Advances in Cancer Research Volume 74

Advances in

CANCER RESEARCH Volume 74

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Advances in

CANCER RESEARCH Volume 74

Edited by

George F. Vande Woude ABL-Basic Research Program National Cancer Institute Frederick Cancer Research and Development Center Frederick, Maryland

George Klein Microbiology and Tumor Biology Center Karolinska lnstitutet Stockholm, Sweden

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper.

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Copyright 0 1998 by ACADEMIC PRESS All Rights Reserved. No part of this publication 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. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center. Inc. (222 Rosewood Drive, Danvers, Massachusetts 0 1923). for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1998 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-230>(/98 $25.00

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Contents

Contributors to Volume 74 vii

Key Effectors of Signal Transduction and GI Progression Martine F. Roussel I. Introduction 1 11. The G1 Phase of the Mammalian Cell Cycle 3 111. Signal Transduction and G1 Progression 5 IV. The RAS/ERK Pathway and the Cell Cycle 5 V. V1. VII. VIII. IX.

RAS, D-Type Cyclins, and RB Connections 9 Cycling with MYC 11 Interplay between MYC and Cyclin D1 13 Signaling and Cell Cycle Roles of the SRC Family of Kinases 14 Concluding Remarks 15 References 16

p53 in Tumor Progression: Life, Death, and Everything Michael R. A. Mowat I. Introduction 25 11. Biochemical Activities of p53 26 111. p53 and Cell Cycle Control 27 IV. p53 and Apoptosis 29 V. p53 and Tumor Progression 37 References 42

Signal Transduction through MAP Kinase Cascades Timothy S. Lewis, Paul S. Shapiro, and Natalie G. Ahn I. The MAP Kinase (MAPK) Module 49 50

11. Mammalian MAPK Pathways

V

vi

Contents

Regulation of MAPK Pathways by Protein Phosphatases 75 Cellular Substrates of MAP Kinases 81 Responses to MAPK Pathways: Growth and Differentiation 91 Yeast MAPK Pathways 100 Intracellular Targeting and Spatial Regulation of MAPK Pathway Components 111 VIII. Future Directions 113 References 114 111. IV. V. VI. VII.

FHIT in Human Cancer Gabriella Sozzi, Kay Huebner, and Carlo M. Croce I. Introduction

141 144 146

11. Cloning and Structural Features of the FHIT Gene

111. The Fhit Protein and Its Biochemical Properties IV. FHIT Abnormalities in Human Cancer 147 V. Conclusions and Perspectives 158 References 159

Phosphoinositide 4- and 5-Kinases and the Cellular Roles of Phosphatldylinositol4,5-Bisphosphate 1. Justin Hsuan, Shane Minogue, and Maria dos Santos I. 11. 111. IV. V.

Introduction 167 Receptor-Linked Phosphoinositide Metabolism 174 Phosphoinositides and the Cytoskeleton 187 Vesicle Biogenesis and Trafficking 195 Roles in Cancer, Summary, and Prospects 206 References 208

Index 217

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Natalie G. Ahn, Department of Chemistry and Biochemistry,Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309 (49) Carlo M. Croce, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107 (141) Maria dos Santos, Ludwig Institute for Cancer Research, University College London Medical School, London W1P 8BT, United Kingdom (167) J. Justin Hsuan, Ludwig Institute for Cancer Research, University College London Medical School, London W1P SBT, and Department of Biochemistry and Molecular Biology, University College London, London WClE 6BT, United Kingdom (167) Kay Huebner, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107 (141) Timothy S. Lewis, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309 (49) Shane Minogue, Ludwig Institute for Cancer Research, University College London Medical School, London W1P SBT, United Kingdom (167) Michael R. A. Mowat, Manitoba Institute of Cell Biology, Winnipeg, Manitoba, Canada R3E OV9 ( 2 5 ) Martine F. Roussel, Department of Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 ( 1 ) Paul S . Shapiro, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309 (49) Gabriella Sozzi, Division of Experimental Oncology A, Istituto Nazionale Tumori, 20133 Milan, Italy (141)

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Key Effectors of Signal Transduction and GI Progression Martine F. Roussel Department of Tumor Cell Biology St. Jude Children’s Research Hospital Memphis, Tennessee 381 0.5

I. Introduction 11. The G1 Phase of the Mammalian Cell Cycle 111. Signal Transduction and GI Progression IV. The RASERK Pathway and the Cell Cycle

V. VI.

VII. VIII. IX.

A. The RASERK Pathway B. ETS Transcription Factors as RAS Targets and Regulators of Proliferation RAS, D-Type Cyclins, and RB Connections Cycling with MYC A. MYC and Its Partners B. MYC and Its Targets Interplay between MYC and Cyclin D 1 Signaling and Cell Cycle Roles of the SRC Family of Kinases Concluding Remarks References

I. INTRODUCTION Cells enter the cell cycle and commit to DNA synthesis in response to extracellular mitogenic signals. Cytokines and polypeptide growth factors, as well as cell-cell contacts and cell-substratum interactions, provide the positive and negative signals that govern cellular proliferation, differentiation, growth arrest, and apoptosis. Cells respond to these signals during the first gap ( G l ) phase of the cell cycle via their surface receptors, which transmit extracellular cues to the inside of the cell. When ligands bind these receptors, they trigger a cascade of events that includes receptor aggregation and the activation of their tyrosine kinases. The receptors for macrophage colonystimulating factor-1 (M-CSF/CSF-1), platelet-derived growth factor (PDGF), or epidermal growth factor (EGF) all possess intrinsic tyrosine kinase activity that is activated in this fashion (Ullrich and Schlessinger, 1990; Schlessinger and Ullrich, 1992). In contrast, the cytokine family of receptors associate with the Janus tyrosine kinases (JAKs), which autophosphorylate and transphosphorylate the receptor (Ihle, 1996). Specific phosphoryAdvances in CANCER RESEARCH 0065-23OW98 $25.00

Copyright 0 1998 by Academic Press All rights of reproduction in anv form reserved.

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Martine F. Roussel

Fig. 1 The mammalian cell cycle. The cell cycle is composed of four distinct phases: G1, S, G2, and M. G1, the first gap phase of the cell cycle, represents the interval during which cells are sensitive to mitogen stimulation; R, the end point of mitogen sensitivity or restriction point defining the point in G1 beyond which cycling is independent of mitogens; S, the DNA synthetic phase; G2, the second gap phase; M, mitosis; GO, the resting or quiescent phase; RB, retinoblastoma gene product; D1,2,3, D-type cyclins; CDK4,6, cyclin-dependent kinase -4 and -6; P,phosphorylated residues. The dark line represents the interval in G1 during which cells are sensitive to extracellular stimuli.

lated tyrosine residues serve as docking sites for effector proteins containing SRC homology-2 and phosphotyrosine-binding (PTB) domains (Schlessinger, 1994; Harrison, 1996). These associations, in turn, activate a complex signaling network that ultimately instructs the cell cycle machinery. Removal of extracellular stimuli during early or mid-G1 before the cells commit to the DNA synthetic (S) phase of the cell cycle disables the cells from entering the next phase of the cell cycle, the S phase. However, once committed to DNA synthesis, or passed the restriction (R)point, cells no longer require extracellular stimuli to complete the first mitosis (Fig. 1) (Pardee, 1989; Sherr, 1994). Persistent receptor activation is therefore required throughout most of G1 to ensure that genes whose products are rate limiting for G1 progression are transcribed. Oncogenes effectively bypass the regulation exerted by extracellular stimuli, which dysregulates cell growth. Effectors of signal transduction are therefore linked to cell cycle regulators. The pathways that connect these effectors and regulators are now emerging, in part as a result of the vast amount of information gained from yeast genetics (Wittenberg and Reed, 1996). This review highlights some of the advances coupling certain key effectors

Key Effectors of Signal Transduction and GI Progression

3

of cytokine and growth factor receptor signaling to the cell cycle clock, thereby connecting these two broad fields of research. Several of the critical effectors that enforce progression through G1 and influence the commitment of the cell to DNA synthesis will be discussed.

11. THE GI PHASE OF THE MAMMALIAN CELL CYCLE Cells are sensitive to extracellular signals during most of the G1 phase of the cell cycle, and these signals ultimately converge to induce the expression and activity of the D-type cyclins (Sherr, 1993, 1994). The D-type cyclins, of which there are three members, D1, D2, and D3, are exquisitely regulated by extracellular stimuli and their expression requires new protein synthesis (Matsushime et al., 1991; Sherr, 1993).Because D-type cyclins are short-lived proteins (half-lives of 15-20 min), the complexes they form with their catalytic partners, cyclin-dependent kinases (CDK4 and CDK6), are dynamic. D cyclins are rate limiting for G1 progression as enforced expression of cyclin D1 into fibroblasts accelerates their G1 phase; conversely, microinjection of antibodies to cyclin D1 in the same cells induces G1 arrest (Quelle et al., 1993; Resnitzky et al., 1994; Lukas et al., 1995). D-type cyclins, therefore, act as growth factor sensors, enforcing G1 progression into S phase (Sherr, 1994). This pivotal role makes D-type cyclins likely targets of signaling effectors that promote proliferation. Active cyclin D/CDK complexes enforce G1 progression in part by phosphorylating the retinoblastoma gene product, RB (Sherr, 1994; Weinberg, 1995). This phosphorylation eliminates the suppressive function of RB by promoting the release of RB-tethered transcription factors, including the E2Fs (Nevins, 1992; La Thangue, 1994). E2Fs are required for the coordinate regulation of genes essential for DNA synthesis (Hollingworth et al., 1993). The principal role of D-type cyclins in G1 appears to phosphorylate RB (Ewen et al., 1993; Sherr, 1994). In fact, microinjection of D1 antibodies into RB null fibroblasts, or into transformed cells that contain a dysfunctional RB, is unable to prevent S phase entry. The same experiment with RB-positive cells leads to G1 arrest (Quelle et al., 1993; Baldin et al., 1993; Lukas et al., 1995). Cyclin D/CDK complexes are positively regulated by growth factors and by phosphorylation by cyclin-activated kinase (CAK) (Morgan, 1995). However, the activity of these complexes is also negatively regulated by two groups of CDK inhibitors that bind to CDKs and inhibit their kinase activity (Hunter, 1993; Elledge and Harper, 1994; Sherr and Roberts, 1995). The first class of inhibitors includes p21WAF-l,p27K*P1,and each of which can bind and inhibit all of the cyclinlCDK complexes. In contrast, a

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second class of CDK inhibitors, termed INK4s for Dhibitors of c d u , is typified by the tumor suppressor ~ 1 6 (Serrano ” ~ ~et al., ~ 1993, 1996; Xiong et al., 1993) and includes four members: p161NK4a,p151NK4b, and ~ 1 9 ” These ~ ~ ~inhibitors . specifically bind to and inhibit CDK4/6 (Elledge and Harper, 1994; Sherr and Roberts, 1995; Hall and Peters, 1996). The ability of the INK4s to arrest cells in G1 and inhibit CDK4/6 activity is dependent on functional RB (Guan et al., 1994; Lukas et al., 1995a,b). The regulation of these inhibitors is under intense investigation and their induction is thought to occur after cells receive antiproliferative signals or as cells differentiate. For example, ~ 1 5 is” synthesized ~ ~ ~ in response to stimulation by tumor growth factor+ (Hannon and Beach, 1994). However, the exact mechanism of this transcriptional regulation has yet to be clearly defined. In addition, INK4 proteins may have additional functions unrelated to their role as inhibitors of D-CDK4/6 complexes (Hirai and Sherr, 1996; Skapek et al., 1996). The remaining G1 cyclin, cyclin E, is expressed later than D cyclins in G1 and is regulated by E2F (Fig. 2 and 5 ) (Botz et al., 1996). Cyclin E associates with CDK2 (Dulic et al., 1992), and this holoenzyme also phosphorylates RB and other substrates likely to be important for the execution of S phase (Fig. 5) (Ohtsubo and Roberts, 1993; Hatakeyama et al., 1994; Resnitzky and Reed, 1995; Ohtsubo et al., 1995). Like cyclin D, cyclin E is required for S-phase entry, and its expression is limiting for G1 progression (Wimmell et al., 1994).

FOS JUN

I\

E

A

MYC

Go

GI

R

S

~///////~~//////////~

Mitogen Sensitivlty

Fig. 2 Transcription regulation during the cell cycle. The dashed bar represents the period during which cells are sensitive to extracellular stimuli. FOS, JUN, and MYC are immediate early genes transcribed in response to extracellular stimuli. Cyclins D1, E, A, and Bare expressed sequentially as cells pass through the cell cycle. G1, S, G2, and M represent the different phases of the cell cycle. GO represents the resting phase following the withdrawal of cells from the cycle.

Key Effectors of Signal Transduction and GI Progression

5

111. SIGNAL TRANSDUCTION AND GI PROGRESSION Signal transduction is the process by which growth factors and extracelM a r stimuli transmit signals from the outside of the cell to the nucleus. Receptor signals usually persist for the first few hours following growth factor or lymphokine binding, receptor aggregation, and internalization of the receptor-ligand complex (Heldin, 1995). Because many of the signaling pathways induced by ligand binding to receptors have been covered in other reviews, only the pathways and effectors relevant for this review will be discussed. The tyrosine kinase growth factor and cytokine family of receptors activate multiple pathways. These include the RAYMAP kinase (RAY ERK) pathway that leads to the expression of the immediate early genes FOS and JUN and the pathways that lead to MYC transcription, which may involve SRC kinase activation (Fig. 3) (Schlessinger and Ullrich, 1992; Schlessinger, 1993). The cytokine family of receptors also activates the JAWSTAT pathway (Darnel1 et d., 1994; Ihle, 1996). The role of these signaling pathways in cell proliferation has started to be elucidated. Pathways leading to FOS and JUN expression were shown to be insufficient to stimulate G1 phase progression. CSF-1 or PDGF-regulated expression of FOS and JUN is insufficient for G1 phase progression (Roussel et al., 1990; Barone and Courtneidge, 1995). Moreover, cytokine receptor activation of RAS, and subsequently of AP-1 and STATs, is dispensable for entry into S phase (Taniguchi and Minami, 1993; Ihle, 1996). In contrast, JAK activation is essential for MYC induction via the “X” pathway (Fig. 3) (Ihle, 1996),which infers that the upstream regulators of MYC in response to cytokine activation are still unknown. In addition, a nonreceptor tyrosine kinase, BCR-ABL, also mediates the transformation of B cells and fibroblasts via both RAS and “MYC” pathways. The constitutively activated kinase V-ABLtransforms B cells only during the early G1 phase of the cell cycle. This suggests that the pathways required for transformation by the ABL kinase are also required for G1 progression (Chen and Rosenberg, 1992); indeed, MYC expression is essential for this transformation process (Sawyers et d.,1992).

1V. THE RASERK PATHWAY AND THE CELL CYCLE A. The RASERK Pathway RAS, which was first identified as an oncogene (Bishop, 1982; Weinberg, 1989), plays an essential role in the proliferation of most cell types as a mediator of ligand-induced receptor signaling (Figs. 3 and 4). Dominant-nega-

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Fig. 3 Signal transduction pathways leading to activation of the immediate early response genes. In response to extracellular stimuli, activated receptors induce several pathways: 1RAS/MAP kinase pathway; 2-SHC, activated RAS/MAPK pathway; 3-JAWSTAT and “X” pathway; 4-SRC/MYC pathway. In the nucleus, the three genes that represent the “immediate early response” are FOS, JUN, and MYC.

tive forms of RAS block G1 progression mediated by receptors for several growth factors, including PDGF, EGF, and CSF-1 (Stacey et al., 1991), and transformation by the cytoplasmic tyrosine kinase BCR-ABL (Sawyers et al., 1995). Similarly, microinjection of anti-RAS antibodies reverses cell transformation by oncogenic forms of growth factor receptors, such as CSF-1R (v-fms),or by the cytoplasmic oncogenic effectors, V-RAS or V-SRC. Therefore, RAS is essential for transformation by most oncogenes (Mulcahy et al., 1985). Conversely, microinjection of activated RAS, Ha-RAS, in NIH3T3 cells leads to transformation (Stacey and Kung, 1984). The signaling pathway initiated by the activation of these small GTP proteins is highly conserved throughout evolution (Schlessinger and Bar-Sagi, 1994). Predictably, these proteins are fundamental to the development of all animal species (Dickson and Hafen, 1994)and control a number of cytoplasmic events that ultimately regulate gene expression (Fig. 4)(Egan and Weinberg, 1993; Segar and Krebs, 1995).

