62 Rac is known to enhance the expression of matrix metalloproteinases which sever the contact of the cell to the matrix and facilitate motility.63,64 It is therefore interesting to speculate that Rac could also induce increased uptake and intracellular degradation of matrix components. The extent to which intracellular matrix degradation contributes to invasion remains to be determined however.

Exocytosis IgE-induced degranulation of secretory granules in mast cells has provided a valuable paradigm for the study of signalling pathways that regulate exocytosis.65,66 A large number of reports over the past several years have indicated a role for Rho, Rac and Cdc42 in regulating secretion in RBL-2H3 cells, an extensively characterized mast cell line (references cited in 67: 68, 69, 70, reviewed in refs 71 and 47). Rac and Cdc42 regulate multiple steps of stimulus-induced degranulation (Fig. 2). Activated Rac1 and Cdc42 stimulate the formation of the secondary messenger IP3 and the subsequent release of intracellular calcium that is necessary for degranulation.71 In addition, in vitro studies showed direct binding of Cdc42 to phospholipase Cγ1 (PLCγ1), suggesting that PLCγ1 may mediate the production of IP3 by Cdc42. Interestingly however, expression of activated Rac1 or Cdc42 can restore calcium mobilization in mutant RBL cells that are defective in antigen-stimulated PLCγ activation, suggesting the existence of additional signalling mechanisms employed by these GTPases to mobilize calcium.72 Candidates for effectors that may be involved in this are type I PIPkinases9 and phospholipase D (PLD).73,74 EGF activation of Rac causes an increase in PLD activity, an effect that is abrogated by dominant negative Rac. In a positive feedback loop PLD hydrolyses PI(4,5)P2 to phosphatidic acid (PA), a cofactor for type I PIPkinases, which catalyse the synthesis of PI(4,5)P2, the substrate of PLC and PLD (Fig. 2). Rac and Rho also participate in antigen-induced degranulation at a point that is downstream of calcium influx.67 At this later stage, Rho appears to fulfill multiple functions that are controlled via divergent pathways. On the one hand, activated RhoA enhances the disassembly of cortical F-actin that is induced by calcium, via a pathway that is mediated by Rock, while on the other, RhoA stimulates secretion itself, in a Rock-independent fashion.75 The precise contribution of cortical F-actin disassembly to degranulation remains controversial however.75 Secretory vesicles are targeted to discrete sites on the plasma membrane in a process that is mediated by a multiprotein complex termed the exocyst.76,77 In the budding yeast S. cerevisiae, the exocyst complex is essential both for targeted secretion and polarization and bud site selection.78 A critical component of the exocyst is Sec3, which is thought to serve as a plasma membrane landmark for exocytosis.77 Recent work showed that Sec3 is a direct effector of Rho1 and that Rho1 is essential for the localization of Sec3 to the bud tip.79 However, other components of the exocyst, such as Exo70, can be targeted correctly in a fashion that is inde-

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pendent of the interaction between Rho1 and Sec3. This suggests that Rho1 drives parallel pathways that control polarized exocytosis. S. cerevisiae Rho3 also performs dual functions in exocytosis. It regulates the delivery of exocytic vesicles via unconventional myosin Myo2, and vesicle docking and fusion with the plasma membrane via Exo70.80,81 An exocyst complex has also been characterized in mammalian cells.82,83 Of all the subunits of the exocyst expressed as GFP fusion proteins, only Exo70 was shown to be recruited to cell-cell contacts in MDCK cells.83 These results suggest that at least some of the essential properties of the mammalian exocyst may differ from that of its budding yeast counterpart.3,84,85 It also remains to be seen if binding of a Rho GTPase is involved in targeting of the complex.

Trafficking in Polarized Epithelial Cells The plasma membrane of polarized epithelial cells is compartmentalized into apical and basolateral domains. The actin cytoskeleton is thought to be an important player in membrane trafficking in polarized epithelial cells. In cytochalasin-treated cells, apical endocytosis is impaired, suggesting a role for actin dynamics in the assembly of the endocytic machinery, the invagination of the plasma membrane and the formation of a contractile ring together with dynamin and myosin which leads to vesicle scission.86 Interestingly, cytochalasin treatment does not affect clathrin-dependent endocytosis from the basolateral membrane. Hence, actin dynamics may differentially affect endocytosis in different membrane compartments (reviewed in ref. 87). Although it is hard to generalize from a limited number of studies, it appears that, at least for endocytic trafficking, the role of Rho GTPases is substantially different from that in nonpolarized cells. Thus, whereas in nonpolarized cells, both activated Rac1 and RhoA inhibit receptor-mediated endocytosis, in polarized MDCK cells, activated RhoA stimulates receptor uptake from both apical and basolateral domains, but activated Rac has an inhibitory effect on these processes. In addition, whereas RhoA controls post-endocytic trafficking on the basolateral side of the cell, Rac1 regulates post-endocytic and biosynthetic membrane trafficking toward the apical membrane.88,89 Identification of downstream effectors that mediate these processes and/or the distinct requirements for specific actin structures should help explain these observations. Cdc42 also plays a role in endocytic and post-endocytic trafficking in MDCK cells. Interestingly, both constitutively active and dominant negative mutants of Cdc42 inhibited apical endocytosis of IgA, suggesting that Cdc42 may need to cycle between the GTP- and GDP-bound states for normal endocytic activity.90 In addition, Cdc42 is an important regulator of biosynthetic trafficking. Earlier studies showed that inhibition of Cdc42 causes depolarization of the basolateral membrane.91 Cohen and coworkers have subsequently addressed the question whether this effect of Cdc42 is due to a general disruption of polarized protein traffic or whether it is confined to proteins targeted to the basolateral membrane. They show that Cdc42 regulates trafficking of membrane proteins to the basolateral membrane, but does not alter the polarized secretion of soluble basolateral proteins. Neither does it affect targeting of apical membrane or soluble proteins.92 Cdc42 can associate with a complex of Par6/Par3 and an atypical PKC that is thought to regulate tight junction assembly.93-95 Cohen et al therefore propose the attractive hypothesis that Cdc42 could coordinate vesicle targeting and fusion at the exocyst complex.92 Interestingly, the above results are in contrast with recent data from Apodaca and coworkers, who find that dominant negative Cdc42 can enhance basolateral membrane protein delivery.90 Whereas this discrepancy could result from differences in the use of experimental tools, it could also be accounted for by the possibility that different proteins may utilize distinct secretory pathways.

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Trafficking Controlled by Rho GTPases in the Regulation of Cell Motility and Cell-Cell Junctions Recent evidence suggests an important role for recycling and exocytosis of plasma membrane-bound proteins as a contributor to cell motility. (reviewed in ref. 96) Rac is one of the key regulators of cell movement and there are different hypothesis as to how Rac stimulates motility. One school of thought envisages lamellipodial extension driven by the actin cytoskeleton,97 another attributes an important role to exocytosis of membrane vesicles as the force behind cell locomotion.98 In neutrophils, there is good evidence that integrins internalize and recycle during cell movement. Integrins are endocytosed at the rear of a moving cell, recycled to the leading lamellae and delivered to sites where they bind extracellular matrix proteins and provide adhesiveness. When intracellular calcium is buffered, integrin detachment and recycling are blocked. Cell motility also depends on changes in free intracelluar calcium and is inhibited by the buffering of calcium.99 This suggests the interesting possibility of a causal connection between integrin recycling and cell movement. The role GTPases play in this process remains to be elucidated. It is conceivable that they regulate the amount of endo- versus exocytosis via a change in actin dynamics. Alternatively, they may function in the targeting of effector proteins which are essential for vesicle budding or scission. Rac plays an important role in the establishment of cadherin-based adherens junctions. E-cadherin is actively internalized and recycled back to the basolateral membrane, processes that are strongly increased upon loss of cell-cell contact. Interference with E-cadherin recycling by various treatments causes an inhibition of junction formation, suggesting that recycling is important for the establishment of adherens junctions.100 Interestingly, disassembly of adherens junctions in keratinocytes induced by expression of constitutively active Rac1 is abrogated by coexpression of a dominant negative version of dynamin.101 These results suggest that Rac could regulate the formation of adherens junctions via its role in vesicular trafficking.

Conclusion In this chapter, we reviewed recent evidence that has indicated an important role for Rho family members in various aspects of vesicular trafficking. One of the main challenges that we now face in this area of research is the dissection of the signalling pathways that mediate the control of membrane trafficking downstream of Rho GTPases. Elucidating these signalling mechanisms also may shed further light on how the control of membrane trafficking by Rho GTPases fits into the larger picture of other functions that are regulated by these GTPases, including cell polarity, motility and proliferation.

Acknowledgement The authors are supported by NIH/NCI grant CA 87567 to M.S.

References 1. Qualmann B, Mellor H. Regulation of endocytic traffic by Rho GTPases. Biochem J 2003; 371:233-241. 2. Symons M, Rusk N. Control of vesicular trafficking by Rho GTPases. Curr Biol 2003; 10:409-418. 3. Novick P, Guo W. Ras family therapy: Rab, Rho and Ral talk to the exocyst. Trends Cell Biol 2002; 12:247-249. 4. Tall GG, Barbieri MA, Stahl PD et al. Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1. Dev Cell 2001; 1:73-82. 5. Lamaze C, Chuang TH, Terlecky LJ et al. Regulation of receptor-mediated endocytosis by Rho and Rac. Nature 1996; 382:177-179. 6. Fujimoto LM, Roth R, Heuser JE et al. Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells. Traffic 2000; 1:161-171. 7. Malecz N, McCabe PC, Spaargaren C et al. Synaptojanin 2, a novel Rac1 effector that regulates clathrin-mediated endocytosis. Curr Biol 2000; 10:1383-1386.

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8. Martin TF. PI(4,5)P(2) regulation of surface membrane traffic. Curr Opin Cell Biol 2001; 13:493-499. 9. Tolias KF, Hartwig JH, Ishihara H et al. Type Ialpha phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly. Curr Biol 2000; 10:153-156. 10. Ren XD, Schwartz MA. Regulation of inositol lipid kinases by Rho and Rac. Curr Opin Genet Dev 1998; 8:63-67. 11. Engqvist-Goldstein AE, Warren RA, Kessels MM et al. The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro. J Cell Biol 2001; 154:1209-1223. 12. Itoh T, Koshiba S, Kigawa T et al. Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. Science 2001; 291:1047-1051. 13. Burger KN, Demel RA, Schmid SL et al. Dynamin is membrane-active: Lipid insertion is induced by phosphoinositides and phosphatidic acid. Biochemistry 2000; 39:12485-12493. 14. Lee E, De Camilli P. Dynamin at actin tails. Proc Natl Acad Sci USA 2002; 99:161-166. 15. Orth JD, Krueger EW, Cao H et al. The large GTPase dynamin regulates actin comet formation and movement in living cells. Proc Natl Acad Sci USA 2002; 99:167-172. 16. Witke W, Podtelejnikov AV, Di Nardo A et al. In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly. EMBO J 1998; 17:967-976. 17. Kessels MM, Engqvist-Goldstein AE, Drubin DG et al. Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin. J Cell Biol 2001; 153:351-366. 18. Qiu RG, Chen J, McCormick F et al. A role for Rho in Ras transformation. Proc Natl Acad Sci USA 1995; 92:11781-11785. 19. Symons M. Rho family GTPases: The cytoskeleton and beyond. Trends Bio Chem Sci 1996; 21:178-181. 20. Sorkin A, Von Zastrow M. Signal transduction and endocytosis: Close encounters of many kinds. Nat Rev Mol Cell Biol 2002; 3:600-614. 21. Lamaze C, Chuang TH, Terlecky LJ et al. Regulation of receptor-mediated endocytosis by Rho and Rac. Nature 1996; 382:177-179. 22. Vogler O, Krummenerl P, Schmidt M et al. RhoA-sensitive trafficking of muscarinic acetylcholine receptors. J Pharmacol Exp Ther 1999; 288:36-42. 23. Gampel A, Parker PJ, Mellor H. Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB. Curr Biol 1999; 9:955-958. 24. Mellor H, Flynn P, Nobes CD et al. PRK1 is targeted to endosomes by the small GTPase, RhoB. J Biol Chem 1998; 273:4811-4814. 25. Slepnev VI, Ochoa GC, Butler MH et al. Role of phosphorylation in regulation of the assembly of endocytic coat complexes. Science 1998; 281:821-824. 26. Adamson P, Paterson HF, Hall A. Intracellular localization of the P21rho proteins. J Cell Biol 1992; 119:617-627. 27. Robertson D, Paterson HF, Adamson P et al. Ultrastructural localization of ras-related proteins using epitope-tagged plasmids. J Histochem Cytochem 1995; 43:471-480. 28. Michaelson D, Silletti J, Murphy G et al. Differential localization of Rho GTPases in live cells: Regulation by hypervariable regions and RhoGDI binding. J Cell Biol 2001; 152:111-126. 29. Teo M, Tan L, Lim L et al. The tyrosine kinase ACK1 associates with clathrin-coated vesicles through a binding motif shared by arrestin and other adaptors. J Biol Chem 2001; 276:18392-18398. 30. Yang W, Lo CG, Dispenza T et al. The Cdc42 target ACK2 directly interacts with clathrin and influences clathrin assembly. J Biol Chem 2001; 276:17468-17473. 31. Hopper NA, Lee J, Sternberg PW. ARK-1 inhibits EGFR signaling in C elegans. Mol.Cell 2000; 6:65-75. 32. Machesky LM, Insall RH. Signaling to actin dynamics. J Cell Biol 1999; 146:267-272. 33. McPherson PS. The endocytic machinery at an interface with the actin cytoskeleton: A dynamic, hip intersection. Trends Cell Biol 2002; 12:312-315. 34. Hussain NK, Jenna S, Glogauer M et al. Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP. Nat Cell Biol 2001; 3:927-932. 35. Jenna S, Hussain NK, Danek EI et al. The activity of the GTPase-activating protein CdGAP is regulated by the endocytic protein intersectin. J Biol Chem 2002; 277:6366-6373. 36. Nobes C, Marsh M. Dendritic cells: New roles for Cdc42 and Rac in antigen uptake? Curr Biol 2000; R739-R741. 37. Ridley AJ, Paterson HF, Johnston CL et al. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992; 70:401-410.