7

Key Effectors of Signal Transduction and G I Progression Growth Factors Cytokines

1

1

RAS

-

FOS, JUN

,-

DllK4 E2F Responsive Genes

Fig. 4 RAS, D-type cyclin, RB, and G1 progression. Summary of the pathways connecting activated receptors to RAS, cyclin D1, and RB. Arrows (-) represent established connections between effectors, whereas dashed arrows (---) represent potential links between FOS, JUN, and cyclin D1. D1, cyclin D1; D l K 4 , holoenzyme complex between cyclin D1 and cyclin-depenmhibitor of C d u ; RB, retinoblastoma gene product; EZFiDPdent kinase CDK4; p l 61NK4a, 1, transcription complex between E2F and DP-1; P, phosphorylated residue.

Microinjection of activated Ha-RAS into quiescent cells can induce DNA synthesis (Stacey and Kung, 1984). Conversely, microinjection of antibodies to RAS into serum-deprived Balb3T3 cells prevents DNA synthesis, demonstrating that RAS is required for S-phase entry (Dobrowolski et al., 1994). Constitutively, activated RAS and RAF can drive the cells through S phase, suggesting a direct link between RAS and the GUS transition (Dobrowolski et al., 1994; Samuel et al., 1993; Marshall, 1995). Similarly, activated MAP kinases (ERK-1 and ERK-2) are required for fibroblast transformation (Pages et al., 1993), and constitutive activation of MEKs is necessary and sufficient for fibroblast transformation (Cowley et al., 1994; Mansour et al., 1994). Interestingly, constitutive activation of each one of the key members of the RAS/ERK pathway leads to the GUS transition, suggesting that this pathway represents a critical link between receptor signaling and G1 progression. RAS regulates a number of downstream effectors (Fig. 3) (Marshall, 1996), including the cytoplasmic serine threonine kinase RAF-1 (McCormick, 1994), the dual specificity mitogen-activated protein kinase kinase (MAPKK or MEK-1 and MEK-2) (Cobb and Goldsmith, 1995), and the ex-

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Martine F. Roussel

tracellular regulated kinases (ERK-1 and ERK-2 or MAPK) (Egan and Weinberg, 1993; Su and Karin, 1996; Davis, 1996).RAF-1 activates MEK-1 (Kyriakis et al., 1992), as well as ERK-1 and ERK-2 (Samuel et al., 1993). In turn, ERKs phosphorylate and modulate the activity of nuclear factors that regulate the transcription of genes required for proliferation and differentiation (Treisman, 1996). In particular, the RAS pathway regulates the transcription of FOS and JUN (Blenis, 1996; Treisman, 1996), which, as dimers, define the AP-1 nuclear transcription factor (Karin, 1995; Su and Karin, 1996).

B. ETS Transcription Factors as RAS Targets

and Regulators of Proliferation Analysis of the FOS promoter has provided the basis for a model in which a complex coined the tertiary complex factor (TCF)contains an ETS domain. TCFs are direct targets of ERKs from the RASERK signaling pathway and are essential for the transient and immediate early transcription of FOS (Marais et al., 1993; Hill and Treisman, 1995; Janknecht et al., 199s). The ETS family of winged-helix-loop-helix transcription factors represents an extended family of genes, some of which were isolated as oncogenes, e.g., ETS-1 and FLI-1 (Macleod et al., 1992; Seth et al., 1989; Klemsz et al., 1993). ETS factors are characterized by a DNA-binding domain, the ETS domain, which recognizes a GGA core sequence surrounded by purine-rich motifs (Wasylyk et al., 1992, 1993; Wang et al., 1992). They often bind cooperatively to promoters with other transcription factors such as AP-1 (Bassuk and Leiden, 1995; Wasylyk et al., 1990) or SP1 (Gegonne et al., 1993) to regulate gene transcription (Janknecht and Nordheim, 1993). In mammalian cells, ectopic expression of a dominant-negative form of ETS inhibits the transformed phenotype imposed by activated RAS (V-RAS) and activated CSF-1R (V-FMS)and blocks CSF-1-dependent proliferation in NIH3T3 fibroblasts expressing human CSF-1R (Langer et al., 1992). Interestingly, this G1 block is associated with a reduction in CSF-l-induced MYC levels. Enforced MYC expression in these cells restores CSF-l-dependent proliferation, suggesting that the RAS pathway, and ETS in particular, regulates the MYC response in this system (Langer et al., 1992; Roussel et al., 1994). An additional connection between ETS and G1 progression has come from studies of the ETS-related transcription factor ELF-1 (Leiden et al., 1992; Davis and Roussel, 1996). ELF-1 activity is regulated, in part, by binding to RB through a pentapeptide (LXCXE) RB-binding domain (Wang et al., 1993),a motif shared by D- and E-type cyclins and by the DNA tumor virus oncoproteins SV40 T antigen, adenovirus ElA, and HPV E7 (Ewen et al., 1993; Ewen, 1994; Weinberg, 1995). This interaction therefore offers a di-

Key Effectors of Signal Transduction and GI Progression

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rect physical link between transcription regulation and the cell cycle, although it is not entirely clear how ELF-l regulates cell growth. The cumulation of these data, combined with the fact that some ETS factors are involved in the etiology of tumors (Zucman et al., 1992, 1993) underscores the role of this family of transcription factors in the regulation of G1 progression, proliferation, and transformation.

V. RAS, D-TYPE CYCLINS, A N D RB CONNECTIONS Cyclin D1 expression is upregulated by both activated RAS (v-Ha-RAS) and ERK-1 and ERK-2 (Lavoie et al., 1996). These data predict that the cyclin D1 promoter is regulated by elements responsive to RAS signals. The D1 promoter contains an AP-1-binding site and a cyclin AMP responsive element (CRE) that are inducible by small t antigen and c-JUN (Watanabe et al., 1996; Herber et al., 1994). Moreover, JUN nullizygous fibroblasts are resistant to RAS-induced transformation (Johnson et al., 1996). Therefore, a likely scenario is that the RASIMAP kinase pathway is linked to cyclin D expression as follows: activated RAS activates ERK kinases, which induce FOS and JUN, which in turn induce cyclin D1 transcription (Fig. 4). These findings suggest that cyclin D1 is in the RAS pathway and would cooperate with MYC, but not RAS, in transformation assays. Paradoxically, Harvey (Ha)-RAS cooperates with cyclin D1 to transform primary rat embryo fibroblasts (REF) (Lovec et al., 1994), although this appears to be cell specific as no synergy is observed if the experiment is done in rat kidney cells (BRK) (Hinds et al., 1994).In agreement with the fact that RAS and D1 are in the same pathway, the inducible expression of activated RAS in epithelial or fibroblast cells induces cyclin D1 expression (Winston et al., 1996) and cyclin D1 levels are increased in Ha-RAS-transformed cells, which accounts for their shortened G1 phase (Liu et al., 1995). RAS-induced transformation of some cells also requires cyclin D/CDK4 activity, and anti-sense cyclin D 1 oligonucleotides reduce the rate at which RAS-transformed cells proliferate (Filmus et al., 1994). Enforced expression of p161NK4"blocks the ability of activated RAS to transform fibroblasts cells (Serrano et al., 1995). However, at least in fibroblast cells, cyclin D1 expression alone is insufficient to drive DNA replication, suggesting that the activation of the DICDK holoenzymes requires mitogenic signals in addition to those involving RAS for Sphase entry (Winston et UE., 1996). This is a common theme as both cyclin D and CDK4 require mitogenic signals to form complexes (Matsushime et al., 1994). Several experiments suggest that D1 is in the RAS pathway. Ectopic expression of a dominant-negative form of RAS (Asnl7) in cells that lack func-

Martine F. Roussel

10 Growth Factors Cytokines

-m IS?C

““p/

rc

---)

”.‘.

FOS, JUN

a

Other Targets CDC25A, 8

D1

I

8 8

Fig. 5 MYC, D1, E2Fs and G1 progression. Summary of signaling pathways linking the major effectors of receptor signaling to those of G1 progression and S-phase entry. Arrows (-+) represent established pathways; whereas dashed arrows (-- +) are potential links between effectors. JAK, Janus kinase; CDC25A, a cell cycle tyrosine phosphatase (25A); D1, cyclin D1; DlK4, complex between cyclin D1 and the cyclin-dependent kinase CDK4; RB, retinoblastoma gene product; S, DNA synthetic phase; P, phosphorylated residue; E, cyclin E; A, cyclin A; DHFR, dihydrofolate reductase; pol 11, DNA polymerase 11; EK2, inactive kinase complex between cyclin E and the cyclin-dependent kinase CDK2; E/K2,* activated kinase complex that phosphorylates cyclin E and RB.

tional RB is unable to prevent S-phase entry, whereas Am17 expression in cells containing wild-type RB blocks both D1 expression and S-phase entry (Peeper et al., 1997). Therefore, RAS is linked to RB in the transition from G1 into S-phase v i s - h i s cyclin D1. Phosphorylation of RB releases its association with the E2F family of transcription factors (Fig. 5). E2F associates with a dimerization partner termed DP-1 and together form a potent transcriptional activator of many genes essential for G1 progression and DNA synthesis (La Thangue, 1994), including cyclin E (Botz et al., 1996), cyclin A (Schulze et al., 1995),cdc2 (Dalton, 1992), dihydrofolate reductase (DHFR) (Blake and Azizkhan, 1989), MYC (Thalmeier et al., 1989; Hiebert et al., 1989), and DNA polymerase a (Pearson et al., 1991).Predictably, enforced E2F expression is sufficient to replace some mitogenic signals, and microinjection of E2F-1 overrides the require-

Key Effectors of Signal Transduction and GI Progression

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ment for cellular RAS in initiating DNA synthesis (Stacey et al., 1994). Thus E2F-1 can substitute for the earlier signaling requirements to progress through G1 and enter S phase.

VI. CYCLING WITH MYC A. MYC and Its Partners MYC was first isolated as a transforming gene from avian acute leukemia viruses, MC-29 and MH-2 (Alitalo et al., 1987), and its cellular counterpart was identified and cloned soon thereafter (Roussel et al., 1979; Bishop, 1982).MYC was the first oncogene found to be overexpressed as a result of a chromosomal translocation (Bishop, 1983).Since this seminal observation, amplification of MYC family members (MYC, N-MYC, and L-MYC) has been shown in many human tumors and is known to deregulate cell growth by promoting continuous, mitogen-independent, cell cycle progression (Eilers et al., 1991; Askew et al., 1991; Evan et al., 1992; Henriksson and Liischer, 1996; Lemaitre et al., 1996). The importance of MYC to cell cycle progression has also been established through studies of its function as a transcription factor (Adkins et al., 1984; Blackwood et al., 1991; Amati and Land, 1984).In concert with its partner MAX (Blackwood and Eisenman, 1991),MYC positively regulates the transcription of genes important in cell growth and homeostasis (Packham and Cleveland, 1995).MAX also forms heterodimers with other members of the HLWbZIP transcription factor family, including MAD1 , MXIl (MAD2), MAD3, and MAD4 (Ayer et al., 1993). MAD/MAX heterodimers actively repress gene transcription and, in so doing, antagonize MYC function (Zervos et al., 1993; Hurlin et al., 1995). Transcriptional repression by MAD/ MAX occurs through the formation of ternary complexes with a conserved, generalized transcriptional repressor (SIN3), which was initially identified in yeast (Schreiber-Aguset al., 1995; Ayer et al., 1995). Enforced expression of MAD inhibits tumor growth (Chen et al., 1995) and induces G1 arrest in serum- or CSF-1-stimulated fibroblasts. These biologic effects require the association of MAD with both MAX and SIN3 (Ayer et al., 1995; Roussel et al., 1996). MYC is normally expressed at a low but constant level in proliferating cells. However, when quiescent cells are stimulated by growth factors or serum, they transiently express much higher levels of MYC (Fig. 2). MYC is downregulated as cells differentiate or in the absence of mitogenic stimuli, whereas MAD genes are induced (Ayer and Eisenman, 1993) and (John Cleveland, personal communication).

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Martine E Roussel

MYC function is necessary for S phase entry as ectopic expression of a dominant-negative form of MYC (Stone et al., 1987; Roussel et al., 1995), MAD (Ayer et al., 1993; Roussel et al., 1996), MAX (Gu et al., 1993), or treatment with anti-sense oligonucleotides (Heikkila et al., 1987; Prochownik et al., 1988) prevents growth factor-induced S-phase entry and leads to G1 arrest. MYC is also limiting for S-phase because its enforced expression in fibroblasts shortens G1 and accelerates cell growth (Keath et al., 1984; Eilers et al., 1989; Roussel et al., 1991; Evan et al., 1984). A mutant form of the CSF-1 receptor that is unable to signal G1 progression (CSF-1R [Y809F]) mediates the normal kinetics of FOS and JUN transcription but fails to induce sustained levels of MYC (Roussel et al., 1990). Similarly, mutants of the p chain of the IL-2 receptor cannot mediate proliferative signals or induce MYC expression, but they do promote normal levels of FOS and JUN (Taniguchi and Minami, 1993). Finally, the nonreceptor tyrosine kinase BCR-ABL mediates transformation via independently activated pathways, including a RAS pathway and a MYC pathway (Sawyers et al., 1992; Goga et al., 1995). CSF-1R and BCR-ABL are tyrosine kinases that mediate mitogenesis and transformation, respectively, by independent and common pathways that involve MYC (Lug0 and Witte, 1989) and cyclin D1 (Afar et al., 1995). Experiments designed to complement either the CSF-1R [Y809F] mutant or a BCR-ABL [SH2] mutant defective in the transformation of B cells or fibroblasts showed that enforced expression of either MYC or cyclin D1 complements both of these defects (Afar et al., 1994,1995). In each system, ectopic MYC expression in the mutants restores receptor-induced proliferation or kinase-induced transformation, demonstrating that the MYC pathway is essential for G1 progression and S-phase entry (Adkins et al., 1984; Roussel et al., 1991; Sawyers et al., 1992).

B. MYC and Its Targets The identification and characterization of the transcriptional targets of MYC have proven to be a difficult task (Fig. 5). Only a relative small number of MYC targets have been identified to date. The following MYC targets have been identified: a-prothymosin (Eilers et al., 1991), ornithine decarboxylase (ODC) (Bello-Fernandez et al., 1993), CAD (Miltenberger et al., 1995), an evolutionary conserved RNA helicase of the DEAD box family, MrDb (Grandori et al., 1996), eIF-4E, a eucaryotic initiation factor of protein synthesis (Jones et d., 1996; Rosenwald et d., 1993), cyclins E and A (Jansen-Durr et al., 19,93), and the tyrosine phosphatases CDC25A and CDC25B (Galaktionov et al., 1996) (Fig. 5). Each of these targets may play a unique role in MYC-mediated S-phase entry, or their combined effects

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might be required to move the cell from G1 into S phase (Packham et al., 1996; Zornig and Evan, 1996). ODC and CDC25A are direct MYC targets. Their promoters contain consensus E box MYC-binding sites whose disruption abrogates the induction of these genes by growth factors (John Cleveland, personal communication). ODC may be directly required for DNA replication because inhibitors of ODC arrest cells in G1 (Packham et al., 1996). The CDC25 phosphatases could activate cyclins E and A expression by regulating the activities of cyclin-CDK complexes. Presumably, this regulation would involve the dephosphorylation of the inhibitory tyrosine phosphate on CDK2 (JansenDurr et al., 1993; Galaktionov et a/., 1996). MYC can alter the expression of several genes, including cyclins E and A (Jansen-Durr et al., 1993; Solomon et al., 1995), although this regulation may be an indirect result of the positive effects of MYC on the cell cycle. MYC also induces the expression of the initiation factor eIF-4E. Phosphorylation of eIF-4E depends on RAS signaling (Frederickson et al., 1992) whereas cyclin D1 levels increase in response to elevated levels of eIF-4E (Rosenwald et al., 1993). Therefore, these are clear links between the regulation of protein synthesis and the cell cycle. Unlike enforced expression of MYC, enforced expression of ODC (Packham et al., 1996) is not sufficient to promote proliferation or to complement a CSF-1R mutant defective in promoting G1 progression ( M.F.R. and John Cleveland, unpublished data). However, CDC25A and CDC25B (but not CDC25C), like MYC, can function as oncoproteins by collaborating with RAS to transform primary mouse embryo fibroblasts (Galaktionov et al., 1995). This suggests that MYC, cyclin D1, and CDC25A may have overlapping functions, at least in this biological setting.

VII. INTERPLAY BETWEEN MYC AND CYCLIN D l Evidence from in vitro and in vivo studies has led to the belief that MYC and D1 act in parallel pathways but cooperate functionally and synergistically to enforce G1 progression. In experiments with transgenic mice, crossing Ep-MYC and E p D 1 mice significantly accelerates the onset of lymphomas (Bodrug et al., 1994; Lovec et al., 1994). Interestingly and surprisingly, the Ep-MYC-induced B-cell tumors express cyclin D1, even though D1 is not normally expressed in B cells (Bodrug et al., 1994). Because cyclin D1 is overexpressed in Ep-MYC-induced tumors only and not in other B cells of Ep-MYC transgenic mice, it suggests that MY C overexpression is necessary but not sufficient to induce cyclin D1 expression. In addition, MYC or D1 is functionally interdependent and each requires the function of the other to

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restore CSF-1-dependent proliferation of fibroblasts engineered to express CSF-lR[Y809F]. For example, microinjection of anti-Dl antibodies into MY C overexpressing cells, or overexpression of a dominant-negative MYC mutant (In373) or MAD proteins in cells expressing CSF-lR[Y809F] and complemented by enforced cyclin D 1 expression, prevents S-phase entry (Roussel et al., 1995, 1996). The interplay between MYC and D1 could be explained by the fact that D/CDK4 complexes phosphorylate RB, thereby releasing E2F/DP-1 complexes that induce MYC transcription (Hiebert et al., 1989; Oswald et al., 1994).Therefore, in cells unable to express MYC, enforced expression of D1 should reinduce MYC expression, albeit with different kinetics from those observed in response to authentic mitogenic stimuli (Roussel et al., 1995). MYC and cyclin D1 seem to enjoy a special relationship that apparently is not shared by D2- and D3-type cyclins. Analysis of the promoter regions and expression patterns of the D-type cyclins (Ewen et al., 1993; Brooks et al., 1996; Wang et al., 1996), coupled with studies of mice in which the D1 or D2 genes are disrupted (Sicinskiet al., 1995,1996), suggests that each D-type cyclin may play specific and overlapping roles in regulating cell proliferation.

W11. SIGNALING AND CELL CYCLE ROLES OF THE SRC FAMILY OF KINASES The SRC family of protein tyrosine kinases, whose founder member SRC was the first oncogene described (Stehelin et al., 1976; Bishop, 1983), has been implicated in the mitogenic response to many growth factors and lymphokines. All SRC kinases contain a conserved SRC homology SH3 domain, a SRC homology SH2 domain, a catalytic domain, and C-terminal regulatory sequences (Superti-Furga and Courtneidge, 1995; Brown and Cooper, 1996). Three members of the SRC kinase family, SRC, FYN, and YES, are closely related and ubiquitously expressed. They mediate signals by physically associating with activated growth factor receptors via their SH2 and their SH3 domains (Erpel et al., 1996). This association is required to activate the activity of the SRC kinases and is dependent on specific tyrosine residues located near the transmembrane-spanning region of such receptors (Mori et al., 1993; Courtneidge et al., 1993; Alonso et al., 1995). A role for the SRC kinases in mitogenic signaling has been established. Microinjection of an anti-SRC antibody that recognizes all three members (anticstl) inhibits EGF-, PDGF-, and CSF-1-induced S-phase entry in fibroblasts expressing equivalent numbers of PDGF-R and ectopically expressed human

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CSF-1R (Twamley-Stein et al., 1993; Roche et al., 1995). However, microinjection of this antibody into cells already committed to S phase (i.e., at least 10 hr after ligand stimulation) has no effect, suggesting that SRC kinases are required to drive cells, at least up to the restriction point, R (Roche et al., 1995).Interestingly, at least in fibroblasts, SRC is also activated in G2, which suggest a requirement for SRC in mitosis (Roche et al., 1995). Microinjection studies have also provided researchers with sufficient data to propose a novel signaling pathway for SRC kinases (Barone and Courtneidge, 1995). Specifically, antibodies to SRC or dominant-negative forms of SRC or FYN prevent MYC mRNA induction and S-phase entry in response to PDGF stimulation. Enforced expression of MYC, but not of FOS or JUN, overcomes this block and restores S-phase entry, thus providing compelling evidence that SRC couples activated growth factor receptors to MYC via a pathway that is independent of RAS. Even though the “SRC/MYC” and/or “X” pathways that lead to MYC expression are dissociated from the RAS signaling pathway, both pathways seem necessary for G1 progression. The situation is further complicated by the fact that these signaling pathways are apparently cell context specific and receptor specific. For example, the IL-2 cytokine receptor activates the SRC kinases LCK, FYN and LYN, which induce pathways that are independent of MY C transcription and insufficient for IL-2-dependent proliferation (Kobayashi et al., 1993; Ihle, 1996). Similarly, BCR-ABL activates the SRC family kinases independently of MYC transcription (Danhauser-Riedl et al., 1996). Nevertheless, the findings of Barone and Courtneidge (1995)provide a link between the SRC kinases and the MYC protooncogene (Eisenman and Cooper, 1995).

IX. CONCLUDING REMARKS Receptor mutants that fail to connect mitogenic signals to cell cycle progression and the enforced expression in cells of key effectors of G1 progression have been useful in identifying many signal transduction pathways. The realization that mammalian D-type cyclins are growth factor sensors has fueled interest in coupling receptor signals to the key effectors that regulate the cell cycle clock. The complexity of these pathways is just beginning to emerge, and novel pathways and effectors likely remain to be identified. Despite the multiplicity of ligands, receptors, and their “wiring” to effectors, most of the identified pathways converge on a few key genes required for G1 progression and S-phase commitment. Strikingly, most of these key players, including RAS, MYC, D-type cyclins, and RB, are frequently targeted in human tumors (Sherr, 1996). One of the major challenges will be to

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understand how these crucial G1 effectors are interconnected. The next few years should yield exciting new discoveries as investigators unravel these pathways.

ACKNOWLEDGMENTS Given the recent explosion of data from the fields of signal transduction and cell cycle, I cannot claim that this review is exhaustive or encyclopedic. Moreover, I must apologize to my colleagues whose work is not included or indirectly cited. I thank Drs. Daniel E. H. Afar, Mangeng Cheng, John L. Cleveland, Jonathan Cooper, Veronika Sexl, and Frederique Zindy for critically reviewing this manuscript and to Dr. Sue Vallance for editorial assistance. My thanks also go to my husband, Dr. Charles J. Sherr, for his continued support, understanding, and encouragement and to the members of my laboratory for their continued efforts and support. MFR is supported by NIH Grants R01-CA-56819, Pol-CA-76907, Cancer Center CORE Grant CA21665 from the National Cancer Institute, and by funds from the American Lebanese Syrian Associated Charities (ALSAC)of St. Jude Children's Research Hospital.

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cap-binding protein (eukaryotic initiation factor 4E) is a target for activation by c-myc. Mol. Cell. Biol. 16,4754-4764. Karin, M. (1995).The regulation of AP-1 activity by mitogen-activated protein kinase. /. Biol. Chem. 270,16843-16846. Keath, E. J., Caimi, P. G., and Cole, M. D. (1984). Fibroblast lines expressing activated c-myc oncogenes are tumorigenic in nude mice and syngenic animals. Cell 39, 339-348. Klemsz, M. J., Maki, R. A,, Papayannopoulou, T., Moore, J., and Hromas, R. (1993). Characterization of the ets oncogene family member, fli-1. J. Biol. Chem. 268,5769-5773. Kobayashi, N., Kono, T., Hatakeyama, M., Minami, Y., Miyazaki, T., Perlmutter, R. M., and Taniguchi, T. (1 993). Functional coupling of the src family protein tyrosine kinases ~ 5 9 ~ y " and p53/56'P with the interleukin 2 receptor: Implications for redundancy and pleiotropism in cytokine signal transduction. Proc. Nutl. Acud. Sci. USA 90,4201-4205. Kyriakis, J. M., App, H., Zhang, X.-F., Banerjee, P., Brautigan, D. L., Rapp, U.R., and Avruch, J. (1992).Raf-1 activates MAP kinase-kinase. Nature 358,421427. La Thangue, N. B. (1994). DRTFlE2F: An expanding family of heterodimeric transcription factors implicated in cell cycle control. Trends Biochem. Sci. 19, 108-114. Langer, S . J., Bortner, D. M., Roussel, M. F., Sherr, C. J., and Ostrowski, M. C. (1992). Mitogenic signaling by colony-stimulating factor 1 and rus is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression. Mol. Cell. Biol. 12,5355-5362. Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouysst?gur,J. (1996). Cyclin D1 expression is regulated positively by the p42/p44MAPKand negatively by the p38/HOGMAPX pathway. J. Biol. Chem. 271,20608-20616. Leiden, J. M., Wang, C., Petryniak, B., Markovitz, D. M., Nabel, G. J., and Thompson, C. B. (1992).A novel ets-related transcription factor, elf-1, binds to human immunodeficiency virus type 2 regulatory elements that are required for inducible trans activation in T cells. J. Virol. 66,5890-5897. Lemaitre, J.-M., Buckle, R. S., and Mkhali, M. (1996). c-Myc in the control of cell proliferation and embryonic development. Adv Cancer Res. 70,96-144. Liu, J.-J., Chao, J.-R., Jiang, M.-C., Ng, S.-Y., Yen, J. J.-Y., and Yang-Yen, H.-F. (1995). Ras transformation results in an elevated level of cyclin D1 and acceleration of GI progression in NIH 3T3 cells. Mol. Cell. Biol. 15, 3654-3663. Lovec, H., Grzeschiczek, A., Kowalski, M. B., and Moroy, T. (1994). Cyclin Dl/bcl-1 cooperates with myc genes in the generation of B-cell lymphoma in transgenic mice. E M B O /. 13, 3487-3495. Lovec, H., Sewing, A., Lucibello, F. C., Muller, R., and Moroy, T. (1994). Oncogene activity of cyclin D1 revealed through cooperation with Ha-rus: Link between cell cycle control and malignant transformation. Oncogene 9, 323-326. Lugo, T. G., and Witte, 0. N. (1989). The BCR-ABL oncogene transforms rat-1 cells and cooperates with v-myc. Mol. Cell. Biol. 9, 1263-1270. Lukas, J., Bartkova, J., Rohde, M., Strauss, M., and Bartek, J. (1995a).Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells, independent of CDK4 activity. Mol. Cell. Biol. 15,2600-2611. Lukas, J,, Parry, D., Aagaard, L., Mann, D. J., Bartkova, J., Strauss, M., Peters, G., and Bartek, J. (199%). Rb-dependent cell cycle inhibition by p16CDKN2tumour suppressor. Nature 375, 503-506. Macleod, K., Leprince, D., and Stehelin, D. (1992).The ets gene family. Trends Biochem. Sci. 17,251-256. Mansour, S . J., Matten, W. T., Hermann, A. S., Candia, J. M., Rong, S., Fukusawa, K., Vande Woude, G. F., and Ahn, N. G. (1994). Transformatiion of mammalian cells by constitutively active MAP kinase kinase. Science 265,966-969. Marais, R., Wynne, J., and Treisman, R. (1993). The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 73,381-393.

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p53 in Tumor Progression: Life, Death, and Everything Michael R. A. Mowat Manitoba Insrirure of Cell Biology, Winnipeg, Manitoba, Canada R 3 E OV9

I. Introduction 11. Biochemical Activities of p53 111. p53 and Cell Cycle Control A. Control of GUS by p53 B. Control of G2/M by p53 C. Nontranscriptional Controls of Cell Cycle by p53 1V. p53 and Apoptosis A. p53 Control of Apoptosis in Tumors B. p53 Transactivation and Apoptosis C. Nontransactivator Function in Apoptosis by p53 V. p53 and Tumor Progression A. Apoptosis and Tumor Progression B. Growth Control and Tumor Progression C. Genornic Instability and Tumor Progression References

I. INTRODUCTION The p53 tumor suppressor protein was first described in 1979 as a protein that binds SV40 virus large T antigen (Linzer and Levine, 1979; Lane and Crawford, 1979) and independently as a tumor antigen (DeLeo et al., 1979). Although the basic outline of p53 protein function in cells has been deciphered, this molecule continues to surprise and baffle scientists. p53 activity has been linked to tumor suppression, cell cycle control, DNA repair, stress responses, cell senescence, genomic stability, and apoptotic cell death. The frequent mutation of the p53 gene in human tumors stresses the importance of trying to understand the function of this protein. Because of the vastness of the p53 literature, this review will concentrate on the role of p53 in tumor progression. In particular, the mechanisms of p53’s control of the cell cycle, genomic stability and apoptosis will be reviewed and how loss of these functions play a role in tumor cell progression. Advances in CANCER RESEARCH 0065-23OW98 $25.00

Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved.

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11. BIOCHEMICAL ACTIVITIES OF p53 p53 primarily functions as a transcriptional transactivator protein with sequence-specific DNA-binding activity (Fields and Jang, 1990; Raycroft et al., 1990; Funk et al., 1992; el Deiry et al., 1992). The transcription transactivating domain of p53, at the amino terminus, interacts with various members of the general transcription initiation complex. These include members of the TFIID complex, such as TATA-binding protein (TBP) (Horikoshi et al., 1995) and TPB associated proteins, TAF,,40 and TAFI160 (Thut et al., 1995; Lu and Levine, 1995). Also, p53 interacts with the TFIIH transcription complex (Xiao et al., 1994; Wang et al., 1995a). The central domain of p53, where most tumor-derived mutations occur, functions in sequence-specific DNA binding (Vogelstein and Kinder, 1992; Cho et al., 1994).The carboxyl terminus of p53 is the location of the oligomerization domain (Shaulian et al., 1992) and the structure of this region has been elucidated (Clore et al., 1994; Lee et al., 1994; Jeffrey et al., 1995). The C-terminal region of p53 can also bind nonspecifically to DNA ends or mismatched DNA and promote annealing of DNA single strands (Lee et al., 1995; Bakalkin et al., 1994; Jayaraman and Prives, 1995; Bakalkin et al., 1995).This region of p53 also functions to control the DNA-binding activity of p53 and can be regulated by phosphorylation (Hupp et al., 1992; Wang and Prives, 1995). The functional domains of p53 referred to in this review are summarized in Fig. 1. A 3' to 5' exonuclease activity intrinsic to p53 protein has been described (Mummenbrauer et al., 1996). Also, p53 binds to and inhibits the DNA re-

53BPl 53BP2 transactivation

TFllH XP-B XP-D

sequence specific DNA binding

N

C

TFllD TBP TAF40 TAFBO TFllH ~ 6 2

50

I

100

I

150

I

200

I

250

I

I

I

u

300

350

393

non-specific DNA binding

Fig. 1 Functional domains of p53 protein. The numbered hatched boxes represent the conserved domains. pS3-binding proteins discussed in the text and their approximate binding regions are shown in bold.

p53 in Tumor Progression

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pair protein Rad51 (Sturzbecher et al., 1996). For more details, the biochemistry of p53 has been reviewed and the reader is referred to KO and Prives (1996).