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38. Dharmawardhane S, Schurmann A, Sells MA et al. Regulation of macropinocytosis by p21-activated kinase-1. Mol Biol Cell 2000; 11:3341-3352. 39. Edwards DC, Sanders LC, Bokoch GM et al. Activation of LIM-kinase by Pak1 couples Rac/ Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol 1999; 1:253-259. 40. Arber S, Barbayannis FA, Hanser H et al. Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 1998; 393:805-809. 41. Yang N, Higuchi O, Ohashi K et al. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 1998; 393:809-812. 42. Sanders LC, Matsumura F, Bokoch GM et al. Inhibition of myosin light chain kinase by p21-activated kinase. Science 1999; 283:2083-2085. 43. Sells MA, Boyd JT, Chernoff J. p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts. J Cell Biol 1999; 145:837-849. 44. Kiosses WB, Daniels RH, Otey C et al. A role for p21-activated kinase in endothelial cell migration. J Cell Biol 1999; 147:831-844. 45. Garrett WS, Chen LM, Kroschewski R et al. Developmental control of endocytosis in dendritic cells by Cdc42. Cell 2000; 102:325-334. 46. West MA, Prescott AR, Eskelinen EL et al. Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation. Curr Biol 2000; 10:839-848. 47. Ridley AJ. Rho proteins: Linking signaling with membrane trafficking. Traffic 2001; 2:303-310. 48. Chimini G, Chavrier P. Function of Rho family proteins in actin dynamics during phagocytosis and engulfment. Nat Cell Biol 2000; 2:E191-E196. 49. Cardelli J. Phagocytosis and macropinocytosis in Dictyostelium: Phosphoinositide-based processes, biochemically distinct. Traffic 2001; 2:311-320. 50. Caron E, Hall A. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 1998; 282:1717-1721. 51. Cox D, Chang P, Zhang Q et al. Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes. J Exp Med 1997; 186:1487-1494. 52. Hackam DJ, Rotstein OD, Schreiber A et al. Rho is required for the initiation of calcium signaling and phagocytosis by Fcgamma receptors in macrophages. J Exp.Med 1997; 186:955-966. 53. Elbashir SM, Harborth J, Lendeckel W et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411:494-498. 54. Albert ML, Kim JI, Birge RB. Alphavbeta5 integrin recruits the CrkII-Dock180-rac1 complex for phagocytosis of apoptotic cells. Nat Cell Biol 2000; 2:899-905. 55. Tosello-Trampont AC, Brugnera E, Ravichandran KS. Evidence for a conserved role for CRKII and Rac in engulfment of apoptotic cells. J Biol Chem 2001; 276:13797-13802. 56. Leverrier Y, Ridley AJ. Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages. Curr Biol 2001; 11:195-199. 57. Leverrier Y, Lorenzi R, Blundell MP et al. Cutting edge: The Wiskott-Aldrich syndrome protein is required for efficient phagocytosis of apoptotic cells. J Immunol 2001; 166:4831-4834. 58. Dharmawardhane S, Brownson D, Lennartz M et al. Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils. J Leukoc.Biol 1999; 66:521-527. 59. Hackam DJ, Botelho RJ, Sjolin C et al. Indirect role for COPI in the completion of FCgamma receptor-mediated phagocytosis. J Biol Chem 2001; 276:18200-18208. 60. Wu WJ, Erickson JW, Lin R et al. The gamma-subunit of the coatomer complex binds Cdc42 to mediate transformation. Nature 2000; 405:800-804. 61. Ahram M, Sameni M, Qiu RG et al. Rac1-induced endocytosis is associated with intracellular proteolysis during migration through a three-dimensional matrix. Exp Cell Res 2000; 260:292-303. 62. Coopman PJ, Do MT, Thompson EW et al. Phagocytosis of cross-linked gelatin matrix by human breast carcinoma cells correlates with their invasive capacity. Clin Cancer Res 1998; 4:507-515. 63. Kheradmand F, Werner E, Tremble P et al. Role of Rac1 and oxygen radicals in collagenase-1 expression induced by cell shape change. Science 1998; 280:898-902. 64. Zhuge Y, Xu J. Rac1 mediates type I collagen-dependent MMP-2 activation. Role in cell invasion across collagen barrier. J Biol Chem 2001; 276:16248-16256. 65. Holt MR, Koffer A. Rho GTPases: Secretion and actin dynamics in permeabilized mast cells. Methods Enzymol 2000; 325:356-369. 66. Sanchez-Mejorada G, Rosales C. Fcgamma receptor-mediated mitogen-activated protein kinase activation in monocytes is independent of Ras. J Biol Chem 1998; 273:27610-27619. 67. Norman JC, Price LS, Ridley AJ et al. The small GTP-binding proteins, Rac and Rho, regulate cytoskeletal organization and exocytosis in mast cells by parallel pathways. Mol Biol Cell 1996; 7:1429-1442.

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68. Brown AM, O’Sullivan AJ, Gomperts BD. Induction of exocytosis from permeabilized mast cells by the guanosine triphosphatases Rac and Cdc42. Mol Biol Cell 1998; 9:1053-1063. 69. Mariot P, O’Sullivan AJ, Brown AM et al. Rho guanine nucleotide dissociation inhibitor protein (RhoGDI) inhibits exocytosis in mast cells. EMBO J 1996; 15:6476-6482. 70. Guillemot JC, Montcourrier P, Vivier E et al. Selective control of membrane ruffling and actin plaque assembly by the Rho GTPases Rac1 and CDC42 in FcepsilonRI-activated rat basophilic leukemia (RBL-2H3) cells. J Cell Sci 1997; 110( Pt 18):2215-2225. 71. Hong-Geller E, Cerione RA. Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells. J Cell Biol 2000; 148:481-494. 72. Hong-Geller E, Holowka D, Siraganian RP et al. Activated Cdc42/Rac reconstitutes Fcepsilon RI-mediated Ca2+ mobilization and degranulation in mutant RBL mast cells. Proc Natl Acad Sci US 2001; 98:1154-1159. 73. Singer WD, Brown HA, Bokoch GM et al. Resolved phospholipase D activity is modulated by cytosolic factors other than Arf. J Biol Chem 1995; 270:14944-14950. 74. Hess JA, Ross AH, Qiu RG et al. Role of Rho family proteins in phospholipase D activation by growth factors. J Biol Chem 1997; 272:1615-1620. 75. Sullivan R, Price LS, Koffer A. Rho controls cortical F-actin disassembly in addition to, but independently of, secretion in mast cells. J Biol Chem 1999; 274:38140-38146. 76. TerBush DR, Maurice T, Roth D et al. The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J 1996; 15:6483-6494. 77. Guo W, Sacher M, Barrowman J et al. Protein complexes in transport vesicle targeting. Trends Cell Biol 2000; 10:251-255. 78. TerBush DR, Guo W, Dunkelbarger S et al. Purification and characterization of yeast exocyst complex. Methods Enzymol 2001; 329:100-110. 79. Guo W, Tamanoi F, Novick P. Spatial regulation of the exocyst complex by Rho1 GTPase. Nat.Cell Biol 2001; 3:353-360. 80. Robinson NG, Guo L, Imai J et al. Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70. Mol Cell Biol 1999; 19:3580-3587. 81. Adamo JE, Rossi G, Brennwald P. The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity. Mol Biol Cell 1999; 10:4121-4133. 82. Kee Y, Yoo JS, Hazuka CD et al. Subunit structure of the mammalian exocyst complex. Proc Natl Acad Sci USA 1997; 94:14438-14443. 83. Matern HT, Yeaman C, Nelson WJ et al. The Sec6/8 complex in mammalian cells: Characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells. Proc Natl Acad Sci USA 2001; 98:9648-9653. 84. Polzin A, Shipitsin M, Goi T et al. Ral-GTPase influences the regulation of the readily releasable pool of synaptic vesicles. Mol Cell Biol 2002; 22:1714-1722. 85. Moskalenko S, Henry DO, Rosse C et al. The exocyst is a Ral effector complex. Nat Cell Biol 2002; 4:66-72. 86. Apodaca G. Endocytic traffic in polarized epithelial cells: Role of the actin and microtubule cytoskeleton. Traffic 2001; 2:149-159. 87. Apodaca G. Endocytic traffic in polarized epithelial cells: Role of the actin and microtubule cytoskeleton. Traffic 2001; 2:149-159. 88. Leung SM, Rojas R, Maples C et al. Modulation of endocytic traffic in polarized Madin-Darby canine kidney cells by the small GTPase RhoA. Mol Biol Cell 1999; 10:4369-4384. 89. Jou TS, Leung SM, Fung LM et al. Selective alterations in biosynthetic and endocytic protein traffic in Madin-Darby canine kidney epithelial cells expressing mutants of the small GTPase Rac1. Mol Biol Cell 2000; 11:287-304. 90. Rojas R, Ruiz WG, Leung SM et al. Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells. Mol Biol Cell 2001; 12:2257-2274. 91. Kroschewski R, Hall A, Mellman I. Cdc42 controls secretory and endocytic transport to the basolateral plasma membrane of MDCK cells. Nat Cell Biol 1999; 1:8-13. 92. Cohen D, Musch A, Rodriguez-Boulan E. Selective control of basolateral membrane protein polarity by cdc42. Traffic 2001; 2:556-564. 93. Lin D, Edwards AS, Fawcett JP et al. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/ Rac1 and aPKC signalling and cell polarity. Nat Cell Biol 2000; 2:540-547. 94. Joberty G, Petersen C, Gao L et al. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat Cell Biol 2000; 2:531-539.

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95. Suzuki A, Yamanaka T, Hirose T et al. Atypical protein kinase C is involved in the evolutionarily conserved par protein complex and plays a critical role in establishing epithelia-specific junctional structures. J Cell Biol 2001; 152:1183-1196. 96. Bretscher MS, Aguado-Velasco C. Membrane traffic during cell locomotion. Curr Opin.Cell Biol 1998; 10:537-541. 97. Mitchison TJ, Cramer LP. Actin-based cell motility and cell locomotion. Cell 1996; 84:371-379. 98. Bretscher MS. Getting membrane flow and the cytoskeleton to cooperate in moving cells. Cell 1996; 87:601-606. 99. Pierini LM, Lawson MA, Eddy RJ et al. Oriented endocytic recycling of alpha5beta1 in motile neutrophils. Blood 2000; 95:2471-2480. 100. Le TL, Yap AS, Stow JL. Recycling of E-cadherin: A potential mechanism for regulating cadherin dynamics. J Cell Biol 1999; 146:219-232. 101. Akhtar N, Hotchin NA. RAC1 regulates adherens junctions through endocytosis of E-cadherin. Mol Biol Cell 2001; 12:847-862. 102. Slepnev VI, Ochoa GC, Butler MH et al. Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites. J Biol Chem 2000; 275:17583-17589. 103. Qualmann B, Kelly RB. Syndapin isoforms participate in receptor-mediated endocytosis and actin organization. J Cell Biol 2000; 148:1047-1062.

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CHAPTER 14

The Dictyostelium Rho Family of Proteins and Effectors David Seastone and James A. Cardelli

Abstract

T

he Rho family of small GTPases is found in all eukaryotic cells and participates in the regulation of a diverse number of different biological processes, including the regulation of motility, phagocytosis, vesicle trafficking, transformation and transcriptional regulation. One of the major functions of Rho GTPases, and the first one identified, is the regulation of F-actin polymerization. Rho proteins act as molecular switches that bind and activate effector proteins to carry out these processes. GTPases are activated and inactivated by exchange factors and GTPase activator proteins, respectively, which in turn are acted on by upstream signals. As described in this review, Dictyostelium discoideum, a genetically tractable professional phagocyte and an organism with a developmental cycle, is an attractive system to investigate the regulation of the actin cytoskeleton and the role of Rho family GTPases. The Dictyostelium Rho family contains a large number of proteins that appear to function in a nonredundant fashion to regulate cell motility, macropinocytosis, phagocytosis, development, cytokinesis and vesicle trafficking. Furthermore, a growing list of guanine nucleotide exchange factors (GEFs), GTPase activator proteins (GAPs) and effector proteins have been identified and have begun to be characterized at a molecular level. The application of both genetic and biochemistry approaches will continue to prove instrumental in deciphering the signalling pathways activated by Dictyostelium Rho proteins to regulate cellular processes critical for all eukaryotic cells.

Choice of Dictyostelium As an Experimental System Dictyostelium has proven to be an excellent organism in which to investigate the molecular mechanisms regulating processes regulated by small GTPases belonging to the Ras superfamily. Dictyostelium is a free-living professional phagocyte, and can internalize fluid and particles at rates higher than those observed for macrophages and neutrophils. Furthermore, the regulation of cell motility, cytokinesis, membrane trafficking and internalization events is comparable to that observed for mammalian cells. The versatility of Dictyostelium as an experimental system has been reviewed recently (BBA (2001) 1525: 197-271), and as a further testament, National Institutes of Health has recently added this organism to its growing list of model system organisms. Dictyostelium strains have been developed that can grow axenically in shaking cultures, and most strains can grow using bacteria as a food source. The generation time for wild-type strains growing axenically is 8 hours and this rapid growth greatly aids biochemical studies. A variety of genetic approaches are also available to dissect cellular processes. Yeast still remains the premier genetic system, however, yeast cells cannot phagocytose or move, and therefore are not particularly relevant models for the study of macrophage or neutrophil function. Since Dictyostelium is haploid, the isolation and characterization of temperature sensitive mutants Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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and null mutants is made easier. Temperature sensitive and null mutants can be rescued by transformation of expression library plasmids, and this has yielded novel second site suppressors that by over-expression can complement a mutant phenotype. The availability of multiple selection markers coupled with the ease of DNA transformation has allowed for the isolation of triple and even quadruple mutants. Gene replacement, gene disruption, and the generation of strains expressing antisense RNA or overexpressing wild-type and mutant proteins or protein domains is also now routine. Restriction enzyme-mediated integration (REMI), first described in yeast, also works in Dictyostelium and allows for the isolation and characterization of genes involved in a wide range of processes. This approach has also been used to isolate second site suppressor mutants that can rescue the original mutant phenotype. Expressed sequence tags and genomic sequencing projects are nearing completion, and the available genetic database for the genome consisting of 34 Mb on 6 chromosomes will greatly aid new gene discovery endeavors (Web site: http://dictybase.org/dicty.html). Dictyostelium has a large family of GTPases belonging to the Ras superfamily, including >18 members of the Rab family, 10 members of the Ras family and 15 members of the Rho family.1-5 Much is currently known about the roles played by these small GTPases in the regulation of actin based cell processes including cell motility, endo-lysosomal trafficking and fluid and particle internalization. This review will focus on the current state of knowledge relating to the Rho family members.