111. p53 AND CELL CYCLE CONTROL

A. Control of GI/S

by p53

DNA damage and other stresses will stabilize p53 protein, resulting in growth arrest (Kastan et al., 1992; Lu and Lane, 1993). It is now generally accepted that a major control by p53 on the cell cycle at GUS is through transcriptional control of the p21wAF11C'P1gene (see Morgan and Kastan, 1997). The p21 (WAF1, CZP1, SDI)gene was discovered about the same time by several groups as a p53-inducible gene (el Deiry et al., 1993), a cyclin-dependent kinase inhibitor (Harper et al., 1993; Xiong et al., 1993; Gu et al., 1993), and a gene expressed at high levels in senescent cells (Noda et al., 1994). p21wAF11C1P1is an effective inhibitor of Gl/S cyclin-dependent kinases (cdk), Cdk2, Cdk3, Cdk4, and Cdk6 kinases, but a weaker inhibitor of G2 active cdc2 kinase (cdkl) (Harper et al., 1995). Studies performed on p21WAF1/C1P1-deficient mice showed a partial defect in G1 arrest after DNA damage or nucleotide pool perturbations compared with the defect in p53deficient mouse cells (Deng et al., 1995; Brugarolas et al., 1995), suggesting that other gene targets or functions of p53 were also needed for growth arrest. p21WAF11CIP1-deficient mice did not show defects in apoptosis induction or spindle checkpoint control (Deng et al., 1995; Brugarolas et al., 1995).These mice also did not show an increased incidence of tumors, suggesting that p21WAF1/C'P1is not important for the tumor suppressor function of p53 (Deng et al., 1995; Brugarolas et al., 1995). Another mechanism of p53 growth arrest in p21WAF1/C1P1-deficient mice may be the GADD45 gene, a transcriptional target of p53 (Kastan et al., 1992). GADD45 is a DNA damage-inducible gene that can cause growth arrests when overexpressed in cells (Zhan et al., 1994b). Another transcriptional target of p53 is cyclin G (Okamoto and Beach, 1994; Zauberman et al., 1995). Unlike GADD45, cyclin G overexpression enhances cell cycle progression and increases cisplatin sensitivity (Skotzko et al., 1995; Smith et al., 1997). The cyclin G protein has been shown to form a complex with the B' regulatory subunits of protein phosphatase 2A (PP2A) (Okamoto et al., 1996). Although the consequence of cyclin G interaction with the By subunits is not known, PP2A regulates many processes, including signal transduction, cell cycle, transcription, and development (for review see Mumby and Walter, 1993).

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B. Control of G2/M by p 5 3 Although p53 was originally thought only to control the Gl/S part of the cell cycle, several groups have shown that p53 may also function at the G2/M part of the cell cycle (Vikhanskaya et al., 1994; Stewart et a/., 1995; Agarwal et al., 1995; Aloni Grinstein et af., 1995).In addition, overexpression of the Ras oncogene in fibroblasts can also cause a G2/M arrest that is dependent on p53 (Hirakawa and Ruley, 1988; Hicks et d., 1991).p53 is also part of the spindle checkpoint control mechanism (Cross et al., 1995). Disruption of the mitotic spindle with drugs normally results in cell arrest at G2/M, but, in p53-deficient cells, multiple rounds of DNA synthesis occur without cell division (Cross et af., 1995).p53 also plays a role in controlling centrosome duplication (Fukasawa et af., 1996). Fibroblasts from p53-deficient mice show multiple copies of centrosomes that result in the abnormal segregation of chromosomes (Fukasawa et af., 1996). Alterations in the spindle checkpoint control and centrosome duplication in p53 mutant cells may play important roles in genetic instability seen in p53-deficient cells (Harvey et af., 1993; Tsukada et al., 1993). The growth arrest at G2/M caused by p53 overexpression may be mimicking the downstream events of the spindle checkpoint control. It is not yet clear whether p53 also functions as part of the G2/M checkpoint control due to unreplicated or damaged DNA. Cells deficient in p53 still show a G2/M arrest after irradiation (Kuerbitz et al., 1992). It is possible that more than one cell cycle checkpoint may be operational at G2/M (see later). How spindle damage activates p53 activity and how p53 controls the G2/M part of the cell cycle are unknown at this time.

C. Nontranscriptional Controls of Cell Cycle by p 5 3 It has become appreciated that nontranscriptional activities of p53 are also important for its cell cycle control. Transcription-defective mutants of p53 were previously shown to still cause growth arrest, suggesting the importance of a nontranscriptional activity of p53 (Sabbatini et al., 1995; Hansen and Braithwaite, 1996). The poly-proline region (PP) of p53 (amino acids 58-93) has been implicated in nontranscriptional control of cell growth (Walker and Levine, 1996).This region of several ProXXPro amino acid repeats can be binding sites for Src homology domain 3 (SH3) proteins, which are involved in tyrosine kinase signal transduction (Walker and Levine, 1996). Deletion of the PP region does not affect the transactivation activity of p53 (Walker and Levine, 1996). Transfection of a PP region deletion mutant into the H1299 and SAOS-2 cell lines reduces colony formation by 2fold compared with 10-fold for wild-type p53 (Walker and Levine, 1996). It has been shown that the PP region of p53 is essential for the growth arrest

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induced by the Gasl gene (Ruaro et al., 1997). The Gasl gene needs a transcription-independent function of p53 to induce growth arrest when ectopically expressed in cells (Del Sal et al., 1995). Growth arrest induced by overexpression of the nuclear c-Abl tyrosine kinase functions through binding to p53 protein (Sawyers et al., 1994; Goga et al., 1995). The proline-rich domain of c-Abl and not the SH3 domain of c-Abl is needed for binding p53 in vitro (Sawyers et al., 1994; Goga et al., 1995). Induction of growth arrest by radiation or genotoxic drugs needs c-Abl kinase activity and p53 binding, but not transactivation of the p21WAF1’C1P1gene (Yuan et al., 1996a,b). This growth arrest is associated with downregulation of cdk2 kinase activity (Yuan et al., 1996a,b). It is unknown whether Abl protein binds to the p53 PP region. Elevated expression of the p210BCR-Ab’gene in hematopoietic cells induces a G2/M arrest and protects cells from radiation and chemotherapeutic-induced apoptosis (Bedi et al., 1995). The Lyn tyrosine kinase also induces a G2/M arrest after the irradiation of hematopoietic cells (Kharbanda et al., 1994; Uckun et al., 1996). The X-irradiation of hematopoietic cells results in an inhibitory tyrosine 1 5 phosphorylation of cdc2 kinase that is dependent on Lyn kinase activity (Kharbanda et al., 1994; Uckun et al., 1996). It is not known if the Lyn kinase also needs p53 for its growth arrest. If Lyn or c-Abl tyrosine kinases are acting on cdc2 function without p53, this may explain why pS3deficient cells still show a G2/M growth arrest after irradiation.

1V. p53 AND APOPTOSIS A. p53 Control of Apoptosis in Tumors Wild-type p53 was initially shown to induce apoptosis by overexpression in myeloid leukemic and colon tumor cell lines (Yonish-Rouach et al., 1991; Shaw et al., 1992). The first evidence that the normal endogenous p53 could induce apoptosis under physiological conditions came from studies expressing the adenovirus E1A oncogene in the REF52 cell line and primary baby rat kidney (BRK) cells (Lowe and Ruley, 1993; Debbas and White, 1993). This E l A-induced apoptosis can be increased by the removal of serum. E1Ainduced apoptosis can be prevented by either coexpression of a dominantnegative mutant p53 or coexpression of the adenovirus E1B 19K or 55K proteins (Lowe and Ruley, 1993; Debbas and White, 1993). The E1B 55K protein binds p53 and inactivates its transactivation function (Yew and Berk, 1992). The E1B 19K protein, a viral homolog of the Bcl-2 oncogene, blocks apoptosis downstream of p53 (Boyd et al., 1994). Wild-type p53 is needed for apoptosis induction by ionizing radiation and

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some chemotherapeutic drugs (Clarke et al., 1993; Lotem and Sachs, 1993; Lowe et af., 1993; Lee and Bernstein, 1993). Thymocytes from p53 gene “knockout” mice demonstrate less radiation (Lowe et al., 1993; Clarke et af., 1993; Lotem and Sachs, 1993) or etoposide (Clarke et al., 1993)-induced apoptosis compared with normal mice. In contrast, lack of p53 does not affect apoptosis induced by synthetic glucocorticoids or Ca2+plus phorbol esters (Clarke et al., 1993; Lotem and Sachs, 1993). In the myeloid bone marrow progenitor cells from p53-deficient mice, increased survival was found at low concentrations of colony-stimulating factors and interleukins (Lotem and Sachs, 1993). Other studies have shown that expression of a dominantnegative mutant p53 in transgenic mice resulted in increased radioresistance in hematopoietic cells, but no difference in resistance to the alkylating agent ethyl methane sulfonate (Lee and Bernstein, 1993). Compared to thymocytes, primary fibroblasts from mice show reduced ionizing radiation or chemotherapeutic drug-initiated apoptosis (Lowe et al., 1993). Treatment of normal fibroblasts with ionizing radiation will normally cause growth arrest and not apoptosis (Kastan et al., 1992). Expression of an oncogene such as the adenovirus E1A is needed to sensitize normal fibroblasts to these treatments and E1A expressing fibroblasts from p53-deficient mice show resistance (Lowe et al., 1993). This resistance to y-irradiation and adriamycin treatments of p53 minus fibrosarcomas has also been demonstrated in vivo (Lowe et al., 1994a).These results lead to the prediction that drug resistance in the c h i c may sometimes be due to the loss of p53-dependent apoptosis in tumors. It has been found that p53 mutation is an independent predictor of clinical drug resistance in relapsed non-Hodgkin’s lymphoma (Wilson et al., 1997).The clinical aspects of p53 mutation and response to therapy have been reviewed by Ruley (1996).

B. p53 Transactivation and Apoptosis How p53 induces apoptosis is still not fully understood at this time. Several studies using various p53 transactivation-defective mutants have shown that induction of apoptosis by p53 is dependent on its transcriptional transactivating activity (Sabbatini et al., 1995; Yonish-Rouach et al., 1995; Ishioka et al., 1995; Attardi et al., 1996; Hansen and Braithwaite, 1996). One potentially important transcriptional target for p53-induced apoptosis is the Bax gene. Bax is a member of the Bcl-2 gene family that promotes cell death (for review see Reed et al., 1996). It has been proposed that cell death agonists of the Bcl-2 family, such as Bax, dimerize with Bcl-2 to promote cell death (Oltvai et a/., 1993). Overexpression of p53 protein or induction of p53 by DNA-damaging agents increases the expression of the Bax gene and decreases the expression of the Bcl-2 gene (Miyashita et al., 1994b; Zhan et

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al., 1994a). p53 DNA-binding sequences have also been found in the Bax gene promoter (Miyashita and Reed, 1995). Transfection of the Bax gene into BRK cell lines, expressing adenovirus E1A and mutant p53 genes, can reduce colony formation and induce apoptosis, supporting a role for Bax downstream of p53 (Han et al., 1996). In another study using E1A expressing fibroblasts from Bax-deficient mice, loss of the Bax gene resulted in partial drug resistance, although the resistance was not as great as p53-deficient fibroblasts (McCurrach et al., 1997). These results suggest that Bax may be part of a p53-dependent apoptosis pathway, depending on the tissue type, and that other factors controlled by p53 may still be needed for full apoptosis induction. Other studies have shown that Bax is not important for p53-dependent apoptosis in some tissues. For example, thymocytes expressing a Bax transgene showed increased levels of y-irradiation-, etoposide-, and dexamethasone-induced apoptosis (Brady et al., 1996). In contrast, expression of the Bax transgene in a p53 minus background did not increase DNA damageinduced apoptosis compared with p53+ thymocytes not carrying the Bax transgene (Brady et al., 1996). In another study, thymocytes from Bax-deficient mice showed similar levels of y-ray-induced apoptosis compared with normal litter mates (Knudson et al., 1995). In Epstein-Bar virus immortalized B cells and an interleukin-3-dependent leukemia cell line with a wildtype p53, there were no changes in the levels of Bcl-2 or Bax proteins after DNA-damaging agents (Shaulian et al., 1995; Canman et al., 1995). Expression of an exogenous Bax gene in E1A expressing p53-'- fibroblasts did not fully restore drug-induced apoptosis (McCurrach et al., 1997). Transfection of a wild-type p53 gene into Saos-2 and H1299 tumor cell lines does not result in any changes in Bax protein levels compared with nontransfected controls (Rowan et al., 1996). These contradictory roles for Bax in p53dependent apoptosis may be due to tissue-specific differences. Zn vivo p53 can alter the expression of Bax in neuronal, prostate, kidney, and small intestinal cell types, whereas Bax and Bcl-2 are not detected in cortical thymocytes (Miyashita et al., 1994b). Because Bcl-2 can block p53-dependent apoptosis (Wang et al., 1993; Wagner et al., 1993; Chiou et al., 1994; Wang et al., 1995c), even in cells without Bax (McCurrach et al., 1997), other death-inducing members of the Bcl-2 family may be possible targets of p53 transactivation, such as Bak and Bik (Boyd et al., 1995; Chittenden et al., 1995; Kiefer et al., 1995). Another transcriptional target of p53 that may be important in apoptosis induction is the insulin growth factor I-binding protein 3 (IGF-BP3) (Buckbinder et al., 1995). Insulin growth factor-1 (IGF-1) can suppress apoptosis induced by Myc oncogene overexpression (Harrington et al., 1994). An increased expression of IGF-BP3 will bind IGF-1 and, in turn, prevent binding to the IGF-1 receptor and promote apoptosis. Interestingly, the apoptosis-

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defective mutants p53Ala-143 and p53Pro-175 failed to activate properly the IGF-BP-3 and Bax gene promoters (Ludwig et al., 1996; Friedlander et al., 1996). The p53Leu-181 allele did not activate IGF-BP3 box B promoter sequences (Ludwig et al., 1996; Friedlander et al., 1996). All of these mutant p53s were still also able to induce growth arrest and activate the WAFZ\CIPI gene. The p53Ala-143 allele was previously shown to be temperature sensitive for growth, but defective for apoptosis induction (Kobayashi et al., 1995). A minority of the tumor-derived mutant p53s at position 175 still show wild-type levels of transcriptional and growth arrest properties (Ory et al., 1994; Crook et al., 1994). It was suggested that this differential induction of apoptotic versus growth arrest genes may reflect differences in the ability of the wild-type p53 protein to modulate expression of these genes, depending on the cellular context (Ludwig et al., 1996; Friedlander et al., 1996). Relevant to this idea is the finding that the substitution of arginine 175 on p53 to alanine will disrupt interaction of the p53-binding proteins 53BP1 and 53BP2, but not alter its transactivation or DNA-binding properties (Thukral et al., 1994; Iwabuchi et al., 1994). The fourth ankyrin repeats of 53BP2 make contact with this region on p53 and its SH3 domain to the loop 3 region of p53 (amino acids 241-248) (Gorina and Pavletich, 1996). It is not known whether the p53 tumor-derived apoptosis mutants Pro-175 and Leu-181 still bind 53BP1 or 53BP2 (Gorina and Pavletich, 1996). The temperature-sensitive mutant p53Ala-143 binds 53BP2 at the permissive temperature, but not at the restrictive temperature (Gorina and Pavletich, 1996). The 53BP2 protein has been shown to bind protein phosphatase 1 ( P P l ) and inhibit its activity (Helps et al., 1995).This interaction may result in changes in p53 phosphorylation and regulation (Helps et al., 1995). It remains to be determined whether the binding of 53BP1 or 53BP2 to p53 plays a role in the modulation of apoptotic versus growth arrest genes. It is interesting that the p53-binding protein 53BP2 has been shown to bind Bcl-2 protein (Naumovski and Cleary, 1996). Overexpression of 53BP2 in cells does not induce apoptosis but causes a G2/M arrest (Naumovski and Cleary, 1996). Another potential target for p53 transactivation in apoptosis is the Fas/APO-1 receptor that is upregulated by p53 (Owen-Schaub et al., 1995). Expression of wild-type p53 in a colon tumor cell line resulted in increased anti-Fas antibody killing (Tamura et al., 1995). Engagement of the Fas/APO1 receptor by the Fas ligand or anti-Fas antibody will induce apoptosis such as in activated T lymphocytes (Nagata and Golstein, 1995). Many tumor types have been shown to express the Fas ligand, and this has been suggested as a tumor defense against infiltrating cytotoxic lymphocytes that express Fas (O’Connell et al., 1996; Saas et al., 1997; Niehans et al., 1997). Loss of wild-type p53 and the resulting downregulation of the Fas/APO-1 receptor

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would then prevent death being induced by cells within the tumor that also express the Fas ligand or to infiltrating cytotoxic lymphocytes.

C. Nontransactivator Function in Apoptosis by p53 The first evidence for a nontransactivator role for p53 in apoptosis came from experiments in SV40 virus large T antigen-transformed somatotropic cell lines (Caelles et al., 1994). Ultraviolet induction of apoptosis was dependent on wild-type p53 activity in these cells and could not be blocked by inhibition of RNA or protein synthesis. In another study, protein synthesis inhibition or cell cycle arrest did not block Myc oncogene-induced apoptosis, which was p53 dependent (Wagner et al., 1994). A carboxyl-terminal deletion mutant of p53, which is transactivation defective, can still induce apoptosis in the HeLa cell line (Haupt et al., 1995b). Also, a p53 transactivation mutant, consisting of substitution of amino acids Glu22,Ser23 in the transactivation domain, would still induce apoptosis in HeLa cells (Haupt et al., 199%). In contrast, a temperature-sensitive variant of the p53(Glu22, Ser23-Va1135) mutant failed to induce apoptosis in baby rat kidney cells (BRK) transformed with the E1A oncogene (Sabbatini et al., 1995). This transactivation mutant of p53 still induced growth arrest, mostly in the S phase (Sabbatini et al., 1995). A human tumor-derived mutant p53prol75 retains sequence-specific transactivation and growth arrest, but was defective for apoptosis induction and suppression of transformation by papilloma virus E 7 and Ras oncogenes (Rowan et al., 1996). These results suggest that the transactivation and induction of apoptosis functions of p53 can be separated genetically. It has been argued that the induction of apoptosis by transactivation-defective p53 mutants may be due to the presence of an endogenous wild-type p.53 in the cell lines used (Attardi et al., 1996). The mutant p53 may bind to the papilloma virus E6 protein in HeLa cells to release wild-type p53 from degradation. Alternatively, the mutant p53 may bind with the endogenous wild-type p53 and allow transactivation. Coexpression of the p53-binding protein mdm2, which inhibits p53 transactivation function, failed to block apoptosis induced by wild-type p53 in HeLa cells, although p53 transactivational activity was blocked (Haupt et al., 1996). mdm2 can block apoptosis in cell lines that require p53 transactivation to induce apoptosis (Haupt et al., 1996; Chen et al., 1996). This argues against the mutant p53 competing for binding to the E6 protein in HeLa cells and releasing the endogenous wild-type p53 protein. Transfection of the p53Gln22,Ser23 mutant into a cell line expressing wild-type p53 results in transactivation of a reporter gene containing a p53 promoter (Roemer and Mueller-Lantzsch, 1996).

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However, the p53Gln22,Ser23 mutant will induce a delayed apoptosis in the p53 minus Saos-2 cell line (Chen et al., 1996).A p53 carboxyl-terminal deletion mutant, defective for transactivation, will also inhibit colony formation in the Saos-2 cell line (Ishioka et al., 1995).p53 induces both apoptosis and growth arrest in Saos-2 cells (Marcellus et al., 1996; Rowan et al., 1996). Therefore, p53 transactivation-dependent and -independent pathways may both be needed for full apoptosis induction. The rate of apoptosis induced by the wild-type p53 is greater than the rate induced by the p53 transactivation mutants alone (Haupt et al., 1995b). Overall, these results suggest that, depending on the cell type and genetic background, the transactivating function of p53 may be needed for apoptosis induction. In other cell types already primed to undergo apoptosis, a nontransactivation function of p53 is sufficient. One possible activity that may play a role in apoptosis is the transcriptional repressor activity of p53. Blocking p53-dependent apoptosis by coexpression of adenovirus E1B 19k or Bcl-2 proteins results in the enhancement of p53-mediated transactivation and inhibition of the transcriptional repressor activity of p53 (Shen and Shenk, 1994; Sabbatini et al., 1995). It is not yet known how Bcl-2 or E1B 19K proteins inhibit the repressor activity of p53. Another study using various p53 mutants showed that the growth suppressing activity of p53 can be genetically separated from its apoptotic functions (Hansen and Braithwaite, 1996). This study also found that the transcriptional activation and repressor activities of p53 are both necessary for apoptosis induction. The Wilms’ tumor suppressor gene product binds p53 and blocks apoptosis (Maheswaran et al., 1995).This binding results in an increase in p53 transactivational activity, but reduces its repressor activity (Maheswaran et al., 1995). What are the target genes for p53 repression of transcription in apoptosis induction? p53 has been shown to negatively regulate the expression of the anti-apoptotic Bcl-2 gene (Miyashita et al., 1994a; Haldar et al., 1994).The 5’-untranslated region of the Bcl-2 gene is important for this p53-dependent repression (Miyashita et al., 1994a). Another target for the repressor activity of p53 is the IGF-1 receptor gene (Werner et al., 1996; Prisco et al., 1997). Loss of p53 activity through mutation results in increased IGF-1 receptors and protects tumor cells from apoptosis through IGF-1 binding to its receptor (Werner et al., 1996; Prisco et al., 1997). The P3 promoter of IGF-I1 is also inhibited by wild-type p53 (Zhang et al., 1996). Wild-type p53 suppresses the expression of the microtubule associated protein 4 gene (MAP4) in cells undergoing apoptosis (Murphy et al., 1996).Expression of the MAP4 gene in cells delays the onset of apoptosis (Murphy et al., 1996). Other studies using p53 mutants defective in transcriptional repressor activity have not shown a correlation with apoptosis. The transactivation-defective mutant p53 Gln22,Ser23 also has impaired repressor activity (Roe-

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mer and Mueller-Lantzsch, 1996), yet it still induced apoptosis in HeLa cells (Haupt et al., 199513). Fusion of the VP-16 transactivator domain to p53 amino acids 100-393 results in a p53 that is defective for transactivation but still able to repress transcription (Pietenpol et al., 1994). The VP-16 p53 fusion protein does not induce apoptosis in E l A-transformed fibroblasts (Attardi et al., 1996). In contrast, a transcription competent fusion mutant VP16 (80-393)~53could induce apoptosis (Attardi et al., 1996). Another p53 mutant could repress transcription and cause growth arrest, but was defective for apoptosis induction (Hansen and Braithwaite, 1996). These results suggest that repression of transcription by p53 may not be sufficient for apoptosis induction, but may enhance apoptosis under certain circumstances. Support for this view has been shown in a study using various p53 mutants that found that both transactivation and repression functions may be needed for apoptosis induction (Hansen and Braithwaite, 1996). It has been suggested that the interaction of p53 with XP-B and XP-D helicases that are members of the TFIIH transcription factorhepair complex may also play a role in apoptosis induction (for a review see Warbrick, 1996). p53 also binds to another member of the TFIIH complex, p62 (TFB1) (Xiao et al., 1994; Leveillard et al., 1996). TFIIH is a general transcription factor complex containing helicase and kinase activities (for a review see Orphanides et al., 1996). Some members of the TFIIH complex are the gene products of the human hereditary DNA repair disorders xeroderma pigmentosum (XP) and Cockayne’s syndrome (CS) (Seroz et al., 1995). Also associated with TFIIH is cyclin-dependent kinase 7 (CDK7)and its partner cyclin H, which is responsible for phosphorylating the carboxyl-terminal domain of RNA polymerase I1 (for a review see Nigg, 1996). It is also responsible for activating cyclin-dependent kinases (CAK),such as cdkl kinase (cdc2). In addition to its role in transcription, the TFIIH complex plays an important role in DNA excision repair, allowing the incision step to continue at the DNA lesion (Sancar, 1996). Both mutant and wild-type p53 proteins bind to the ERCC3 (XPD)and ERCC2 (XPB)helicases and inhibit both 5‘-3’ and 3’-5’ helicase activities in vitro (Wang et al., 1994; Leveillard et al., 1996). Fibroblasts from patients with mutations of the XP-D and XP-B genes showed reduced apoptosis after injection of p53 expression vectors compared with normal fibroblasts (Wang et al., 1996). This apoptosis-defective phenotype can be rescued by coexpression of the wild-type XP-B and XP-D genes (Wang et al., 1996).The XP-D and XP-B mutations did not prevent p53 transactivation of the WAFlICIPl gene (Wang et al., 1996). It is possible that the interaction of p53 with the TFIIH complex may influence the control of expression of apoptosis-inducing genes such as Bax or ZGFlBP3, which appear to have a different level of control. It has been shown that TFIIH will relieve p53-mediated transcription inhibition at the IgH promoter in uitro (Leveillard et d., 1996).

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The carboxyLterminal26 amino acids of p53 are needed for binding XPD and XP-B (Wang et al., 1995b), and microinjection of this region of p53 into cells can induce apoptosis (Wang et al., 1996). In contrast, transfection of a p53 gene containing only the first 214 amino acids into HeLa cells can still induce apoptosis (Haupt et al., 1995b). The alternatively spliced form of p53, which has the XP-D- and XP-B-binding domain removed, induces apoptosis, but with slower kinetics compared with the regularly spliced form of p53 (Almog et al., 1997). The effect of p53 interaction with TFIIH on excision repair is not clear at this time. Consistent with the inhibition of p53 of the XP-D and XP-B helicases, fibroblasts from Li-Fraumeni patients heterozygous for a p53 codon 145 mutation showed reduced repair of pyrimidine dimers (Wang et al., 1995b). Another study using Li-Fraumeni syndrome fibroblasts found that loss of p53 did not affect excision repair of pyrimidine dimers from the transcribed stand (transcription-coupled repair) (Ford and Hanawalt, 1995). p53-deficient cells showed a reduced removal of dimers from the nontranscribed strand, which is indicative of a global defect in excision repair (Ford and Hanawalt, 1995). This phenotype resembles xeroderma pigmentosum complementation group C (XP-C)cells (Venema et al., 1991; van Hoffen et al., 1995). Crosses of XP-C and p53-deficient mice result in a synergistic increase in severe solar keratosis and increased skin cancer after UV irradiation (Cheo et al., 1996).These mice also showed a variable spectrum of neural tube abnormalities (Cheo et al., 1996). This genetic interaction of p53 and XP-C suggests that they may be acting at a common DNA lesion or that the proteins may be in direct contact in the repair complex. The XP-C protein, along with its partner HHR23B, is needed for excision repair of pyriniidine dimers but not bulky DNA lesions in vitro (Mu et al., 1996). In transcribed DNA, the XP-C protein may not be needed for excision repair of lesions next to a stalled RNA polymerase (Sancar, 1996; Mu et al., 1996). Because of its nonspecific binding to single-stranded DNA, it was suggested that XP-C may play a role in stabilizing unwound DNA for incision and protecting the damaged DNA from nuclease digestion (Mu et d., 1996; Sancar, 1996).Interestingly, fibroblasts mutant for CS-A, CS-B, and XP-A, which are defective in preferential repair of the transcribed strand, show increased UV induction of p53 and apoptosis compared with XP-C and normal fibroblasts (Ljungman and Zhang, 1996). The stalling of RNA polymerase at DNA lesions was suggested as an important trigger for p53-dependent apoptosis induction (Ljungman and Zhang et al., 1996). It is not known whether p53 binding to the TFIIH repair complex at the stalled RNA polymerase complex plays a role in this induction. It has also been reported that Li-Fraumeni fibroblasts show a defect in long-patch excision repair after exposure to UV or y-radiation (Mirzayans et al., 1996). Other studies have failed to show a direct inhibition of excision repair by

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pS3 in uitro assays that is dependent on TFIIH activity (Leveillard et al., 1996; Sancar, 1996).Fibroblasts from p53 knockout mice do not show any differences in the removal of UV-induced pyrimidine dimers or (6-4) photo products compared with normal mice, but do show an increase in sister chromatid exchange (Ishizaki et al., 1994). Although p53 may not be directly involved in excision repair, its association with members of the TFIIH complex may play associated roles in DNA repair, such as repair of nontranscribed regions of the genome. More work is still needed to clarify the role of p.53 interaction with the TFIIH complex with respect to DNA repair, transcription, and apoptosis. Another region of pS3 implicated in nontranscriptional control of apoptosis by p.53 is the poly-proline region. Deletion of the PP region in the mouse pS3 gene is critical for the induction of apoptosis in E1A expressing cells (Sakamuro et al., 1997). This deletion does not affect the ability of pS3 to genes (Sakamuro et al., 1997). The inability of induce Bax or p21WAF1’CrP1 a p.53 transactivation region deletion mutant (AA 11-69) to induce apoptosis in HeLa cells (Yonish-Rouach et al., 1995) may be due to the partial removal of the PP region. These results show that a nontranscriptional activity of p53, presumably through binding to an SH3 containing protein, is necessary for pS3 induction of apoptosis and that transcription may be necessary but not sufficient for apoptosis induction. The identity of this protein(s) is unknown at this time. Whatever the mechanisms of p53-induced apoptosis, the events downstream from pS3 result in activation of the ZCEICed3 family of caspase proteases to manifest the late events in apoptosis (Sabbatini et al., 1997).

V. p53 AND TUMOR PROGRESSION

A. Apoptosis and Tumor Progression It is now accepted that tumor cell progression results from the accumulation of multiple mutations in both tumor suppressor genes and oncogenes. These mutations occur randomly, but to be selected for they must give a growth advantage to the tumor cell. The increased expression of many oncogenes or inactivation of tumor suppressor genes can make a cell susceptible to apoptosis that is dependent on p53. Expression of the adenovirus E1A oncogene (Lowe and Ruley, 1993; Debbas and White, 1993), inactivation of the retinoblastoma (Rb) gene by mutation or binding to the papilloma virus E7 protein (Morgenbesser et al., 1994; Pan and Griep, 1994; Almasan et al., 1995), expression of the E2F transcription factor (Ramqvist et al., 1993; Wu and Levine, 1994; Qin et al., 1994), and M y c oncogene expression

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(Ramqvist et al., 1993; Hermeking and Eick, 1994) can increase apoptosis in cells that can be attenuated by p53 inactivation. These genes either disrupt or bypass the RbE2F complex that controls GUS cell cycle progression (for review see Harper and Elledge, 1996). Release of the E2F transcription factor from the phosphorylated Rb protein allows activation of cell cycleregulated genes such as c-myc, dihydrofolate reductase, and thymidylate synthase (Lam and La Thangue, 1994). Consistent with this view is the finding that overexpression of the Rb protein can induce cell cycle arrest and prevent p53-dependent apoptosis (Qin et af., 1994; Haupt et af., 1995a). Disruption or Rb function allows cells to overcome p53 G1-dependent growth arrest (Slebos et al., 1994). It should also be noted that oncogene and Rbdependent apoptosis can also occur by a p53-independent mechanism (Teodoro et a1.,1995; Sakamuro et al., 1995; Macleod et al., 1996). The ability of p53 to prevent oncogene-mediated apoptosis was shown as an important selective advantage for tumor progression (Lowe et al., 1994b). Cells containing the E1A and Ras oncogenes in a wild-type p53 background still formed tumors in nude mice, despite showing increased apoptosis (Lowe et al., 1994b). Absence of p53 in these tumors resulted in shorter tumor latency. Loss of p53 in an in vivo choroid plexus tumor model induced by a truncated large T antigen that binds Rb protein but not p53 resulted in aggressive tumor outgrowths and reduced apoptosis (Symonds et af., 1994).In this tumor model, crossing of truncated large T antigen mice to Bax-deficient mice resulted in a more rapid tumor growth rate and reduced overall survival (Yin et al., 1997).However, this effect was not as great as seen in p53deficient mice. The apoptotic index was reduced by approximately 50% in CP tumors from Bax-deficient mice compared to 90% in tumors from p53deficient mice (Yin et af., 1997). These data suggest that Bax may be responsible for about 50% of the p53-dependent apoptosis seen in these tumors, but that other p53 targets are also important for apoptosis. These data support the idea that p53 inactivation is not essential for tumor growth, but that loss of p53 apoptotic function results in more aggressive tumor outgrowths in cells with oncogene activation or tumor suppressor loss. Environmental factors can also alter the response of a transformed cell to apoptotic signals. The presence of growthhurvival factors can greatly reduce p53 and oncogene-mediated apoptosis (Yonish-Rouach et al., 1991; Collins et af., 1992; Canman et af., 1995; Lin and Benchimol, 1995b).Transfection of oncogenes on growth factor signal transduction pathways such as Src, Ras, and Raf can rescue cells from p53-dependent apoptosis (Cleveland et al., 1994; Lin et al., 1995a; Canman et al., 1995). Interestingly, although oncogenes such as Ras can overcome apoptosis, it cannot overcome p53 growth arrest without the coexpression of the E l A gene (Lin et af., 1995a). Rat fibroblasts can be transformed with the papilloma E 7 and Ras oncogenes with or without p53 mutation (Peacock and Benchimol, 1994). Clones with

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a mutated p53 gene can grow in low serum conditions and respond to autocrine growth factors. In contrast, transformed cells with a wild-type p53 gene need external growth factors and do not respond to autocrine factors (Peacock and Benchimol, 1994). This difference in response to autocrine or external growth factors by tumors may be an important selective pressure for loss of p53 in vivo. Hypoxic conditions within the tumor may be another important environmental trigger for loss of p53 activity. p53 is induced by hypoxic conditions, and transformed cells lacking p53 are more resistant to hypoxia-induced apoptosis (Graeber et al., 1996).