An Overview of the Dictyostelium Rho Family of GTPases

To date, 15 genes encoding Rho family GTPases have been identified in Dictyostelium.4-6 Genome and cDNA sequence analyses indicate that additional genes encoding Rho family members probably do not exist. The original characterization of GTPases in this organism was accomplished by probing a cDNA library with an oligonucleotide complementary to the highly conserved DTAGQE region of GTPases.4 In this first study, seven Rho family genes were reported and termed: Rac1A, Rac1B, Rac1C (these shared at least 90% identity with each other and 85% identity to human Rac1), RacA, RacB, RacC, RacD (these shared much less identity and similarity to human Rac1 and were considered to be novel Rac GTPases). Subsequently, another Rho family GTPase was discovered by during a genetic screen for cytokinesis mutants.7 In keeping with the nomenclature first implemented by Bush et al., this newly discovered GTPase was named RacE. Another group used a PCR-based screen to identify two more Rho family GTPases they termed RacF1 and RacF2.6 Finally, using a computer-based search of the Dictyostelium database, the same group published the sequences for five more Rho family GTPases; RacG, RacH, RacI, RacJ, RacL, and one pseudogene RacKΨ.5 For a comparison of the amino acid sequence of the Dictyostelium rho family of proteins, see supplementary information on line. The Rho subfamily of GTPases in Dictyostelium appears quite unique relative to Rho families in other organisms. Whereas in most other eukaryotes there seem to be distinct classes of Rho-, Rac-, and Cdc42-like proteins, in Dictyostelium, the class separation is not as clear. A very thorough phylogenetic analysis was performed and indicated that the Rho family of GTPases in this organism is quite divergent.5 Thus, although the nomenclature for the Dictyostelium Rho family GTPases indicates they all might function like Rac proteins, it remains to be determined if this is true. For a dendogram indicating the relatedness of each Dictyostelium Rho family protein, see supplementary information on line.5 What is evident is that Rac1A/1B/1C, RacF1/F2 and RacB and the GTPase domain of RacA can be grouped in the Rac subfamily. None of the remaining Dictyostelium rho family members belong to any of the well-defined subfamilies including Rac, Cdc42 and Rho. All of these proteins contain the Rho insert, a characteristic 13 amino acid insertion between the α-strand B5 and the ?-helix A4, suggesting they indeed are members of the Rho family. As this review will point out, the current studies of the Rho-family GTPases indicate a great diversity of function with minimal redundancy between different family members. The next

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section will summarize the available information about function of each Dictyostelium Rho family member (also see Table 1). A discussion of individual Rho-family GTPases would be difficult without also mentioning some of the proteins that regulate and/or mediate the function of these proteins (GAP’s, GEF’s and effector proteins), therefore, some of these accessory proteins will be mentioned in this section. However, all of these other proteins will be discussed in greater detail later in the review.

Rac1A/Rac1B/Rac1C These three GTPases share the highest degree of amino acid sequence identity with human Rac1, and they have been characterized in greater detail than the other Rho GTPases. Since Rac1A/B/C share at least 90% identity to each other, it is also not surprising that they appear to share completely overlapping functions, although more comprehensive studies will be required to prove this.8 Three recent studies demonstrated that the Rac1A/B/C group (hereafter collectively referred to as DdRac1) regulates the organization of the cortical F-actin cytoskeleton.8-10 These results were not unexpected because for at least 10 years, human Rac1 has been considered to function in part in the same fashion.11 In each of the studies performed in Dictyostelium, constitutively activated (CA) and dominant negative (DN) mutants were generated and expressed in stable cell lines to test for various physiological functions. It appears that DdRac1s are important for regulating cytokinesis, cell growth, endocytosis, motility, chemotaxis, development, and F-actin polymerization leading to filopodia and membrane ruffle formation. Importantly, it was also observed that proper cycling between the GDP- and the

Table 1. The dictyostelium Rho family and associated proteins Protein

Proposed Function

Approaches

Interacting Proteins

Rac1A/B/C

actin, motility, cytokinesis endocytosis

KO, overexpression

DRG, Darlin, MIHCK 8-10 DdLIM, Dgap1, GapA, Paka unknown 51 Scar, Darlin 23

RacB RacC

membrane blebs, actin actin, petalopodia, phagocytosis RacE cytokinesis RacF1/F2 unknown RacA,D,G-H unknown DRG GEF for Racs and GAP for Racs and Rabs MIHCK myosin phosphorylation Dgap1 actin bundling, cytokinesis GapA actin bundling, cytokinesis Darlin cell aggregation Scar actin polymerization, phago, macro, endo trafficking DdLIM cytokinesis, actin Paka myosin-II assembly, chemotaxis MyoM actin polymerization, ramopodia

overexpression overexpression

Refs

KO DRG, Darlin truncated mutants unknown unknown KO Rac1, RacE, RabD

20 6

KO KO KO KO overexpression

DdRac1 DdRac1 DdRac1 RacE, RacC RacC, profilin

41,43 35,36 27 16 38

overexpression KO, overexpression KO, overexpression

DdRac1 (weakly) Paka

17 18

DdRac1

19,48

Overexpression studies involved using wild-type and/or mutant forms of proteins. a. Seastone, Saxe and Cardelli (unpublished results)

28

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GTP-bound forms of DdRac1 was required for proper protein function.8-10 Thus, expression of CA-DdRac1 blocked many of the processes mentioned above, while the expression of DN-Rac1 gave results similar to wild-type overexpressing cells.8 This is in contrast to the situation observed in mammalian cells, where constitutively activated Rac1 usually induces actin-based effects, whereas dominant negative Rac1 blocks actin polymerization.11 Based on these studies a reasonable argument can be made that DdRac1s are involved processes in Dictyostelium comparable to those regulated by mammalian Rac1 through the ability to regulate F-actin organization. GTPases act as molecular switches and mediate their biological effects through downstream effector proteins. In addition, regulatory proteins are usually present to guide the GTPases into an active or inactive conformation. A number of candidate effector and regulatory proteins for DdRac1s have been identified. These include: DGap1, myosin I heavy chain kinase (MIHCK), DdRacGAP, GapA, darlin, DdLim, pakA and myoM.9,12-19 As will be discussed later, for some of these proteins, it is still unclear whether they are regulators of DdRac1 function, effectors of DdRac1 function, or both.

RacE RacE, identified during a REMI-screen for cytokinesis-defective mutants, was the first Rho family member of Dictyostelium to be characterized.20 RacE is 47% homologous to human Rac1 and it is most closely related Dictyostelium Rho-family GTPase is RacC, with which it share 50% identify. RacE null cell-lines are severely defective in cytokinesis when grown in shaking suspension; however, the RacE null cells are able to divide normally on a solid surface, through a process termed traction-mediated cytofission.21 Interestingly, when F-actin cytoskeleton distribution was examined in the RacE-null cells, no defects were found. In addition, phagocytosis, pinocytosis, and development, all actin-dependent processes, were also unaffected. Subsequent studies using GFP-tagged RacE indicated that it was primarily localized to the cortical region of the cell.7 Moreover, these studies also showed that a constitutively activated form of RacE was capable of rescuing the RacE null cells, indicating that GTP-cycling on RacE is not required for cytokinesis cells growing in shaking suspension. More recently it has been demonstrated that RacE null cells formed contractile rings, and began to form a cleavage furrow, but were unable to complete cytokinesis. These results indicate that RacE is required for a specific stage of cytokinesis; specifically, contraction of the cleavage furrow.22 The authors propose that in the absence of an attachment signal, RacE is necessary to properly organize the F-actin cytoskeleton so that adequate cortical tension is maintained. However, cells growing on a solid surface complete cytokinesis in the absence of RacE because proper cortical tension is maintained via attachment signal-dependent actin polymerization.

RacC As with mammalian Rho family GTPases, RacC appears to play a major role in regulating cortical F-actin organization.23 Slight overexpression of wild-type-forms of (WT) RacC induced the formation of unusual cortical F-actin structures termed petalopodia. In addition, overexpression of WT-RacC stimulated the rate of phagocytosis 3-fold, while it reduced the rate of macropinocytosis and fluid phase exocytosis by 50%. RacC-mediated increases in phagocytosis required the function of a DAG binding protein, as previously observed for Rap1-mediated increases in phagocytosis.24 Interestingly, the formation of petalopodia occurred in a PI 3-kinase-dependent fashion, whereas the effects of RacC on phagocytosis and macropinocytosis were clearly PI 3-kinase independent. These results demonstrated that the RacC-induced morphological changes were independent of the effects of overexpression on phagocytosis/macropinocytosis, suggesting that WT-RacC is involved in two separate pathways in Dictyostelium.23 Current studies are underway to determine the effector proteins that interact with RacC (also see below).

212

Rho GTPases

RacF1/2 RacF1 and RacF2 were identified during a PCR-based screen for additional Dictyostelium Rho family GTPases, and these two GTPases are considered to belong to the Rac subfamily of GTPases.6 Studies with GFP-tagged RacF1 demonstrated that although it was distributed throughout the cytosol, it was particularly enriched in macropinosomes, phagocytic cups, and areas of cell-cell contacts.6 Interestingly, RacF1 associated with the phagocytic cup early-on during particle internalization, but dissociated within 45 seconds, suggesting that this association is transient. Cell lines expressing only a C-terminally truncated form of the RacF1 protein showed no inhibition in phagocytosis or pinocytosis, suggesting that RacF1 and RacF2 are not required for internalization or play redundant roles, or that, in RacF1’s absence, its function is carried out by another Rac protein.

RacB Recent studies by Knecht and colleagues have demonstrated that RacB is also important for regulating the F-actin cytoskeleton of Dictyostelium. Cell lines were generated that overexpressed wild-type, constitutively active, or dominant negative forms of RacB and were analyzed using a variety of methods. These studies showed that CA-RacB expressing cells formed unusual actin cytoskeleton structures that were distinct from those seen in cells overexpressing WT-RacC and CA-DdRac1.5 In addition, cells overexpressing dominant negative and constitutively active forms were defective in phagocytosis, pinocytosis, and exocytosis. Furthermore, the CA-RacB expressing cells displayed a defect in cell growth at higher titers and became multinucleate. The authors concluded that the RacB-induced effects on morphology were independent of myosin II function, as myoII null cell-lines did not respond in a similar fashion.

Additional Rac Family Members The Rho family of GTPases in Dictyostelium is quite large, and little is known about many of these proteins. For instance, other than expression patterns throughout Dictyostelium development and cladistic analyses, virtually nothing is known concerning the roles played by RacA, RacD, RacG, RacH, RacI, RacJ, and RacL.4,5 Future investigations will involve the functional characterization of these proteins and the identification of their requisite regulatory and effector proteins. Notably, the RacA protein is an unusually long small GTPase of 598 residues containing a GTPase domain at the N-terminus and a novel C-terminal region. The C-terminal region contains domains related to BTB or POZ domains involved in protein-protein interactions, and an SH3 domain linking the GTPase domain with the first BTB domain.25 Based on this C-terminal sequence information, RacA has been therefore grouped in a novel subfamily of Rho GTPases named RhoBTB, containing proteins first described in yeast.5 Note that the phylogenetic tree (supplementary information on line) is based only on amino acid sequences in the GTPase core and accordingly RacA is closer to Rac1 based on this information. It is not surprising that Dictyostelium appears not to express Rho-subfamily proteins, since RhoA is involved in the regulation of actin stress fiber formation, and these structures do not exist in Dictyostelium. However, amoebae are very motile and undergo cycles of actin-based contraction and expansion, suggesting other proteins are assuming the role played by Rho proteins in mammalian cells. Also, although filopodia are common actin-based structures observed in Dictyostelium, no Cdc42-like GTPase has been discovered. Dictyostelium cells can also become polar suggesting that Cdc42 is not necessary for polarity or formation of filopodia and that the function of this GTPase in these processes appeared more recently in evolution than perhaps thought.

Rho Family GTPase Regulators and Effectors In addition to investigations involving the Rho family members of Dictyostelium, many other studies of the proteins that regulate and/or mediate the functions of these proteins have

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been initiated. In many cases, more is known about the regulators of individual Rho family proteins than is known about the GTPases themselves. Following below is a description for each of these regulators and/or effectors that have been described.

GTPase Activating Proteins (GAPs) and Guanine Exchange Factors (GEFs) Several GAP-related proteins have been identified in Dictyostelium, including Dgap1, GapA, and DdRacGap1 (also referred to as DRG).12,14,15 Two of these proteins, GapA and Dgap1, share homology with the mammalian protein IQGAP1, which is postulated to be an effector protein for Rac and Cdc42.26,27 So, although IQGAP1-related proteins all share a GTPase activating (GAP) domain, none of them possesses significant GAP activity. In fact, GapA and Dgap1 show no GAP activity for any of the known Dictyostelium Rho family members. Instead, it appears that these proteins act as effectors that mediate the functions of a subset of Dictyostelium Rho family GTPases. On the other hand, DdRacGap (DRG), displays GAP activity for specific GTPases in Dictyostelium that are not limited to the Rho subfamily (see below).

DdRacGap1 (DRG)—A Bifunctional Protein DdRacGap1 (DRG for short), identified during a PCR-based screen for members of the Dictyostelium Rho-GAP family, is a bifunctional protein that possesses both GAP and GEF domains.14 In vitro studies showed that DRG had the ability to bind to DdRac1, so it was hypothesized that this protein could stimulate GTPase activity on Rac1 (through its GAP domain), and thus lower the levels of activated Rac1. It was predicted that the absence of DRG would lead to an increase in Rac1 activity and would subsequently lead to an increase in F-actin assembly. Studies of DRG-null cell-lines were performed independently by two groups.9,28 According to Chung and colleagues, DRG-null cells displayed increased F-actin levels, and most of the actin was organized into crown structures in these cells. Knetsch colleagues, also reported an increase in cortical F-actin levels for DRG-null cells, however the expression of the GAP domain of DRG was not sufficient to rescue the actin cytoskeletal defects of the DRG-null cells. Furthermore, the authors demonstrated that DRG acted as a GEF and stimulated GDP/ GTP exchange on Rac1. Taken together, these data suggest that DdRac1s are not the preferred in vivo substrate for the GAP activity of DRG and instead may be one of the in vivo substrate for the GEF activity associated with DRG.28 The increase in F-actin in the absence of DRG may be due to the activation of Racs other than Rac1. Interestingly, DRG displayed GAP activity towards the Rho family GTPase RacE and the Dictyostelium Rab family GTPase RabD, an orthologue of mammalian Rab14.28,29 In vitro studies revealed that DRG increased the GTPase activity of RabD by 30-fold, while it increased the GTPase activity of RacE by 5-fold. As discussed earlier, RabD is important for the proper functioning of the contractile vacuole system of Dictyostelium and regulates endocytic traffic, while RacE is necessary for cleavage furrow formation during cytokinesis.20,30 In support of the above results, DRG null cells were defective in contractile vacuole function and cells expressing only the GAP-SH3 domains of DRG displayed cytokinesis defects similar to those observed in RacE null cells.28 One of the remarkable aspects of the DRG protein is that it possesses both GAP and GEF (dbl) domains in addition to a pleckstrin homology (PH) domain and an SH3 domain. It shares these properties with some earlier described proteins, including human BCR, Abr, and the Drosophila 23E12 protein.31-33 A current model proposes that DRG influences the nucleotide state of at least three Dictyostelium GTPases, through distinct pathways (see Fig. 1) and may coordinately regulate actin-based and vesicle-based processes.