B. Growth Control and Tumor Progression Another possible selection for loss of p53 in tumors is to overcome growth arrest caused by oncogene overexpression. Previous work had shown that the elevated Ras oncogene expression in the REF52 cell line induces a block in the cell cycle at the GUS and G2/M boundaries that can be overcome by large T antigen expression (Hirakawa and Ruley, 1988; Franza, J . et al., 1986). This Ras growth arrest was also associated with an accumulation of neutral lipids within cells (Hirakawa et al., 1991). Similarly, in Schwann cells, transfection of Ras can cause growth arrest at GUS and G2/M, and the SV40 large T antigen can overcome this growth arrest (Ridley et al., 1988). It has been shown previously that inactivation of p53 function through transfection of dominant-negative mutant p53 allows elevated Ras expression and increased tumorigenicity in REF52 cells (Hicks etal., 1991).In another study using mouse primary prostate cells, transfection of the Ras oncogene always resulted in selection for p53 mutations (Lu et al., 1992). In contrast, transfection of Myc and Ras oncogenes induced the formation of carcinomas that expressed elevated levels of the wild-type p53 protein (Lu et al., 1992).These Myc/Ras cells also showed evidence of increased apoptosis. Another study showed that transformation by EJ-Ras is susceptible to wild-type p.53 suppression (Hansen et al., 1995). It was also in this study that the function of large T antigen responsible for overcoming p53 suppression of transformation was the Rb-binding. In a MMTV-Ras transgenic mouse model, crosses to p53-deficient mice showed an increase in salivary tumors without an alteration of apoptosis rates, but increased genomic instability compared with Raslp53”’ mice (Hundley et al., 1997). It has been shown that Ras oncogene expression can induce growth arrest and premature senescence in primary fibroblasts (Serrano et al., 1997). This premature senescence could be overcome by inactivation of either p53 or cdk4 inhibitor ~ 1 6 in” rodent ~ ~cells. ~ In human primary cells, neither p53 nor ~ 1 6 inactivation ” ~ ~ was ~ sufficient to overcome the Ras growth arrest. However, E1A oncogene coexpression could overcome Ras-induced growth

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arrest in human cells (Serrano et al., 1997).It has also been shown that overexpression of the Mos oncogene and members of the MAP kinase pathway, including activated Ras, Raf, and M E K oncogenes, induce growth arrest and apoptosis in fibroblasts (Fukasawa and Vande Woude, 1997). This lethality was greatly reduced in fibroblasts from p53-I- mice. In primary rat Schwann cells, Raf oncogene expression induces growth arrest that is mediated by p21wA"1'c1p1, which can be overcome by mutant p53 or large T antigen (Lloyd et al., 1997). Collectively, these studies strongly suggest that there is a strong selective pressure to inactivate p53 in a cell that overexpresses activated members of the RasIMAP kinase pathway. This Ras-induced growth arrest resembles the premature senescence seen in normal diploid fibroblasts induced by y-radiation that is dependent on p53 (Di Leonardo et al., 1994; Linke etal., 1997).Radiation-induced growth arrest is presumably due to unrepaired double strand breaks causing p53 stabilization and p21WAF1/C1P1 induction or other p53-dependent factors (Di Leonardo et al., 1994; Linke et al., 1997). Induction of the cdk inhibitor p21WAF1/C1P1 (SDIl) was previously associated with senescent cells (Noda et al., 1994). It has been hypothesized that telomere erosion seen in aging cells may trigger p53 growth arrest (Bond et al., 1994; Wynford-Thomas et al., 1995). p53 arrest can be reversible when it is induced by a decrease in ribonucleotide pools or when using an inducible system (Agarwal et al., 1995; Linke et al., 1996). This suggests that p53 may not be inducing senescence directly, but that it is important for permanent growth arrest. In cases of unrepaired DNA breaks or constitutive Ras oncogene expression, this arrest becomes permanent, leading to senescence unless p53 is inactivated. It is not known at this time if Ras induces DNA breaks directly. However, several studies have shown that induced expression of members of the Ras/MAPK pathway can lead to genomic instability and apoptosis (Denko et al., 1994, 1995; Fukasawa and Vande Woude, 1997). Alternatively, high levels of constitutively activated Ras may elevate p53 or p161NK4activity directly. As shown previously, pS3 can be phosphorylated by MAP kinase (Milne et al., 1994). However, in normal nonimmortal cells, Ras signaling functions to cause phosphorylation of Rb protein and E2F release by altering cyclin Ddependent kinases and p161NK4(Peeper et al., 1997; Mittnacht et al., 1997). Understanding how high levels of constitutively activated Ras leads to p21WAF1/C1P1 and p 161NK4expression and growth arrest will be important for understanding the basis of oncogene cooperativity. The possible pathways for p53 tumor suppression are shown in Fig. 2.

C. Genomic Instability and Tumor Progression Germline inactivation of p53 on its own can also result in spontaneous tumors in mice or Li-Faumeni patients (Donehower et al., 1992; Jacks et al.,

p53 in Tumor Progression

41

Fig. 2 Possible pathways for the induction of the tumor suppressor function of pS3. Input signals to p53 are shown in italics and downstream pathways are shown in bold. Various inputs from activated oncogenes and DNA damage to hypoxia can activate p53 function to cause either growth arrest and senescence or apoptosis.

1994; Malkin, 1994). Spontaneous mutation of oncogenes in a cell without a functional p53 gene would have an automatic growth advantage compared with a cell with a wild-type p53 gene by the previously mentioned mechanisms. However, inactivation of p53 may also directly play a role in tumor progression through its role in controlling genomic stability. Loss of p53 has been previously associated with increased gene amplification (Yin et al., 1992; Livingstone et al., 1992). Increased expression and amplification of the Met and Myc oncogenes were seen in tumors and primary cells from p53deficient mice and Li-Fraumeni patients (Tainsky et al., 1995; Rong et al., 1995; Fukasawa et al., 1997). The increased aneuploidy found in pS3-Icells could reveal other tumor suppressor mutations through chromosome loss or increased oncogene expression due to a gain in chromosome numbers. The increased propensity for gene amplification seen in p53-deficient cells may also increase the likelihood of amplification of drug-resistant genes following chemotherapeutic treatment of tumors (Yin et al., 1992; Livingstone et al., 1992; Perry et ul., 1992).

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As knowledge of the p53 status in tumors becomes more routine in the clinic, knowledge of the context in which p53 mutations are found will be important. Depending on the order of oncogene activation, p53 may have been selected to prevent apoptosis or senescence in the tumor, which in turn may influence the response of the tumor to certain therapies. Therefore, the context in which pS3 mutations occur will be important to understand. Also, the many genetic variants within the tumor may lead to outgrowths of drugresistant or metastatic clones. It remains to be seen whether the knowledge of p53 status in tumors can be successfully exploited for treatment and if this will also lead to the discovery of new treatments or strategies. More research is still needed to give answers to these questions.

ACKNOWLEDGMENTS I thank my colleagues Sabine Mai, Jennifer Brown-Gladden, Dan Gietz, and Arnold Greenberg for comments and reading the manuscript. I also thank George Prendergast, Eileen White, and Sabine Mai for sharing papers before publication and Steve Linke for discussions via e-mail.

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Signal Transduction through MAP Kinase Cascades Timothy S. Lewis, Paul S. Shapiro, and Natalie G. Ahn Department of Chemistry and Biochemistry, Howard Hughes Medical Institute University of Colorado, Boulder, Colorado 80309

I. The MAP Kinase (MAPK) Module 11. Mammalian MAPK Pathways A. ERKlR and MKKlR Pathways B. Stress-Activated Protein Kinase Pathways 111. Regulation of MAPK Pathways by Protein Phosphatases A. Dual Specificity Phosphatases B. Serinenhreonine Phosphatases C. Protein Tyrosine Phosphatases IV. Cellular Substrates of MAP Kinases A. Protein Kinase Substrates for MAPKs B. Nuclear Transcription Factors C. Signaling Components D. Cytoskeletal Proteins V. Responses to MAPK Pathways: Growth and Differentiation A. Regulation of Cell Growth and Transformation B. Regulation of Cell Differentiation and Development VI. Yeast MAPK Pathways A. Saccharomyces cereuisiae (Budding Yeast) B. Schizosaccharomyces pombe (Fission Yeast) VII. Intracellular Targeting and Spatial Regulation of MAPK Pathway Components A. Signaling Complexes B. Nuclear Translocation of MAPK and MKK VIII. Future Directions References

I. THE MAP KINASE (MAPK) MODULE The identification of MAP kinase pathways exemplifies the power of combining biochemical and genetic approaches to molecular problems. Components in these pathways were first discovered in genetic studies of the pheromone-regulated mating response in Saccharomyces cerevisiae as genes that showed homologies with protein kinases (Errede and Levin, 1993). Although these enzymes could be ranked genetically, evidence of their protein kinase activities and the realization that they functioned in the context of a Advances In CANCER RESEARCH

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kinase cascade awaited biochemical studies of growth regulation in mammalian cells. As a result of earlier work establishing the importance of protein phosphorylation in cyclic nucleotide and calcium and phospholipid-regulated pathways, studies carried out during the 1970s focused on identifying kinases mediating growth factor and hormone action, particularly insulin. A key finding was the observation of growth factor-dependent phosphorylation of ribosomal protein S6 implicating factor-stimulated protein kinases in growth regulatory pathways. Combined with the discovery of tyrosine phosphorylation and receptor tyrosine kinases around 1980, protein kinase cascades in growth regulation were anticipated based on the paradigm of hormone regulated kinase cascades in the regulation of glycogen breakdown. Biochemical studies on hormone signaling in mammalian cell growth and oocyte maturation utilized strategies of cell stimulation followed by screening for regulated protein kinase activity. From these experiments, growth factor-stimulated pp90 ribosomal S6 kinases (Rsk)as well as MAP kinases, now known as ERKl and ERK2, were identified (Erikson and Maller, 1986; Ray and Sturgill, 1987; Jones et al., 1988; Hoshi et al., 1988; Ahn et al., 1990; Boulton et al., 1990), and the enzyme-substrate relationship between ERKs and Rsk was established (Sturgill et al., 1988; Ahn and Krebs, 1990). This was soon followed by biochemical identification of an upstream kinase regulator of ERK, called MAP kinase kinase (MKK), also known as MAPERK kinase (MEK) (Ahn et al., 1991; Gomez and Cohen, 1991). Molecular cloning of ERKsl/2 (Boulton et al., 1990, 1991) and MKKl (Crews et al., 1992; Kosako et al., 1993) established the homology of these enzymes with yeast FUS3KSSl and STE7, respectively, followed by confirmation that STE components were also regulated by direct phosphorylation. Homologous pathways have been identified in all eukaryotic organisms inspected so far. In nearly every case, a MAP kinase homolog regulated by a MKK homolog has been identified, thus the term “MAPK module” was coined to refer to the MAPIUMKK pair (Errede and Levin, 1993). At this point, evidence exists for at least three pathways involving MAPK modules in mammalian systems, five pathways in cerevisiae, and three pathways in Schizosaccaromyces pornbe (Figs. 1and 2). Functional MAPK modules have also been identified and characterized in frogs, fruit flies, nematodes, slime mold, and plants.

11. MAMMALIAN MAPK PATHWAYS In mammals, 11 distinct MAPK and 7 MKK genes have been identified to date. Members of the MAPK family include (i) extracellular signal-regulat-

cdl Growth and Differentiation

Stress Responses

Undefined

MEKK

MEK MAPK

\ J Mitosis. Meiosis. Diflerentiation, Development

I

1

1

Inflammation, Apcptosis

?

?

Fig. I Mammalian MAPK pathways. Positive signaling events are represented by solid pointed arrows (1)and inhibitory signaling is represented by blunted arrows (1) Signaling . events that are indirect or mechanistically uncharacterized are denoted by dashed arrows.

S. pornbe

S. cerevisiae Pheromone Response

Pseudohyphal or lnvasive Growth

a or a Factor

Nitrogen Starvation

High Osmolarity High Salt

Cell Wall Integrity

SpoNlatiOn

Hypotonic Shock Nitrogen Heat Shock Starvation Polarized Growth

Pheromone Response P or M

Stress Response

Cell Wall Integrity

Osmotic Stress Oxidative Stress

?

t

Cell Cycle Arrest

Mating Specific Genes

Filamentous Growth Genes

t

Osmotic Shock Genes

Cell Wall Biosynthesis Genes

Late Sporulation Genes

Mating, Stress Response, Cell Wall Sporulation Mating Specific Biosynthesis Genes Genes Genes?

Fig. 2 MAPK pathways of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Positive signaling is represented by The . dashed line signifies a protein-protein insolid pointed arrows ( 1) and inhibitory events are denoted by solid blunted arrows (1) teraction between STES and G, (STE4). The dashed pointed and blunt arrows in the HOG pathway represent inhibited two-component signaling among SLN1, YPD1, and S S K l as caused by high extracellular salt concentrations.

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ed kinases (ERKs) 1 and 2, (ii)NH,-terminal Jun kinasehtress-activated protein kinases (JNWSAPK) a, p, and y, and (iii) p38 MAPKs a,p, y, and 8. These are specifically recognized and phosphorylated by: (i) MKKl and MKK2 (also known as MEKs 1 and 2), (ii) MKK4 and MKK7, and (iii) MKK3 and MKK6, respectively. Less well characterized is ERK3, for which upstream regulators have not been identified, ERK4, a protein immunocrossreactive with antibodies to ERKs 1and 2, and ERK5 (also known as big molecular weight kinase, BMK), which associates with MKKS. MAPKs and MKKs are closely related to each other by sequence, suggesting that these multicomponent kinase cascades arose through gene duplication of the MAPK module. MAPKs are activated by MKKs through common mechanisms involving phosphorylation at two regulatory phosphorylation sites with sequence T(P)-X-Y(P) located in the “activation lip” between subdomains 7 and 8 of the conserved kinase core sequence. Thus MKKs fall within a relatively rare class of protein kinases with dual specificity toward Ser/Thr and Tyr residues on exogenous substrates. MKKs are also activated by phosphorylation at Ser/Thr residues within the activation lip. Unlike MAPKs, which are specifically recognized by their corresponding MKKs, each of the MKKs can be phosphorylated and activated by several different MAP kinase kinase kinases (MKKKs), including Raf family members, C-MOS,MEK kinases (MEKKs), and multilineage protein kinases (MLKs).These MKKKs recognize different MKKs, enabling diversity in the activation of MAPK pathways upstream of MKK. As required for switching mechanisms, MAPK cascades transform graded effector signals into cooperative responses. This is a theoretical consequence of pathways involving sequential protein kinases, each regulated by dual phosphorylation, as demonstrated by Huang and Ferrell (1996b).

A. ERK1/2 and MKK1/2 Pathways ERKl and ERK2 and their upstream regulators MKKl and MKK2 are acutely stimulated by growth and differentiation factors, in pathways mediated by receptor tyrosine kinases, heterotrimeric G protein-coupled receptors, or cytokine receptors. At the moment, it is unclear why two forms of each enzyme exist, although conservation of two forms throughout eukaryotic species suggest nonredundant functions. These enzymes are expressed ubiquitously in mammalian cells at micromolar levels (Huang and Ferrell, 1996b), although some variation in expression between different tissues has been noted (Boulton and Cobb, 1991; Moriguchi et al., 1995a). 112 vitro, MKKl and MKK2 show comparable activity toward ERKl and ERK2 (Zheng and Guan, 1993a; Dent et al., 1994). A few examples exist where

Timothy S.Lewis et al.

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ERKl and ERK2 are activated with distinct magnitudes or kinetics following factor stimulation of intact cells (Papkoff et al., 1994; Kalb et al., 1996); however, in most instances, both enzymes are robustly activated on cell stimulation. 1.

ERKl AND ERK2

ERKl and ERK2 are 44- and 42-kDa enzymes, respectively; in mammals, these have 90% sequence identity (Boulton et al., 1991). In addition to the conserved catalytic core, both enzymes contain C-terminal extensions and a 25 amino acid insert between subdomains 9 and 10. Both enzymes are activated by phosphorylation within their activation lip at Thrl8,-Glu-TyrI8, (numbered as in rat ERK2) in a reaction that is partially ordered with Tyr preceding Thr phosphorylation (Haystead et al., 1992). Both phosphorylation events are required to fully activate wild-type ERKl and ERK2, although 10% activation can be achieved with mutants containing a Thr18,-Glu amino acid substitution, presumably due to the substitution of this phosphorylation site with a negatively charged residue (Robbins et al., 1993).Activation by phosphorylation leads to increased V, and decreased Km for peptide substrate, with little effect on KmATp(Robinson etal., 1996b). The crystal structure of ERK2 in its inactive, unphosphorylated form was solved by Goldsmith and colleagues to 2.3 A resolution (Zhang et al., 1994). Similarities within the conserved kinase domain to the structures of other protein kinases were observed, with the inclusion of an additional P-sheet structure at the N-terminal lobe (residues 1-20), a novel linker between p4 and PS (residues 91-96), and an a-helical insert between helices G and H in the C-terminal lobe (residues 247-266). Thirty-five C-terminal amino acids wrap in a random coil spanning the C-terminal to N-terminal lobes on the side of the molecule opposite to the catalytic cleft. Recombinant C-terminal residue truncations of ERK2 are poorly expressed (J. Means and N. Ahn, unpublished observations), suggesting that this region may play a role in enzyme stabilization. The two domains of ERK2 are rotated into an open configuration with respect to each other, and the activation lip is distorted with access to the catalytic cleft blocked by the Tyrlss phosphorylation site, thus providing an understandable rationale for the low basal activity. In this state, TyrlS5 is buried whereas Thr,,, is solvent accessible, suggesting that MKK binding induces a major conformational change in ERK to expose and enable preferential phosphorylation of the Tyr residue. Interestingly, substitution of Tyrls5 with acidic amino acids led to marked disorder within the activation lip (Zhang, F. et al., 1995). A model to account for these observations postulates induced fit binding of MKK and initial TyrlFs phosphorylation on ERK followed by substantial reconfiguration of the lip on Thr,,, phosphorylation. This stabilizes interactions between phosphorylated

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residues and catalytic site residues that favor the active conformation (Zhang, F. et al., 1995). Crystallographic determination of the activated ERK2 phosphorylated at Thr,,, and Tyrl,, supports this prediction by showing a reorientation of the activation lip, with phosphorylated Thr, bridging residues from both N- and C-terminal lobes and phosphorylated Tyr,,, forming the substrate binding pocket (Canagarajah et al., 1997).

,,

2. M K K l

AND MKK2

MKKl and MKK2 are both 44-kDa enzymes, related by 80% sequence identity (Zheng and Guan, 1993a; Wu et al., 1993a). Sequences outside the conserved catalytic core of both enzymes include an additional 60 amino acids at the N terminus and a 40 amino acid insert between subdomains 9 and 10. Activation occurs through phosphorylation at the sequence Ser,,8(P)-Met-Ala-Asn-Ser222(P) (numbered as in human MKKl ) within the activation lip (Zheng and Guan, 1994a; Alessi et al., 1994; Gotoh et al., 1994; Resing et al., 1995). Unlike ERKs, MKKs can be partially activated by phosphorylation at either serine phosphorylation site (Gotoh et al., 1994; Resing et al., 1995). In addition, substitution of these sites by acidic amino acids enhances the basal activity (Zheng and Guan, 1994a; Alessi et al., 1994; Cowley et al., 1994; Pages et al., 1994; Mansour et al., 1994a). Further enhancement of activity on substituting adjacent nonphosphorylatable residues with acidic amino acids indicates that the activation can be, in part, ascribed to electrostatic effects of the negatively charged residues (Mansour et al., 1996a). The N terminus of MKK outside the conserved kinase subdomain appears to be important in regulating MKK activity, as deletion of residues 44-51 or proline substitution within this region leads to significant activation (Mansour et al., 1994a, 1996a; Bottorff et al., 1995). Alignment of this domain with the A-helix of CAMP-dependent protein kinase suggests potential long range interactions with the activation lip (Herberg et al., 1997). This is supported by the synergistic enhancement of rates between Nterminal deletion and phosphorylation site mutations (Mansour et al., 1996a), as well as by deuterium exchange studies showing that both types of mutations enhance the flexibility within the N-terminal ATP-binding lobe (Resing and Ahn, 1997). Sequences in MKKl and MKK2 inserted between subdomains 9 and 10 of the conserved core are proline rich, containing consensus sequences for potential SH3 domain interactions. In MKK1, this domain is important for interactions between MKK and Raf-1 (Jelinek et al., 1994; Papin et al., 1996). Deletion of these residues in a constitutively active MKK background interferes with ERK activation and growth signaling in cells (Catling et al., 1996), suggesting that this domain is involved in substrate recognition in vivo, despite the fact that the deletion mutants still phosphorylate ERK in vitro. In

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MKKl, the insert contains four phosphorylatable residues that are targets for autophosphorylation ( Ser,,,, Tyr300)as well as heterologous phosphorylation by ERK1/2 and cyclidcdk2 (Thr2,6, Thr,,,) or PAKl (Ser298) (Brunet et al., 1994a; Saito et al., 1994; Mansour et al., 1994b; Rossomando et al., 1994; Resing et al., 1995; Frost et al., 1997). In MKK2, phosphorylatable residues are only found at Thr,,, and Ser,,,. Phosphorylation of these sites could potentially mediate MKURaf-1 interactions (Jelinek et al., 1994), feedback regulation of MKK activity by ERK (Brunet et al., 1994a), or cross-regulation of MKKl with PAK-dependent signaling pathways (Frost et al., 1997). In vitro, MKKl and MKK2 are targets for phosphorylation and activation by at least three protein kinase families. Raf-1 and B-Raf phosphorylate MKKl and MKK2, whereas A-Raf is a weak activator of MKK1/2 in intact cells and shows greater selectivity for MKKl in vitro (Wu, X. et al., 1996; Pritchard et al., 1995).MKKl and MKK2 are also activated by the germ cellspecific protein kinase, C-MOS,which controls meiotic cell division (Posada et al., 1993; Nebreda et al., 1993). Because MEKK and several isoforms are potent regulators of stress-activated protein kinase pathways, the discussion of MEKKs will be deferred to the next section. However, MEKKs and Tpl2 phosphorylate MKKl in vitro and under some conditions of expression in vivo (Yan and Templeton, 1994; Xu et al., 1995; Blank etal., 1996; Salmer6n et al., 1996). These enzymes phosphorylate both of the serine residues within the activation loop, although Mos preferentially phosphorylates Ser222, MEKKl and STEll prefer Ser218,and Raf-1 phosphorylates both sites equally well (Yan and Templeton, 1994; Alessi et al., 1994; Gotoh et al., 1994; Resing et al., 1995).In vitro, this activation is followed by intramolecular autophosphorylation at Thr23, Ser298,and Tyr300(Resing et al., 1995).

3. Raf Raf can be regulated by several mechanisms, the combination of which leads to full activation by growth regulatory signals. In addition to the Cterminal catalytic domain (amino acids 335-627), Raf-1, A-Raf, and B-Raf contain N-terminal regulatory domains within a conserved region 1 (CR1, amino acids 61-194 numbered as in Raf-1), which include a Ras- binding domain (RBD, amino acids 51-131) and a cysteine-rich domain containing a zinc finger motif (amino acids 139-184). A conserved region 2 (CR2, amino acids 254-269) and the very C terminus are important for binding the regulatory 14-3-3 protein. A basic model for Raf-1 regulation involves membrane recruitment, enabling its interactions with upstream regulators. Raf-1 recruitment is mediated by Ras-GTP (Marais et al., 1995), involving interactions between the Ras-binding and Cys-rich domains of Raf, respectively, with Switch I (amino

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acids 30-38) and Switch I1 (amino acids 60-76) effector regions of Ras-GTP (Moodie et al., 1993; Wojtek et al., 1993; Hu, C. et al., 1995; Brtva et al., 1995; Drugan et al., 1996). The GTP/GDP exchange of p21Ras is catalyzed by Ras guanine nucleotide exchange factors, including mammalian son-ofsevenless (Sos). Growth factor stimulation of GTP/GDP exchange entails recruitment of Grb2-Sos to membrane-bound Ras via direct interactions between receptor and Grb2 or indirect interactions through adaptor proteins such as Shc or IRS-1 (reviewed by Pawson, 1995). Disruption of either interaction leads to the suppression of transformation by v-ras, thus both interactions are important for Ras signaling (Fridman et al., 1994; Brtva et al., 1995). The importance of membrane recruitment was illustrated by the observation that membrane targeting of Raf-1 by C-terminal farnesylation or N-terminal myristylation transforms cells without participation of Ras (Leevers et al., 1994; Stokoe et al., 1994). A cocrystal structure of Raf-RBD and Rap complexed with a nonhydrolyzable GTP analog reveals close contacts between residues in the Raf-RBD and residues conserved between Ras and Rap (Nassar et al., 1995), suggesting how analogous interactions between Ras and Raf might be structured. Phosphorylation of Raf-1 appears to be a key regulatory mechanism. After purification from Sf9 cell expression, Raf-1 is phosphorylated at Ser,,, Ser,,,, Ser,21, Tyr340and Tyr341, and autophosphorylated at Thr,,, (Fabian et al., 1993a; Morrison et al., 1993). Raf-1 activation appears to require phosphorylation at one or both tyrosine residues because mutation of these sites blocks activation by Ras and because protein tyrosine phosphatases inactivate Raf-1 (Dent et al., 1995; Jelinek et al., 1996). Likely candidates for regulatory tyrosine kinases are Src and Fyn, based on studies demonstrating Raf-1 activation on coexpression with Src in a manner dependent on Tyr340/341(Fabian et al., 1993; Cleghon et al., 1994; Marais et al., 1995). Raf-1 activation by Src requires a functional RBD, suggesting that membrane recruitment is required for tyrosine phosphorylation (Marais et al., 1995). Ras-Raf interactions are not as important in Sf9 coexpression systems, presumably due to high expression levels (Fabian et al., 1994). Other candidates are the JAK tyrosine kinases, which appear to regulate Raf-1 in systems involving signaling through receptors for cytokines such as erythropoetin, interferon-y, and growth hormone (Miura et al., 1994; Winston and Hunter, 1995; Xia et al., 1996). JAK2 directly phosphorylates Raf-1 in vitro, although Tyr340/341account for only 75% of the incorporated label from 32P-ATP (Xia et al., 1996). Involvement of serinekhreonine kinases is indicated by reports of Raf-1 activation following its direct phosphorylation by protein kinase C (PKC) at Ser,,, (Sozeri et al., 1992; Kolch et al., 1993; Carroll and May, 1994)or by a ceramide-activated protein kinase at Thr,,, (Yao et al., 1995). Inhibition of Raf-1 on phosphorylation by CAMP- dependent protein kinase at Ser,, or Ser,,, has also been reported (Wu et al., 1993b;

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Mischak etal., 1996);phosphorylation at Ser,,, may be required for Raf activation through its interaction with 14-3-3 (Muslin et al., 1996). ERK also phosphorylates Raf-1, with no clear-cut effect in vitro, although it may suppress Raf-1 activity in viuo (Anderson et al., 1991; Ueki et al., 1994). In support of this, cellular stimulation of ERK leads to retardation of Raf gel mobility, which correlates with Ser621,624phosphorylation and lowered Raf activity (Ueki et al., 1994; Ferrier et al., 1997). Raf-1 heterocomplex formation has been proposed to be an important aspect of regulation. Physiologically, Raf-1 oligomerization may be accomplished through its interaction with 14-3-38, q, 5 and 0 (Fantl et al., 1994; Fu et al., 1994; Irie etal., 1994; Aitken et al., 1995; Papin et al., 1996),members of a large conserved acidic protein family that interact with many targets, including Raf-1, B-Raf, Bcr, polyomavirus middle T antigen, cdc25 phosphatases, phosphatidylinositol 3' kinase, Cbl, and protein kinase C-8 (reviewed by Aitken 1995; Meller et al., 1996). Residues within the C terminus of 14-3-3 interact with CR2 and the C terminus of Raf-1, recognizingphosphoserine at SerZs, and Ser,,, (Luo etal., 1995; Muslinetal., 1996). The importance of this interaction on Raf-1 regulation is indicated by several studies demonstrating activation of Raf-1 by 14-3-3 on coexpression in cells or in cell-free extracts (Freed et al., 1994; Fantl et al., 1994; Fu et al., 1994; Irie et al., 1994; Li et al., 1995). However, activation has not been observed in all cases (Michaud et al., 1995; Suen et al., 1995), and, in fact, 143-3 binding may stabilize an inactive form of Raf, based on Raf activation by disruption of 14-3-3 interactions. The ability of 14-3-3 to dimerize raises the possibility that 14-3-3 proteins may mediate the heterocomplex formation of Raf-l with other signaling molecules. For example, Raf-l association with Bcr has been demonstrated, mediated by their mutual interaction with 14-3-3 (Braselmann and McCormick, 1995). A 14-3-35 mutant that is unable to dimerize binds efficiently to Raf-1, but favors the inactive form of the kinase, suggesting that dimerization may be needed for Raf activation (Luo et al., 1995). Consistent with this, activation of basal Raf-1 and MKK1/2 activity was demonstrated on homodimerization of FKBP-Raf-Ucyclophilin or bacterial DNA gyrase-Raf-1 chimeras in response to bifunctional ligands FK1012A or coumermycin, respectively (Luo et al., 1996; Farrar et al., 1996). In vitro, Raf-1 elutes by gel filtration within protein complexes greater than 300 kDa containing several proteins with chaperone function, such as hsp90, Cdc37, and hsp56 (Wartmann and Davis, 1994; Stancato et al., 1994). Formation of hsp90 complexes appears to be important for Raf-1 signaling because the hsp90 ligand, geldanamycin, blocks growth factor signaling through the disruption of Raf-UMKK complexes (Schulte et al., 1996). However, no evidence exists for growth factor regulation of Raf-l:hspSO association or dissociation. Most likely, hsp90 interacts with Raf during folding, although hsp90 and 14-3-3 binding to Raf