Dgap1/DdRasGAP This molecule was first described by and was identified independently by, who termed the protein DdRasGAP, because it was found during a yeast two-hybrid screen for proteins that

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Figure 1. Model describing the function of MyoM and DRG in coordinating the activating of Rho and Rab proteins with changes in the actin cytoskeleton and vesicle trafficking.

preferentially interacted with Ras-GTP and not Ras-GDP.12,34 To avoid confusion this molecule will hereafter be referred to as Dgap1 in accord with its initial discovery. Dgap1 is most closely related to another Dictyostelium IQGAP-related protein, GapA.15 Dgap1 preferentially binds to the GTP-bound form of Rac1;35 and the developmental expression pattern of Dgap1 was similar to that of Rac1.8 In addition, disruption of the Dgap1 gene caused a shift in the G/ F actin ratio; Dgap1 null cells contain increased F-actin content, leading to the possibility that Dgap1 could be a GAP protein for Rac1. However, Dgap1 does not possess GAP activity, so it is most likely an effector protein for Rac1. 12 Dgap1 also binds to RacE, but in a nucleotide-independent manner.35 Further studies of the Dgap1 null cells underscore its importance for many actin-based processes including: cell motility, cytokinesis, and development.35 Using GFP-tagged Dgap1, the most recent investigations have indicated that Dgap1, Rac1, and the actin bundling protein cortexillin can form a complex at the cleavage furrow of dividing cells and in the cell cortex of vegetative cells.36 It is hypothesized that both Dgap1 and GapA act as a scaffold that brings Rac1, cortexillin, and other proteins together in order to relay signals from Rho-family GTPases to the actin cytoskeleton.

GapA GapA, identified in a REMI-screen for Dictyostelium mutants that grew as giant, multinucleate cells is also homologous to IQGAP-related proteins.15 It was called GapA because it shares close homology with the Gap1/Sar1 protein from S. pombe.37 Both GapA and Gap1/ Sar1 contain a Gap-related domain (GRD), but interestingly, none of these proteins possesses GAP activity for GTPases. Studies of a Dictyostelium GapA null cell-line demonstrated that it

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was required for cleavage of the mid-body during later stages of cytokinesis, but was not required for development or phagocytosis. Therefore, although GapA and RacE participate in cytokinesis, they appear to do so in a distinct manner.

Other Proteins that Interact with Rho Family GTPases Scar Scar, a Dictyostelium protein belonging to the WASP family of proteins, has been demonstrated to regulated phagocytosis and F-actin polymerization at the plasma membrane and on endosomes.38,50 The WASP proteins contain multiple domains enabling these proteins to bind to profilin, G-actin and the arp2/3 complex to regulate the polymerization of actin; WASP but not Scar also contains a Cdc42 binding domain.39 Interestingly, WAVE, related to Scar, binds Rac1 via interaction with a linker protein called IRS-53.40 Yeast two hybrid studies have indicated that RacC interacts with Scar, although it has not been confirmed that this interaction is direct (Seastone and Cardelli, unpublished results). Overexpression of WT-RacC in Scar null cells does not result in an increase in phagocytosis rates as observed in wild-type cells, suggesting Scar is a downstream effector for RacC (Seastone and Cardelli, unpublished results).

Myosin I Heavy Chain Kinase (MIHCK) The MICHK protein was purified, the gene was cloned and sequence analysis revealed that it shared significant similarity to human PAK, an effector protein for the mammalian GTPases cdc42 and Rac1.13,41,42 Because MIHCK contains a CRIB motif, it seemed like a good candidate effector protein for Dictyostelium Racs. Phosphorylated forms of MIHCK preferentially interacted with both mammalian Cdc42 and Rac1;43 however, no evidence that MIHCK interacts with Dictyostelium Rac proteins has been published.

Darlin Darlin (for Dictyostelium Armadillo-like protein) is an 88 Kd protein that was identified during a screen for in vitro interaction partners of GST-RacE.16 It is also able to bind to RacC, DdTCRan, and HsCdc42 in vitro. Darlin contains several armadillo repeats, which have been described in other GEF domain-containing proteins. Unexpectedly, Darlin did not show GEF activity for any of the Dictyostelium GTPases examined, which was not totally surprising since other armadillo repeat-containing proteins, such as smgGDS (Yamamoto et al., 1990), are also poor GEFs for GTPases. The Darlin gene was isolated using reverse genetics and null mutants were generated. Darlin null cells were normal in terms of cytokinesis, actin cytoskeleton distribution, pinocytosis, and phagocytosis. However, aggregation was completely ablated in the Darlin null cells, suggesting it may play a role in cAMP signalling, necessary for cells to aggregate. In conclusion, Darlin is thought to be an adaptor molecule that function to bring GTPases (such as RacE or RacC) into close proximity with the proper effector molecules. Another Dictyostelium GEF (Ras-GEF or AIMLESS) is thought to function in a similar manner.44

DdLim DdLim is a 22.1 Kd protein that was identified based on its ability to colocalize with the actin cytoskeleton.17 When DdLim was overexpressed, cells became defective in cytokinesis, and the morphology of the cells changed. DdLim overexpressors formed many lamellae, which resembled the lamellapodia induced by expression of Rac1 in mammalian cells.45 Because of this, the authors tested the ability of DdLim to interact with Rho family proteins. DdLim interacted strongly with mammalian Rac1, but weakly with DdRac1A.17 Therefore, the authors concluded that DdLim probably interacts with a Dictyostelium GTPase that is closely related to human Rac1 in order to control actin polymerization and the formation of lamellipodia at the leading edges of cells.

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PAK Using degenerate primers in a PCR screen for PAK/Ste20 homologues, discovered a new Dictyostelium protein they referred to as PAKa.18 The structure of PAKa is very similar to mammalian PAK1 and Dictyostelium MIHCK, all of which contain a CRIB motif.13,46 PAKa was demonstrated to bind preferentially to GTP-bound forms of DdRac1 and HsCdc42 using both yeast two-hybrid analysis and GTP-overlay assays.18 When PAKa was deleted, cells became defective in cytokinesis, cell polarity, development, and chemotaxis. In addition, expression of a kinase dead form of PAKa led to defects in the actin cytoskeleton. Interestingly, PAKa was localized to the cleavage furrow of dividing cells, similar to the GTPase DdRacE, suggesting it plays a specific role in cytokinesis. The most recent data suggests that PAKa is a substrate for the PI 3-kinase and PKB signalling pathways in order to coordinate myosin assembly.47

MyoM MyoM is a divergent unconventional myosin that was discovered during an exhaustive screen for Dictyostelium myosin family members and was later functionally characterized.19,48 Structural analysis of the MyoM protein revealed that it contains a fusion between a myosin motor in the head domain of the protein, and a dbl homology (GEF) domain in the tail portion of the molecule.19 In addition, a PH domain lies adjacent to the GEF domain, indicating MyoM may bind to phospholipids, where it could exert its GEF function on GTPases. However, 3D modeling indicated that MyoM may not bind to phosphoinositides as has been historically shown for PH domain-containing proteins.19 Instead, MyoM may partner with more proteinaceous molecules (such as Rho family GTPases). Disruption of the entire MyoM gene produced no readily observable phenotype; phagocytosis rates, cell viability, development, and cell morphology were unaffected. However, expression of only the tail GEF domain of MyoM was sufficient to produce novel actin-based structures termed ramopodia when cells were subjected to hypo-osmotic stress. Ramopodia are actually protrusions of the plasma membrane that are filled with actin, and are collared by the protein coronin.49 In addition, the GEF domain of MyoM was localized at the tip of the ramopodia, suggesting that it may activate a GTPase at that particular location. MyoM displayed significant GEF activity for DdRac1, but not RacC or RacE, indicating that it activates a subgroup of Dictyostelium Rho family GTPases.

Summary Figure 1 represents a speculative model showing the potential interactions between MyoM and DRG as they modulate the activation state and/or function of multiple Racs and at least one Rab to coordinate changes in F-actin polymerization and vesicle trafficking. Many of the other proteins described above will undoubtedly functionally interact with different elements outlined in this schematic pathway. It will be interesting to determine if the other uncharacterised Rho family proteins RacA and RacG – RacL, function in a nonredundant fashion. Also, future studies will involve examining the roles of Rho proteins not only during vegetative growth but also during the developmental cycle. Development involves many processes in mammalian systems including cell signalling, motility, morphogenesis and cell differentiation. These studies will become more rapid now that the genome has been sequenced.

References 1. Bush J, Franek K, Daniel J et al. Cloning and characterization of five novel Dictyostelium discoideum rab-related genes. Gene 1993; 136:55-60. 2. Rupper A, Cardelli J. Regulation of Phagocytosis and Endo-Phagosomal Trafficking Pathways in Dictyostelium discoideum. In Dictyostelium: A Genetic Model for the Study of Professional Phagocytes. Cardelli J, ed. Biochim Biophys Acta 2001; 1525: 205-216. 3. Chubb JR, Insall RH. Dictyostelium: An ideal organism for genetic dissection of Ras signalling networks. Biochim Biophys Acta 2001; 1525:262-271. 4. Bush J, Franek K, Cardelli J. Cloning and characterization of seven novel Dictyostelium discoideum rac-related genes belonging to the rho family of GTPases. Gene 1993; 136:61-68.

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5. Rivero F, Dislich H, Glockner G et al. The Dictyostelium discoideum family of Rho-related proteins. Nucleic Acids Res 2001; 29:1068-1079. 6. Rivero F, Albrecht R, Dislich H et al. RacF1, a novel member of the Rho protein family in Dictyostelium discoideum, associates transiently with cell contact areas, macropinosomes, and phagosomes. Mol Biol Cell 1999; 10:1205-1219. 7. Larochelle DA, Vithalani KK, De Lozanne A. Role of Dictyostelium racE in cytokinesis: Mutational analysis and localization studies by use of green fluorescent protein. Mol Biol Cell 1997; 8:935-944. 8. Dumontier M, Hocht P, Mintert U et al. Rac1 GTPases control filopodia formation, cell motility, endocytosis, cytokinesis and development in Dictyostelium. J Cell Sci 2000; 113:2253-2265. 9. Chung CY, Lee S, Briscoe C et al. Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells. Proc Natl Acad Sci USA 2000; 97:5225-5230. 10. Palmieri SJ, Nebl T, Pope RK et al. Mutant Rac1B expression in dictyostelium: Effects on morphology, growth, endocytosis, development, and the actin cytoskeleton [In Process Citation]. Cell Motil Cytoskeleton 2000; 46:285-304. 11. Ridley AJ, Paterson HF, Johnston CL et al. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992; 70:401-410. 12. Faix J, Dittrich W. DGAP1, a homologue of rasGTPase activating proteins that controls growth, cytokinesis, and development in Dictyostelium discoideum. FEBS Lett 1996; 394:251-257. 13. Lee SF, Egelhoff TT, Mahasneh A et al. Cloning and characterization of a Dictyostelium myosin I heavy chain kinase activated by Cdc42 and Rac. J Biol Chem 1996; 271:27044-27048. 14. Ludbrook SB, Eccleston JF, Strom M. Cloning and characterization of a rhoGAP homolog from Dictyostelium discoideum. J Biol Chem 1997; 272:15682-15686. 15. Adachi H, Takahashi Y, Hasebe T et al. Dictyostelium IQGAP-related protein specifically involved in the completion of cytokinesis. J Cell Biol 1997; 137:891-898. 16. Vithalani KK, Parent CA, Thorn EM et al. Identification of darlin, a Dictyostelium protein with Armadillo-like repeats that binds to small GTPases and is important for the proper aggregation of developing cells. Mol Biol Cell 1998; 9:3095-3106. 17. Prassler J, Murr A, Stocker S et al. DdLIM is a cytoskeleton-associated protein involved in the protrusion of lamellipodia in Dictyostelium. Mol Biol Cell 1998; 9:545-559. 18. Chung CY, Firtel RA. PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis. J Cell Biol 1999; 147:559-576. 19. Geissler H, Ullmann R, Soldati T. The tail domain of myosin M catalyses nucleotide exchange on Rac1 GTPases and can induce actin-driven surface protrusions. Traffic 2000; 1:399-410. 20. Larochelle DA, Vithalani KK, De Lozanne A. A novel member of the rho family of small GTP-binding proteins is specifically required for cytokinesis. J Cell Biol 1996; 133:1321-1329. 21. Fukui Y, De Lozanne A, Spudich JA. Structure and function of the cytoskeleton of a Dictyostelium myosin-defective mutant. J Cell Biol 1990; 110:367-378. 22. Gerald N, Dai J, Ting-Beall HP et al. A role for Dictyostelium racE in cortical tension and cleavage furrow progression. J Cell Biol 1998; 141:483-492. 23. Seastone DJ, Lee E, Bush J et al. Overexpression of a novel rho family GTPase, RacC, induces unusual actin-based structures and positively affects phagocytosis in Dictyostelium discoideum. Mol Biol Cell 1998; 9:2891-2904. 24. Seastone DJ, Zhang L, Buczynski et al. The small Mr ras-like GTPase rap1 and the phospholipase C pathway act to regulate phagocytosis in dictyostelium discoideum [In Process Citation]. Mol Biol Cell 1999; 10:393-406. 25. Collins T, Stone JR, Williams AJ. All in the family: The BTB/POZ, KRAB, and SCAN domains. Mol Cell Biol 2001; 21:3609-3615. 26. McCallum SJ, Erickson JW, Cerione RA. Characterization of the association of the actin-binding protein, IQGAP, and activated Cdc42 with Golgi membranes. J Biol Chem 1998; 273:22537-22544. 27. Kuroda S, Fukata M, Kobayashi K et al. Identification of IQGAP as a putative target for the small GTPases, Cdc42 and Rac1. J Biol Chem 1996; 271:23363-23367. 28. Knetsch ML, Schafers N, Horstmann H et al. The Dictyostelium Bcr/Abr-related protein DRG regulates both Rac-and Rab-dependent pathways. EMBO J 2001; 20:1620-1629. 29. Bush J, Nolta K, Rodriguez-Paris J et al. A Rab4-like GTPase in Dictyostelium discoideum colocalizes with V-H(+)-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes. Journal of Cell Science 1994; 107:2801-2812. 30. Bush J, Temesvari L, Rodriguez-Paris J et al. A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum. Molecular Biology of the Cell 1996; 7:1623-1638.

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31. Heisterkamp N, Morris C, Groffen J. ABR, an active BCR-related gene. Nucleic Acids Res 1989; 17:8821-8831. 32. Tan EC, Leung T, Manser E et al. The human active breakpoint cluster region-related gene encodes a brain protein with homology to guanine nucleotide exchange proteins and GTPase-activating proteins. J Biol Chem 1993; 268:27291-27298. 33. Chuang TH, Xu X, Kaartinen V et al. Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family. Proc Natl Acad Sci USA 1995; 92:10282-10286. 34. Lee S, Escalante R, Firtel RA. A Ras GAP is essential for cytokinesis and spatial patterning in Dictyostelium. Development 1997; 124:983-996. 35. Faix J, Clougherty C, Konzok A et al. The IQGAP-related protein DGAP1 interacts with Rac and is involved in the modulation of the F-actin cytoskeleton and control of cell motility. J Cell Sci 1998; 111:3059-3071. 36. Faix J, Weber I, Mintert U et al. Recruitment of cortexillin into the cleavage furrow is controlled by Rac1 and IQGAP-related proteins. EMBO J 2001; 20:3705-3715. 37. Imai Y, Miyake S, Hughes DA et al. Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe. Mol Cell Biol 1991; 11:3088-3094. 38. Bear JE, Rawls JF, Saxe CL, III. SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development. J Cell Biol 1998; 142:1325-1335. 39. Zigmond SH. How WASP regulates actin polymerization. J Cell Biol 2000; 150:F117-F120. 40. Miki H, Yamaguchi H, Suetsugu S et al. IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature 2000; 408:732-735. 41. Lee SF, Cote GP. Purification and characterization of a Dictyostelium protein kinase required for actin activation of the Mg2+ ATPase activity of Dictyostelium myosin ID. J Biol Chem 1995; 270:11776-11782. 42. Bokoch GM. Regulation of cell function by Rho family GTPases. Immunol Res 2000; 21:139-148. 43. Lee SF, Mahasneh A, de la RM et al. Regulation of the p21-activated kinase-related Dictyostelium myosin I heavy chain kinase by autophosphorylation, acidic phospholipids, and Ca2+-calmodulin. J Biol Chem 1998; 273:27911-27917. 44. Tuxworth RI, Cheetham JL, Machesky LM et al. Dictyostelium RasG is required for normal motility and cytokinesis, but not growth. J Cell Biol 1997; 138:605-614. 45. Nobes CD, Hawkins P, Stephens L et al. Activation of the small GTP-binding proteins rho and rac by growth factor receptors. J Cell Sci 1995; 108:225-233. 46. Buczynski G, Grove B, Nomura A et al. Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization. Journal of Cell Biology 1997; 136:1271-1286. 47. Chung CY, Potikyan G, Firtel RA. Control of cell polarity and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa. Mol Cell 2001; 7:937-947. 48. Schwarz EC, Geissler H, Soldati T. A potentially exhaustive screening strategy reveals two novel divergent myosins in Dictyostelium. Cell Biochem Biophys 1999; 30:413-435. 49. Maniak M, Rauchenberger R, Albrecht R et al. Coronin involved in phagocytosis: Dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag. Cell 1995; 83:915-924. 50 Seastone D, Harris, E, Temesvari L et al. The WASP-like protein Scar regulates macropinocytosis, phagocytosis and endosomal membrane flow in Dictyostelium. J Cell Sci 2001; 114:2673-2683. 51. Lee E, Seastone D, Harris E et al. RacB regulates cytoskeletal function in Dictyostelium. Eukaryotic Cell 2003 in press.

CHAPTER 15

Rho GTPases in Plants Aline H. Valster, Peter K. Hepler and Jonathan Chernoff

Abstract

P

lants contain a unique group of Rho family GTPases called Rop. Members of the Rop subgroup are numerous and highly similar to each other. They share some basic signalling properties with Rho family GTPases found in animals and yeasts, such as the guanine nucleotide-dependent cycling between the active and inactive form and the regulation of this cycle by other proteins. However, some striking differences in mechanisms of action of these signalling modules have recently surfaced. For example the absence of classic guanine nucleotide exchange factor in plant sequences, and the presence of CRIB domains in plant GTPase activating proteins suggest that Rops are regulated in a novel manner. As in animals and yeasts, a plethora of processes have been identified in which Rop appears to play a key role in plants. While the full range of Rop activity is not yet known, it is clear that it plays a central role in plant physiology, affecting processes such as vacuole development, cell polarity establishment, cell wall formation, and hormone responses. Current efforts are geared towards elucidating details regarding the Rop-regulated signalling cascades in these physiological and developmental processes in plants.

Introduction

The initial report on the presence of a Rho-related protein in plants1 was published 8 years after the identification of the first member of the Rho subfamily of the Ras superfamily of small GTPases in Aplysia.2 Today, it appears that this first Rho-like protein in plants is the founding member of a group of proteins within the Rho subfamily that is unique to plants. The group is termed Rop (for Rho family GTPases in plants) and is distinct from the groups that already had been identified within the Rho subfamily in animals and yeasts, namely Rho, Rac and Cdc42. Whereas the initial distinction of these already existing groups has its basis in functional differences (Rho causes stress fiber formation; Rac is implicated in lamellipodia formation, and Cdc42 functions in filopodia formation in human fibroblasts),3 the Rops have been identified and named on the basis of sequence homology with other Rho proteins. Since Rops share the highest sequence homology with Racs, they are sometimes referred to as plant Racs (for example AtRac or ARAC refers to a Rac-like protein from Arabidopsis thaliana). However, since phylogenetic analysis indicates that they form a clearly distinct subgroup among the Rho GTPases (Fig. 1), referring to them as “Rops” seems appropriate.

Rop Identity While no small GTPases in plants have been identified that belong to the Rho, Rac or Cdc42 subgroups found in animals and fungi, the single Rop family in plants contains more members than any of the above mentioned subgroups. In addition, the Rops are highly similar to each other; on average they share more than 90% identity. However, there is one exception, namely a Rop related protein from Arabidopsis (AtRho; GenBank accession number U88402)4 Rho GTPases, edited by Marc Symons. ©2003 Eurekah.com and Kluwer Academic / Plenum Publishers.

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Figure 1. Diagram showing phylogenetic relationships between Rho family GTPases from plants, humans, yeasts and flies. The Rop subfamily is found exclusively in plants and is most closely related to the Rac subfamily. Several distinct groups can be distinguished within the Rop subfamily according to Winge et al.5 The Rop group in this diagram contains members from the dicotyledon Arabidopsis. Group Ia (containing Rop6, Rop 2, Rop4 etc) is the largest group and contains Rops found in dicotyledons. Group Ib contains Arac2 and group II (containing Arac 7, 8 and 10) is found in both mono- and dicotyledons. Slash symbols indicate foreshortened branches of the Rho subfamily. Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe.

that has only ~50% sequence similarity as compared to other Rop proteins and is more closely related to Rho than to Rac.5 Overall amino acid sequence similarity, and in particular sequence similarity in the GTP-binding domain, shows that the Rop subfamily is most closely related to the Rac subfamily of GTPases (Fig. 1). Cdc42, RhoA and Rac show considerable sequence variation in their GTP-binding site and insert region, but their effector loops are similar. All Rops, on the other hand, share Rac’s GTP binding motif (TKLD) and show close sequence similarity to the effector loop region of Racs.6 Overall, the Rops share about 70% sequence similarity at the amino acid level with Racs, whereas the levels of sequence identity with Cdc42 and Rho are 55% and 45-50% respectively. In Arabidopsis 11 Rops have been identified5,7 which can be further divided into 2 distinct groups (Table 1). Members of Group I (Rop1, ARAC1/Rop3, ARAC2, ARAC3/Rop6, ARAC4/ Rop2, ARAC5/Rop4, ARAC6, ARAC9 and ARAC11) all have a C-terminal geranylgeranylation signal (CXXL) that is also characteristic for Rac proteins in mammals. Group I Rops can be further divided into three distinct subgroups; Group Ia containing the Group I Rops from dicotyledons, Group Ib containing the Group I Rops from monocotyledons, and a third group (Group Ic) which is composed of members that are all very similar to ARAC2. Group II Rops

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(ARAC7, ARAC8 and ARAC10) are found in both monocotyledons and dicotyledons, and lack the geranylgeranylation site but have a C-terminal aaCG (a stands for aliphatic amino acid) signal that resembles the palmitoylation site commonly found in Ras proteins. Whether the distinction of these various subgroups of Rop proteins, based on sequence similarity, is reflected in functional or specificity differences at the cellular level, remains elusive and will certainly be a focus of future research.

Regulation of Rop Given that plants encode a single large subgroup of nearly identical small GTPases, the question as to how these enzymes are regulated promises to be an interesting one and could illustrate important differences in signalling mechanisms between plants, fungi and animals. Like all members of the small GTPase superfamily, the Rop GTPases cycle between GTP and GDP bound states, which renders their action as molecular switches in signal transduction cascades ‘on’ and ‘off ’ respectively. One level of regulation occurs through differential expression of the various Rops in different plant tissues (Table 1). Based upon expression patterns a reproductive and a vegetative class of Rops can be identified.8 Rop1At (and Rop1Ps), Rop3At and Rop5At are all expressed in mature pollen and form the reproductive class. Rop3At and Rop5At, however, are also expressed in vegetative tissues, whereas Rop1At is only expressed in pollen. Rop3At is highly expressed in roots, whereas Rop5At transcription is very low.8 Members of the vegetative class (Rop2At, Rop4At, Rop6At, and Arac2) are not expressed in pollen. Rop2At and Rop4At are expressed in all organs and Rop6At expression is found in open flowers, leaves, roots and stems but not in closed flowers and siliques.8 In addition, a general high level of expression of Rops is found in meristematic tissues.9 In animal and fungal cells, Rho function is regulated by three different classes of regulatory proteins (see Chapter 1 for details), namely guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). These proteins are able to regulate the intrinsically low GTPase activity of the Rho GTPases. Little is known about the biochemical properties of most Rops, but kinetic measurements for AtRop4 and AtRop6 have recently been reported. The intrinsic GTPase rate for these enzymes (0.04/ min)10 is about ten-fold lower than that reported for mammalian Rac1.11 This slow GTPase rate makes it likely that Rop activity, like that of Rho family GTPases in fungi and animals, is modulated in vivo by accessory proteins that affect GTP binding and hydrolysis such as the GEFs, GAPs and GDIs (see below). GEFs are positive regulators that promote the exchange of GDP for GTP, which renders the Rho GTPase in an active conformation, capable of physically interacting with downstream effectors. Classically, GEFs are characterized by the presence of Dbl-homology (DH) domains.12 Interestingly, in the recently completed Arabidopsis genome sequence, no proteins can be found with DH-domains. Do plants then encode a different class of GEF? Hardt et al13 have shown that the bacterium Salmonella typhimurium encodes a protein, SopE, that lacks a recognizable DH-domain, yet acts as an efficient GEF for Rac and Cdc42.13,14 While plants lack proteins similar to SopE, the discovery of nonclassical GEFs in bacteria means that there may be additional ways to construct GEFs, and that plants may encode GEFs that we are not currently able to recognize by sequence. Alternatively, it is possible that plants lack GEFs altogether, and that Rops are regulated by other means. For example, a ‘fast-cycling’ Cdc42 mutant has been created that does not require GEF activity for successful cycling between GDP and GTP-bound states.15 If Rop GTPases have an intrinsically high off-rate, it is conceivable that they might not require GEFs for activation. The lack of classical Rop GEFs in plants also poses the question about the mechanism of action of the dominant negative mutant forms of Rop. In animals and fungi it is thought that the dominant negative mutant forms of the Rho GTPases sequester GEFs. The resulting lack of GEF activity keeps the native Rho GTPase in its GDP-bound and inactive form.16 As dis-

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cussed in detail below, overexpression of dominant negative Rop mutations in pollen tubes causes the inhibition of growth, whereas overexpression of the constitutively active mutant Rop causes delocalized growth.17,18 The dominant negative mutant shows the same effect as does the microinjection of anti-Rop antibodies,19 which indicates that this phenotype is likely due to loss of function of Rop. These observations lend weight to the probability that a novel GEF activity in fact exists in plants and that this activity is likely to be sequestered by the dominant negative mutant Rop to cause a loss-of-function phenotype. GAPs and GDIs are both negative regulators of Rho GTPases. GAPs stimulate the intrinsic GTPase activity of Rho, thereby turning the signal transduction switch to the ‘off ’ position. In a search for Rop-interacting proteins using a two-hybrid approach, several RopGAPs were identified in Lotus japonicus and Arabidopsis.20,21 In addition to a recognizable GAP domain, these proteins contain a CRIB-domain (Cdc42/Rac interactive binding) that is a signature for effector molecules of Cdc42 and/or Rac.22 This feature has not been found in any GAP protein outside the plant kingdom, further pointing to the probability that Rops are regulated by novel mechanisms. Although these GAPs have been shown to possess bona-fide GAP activity in vitro,20,21 it is also possible that they play an effector role in mediating Rop functions, as IQ-GAP (which lacks true GAP function) does in mammalian cells.23 The other class of negative regulators for Rho family proteins, the Rho-GDIs, which inhibit the dissociation of GDP and prevent its exchange for GTP and the resulting activation of the GTPase, are also found in plants. Two putative Rop-GDIs have been identified in the Arabidopsis genome database and one (AtRhoGDI1) has been cloned.24 AtRhoGDI1 shows 29% to 37% similarity to mammalian RhoGDI homologues and is expressed ubiquitously in Arabidopsis. Furthermore, binding of AtRhoGDI1 to AtRop4 and AtRop6 has been shown in vitro as well as in vivo.24 Interestingly, in another study a heterologous two hybrid approach has led to the discovery of a GDI from tobacco.25 In this two hybrid experiment, a mammalian RhoGDI-bait was able to interact with a tobacco Rop, which was then used as the bait in a subsequent screen to identify a RhoGDI from tobacco.25 These findings emphasize the similarities in the regulation of the small GTPases from different kingdoms. In conclusion it appears that, when compared with the regulation of Rhos in organisms across kingdoms, the regulation of Rops shares the basic principles of cycling in a nucleotide dependent manner and the control by regulatory proteins. However, the absence of putative GEFs with a DH-domain, found in GEFs in animals and fungi, and the presence of a CRIB domain in Rop-GAPs point to the possibility of intriguing differences. It is likely that further biochemical analysis of Rop properties will yield new insights into the regulation of these small GTPases.

Rop Localization Information about the intracellular localization of different Rop proteins is rather limited, although the localization of several Rops to plasma membranes is well established (Table 1). Anti Rop1Ps antibodies have localized Rop1 to the plasma membrane of the tip of the pollen tube,26 an observation that has been confirmed by expression of a GFP-tagged AtRac2 in tobacco pollen tubes.18 Similarly, anti-AtRop4 antibodies and GFP-AtRop4 expression localized to the apical plasma membrane of developing and growing root hairs.10 When expressed in tobacco suspension cells (BY-2 cells), GFP-AtRop4 also associates with developing cell plates during cytokinesis and the plasma membrane.10 Wildtype OsRac1 and its constituively active and dominant negative mutants, all localize to the plasma membrane of rice protoplasts as well. Interestingly, when the cysteine in the prenylation site (CXXL) is replaced by a serine, the association with the plasma membrane is lost and a general cytoplasmic staining results, indicating the importance of the cysteine residue in proper localization of OsRac1.27 Surprisingly, AtRop 4 has been reported to be localized to the plasma membrane,10 whereas in another study it was reported to localize to the perinuclear region in BY-2 cells.24 AtRop 6 and Rop7 have both been localized to plasma membranes as well.24-28

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Table 1. Summary showing Rop proteins and their expression patterns, intracellular localization and the processes they regulate, as far as known GroupI

Expression

Localization

Implicated in

References

Rop1Ps/ Rop5At Rop2/Arac4

pollen exclusively not in pollen

plasma membrane tonoplast —

1,17-19,26 29 5,9

Rop3/Arac1

pollen, vegetative tissue, roots not in pollen

—

pollentube growth vacuole development embryo development seed dormancy apical dominance —

Rop4/Arac5 Rop6/Arac3/ AtRac1

Arac2/similar to Rac13 from cotton Arac6/AtRac2 Arac9 Arac11

5,8

plasma membrane cell plate plasma membrane

root hair growth and polarity stomatal closure

5,8,10

—

cell wall formation

5,42,43

pollen specific — —

— — —

— — —

5,18 5 5

—

—

disease resistance apoptosis

5,27,41

— —

— —

— —

5 5

not in pollen, open flowers, leaves, roots, stems, guard cells —

5,8,32

Group II Arac7/similar to OsRac1 from rice Arac8 Arac10

— means no information available.

Immunolocalization studies using anti-Rop1Ps antibodies in pea tapetal cells, label specifically the tonoplast of developing vacuoles. Tonoplasts of mature vacuoles are not stained.29 Together, these localization data indicate specific associations of Rops with distinct endomembrane systems. With regard to the immunolocalization studies it is important to note that, based on the high degree of similarity among Rops, it is to be expected that the antibodies display broad specificity concerning Rops.

Rop-Regulated Processes Like Rho proteins in animal cells, Rops appear to regulate a variety of different processes. More specifically, involvement of various Rops have been shown in establishment of polarity, regulation of the actin cytoskeleton, pathogen defense responses, secondary cell wall formation, meristem signalling, vacuole development, and auxin signalling (Table 1 and Fig. 2). Although Rop involvement in these processes has been clearly shown using different experimental approaches, many details regarding events upstream and downstream of Rop activation are still unknown.

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Figure 2. Rop regulated signalling cascades. Rop has been correlated with the processes indicated in the boxes. Our current knowledge about up- and down stream events from Rop function is indicated. Arrows do not necessarily indicate direct interactions and question marks indicate hypothetical aspects of this model or lack of knowledge. ABA, abscisic acid; CLV receptor, CLAVATA receptor; MAPKs, mitogen activated protein kinases; PI, phosphatidylinositol; PIP2, phosphatidylinositol (4,5)-biphosphate; WUS, WUSCHEL protein.

Cell Polarity To date, the most well studied cellular role for Rops are in polarity establishment and tip growth in pollen tubes. The pollen tube plays a crucial role in sexual reproduction in flowering plants where it is responsible for the delivery of the sperm nuclei to the ovule. A pollen tube is a long cylindrical cell that germinates from a pollen grain after it lands on the stigma. The tube grows through the tissue of the style to the ovule by tip growth in which localized secretion occurs only at the extreme apex of the pollen tube. This secretion is in part regulated by a steep intracellular calcium gradient also localized at the extreme apex of the tube. Conditions or events that alter the calcium gradient also inhibit growth thus establishing a close interrelationship between calcium and pollen tube growth.30 The first indication of Rops function in pollen tube growth was the localization of the pollen specific Rop1Ps to the tip of the pollen tube.26 Evidence for Rops function in pollen tube growth derives from subsequent studies in which antibodies, raised against the Rop1Ps protein were microinjected in pea pollen tubes and were shown to inhibit tube elongation.19 Further evidence resulted from studies in which dominant negative and constitutively active mutants of Rops were overexpressed in pollen tubes of Arabidopsis and Nicotiana.17,18,31 Overexpression of the dominant negative Rop resulted in an inhibition of growth of the pollen tube, indicating that active Rop is necessary for growth. This inhibition could be partially reversed by increasing the extracellular calcium concentration.17 Although a scheme where calcium influx is downstream of Rop function is tempting to contemplate, it is important to keep in mind that the rescue is only partial. Therefore, it might be more likely that active Rop increases the efficiency of the mechanism that establishes the intracellular calcium gradient. It is conceivable that a calcium channel activity localized at the extreme tip in the plasmamembrane, is enhanced by Rop activation. Overexpression of constitutively active Rop results in swelling of the pollen tube tip,17,18,31 which is due to an isodiametric growth as opposed to the localized tip growth in control cells. Wildtype Rop1At, overexpressed in Arabidopsis pollen tubes17 also causes depolarized growth but the defect is less severe than that seen with the constitutively activated form of the protein.

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These results indicate that inactivation of Rop is necessary for polarity establishment in the tip growth phenomenon. This notion is further supported by the observation that overexpression of Rop1At in fission yeast (Schizosaccharomyces pombe) results in isotropic cell growth and that a GFP fusion of Rop1At localizes to the sites of expansion.8 Rop function in establishing cell polarity is not confined to pollen tubes. In a recent study it was shown that root hair development in Arabidopsis is dependent on Rop in a manner similar to it’s role in pollen tube polarity.10 Root hairs are single, tubular cells that originate from a root epidermal cell and elongate by means of tip growth, as do pollen tubes. Overexpression of constitutively active GFP-AtRop6 results in a swollen morphology of the root hair tip10 reinforcing the notion that constitutive activation of Rop causes de-localized growth, as seen in pollen tubes. Moreover, antibodies to AtRop4 immunolocalize to the site of roothair emergence during initiation and early bulging of the root hair, and to the plasma membrane at the tip of the elongating root hair.10 Together, these results make it clear that a proper balance between activation/deactivation of Rop is key for the successful establishment of cell polarity and growth in pollen tubes and root hairs, although the specific underlying processes that are being regulated are not so evident.

Regulation of the Actin Cytoskeleton In animal and yeast cells the Rho family of proteins are well known for their role as regulators of the actin cytoskeleton.3 Indications of Rop’s possible involvement in regulating the plant actin cytoskeleton come from several lines of evidence. Aberrant pollen tube growth and morphology due to overexpression of mutant forms of Rop, are associated with changes in the organization of the actin cytoskeleton. Coexpression of a GFP tagged F-actin binding domain of mouse talin and the constitutively active At-Rac2 in tobacco pollen tubes, shows a dramatic change in the organization of the actin cytoskeleton in the swollen pollen tube tip.18 Actin filaments spiral the periphery of the tip transversely, whereas in control cells actin filaments are oriented longitudinally within the interior of the tube with a fine network of short actin filaments towards the tip. In this study, overexpression of dominant negative At-Rac2 shows no dramatic changes in the actin cytoskeleton.18 A similar actin transverse band is observed in a study by Fu et al31 that also relies on a GFP-bound talin construct to visualize the actin cytoskeleton. Surprisingly, in this study it is overexpression of dominant negative and wild type Rop (Rop1At) that causes the transverse actin band.31 Immunofluorescence detection of the actin cytoskeleton (after using a freeze-shattering procedure) in the swollen root hair tips, due to overexpression of constitutively active AtRop6, is not associated with a transverse actin filament band.10 Taken together, these seemingly inconsistent observations are difficult to interpret, especially given the fact that actin transverse bands can sometimes be observed as a result of high levels of transient expression of the GFP- tagged F-actin binding domain of talin alone (K. Wilsen, pers. com.). Lack of detailed knowledge about the dynamic behavior of the actin cytoskeleton in pollen tubes and root hairs hampers interpretation of the above data. Dramatic changes in the actin cytoskeleton are also associated with stomatal closure that can be induced by the hormone abscisic acid or overexpression of dominant negative AtRac1 (a.k.a. ARAC3 or Rop6At).32 In open stomates, transgenic Arabidopsis plants expressing GFP-mTn (GFP-tagged F-actin binding domain of mouse talin) show many actin filaments in the guard cells. Overexpression of dominant negative AtRac1 causes the breakdown of those filaments and stomatal closure (for more details about these experiments see below) while microtubules remain and keep their transverse orientation.32

Pathogen Defense An important physiological role for Rop has been shown in disease resistance. Rop is involved in the elicitor-induced production of reactive oxygen species (ROS), which is the product of an oxidative burst reaction that induces cell death of infected host-cells. Cell death is part

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of the hypersensitive response of resistant plants to pathogen attack.33,34 As in the oxidative burst in mammalian neutrophils, ROS production in plants is carried out by the NADPH oxidase enzyme complex. The mammalian complex contains several subunits, some homologs of which have been identified in plants as well (for example the ‘phox’ proteins).35-38 Analogous to the regulation of the enzyme complex in neutrophils by Rac1 and Rac2,39 ROS production in plants is regulated by Rop small GTPases. A Rop protein was identified as part of the NADPH enzyme complex in tobacco cells.40 Functional evidence for a role of Rop in ROS production results from studies that show induction of ROS production and cell death due to the over-expression of constitutively active OsRac1 (from rice; most similar to Arac7) in rice, whereas the over-expression of the dominant negative OsRac1 inhibits ROS production and cell death in cells treated with the protein phosphatase inhibitor calyculin A.41 Futhermore, it was shown that constitutively active OsRac1 induces resistance of rice plants against a virulent race of the rice blast fungus and alters expression of defense-related genes.27 These findings imply a role for Rop in general disease resistance in rice.

Cell Wall Formation Another process that involves the production of ROS is the formation of the secondary cell wall. Also in this process Rop appears to play a regulatory role. It was shown that in cotton fibers (a model system for studies of secondary wall formation) the switch between primary to secondary wall formation is accompanied by the production of H2O242 H2O2 production and the start of secondary wall deposition coincide with an elevation in the expression of Rac13.43 Rac 13 from cotton is 90% identical to Arac2 from Arabidopsis, and is able to regulate H2O2 levels as has been shown by expression of mutant forms of the protein in cell cultures from Arabidopsis and soy bean. Constitutive active Rac13 from cotton results in stimulated H2O2 production, whereas the dominant negative form resulted in decreased H2O2 levels.42 From these results it seems evident that Rac13 has the potential to regulate cell wall formation through regulation of H2O2 production.

Meristem Signalling At the morphological and developmental level, the importance of Rop is illustrated by its role in meristem signalling. A careful balance between cell proliferation and differentiation is required in a meristem.44 CLAVATA1 (CLV1) is a receptor-like kinase that is required for maintaining this balance within the shoot and floral meristems of Arabidopsis.45 In mutant clv1 plants meristem size is increased up to a 1000-fold due to a lack of cell differentiation after multiplication. Subsequent coimmunoprecipitation experiments have shown that the CLV1 receptor is part of a large signalling complex that also contains CLV3, KAPP (a kinase-associated protein phosphatase and a negative regulator of CLV1 signalling) and Rop.46 This signalling complex exists in two distinct forms with different molecular weights (185 kDa and 450 kDa). The 450 kDa complex is thought to be the activated form and Rop is specifically associated with this larger complex.46 The activated complex possibly acts upstream of a mitogen-activated protein kinase (MAPK) cascade.47 The MAPK cascade, in turn, is thought to regulate the WUSCHEL gene, which is required for normal meristem integrity.48 The possible direct link between the receptor-like kinase CLV1 and Rop is interesting for the reason that it is generally believed that several layers of signalling modules separate the receptor and the small GTPase in mammalian and yeast systems. This observation might indicate yet another distinction in the regulation of plant Rho GTPases.

Vacuole Development

A novel role for Rop in vacuole development has recently been reported by Lin et al.29 Anti-Rop1Ps antibodies recognize epitopes preferentially in tapetal cells, microsporogenic cells, and microspores in the anther. Although these antibodies are raised against the pollen specific Rop1Ps, it is assumed that different Rops will be recognized, based on the high degree of

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similarity among the Rop family. At the subcellular level, Rop appears to be localized to the tonoplast of developing vacuoles, but not to the tonoplast of mature vacuoles.29 The specific role of Rop in the biogenesis of vacuoles is still unknown but speculation about Rops role in regulation of membrane fusion events is tempting.

Hormone Responses Recently, reports are accumulating that indicate the importance of Rops in processes involving hormone responses. One such report deals with the regulation of stomatal closure in Arabidopsis.32 To regulate water homeostasis and prevent descication, the Arabidopsis plant closes its stomates in response to drought and elevated levels of the hormone absissic acid (ABA). It was shown that ABA-induced stomatal closure coincides with AtRac1 inactivation and disruption of the actin cytoskeleton in guard cells.32 Overexpression of constitutively activated AtRac1 resulted in failure of stomates to close in response to ABA and the actin cytoskeleton in the guard cells appeared unaltered. Dominant negative AtRac1 was able to restore the defect in stomatal closure in ABA-insensitive mutant (abi1-1) Arabidopsis plants.32 It can be concluded from these studies that inactivation of AtRac1 is necessary for stomatal closure in Arabidopsis and plays therefore an important role in drought tolerance. Recently, Li et al9 have created transgenic plants, expressing either constitutive active and dominant negative forms of Rop2. Many aspects of plant growth and development were affected in the mutant rop2 plants, such as embryo development, seed dormancy, seedling development, shoot apical dominance and lateral organ orientation. Interestingly, behavior of rop2 plants in response to several hormones is altered as well. More specifically, lateral shoot initiation (mediated by auxin), hypocotyl elongation (mediated by brassinolide), and seed dormancy (mediated by ABA) are affected.9 The characterization of the mutant rop2 Arabidopsis plants indicates the importance of Rops in general regulation of growth and development in plants.

Concluding Remarks The field of Rho GTPases in plants is rapidly expanding. Like Rho-related small GTPases in other kingdoms, it has become increasingly clear that Rops mediate a variety of basic regulatory and developmental processes in plants. Besides parallels regarding the regulation and action of the Rho-related small GTPases in plants, animals and yeasts, interesting diversions have come to light as well. These diversions are important in our understanding of the basic signal-transducing principles and warrant further exploration. Careful biochemical analysis and kinetic studies of different Rops and their regulators are necessary and are likely to provide in-depth understanding regarding the evolution/adaptation of different signalling schemes. At present, a variety of physiological processes have been identified in plants in which Rop plays a crucial role. Over the next few years, efforts will need to be made to gain knowledge about upstream and downstream events from Rop function at the biochemical and cellular level.

Acknowledgment This work is supported by a Constantine Gilgut Post Doctoral Fellowship awarded to AHV, from the Constantine Gilgut Endowed Professorship at the University of Mass.

References 1. Yang Z, Watson JC. Molecular cloning and characterization of rho, a ras-related small GTP-binding protein from the garden pea. Proc Natl Acad Sci USA 1993; 90:8732-8736. 2. Madaule P, Axel R. A novel ras-related gene family. Cell 1985; 41:31-40. 3. Mackay DJG, Hall A. Rho GTPases. J Biol Chem 1998; 273:20685-20688. 4. Collins CC, Johnson DI. An Arabidopsis thaliana expressed sequence tag cDNA that encodes a Rac-like protein (Accession No. U88402). Plant Physiol 1997; 113:1463-1465. 5. Winge P, Brembu T, Kristensen R et al. Genetic structure and evolution of RAC-GTPases in Arabidopsis thaliana. Genetics 2000; 156:1959-1971. 6. Valster AH, Hepler PK, Chernoff J. Plant GTPases: The Rhos in bloom. Trends Cell Biol 2000; 10:141-146.

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7. Winge P, Brembu T, Bones M. Cloning and characterization of rac-like cDNAs from Arabidopsis thaliana. Plant Mol Biol 1997; 35:483-495. 8. Li H, Wu G, Ware D et al. Arabidopsis Rho-related GTPases: Differential gene expression in pollen and polar localization in fission yeast. Plant Physiol 1998; 118:407-417. 9. Li H, Shen J-J, Zheng Z-L et al. The Rop GTPases switch controls multiple developmental processes in Arabidopsis. Plant Physiol 2001; 126:670-684. 10. Molendijk AJ, Bischoff F, Rajendrakumar CSV et al. Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth. EMBO J 2001; 20:2779-2788. 11. Ménard L, Tomhave E, Casey PJ et al. Rac1, a low-molecular-mass GTP-binding-protein with high intrinsic GTPase activity and distinct biochemical properties. Eur J Biochem 1992; 206:537-46. 12. Quilliam LA, Khosravi-Far R, Huff SY et al. Guanine nucleotide exchange factors: Activators of the Ras superfamily of proteins. Bioessays 1995; 17:395-404. 13. Hardt WD, Chen LM, Schuebel KE et al. S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell 1998; 93:815-826. 14. Rudolph MG, Weise C, Mirold S et al. Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for RhoGTPases. J Biol Chem 1999; 274:30501-30509. 15. Lin R, Bagrodia S, Cerione RA et al. A novel Cdc42Hs mutant induces cellular transformation. Curr Biol 1997; 7:794-797. 16. Feig LA. Tools of the trade: Use of dominant-inhibitory mutants of Ras-family GTPases. Nat Cell Biol 1999; 1:25-27. 17. Li HL, Lin Y, Heath RM et al. Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell 1999; 11:1731-1742. 18. Kost B, Lemichez E, Spielhofer P et al. Rac homologues and compartmentalized phosphatidylinositol 4,5-biphosphate act in a common pathway to regulate polar pollen tube growth. J Cell Biol 1999; 145:317-330. 19. Lin Y, Yang Z. Inhibition of pollen tube elongation by microinjected anti-Rop1Ps antibodies suggests a crucial role for Rho-type GTPase in the control of tip growth. Plant Cell 1997; 9:1647-1659. 20. Borg S, Podenphant L, Jensen TJ et al. Plant cell growth and differentiation may involve GAP regulation of Rac activity. FEBS Lett 1999; 453:341-345. 21. Wu G, Li H, Yang Z. Arabidopsis RopGAPs are a novel family of Rho GTPase-activating proteins that require the Cdc42/Rac-interactive binding motif for Rop-specific GTPase stimulation. Plant Physiology 2000; 124:1625-1636. 22. Hoffman GR, Cerione RA Flipping the switch: The structural basis for signalling through the CRIB motif. Cell 2000; 102:403-406. 23. Brill S, Li S, Lyman CW et al. The Ras GTPase-activating-protein-related human protein OQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases. Mol Cell Biol 1996; 16:4869-4878. 24. Bischoff F, Vahlkamp L, Molendijk A et al. Localization of AtRop4 and AtRop6 and interaction with the guanine nucleotide dissociation inhibitor AtRhoGDI1 from Arabidopsis. Plant Mol Biol 2000; 42:515-530. 25. Kieffer F, Elmayan T, Rubier S et al. Cloning of Rac and Rho-GDI from tobacco using an heterologous two-hybrid screen. Biochimie 2000; 82:1099-1105. 26. Lin Y, Wang Y, Zhu J et al. Localization of a Rho GTPase implies a role in tip growth and movement of the generative cell in pollen tubes. Plant Cell 1996; 8:293-303. 27. Ono E, Wong HL, Kawasaki T et al. Essential role of the small GTPase Rac in disease resistance of rice. PNAS 2001; 98:759-764. 28. Ivanchenko M, Vejlupkova Z, Quatrano RS et al. Maize Rop7 GTPase contains a unique, CaaX box-independent plasma membrane targeting signal. Plant J 2000; 24:79-90. 29. Lin Y, Seals DF, Randall SK et al. Dynamic localization of Rop GTPases to the tonoplast during vacuole development. Plant Physiol 2001; 125:241-251. 30. Feijo JA, Sainhas J, Holdaway-Clarke T et al. Cellular oscillations and the regulation of growth: The pollen tube paradigm. Bioessays 2001; 23:86-94. 31. Fu Y, Wu G, Yang Z. Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes. J Cell Biol 2001; 152:1019-1032. 32. Lemichez E, Wu Y, Sanchez J-P, et al. Inactivation of AtRac1 by abscisic acid is essential for stomatal closure. Genes & Development 2001; 15:1808-1816. 33. Levine A, Tenhaken R, Dixon R et al. H2O2 production from the oxidative burst orchestrates .the plant hypersensitive disease resistance response. Cell 1994; 79:583-593. 34. Mehdy MC. The role of activated oxygen species in plant disease resistance. Physiol Plant 1996; 98:365-374.

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35. Dwyer SC, Legendre L, Low PS et al. Plant and human neutrophil oxidative burst complexes contain immunologically related proteins. Biochim Biophys Acta 1996; 1298:231-237. 36. Groom QJ, Torres MA, Fordham S et al. RbohA, a rice homolog of the mammalian gp91 phox resporiratory burst oxidase gene. Plant J 1996; 10:515-522. 37. Keller T, Damude HG, Werner D et al. A plant homolog of the neutrophil NAPDH oxidase gp91phox subunit gene encodes a plasmamembrane protein with Ca2+ binding motifs. Plant Cell 1998; 10:255-266. 38. Torres MA, Onouchi H, Hamada S et al. Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox). Plant J 1998; 14:365-370. 39. Bokoch GM. Regulation of phagocyte respiratory burst by small GTP-binding proteins. Trends Cell Biol 1995; 5:109-113. 40. Kieffer F, Simon-Plas F, Maume BF et al. Tobacco cells contain a protein, immunologically related to the neutrophil small G protein Rac2 and involved in elicitor-induced oxidative burst. FEBS Lett 1997; 403:149-153. 41. Kawasaki T, Henmi K, Ono E et al. The small GTP-binding protein Rac is a regulator of cell death in plants. Proc Natl Acad Sci USA 1999; 96:10922-10926. 42. Potikha T. The involvement of hydrogen peroxide in the differentiation of secondary walls in cotton fibers. Plant Physiol 1999; 119:849-858. 43. Delmer DP, Pear JR, Andrawis A et al. Genes encoding small GTP-binding proteins analogous to mammalian Rac are preferentially expressed in developing cotton fibers. Mol Gen Genet 1995; 248:43-51. 44. Clark SE. Cell signalling at the shoot meristem. Nat Rev Mol Cell Biol 2001; 2:276-284. 45. Clark SE, Williams RW, Meyerowitz EM. The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis. Cell 1997; 89:575-585. 46. Trotochaud AE, Hao T, Wu G et al. The CLAVATA1 receptor-like kinase requires CLAVATA3 for its assembly into a signalling complex that includes KAPP and a Rho-related protein. Plant Cell 1999; 11:393-405. 47. Kohorn BD. Shuffling the deck: Plant signalling plays a club. Trends Cell Biol 1999; 9:381-383. 48. Laux T, Mayer KF, Berger J et al. The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis. Development 1996; 122:87-96. 49. Novokova GV, Moshkov IE, Smith AR et al. The effect of ethylene and cytokinin on guanosine 5'-triphosphate binding and protein phosphorylation in leaves of Arabidopsis thaliana. Planta 1999; 208:239-246.

Index A Aarskog-Scott Syndrome 61 Abl 62, 63, 74, 75, 80, 84, 165, 170, 172, 174, 175 Actin cytoskeleton 1, 17, 28, 29, 32, 41, 46, 56-58, 60, 63, 75, 79, 85, 95, 96, 102, 104, 107, 108, 112, 115, 116, 119, 120, 124, 125, 127, 128, 139, 140, 148-150, 188, 196-200, 202, 203, 208, 210-212, 214-216, 223, 225, 227 Actin stress fiber 77, 96, 101, 103-105, 157, 169, 171, 212 Acute myelogenous leukemia (AML) 62 Adhesion 1, 8, 10, 68, 75-78, 96, 102, 104, 107-109, 112, 115-132, 141, 142, 148-152, 155, 156, 158, 159, 169 ADP-ribosyltransferase 85, 99 Apoptosis 1, 10, 11, 79, 131, 170-172, 176, 187, 223 Arabidopsis thalia 68, 72, 81, 219 Arabidopsis thaliana 46, 219 Arachidonic acid 185, 187-190, 193 Arginine finger 72, 73 ARHGAP6 Subfamily 80

B 3BP-1 subfamily 80 Bacterial toxin 48, 99 Bardet-Bield syndrome (BBS) 78 Breakpoint cluster region gene (Bcr) 9, 10, 47, 62, 63, 68, 70, 73-75, 83

C C1 subfamily 71, 80 Cadherins 105, 115, 117-132, 155, 203 Caenorabditis elegans 3, 46, 48, 57, 68, 70, 77, 79, 80, 86, 127, 142, 198 Calcium 50, 101, 102, 115, 117, 118, 120, 123, 130-132, 188, 191, 201, 203, 224 Calcium influx 188, 201, 224 Calcium sensitization 102 Cancer 3, 4, 11, 63, 76, 83, 98, 103, 104, 163, 167, 169, 170, 172-175, 177, 182, 183, 198

Symons INDEX

231

Catenin 117-119, 124, 127-130 CDC42 60 Cdc42 1-5, 7-11, 20, 26-28, 35-38, 40, 41, 43, 47-49, 51, 53, 60-63, 69, 72, 74, 75, 77-81, 83-85, 96, 102, 105, 107-112, 120, 122-124, 126-130, 141, 142, 145, 146, 149, 150, 163-167, 169-173, 175-177, 185, 187, 191, 197-202, 209, 212, 213, 215, 219-222 CdGAP subfamily 80 CeGAP subfamily 68, 80 Cell adhesion kinase β (CAKβ) 77 Cell morphology 8, 11, 56, 116, 117, 146, 175, 216 Cell polarity 2, 32, 46, 48, 103, 104, 127, 128, 142, 145, 169, 176, 203, 216, 219, 224, 225 Cell proliferation 1, 4, 103, 169, 175, 196, 198, 226 Cell wall 28, 81, 84, 219, 223, 226 Cell-cell adhesion 115-120, 123-132, 155 Chimaerin subfamily 76, 77 Chp 2, 5, 7, 8, 10 Chronic myelogenous leukemia (CML) 47, 62, 74 Citron kinase 19, 28 Clathrin-mediated endocytosis 196, 198 Crosstalk 1, 8, 11, 83, 198 Cytokinesis 7, 46-48, 56, 58, 59, 68, 77, 83, 105, 124, 145, 169, 208-211, 213-216, 222 Cytoskeleton 1, 17, 28, 29, 32, 41, 46, 56-58, 60, 63, 68, 75, 79, 85, 96, 99, 101, 102, 104, 105, 107, 108, 112, 115-117, 119, 120, 124, 125, 127, 128, 141, 146, 148-150, 152, 155, 188, 191, 196-200, 202, 203, 208, 210-212, 214-216, 223, 225, 227

D Darlin 211, 215 Dbl 9, 17, 38, 41, 47, 49, 51-54, 61, 69, 74, 165, 169, 170, 172-174, 176, 221 Dbl-homology (DH) domains 49, 69, 165, 74, 221, 222 Dbs 47, 49-52, 61, 176

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232

Rho GTPases

DdLim 211, 215 DdRacGap1 (DRG) 48, 49, 56, 81, 211, 213, 215, 216 Dendritic cells (DC) 199 Dgap1/DdRasGAP 213 Dictyostelium 72, 81, 208-216 Dock 57, 145 Domain 3, 4, 9, 10, 17, 20-27, 32, 35-38, 40, 41, 46-57, 60-63, 68-81, 85, 101-105, 107, 110-112, 115-120, 128, 130, 132, 145, 146, 148, 165, 167-169, 171, 172, 174-176, 197-199, 202, 209, 212-216, 219-222, 225 Dominant negative 5, 7, 8, 10, 11, 99, 101, 103, 104, 123, 125, 127-129, 142, 149, 166-173, 188, 191, 199-203, 210-212, 221, 222, 224-227 Drosophila 1, 48, 57-59, 62, 68, 70, 74, 77, 119, 127, 128, 142, 156, 213, 220

E Ect2 53, 54, 58-60, 173 Effector protein 32, 55, 56, 61, 109, 128, 141, 165 EGF 3, 102, 196-198, 201 Epithelial phenotype 115, 116, 120, 122, 127, 130 Epithelial polarity 128 Exchange factor 8, 11, 17, 32, 38, 46, 49, 55, 61, 63, 69, 103, 105, 122, 124, 128, 130, 142, 148, 164, 165, 172, 187, 208, 213, 219, 221 Exocytosis 80, 84, 196, 201-203, 211, 212

F Faciogenital dysplasia (FGDY) 61, 62 Farnesylation 96, 98 Fibroblasts 4, 5, 9, 60, 74-76, 80, 83, 86, 96, 101, 102, 108, 112, 116, 117, 120-123, 125, 127-129, 131, 141, 142, 149, 156, 158-160, 169-171, 173, 174, 187, 188, 191, 199, 201, 219 Focal adhesion 10, 76-78, 96, 102, 109, 129, 141, 142, 148-151, 156, 158, 169 Focal adhesion tyrosine kinase (FAK) 77, 78

Symons INDEX

232

G G protein-coupled receptor (GPCR) 54, 55, 157, 168, 170, 174, 175 GAP 9, 10, 17, 19, 32, 35, 38, 40, 47, 48, 55, 56, 62, 63, 68, 72-81, 83-86, 101, 103, 104, 122, 124, 127, 160, 164-167, 169, 172, 190, 199, 208, 211, 213, 214, 221, 222 GAP for Rho associated with focal adhesion kinase (GRAF) 68, 71, 77, 83 GapA 211, 213-215 GDI 17, 22, 23, 25, 27, 28, 32, 33, 35-38, 40-43, 49, 164-166, 193, 221, 222 GIP 35, 38, 43 GRAF subfamily 77 Grb7 102 Green fluorescent protein (GFP) 21-25, 27, 28, 146, 202, 211, 212, 214, 222, 225 GTP hydrolysis 32, 36, 38, 40, 72, 74, 98, 101, 129, 164, 166, 167 GTPases 1, 3-6, 8-11, 17, 19, 22-26, 28, 29, 32, 33, 35-38, 41-43, 46, 49-53, 55, 56, 59-63, 68-69, 71-75, 77, 81, 84, 86, 96, 98, 99, 101, 107-110, 112, 120-124, 126, 127, 129-132, 141, 142, 145, 146, 148, 150, 152, 155-158, 160, 163-177, 185-188, 190-192, 196-203, 208-216, 219-222, 226, 227 Guanine nucleotide 1, 17, 32, 36, 46, 49, 50, 53, 55, 56, 63, 69, 75, 122, 142, 164, 165, 167, 172, 175, 208, 219, 221 Guanine nucleotide exchange factor (GEF) 9, 10, 17, 19, 26, 28, 32, 33, 36, 38, 41, 46, 48-51, 53-56, 59-61, 63, 69, 74, 81, 85, 99, 122, 124, 142, 148, 160, 164-166, 168, 169, 171-174, 176, 197, 199, 200, 208, 211, 213, 215, 216, 219, 221, 222

H Hypervariable region 17-28

I Integrin 76, 86, 103, 108, 112, 124, 125, 129, 130, 141, 148, 149, 158, 159, 168, 200, 203 Intracellular signalling 55, 130 Invasion 8, 46, 76, 83, 108, 128, 155-157, 159, 160, 169-176, 201

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233

Index

L

N

Lamellipodia 5, 7, 8, 11, 77, 107, 108, 110, 111, 122, 125, 141, 142, 145, 146, 148, 149, 155-157, 169, 174, 176, 199, 203, 215, 219 Leukemia associated Rho guanine nucleotide exchange factor (LARG) 48, 55, 63, 172, 174 Leukotrienes 188 LIM-kinase 109-111 Lipid-binding 36, 54, 68, 69, 77, 79 Localization 7, 17, 19, 21-29, 32, 35, 40, 41, 43, 52, 53, 59, 61, 72, 79, 86, 98, 101, 119, 122, 123, 125, 127, 128, 145, 151, 197, 198, 201, 222-224

Neurite 4, 5, 9, 26, 48, 76, 102, 188 Neurons 4, 9, 57, 58, 80, 84, 99, 102, 105, 170 Nucleotide dissociation 32, 33, 36, 165, 166, 172, 221

OCRL Subfamily 79 Oligophrenin-1 77, 78 Oncogene 41, 47, 48, 60, 74, 120, 177 Oncoproteins 53, 163, 165, 166, 169, 170, 172, 173, 176, 177

P

M Macropinocytosis 199, 208, 211 Membrane association 43, 52, 61, 164 Mental retardation 48, 61, 62, 77, 78, 83 Meristem signalling 223, 226 Metastasis 112, 155, 169-171, 174, 175 Microphthalmia with linear skin defects (MLS) 80 Microtubule organizing center (MTOC) 145 Migration 7, 46, 48, 68, 76, 83, 102, 104, 105, 108, 115, 122, 127-130, 149, 155-160, 171, 196, 199 Mixed lineage leukemia (MLL) 48, 63, 172, 174 Model system 56, 103, 120, 208, 226 Motility 1, 6, 10, 11, 46, 111, 130, 141, 142, 145, 146, 148, 149, 155, 163, 170, 196, 199, 201, 203, 208-211, 214, 216 MRX see X-linked mental retardation Multifunctional 9, 32, 33, 68, 72, 74 Multimodular 68, 70, 78 MyoM 48, 211, 215, 216 Myosin 9, 48, 58, 68-71, 78, 83, 101, 102, 108-110, 112, 119, 124, 125, 141, 145-147, 149-151, 159, 199, 202, 211, 212, 215, 216 Myosin I heavy chain kinase (MIHCK) 211, 215, 216 Myosin-IX subfamily 70, 71, 78

Symons INDEX

O

233

P21 activated kinase (PAK) 2, 4, 9, 10, 102, 109, 111, 112, 165, 176, 199, 200, 215, 216 P38 MAP kinase 188, 189 P50-RhoGAP subfamily 70, 79 P85 subfamily 71, 79 P122-RhoGAP subfamily 79 P190-RhoGAP subfamily 68, 75 Pak 57, 62, 142, 145-148, 211 Pathogen 68, 81, 84, 108, 109, 111, 199, 223, 225, 226 Pebble 48, 58, 59 PH domains 46, 48-56, 61, 63, 77, 78, 81, 165, 172, 216 Phagocytosis 8, 17, 84, 85, 124, 158, 199, 200, 201, 208, 211, 212, 215, 216 Philadelphia chromosome (Ph1) 62, 63, 74, 165, 174 Phosphatidylinositol 3-kinase (PI3-kinase) 4, 123, 131, 159, 171, 185-187 Phosphatidylinositol-3,4,5-triphosphate (PIP3) 172, 185, 187, 188, 192 Phosphoinositides 50, 52-54, 56, 61, 63, 187, 188, 191, 216 Phospholipase A2 185, 187-190, 192 Phospholipase C 79, 187, 188, 190, 192 Phospholipase D 185, 187, 191, 192, 201 PIP3-dependent protein kinase-1 (PDK1) 187 PIX 9, 51, 148 Pix 48

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Rho GTPases

Plants 3, 81, 164, 219-222, 224-227 Platelet-derived growth factor (PDGF) 23, 76, 79, 145, 148, 187 Pleckstrin homology 9, 49, 68, 69, 165, 213 PlexinA1 103 Polarity 2, 28, 32, 46, 48, 103, 104, 127, 128, 141, 142, 145, 149, 152, 169, 176, 203, 212, 216, 219, 223-225 Prenylation 19, 26, 35, 61, 96, 101, 164, 166, 167, 222 Protein module 69, 72 Protrusion 5, 8, 141, 142, 145, 146, 148, 149, 152, 158, 159, 169, 199, 216 Pseudomonas aeruginosa ExoS 73, 74

R Rac 3-5, 7, 8, 17, 22, 35, 36, 38, 41, 43, 56, 57, 62, 69, 75, 77, 81, 96, 99, 102, 103, 105, 107-109, 111, 112, 120, 122-130, 132, 141, 142, 145-150, 155-158, 160, 163, 168, 170-172, 174-177, 185-191, 196-203, 209, 212, 213, 215, 219-222, 226 Rac1/GDI2 35, 36 Rac1A/B/C 210, 211 Rac1A/Rac1B/Rac1C 210 RacB 209, 211, 212 RacC 209, 211, 212, 215, 216 RacE 209, 211, 213-216 RacF1/2 212 Ras 1, 2, 8, 10, 17, 19-23, 25-28, 38, 43, 55, 56, 59, 60, 68, 69, 74, 75, 78, 80, 81, 83-85, 96, 98, 99, 101, 103, 129, 130, 146, 157, 158, 163-173, 176, 188, 196, 198, 208, 209, 214, 215, 219, 221 Rho 1-11, 17-29, 32, 33, 35-38, 40-43, 46, 49, 51, 55, 56, 58, 60-63, 68, 69, 72-74, 77, 81, 83, 84, 86, 96, 98, 99, 102-105, 107-112, 120, 122-126, 129-132, 141, 142, 145, 148-152, 155-160, 163-177, 185-188, 190-193, 196-203, 208-216, 219-223, 225-227

Symons INDEX

234

Rho effector 7, 10, 28, 102-104, 108-111, 150, 160, 190, 191, 198 Rho GTPase 1, 5, 6, 8-11, 17, 19, 24, 28, 29, 32, 33, 35-38, 41, 43, 46, 49, 51, 55, 60, 61, 69, 72-74, 77, 81, 84, 86, 107, 109, 110, 112, 141, 142, 152, 155, 156, 159, 163-177, 186, 191, 196, 197, 199-203, 208, 210, 212, 219, 221, 222, 226, 227 Rho protein 1, 3, 4, 8, 10, 17-19, 21-23, 25, 27-29, 32, 33, 35, 36, 38, 40-43, 46, 49, 56, 58, 96, 99, 102, 120, 122, 124, 130, 131, 163-169, 170, 175, 177, 185, 190, 191, 193, 208, 212, 216, 219, 223 RhoA 1, 3, 5, 7-11, 17, 21, 22, 24-28, 35, 41, 47, 48, 54, 55, 58-61, 63, 72, 74-81, 83-86, 96, 98, 99, 102-104, 107, 112, 122, 124, 156-159, 164-167, 169-177, 198, 201, 202, 212, 220 RhoB 3, 7, 10, 22, 23, 27, 28, 35, 83, 124, 166, 167, 170, 198 RhoBTB cluster 2-4 RhoC 1, 3, 7, 10, 11, 22, 83, 124, 156, 159, 160, 170, 172, 175, 177 RhoD 2-8, 10, 22, 23, 35, 96, 163, 166 RhoE 35, 96, 98, 99, 101, 103, 104, 129, 164, 166, 169 RhoG 2, 3, 5, 7-10, 35, 47, 163, 170 RhoGAP 6, 9-11, 50, 62, 68-81, 83-86, 98, 102, 104, 105, 173, 190, 193 RhoGDI 9, 10, 25, 27, 28, 32, 35, 36, 38, 41, 43, 102, 166, 172, 174, 222 RhoGEF 6, 9-11, 46-63, 174, 175 RhoH 2, 3, 5, 7, 10, 172, 175 Rif 3-10 RLIP subfamily 71, 78 Rnd 2-4, 8-10, 96, 98, 99, 101-105, 129, 163 ROCK 61, 102, 125, 142, 149, 151, 156-160, 165, 176 Rop 3, 219-227

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235

Index

S

V

Saccharomyces cerevisiae 2, 46, 59, 68, 69, 72, 81, 84, 142, 145, 201, 202, 220 Salmonella typhimurium SptP 74 Scar 146, 211, 215 Serum response factor (SRF) 10, 17, 169, 176 Signalling 1, 4, 7-9, 17, 26, 28, 29, 32, 33, 35, 38, 41-43, 49, 50, 52, 55, 61-63, 75, 80, 84, 86, 104, 111, 112, 115, 116, 119-124, 129-132, 141, 142, 145, 148, 152, 155, 158, 160, 163-171, 173-177, 185, 187, 189-191, 198, 201, 203, 208, 215, 216, 219, 221, 223, 224, 226, 227 Small GTPase 46, 49, 50, 56, 60, 68, 75, 120-124, 126, 127, 129-132, 146, 148, 163, 172, 177, 187, 196, 208, 209, 212, 219, 221, 222, 226, 227 SMART 68-70 Smooth muscle 101, 102 Sos1 49-53, 55, 56, 172, 173 SRF 10, 169, 176 Stress fiber 1, 3, 4, 6-11, 77, 80, 96, 98, 101-105, 116, 117, 122, 124, 125, 129, 147, 149-152, 156-158, 169, 171, 176, 190, 191, 212, 219

Vacuole development 219, 223, 226 Vav 9, 41, 49, 51, 53, 54, 56, 124, 130, 172-174, 187

W WAVE 109-111, 142, 146, 147, 215 Wiscott–Aldrich syndrome protein (WASP) 10, 26, 109-111, 142, 145, 146, 148, 152, 197, 198, 200, 215 Wound healing 104, 108, 145 Wrch 3, 5, 7, 8, 163, 175

X X-linked mental retardation (MRX) 62, 77, 83 Xenopus development 104

Y Yersinia spp. YOPE 74

T TC10 2-5, 7, 8, 10, 17, 22-28, 163, 170 TGF-β 3, 130, 157 Tiam1 9, 36, 38, 47, 49-54, 172-174, 187 Toxin 1-3, 48, 81, 99, 150, 171 Transformation 1, 9, 11, 17, 32, 46, 51, 56, 60, 61, 63, 103, 112, 163, 169-177, 198, 201, 208, 209 Trio 9, 10, 47, 49, 50, 53, 56-58

Symons INDEX

235

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MOLECULAR BIOLOGY INTELLIGENCE UNIT

INTELLIGENCE UNITS Biotechnology Intelligence Unit Medical Intelligence Unit Molecular Biology Intelligence Unit Neuroscience Intelligence Unit Tissue Engineering Intelligence Unit

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Rho GTPases

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