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also protect against Raf-1 dephosphorylation and inactivation by PTPlB (Jelinek et af., 1996). Two additional interacting proteins may be important for Raf-1 activation. The kinase suppressor of Ras (KSR), a protein kinase closely related to Raf, was identified as a positive modifier of Ras signaling in Drosophila melanogaster and Caenorhabditis elegans (Kornfeld et al., 1995a; Sundaram and Han 1995; Therrien et af., 1995). Although direct regulation of Raf-1 by KSR has not been observed, biochemical experiments show association of human KSR with Raf, MKK1, and ERK and enhancement of signaling through the MKKERK pathway on coexpression with v-Ras in mammalian cells or Xenopus oocytes (Therrien et al., 1996). Direct stimulation of Raf1 by BAG-1, a protein that binds to and enhances the antiapoptotic effects of Bcl-2, has been demonstrated in vitro (H. H. Wang et al., 1996). Thus, BAG-1 may bind to and recruit Raf to Bcl-2. Involvement of Raf-1 in Bcl-2 regulation of apoptosis is suggested by biochemical interactions between Raf-1 and Bcl-2 and the observation that Bcl-2 and the proapoptotic Bc12 homolog, BAD, are both substrates for phosphorylation by Raf-1 (Wang et af., 1994; Blagosklonny et al., 1996; Zha et af., 1996). Other potential membrane activators of Raf-1 include lipids and lipid metabolites. In vitro, Raf-1 can be activated by direct phorbol ester binding, mediated through its zinc finger domains, which are homologous to those on several PKC isoforms (Luo et al., 1997). Raf-1 also binds to phosphatidylserine, phosphatidic acid, and ceramide (Ghosh et af., 1996; Huwiler et al., 1996). Binding of ceramide enhances Raf-1 activity in vitro and in vivo, and inhibition of phosphatidic acid synthesis in cells blocks Raf-1 membrane translocation. Finally, Raf-1 activation has been shown under certain conditions to be retarded by the inhibition of phosphatidylinositol 3' kinase, suggesting a possible role for lipid products of this enzyme in regulating Raf1 activity (Cross et af., 1994; J. Huang et al., 1995). In contrast to Raf-1, which is ubiquitous, other forms of Raf show more restricted tissue expression. A-Raf is mainly expressed in steroid responsive ( e g , urogenital) tissues (Winer and Wolgemuth, 1995; Lee et al., 1996).Several isoforms of B-Raf exist, many of which are selectively expressed in neuronal tissue (Barnier et af., 1996). Comparison of B-Raf to Raf-1 reveals similar but nonidentical mechanisms of regulation. Like Raf-1, B-Raf interacts with Ras-GTP and forms large molecular weight complexes that include hsp90 and 14-3-3 (Moodie et af., 1994; Jaiswal et al., 1996; Papin et al., 1996);however, unlike Raf-1, B-Raf complexed with 14-3-3 proteins retains its activity (Yamamori et al., 1995). B-Raf has a higher basal activity than Raf-1,ascribed to negatively charged Asp residues at positions equivalent to Tyr340and Tyr341 in Raf-1, and it is not as highly phosphorylated on tyrosine in response to factor stimulation, although it is still phosphorylated on Ser/Thr (Stephens et af., 1992; Jelinek et af., 1996). Moreover, B-Raf is stim-

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ulated by interaction with Rapl-GTP (Ohtsuka et al., 1996; Vossler et al., 1997), whereas RaplB-GTP binds but does not lead to Raf-1 activation (Zhang et a/., 1993). Dependence on phospholipid activators also differ; thus, phosphatidylserine stimulates B-Raf activity in a Ras-dependent manner, whereas phosphatidylinositol, phosphotidylserine, and phosphatidylethanolamine all inhibit RaplB-stimulated B-Raf activation (Kuroda et al., 1996). 4. Mos

The serinekhreonine protein kinase c-Mos regulates meiosis in vertebrate oocytes (Singh and Arlinghaus 1992; Yew et al., 1993). Its specific expression in germ cells is regulated by somatic cell repressor DNA-binding elements within the Mos promoter (Xu and Cooper, 1995), although somatic cell expression has also been reported (Leibovitch et al., 1993; Gao et al., 1996). Xenopus laevis oocytes stimulated to undergo meiotic cell division upregulate Mos protein levels by cytoplasmic 3’ polyadenylation and stabilization of message (Sheetset al., 1994,1995; Gebauer et al., 1994; StebbinsBoaz et al., 1996). Meiotic cell division, which can be induced by injection of Mos mRNA or protein into oocytes, requires activation of MKK and ERK; these are maximally active at metaphase I and I1 (Kosako et al., 1994a; Gotoh et al., 1995a; Roy et al., 1996). In vitro, Mos phosphorylates MKKl at regulatory Ser,,, and Ser,,, phosphorylation sites (Posada et al., 1993; Nebreda et al., 1993; Resing et al., 1995), and maturation through both meiosis I and I1 can be mimicked by the injection of constitutively active mutant MKKl or thiophosphorylated ERKl (Haccard et d., 1995; Gotoh et al., 1995a), thus MKK is a key target for Mos in this process. Regulation of Mos- or progesterone-dependent meiosis also involves Raf-1 activation, as indicated by the interference of germinal vesicle breakdown on the expression of inactive dominant negative Raf mutants (Muslin et al., 1993; Fabian et al., 1993); however, the ability of both Raf-1 and Mos to directly activate MKK suggests convergence of Raf and Mos signaling rather than sequential activation. Maturation of oocytes into meiosis I1 is inhibited by cycloheximide. This is due in part to a requirement for further synthesis of Mos protein, in a secondary induction that appears to require ERK activity (Yewet al., 1992; Gotoh et al., 1995a; Roy et al., 1996; Matten et al., 1996). Phosphorylation of Mos at Ser, enhances Mos protein levels (Nishizawa et al., 1992) and may lead to stable MKK interactions (Chen and Cooper, 1995). Ser, is phosphorylated by ERK in vitro, suggesting that in addition to message stabilization, Mos is regulated by positive feedback involving direct phosphorylation by ERK (Matten et al., 1996). Importantly, Mos functions as a cytostatic factor, elevating MPF activity and thus maintaining metaphase I1 arrest prior to fertilization. Following fer-

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tilization, calpain-mediated degradation of Mos is followed by a rapid decay of ERK activity, releasing cell cycle arrest. That ERK is a key component of cell cycle arrest is indicated by the cytostatic arrest of two cell embryos on microinjection of constitutively active MKK or thiophosphorylated ERK (Haccard et al., 1993; Kosako et al., 1994b). Targeted gene disruption of Mos in mice yields mouse oocytes that are unable to arrest in metaphase I1 and undergo parthenogenetic activation (Colledge et al., 1994; Hashimoto et al., 1994). Based on studies examining the effects of Mos on meiosis, ERK is implicated in metaphase arrest, meiotic spindle formation, and polar body degradation, and in the prevention of pronuclear envelope formation until fertilization (Verlhac et al., 1993; Moos et al., 1996; Choi et al., 1996 a&). MPF activation by Mos likely involves protection of cyclin B from proteolysis. Although some studies have shown suppression of cdc2kyclinB activation by dominant-interfering ERK mutants, the results have been mixed and a direct link with the MAPK pathway has not been established (Nebreda and Hunt, 1993; Pomerance et al., 1996; Huang and Ferrell 1996a). However, egg extracts induced to undergo spindle checkpoint arrest show high ERK activity and cyclin B levels. Arrest is released on inactivation of ERK by MAP kinase phosphatase-1, suggesting that ERK may also affect the mechanisms involved in cyclin degradation (Minshull et al., 1994).

5. SIGNALING THROUGH INTRACELLULAR CALCIUM MKK/ERK can be stimulated by elevation of intracellular calcium with calcium ionophores, thapsigargin, elevated extracellular calcium, or membrane depolarization (Chao et al., 1994; Rosen et al., 1994; S. Huang et al., 1995; Kurino et al., 1995; Bogoyevitch et al., 1996). In many cases, activation of Raf-1 and/or Ras in addition to MKKERK can be demonstrated, suggesting regulation of targets upstream of Ras. One candidate is Pyk2, a member of the focal adhesion kinase (FAK)family of nonreceptor protein tyrosine kinases, which is expressed in neuronal cells and tyrosine phosphorylated in response to calcium influx and membrane depolarization (Lev et al., 1995; Dikic et al., 1996).Forced expression of Pyk2 activates ERK, and dominant negative mutant Pyk2 blocks ERK activation by carbachol. Pyk2 also associates with Grb2 and Sos in intact cells following membrane depolarization or carbachol treatment of PC12 cells, suggesting a mechanism for its activation of Ras. An alternative (but potentially related) means of activating MKK/ERK by calcium involves Ca2+-calmodulin (CaM)-dependent protein kinases. Overexpression of Ca2+-CaM kinase IV in combination with its activator, CaM kinase kinase, led to a two- to fivefold activation of several MAPKs in PC12 cells, including JNWSAPK, p38, and ERK2 (Enslen et al., 1996). An involvement of Ca2+-CaMkinase I1 in the regulation of MKWERK is also sug-

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gested by the inhibition of norepinephrine activation of ERK by the Ca2+CaM kinase I1 inhibitor KN-93 in aortic smooth muscle cells (Muthalif et al., 1996). However, the involvement of calmodulin-regulated kinases in activating upstream regulators such as Raf-1 or Ras has not been established.

6. SIGNALING THROUGH HETEROTRIMERIC G PROTEINS Activation of MKWERK by heterotrimeric Gai, Gao, or Gaq proteincoupled receptors occurs in response to many agonists (e.g., lysophosphatidic acid, carbachol, endothelin, prostaglandin F,, bombesin, and thrombin). Overexpression of constitutively active GTPase-deficient Gai, Ga,, or G, mutants in various cell lines also leads to MKK/ERK activation (Gupta eta?., 1992; Faure et al., 1994; Pace et al., 1995). G,, subunits appear to be involved in the mechanism of Gai-coupled ERK regulation because activation can be suppressed by peptides corresponding to the C-terminal py-binding domain of the P-adrenergic receptor kinase (Koch et al., 1994) and because ERK is activated on expression of GP, subunits (Faure et al., 1994; Crespo et al., 1994; Ito et al., 1995; Hawes et al., 1995). G subunits appear to activate Ras, Raf, MKK, and ERK through a Pd?-independent mechanism that involves tyrosine phosphorylation of Shc and signaling to Ras through Shc-Grb2-Sos interactions (Van Biesen et al., 1995; Hawes et al., 1995). The tyrosine kinases Src and Syk are implicated in this process (Luttrell et al., 1996; Wan et al., 1996). In addition, the sensitivity of this pathway to wortmannin suggests a role of phosphatidylinositol 3' kinase downstream of G,, and upstream of Ras (Hawes et al., 1996). G-proteincoupled a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) activation of ERK resulted in association of G,, with a complex containing Ras, Raf, MEK, and ERK (Wang and Durkin, 1995), indicating that heterocomplex formation may occur between kinases and heterotrimeric G proteins. In contrast to Gai, stimulation of MKKERK through Gaq-or Gao-coupled receptors is independent of G, or Ras and instead activates Raf/MKK/ERK through a PKC-dependent meckanism (Van Biesen et al., 1996; Hawes et al., 1996). This may be a cell type-specific result because signaling through Gag in cardiac myocytes or CCL39 fibroblasts led to Ras/Raf activation through Shc-Grb2-Sos interactions in a PKC-independent manner (Y. Chen et al., 1996b; Sadoshima and Izumo, 1996). Candidate tyrosine kinase mediators of ERK activation through Gffqcoupled signaling are Lyn or Syk (Wan et al., 1996). Pyk2 appears to play an important role in heterotrimeric G protein signaling as well because ERK stimulation by agonists of Gai- or Gaq-linked pathways was suppressed by the dominant negative mutant Pyk2 in a process dependent on Src activation (Dikic et al., 1996). Under these conditions, Grb2 and Sos were required for ERK activation, indicating that signaling is Ras dependent.

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Gas-mediated signaling through CAMP-dependent protein kinase suppresses MKWERK activation in many systems (Wu et al., 1993b; Sevetson et al., 1993; Graves et al., 1993; Cook and McCormick, 1993; Hordijk et al., 1994), providing an explanation for the commonly observed antagonistic effects of cAMP on cell growth. One target for CAMP-dependent protein kinase inhibition is Raf-1, which is directly phosphorylated at Ser,, and Ser,,, in vitro (Wu et al., 1993b; Mischak et al., 1996).However, inhibition of ERK signaling is not observed in all cells and cAMP modulates the kinetics but not the magnitude of ERK activation under some conditions (McKenzie and PouyssCgur, 1996). In PC12 cells, agonists of cAMP signaling actually stimulate MKKERK (Frodin et al., 1994; Young et al., 1994; Vossler et al., 1997). This appears to result from the predominant role of B-Raf in regulating the MKKERK pathway in PC12 cells in that cAMP activates guanine nucleotide exchange of Rapl, which in turn activates B-Raf in a Rasindependent manner (Vossler et al., 1997).

7. SIGNALING THROUGH PROTEIN KINASE C A long-standing problem that is still unresolved is the mechanism by which PKC regulates MKK and ERK. Phorbol esters activate ERK in nearly every cell, and many reports exist of agonist stimulation of ERK that is at least partially blocked on PKC inhibition by extended phorbol ester treatment, low molecular weight inhibitors, or dominant negative PKC mutants (Hoshi et al., 1989; Ueda et al., 1996; Hundle et al., 1995; Berra et al., 1995). In lymphocytes, phorbol 12-myristate 13-acetate (PMA) leads to Ras-GTP elevation (Downward et al., 1990), and activation of ERK by PKC in broken cell extracts has been shown to require Ras (VanRenterghem et al., 1994).Therefore, one effect of PMA is to probably regulate Raf through the activation of Ras. The Pyk2 tyrosine kinase is a candidate for a Ras activator because it is also stimulated by phorbol ester (Lev et al., 1995).This may explain why PMA-dependent ERK activation is sensitive to tyrosine kinase inhibitors (Seger et al., 1995). However, Ras-independent activation of Raf-1 has also been observed (Ueda etal., 1996).An alternative mechanism to account for this involves direct phosphorylation of Raf by PKC-y, resulting in Raf-1 activation in vitro, as mentioned earlier (Sozeri et al., 1992; Kolch et al., 1993; Carroll and May, 1994). Interestingly, a Raf-1 chimera substituting the zinc finger domain with the analogous region from PKC is strongly responsive to phorbol ester in a manner that is unaffected by phorbol ester downregulation of PKC, suggesting that the zinc finger in Raf may be involved in the response to PMA (Luo et al., 1997). Although a direct comparison of the effects of individual PKC isoforms on MKWERK activation is still lacking, various reports have documented the involvement of PKC a,PI, y, 6 , E, and L in MKWERK activation (Ueda et al., 1996; Yamaguchi et al., 1995a; Hundle et al., 1995; Berra et al., 1995; Young et al., 1996).

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8. SIGNALING THROUGH JAWSTAT PATHWAYS Cytokine signaling pathways lead to interactions between receptors and members of the Janus family of tyrosine kinases (JAKs1, 2, and 3, TYK2), resulting in transphosphorylation and activation of kinase activity. STAT transcription factors are recruited to the receptor complex via SH2 domain interactions where they become tyrosine phosphorylated and dimerize through intermolecular interactions between N-terminal SH2 domains and C- terminal phosphotyrosine motifs (reviewed by Ihle, 1996). The resulting hetero- or homodimers translocate to nuclei where they mediate transcription by interaction with DNA response elements within cytokine-regulated enhancers, such as the interferon-? site (GAS) response element. The cytokine regulation of MKWERK in some cases may be explained by Janus kinase activation because growth hormone stimulation of ERK2 is blocked by dominant interfering mutants of JAK2 (Winston and Hunter, 1996). Expression of dominant negative mutant ERK2 blocks interferon-P-induced transcription, and treatment of cells with the MKKl inhibitor, PD98059, blocks IL-6-induced transcription, indicating a role for ERK in cytokine signaling through STAT1 or STAT3 (David et al., 1995; Bhat et af., 1996). A reasonable model would involve JAK-dependent activation of Ras through a Shc/Grb2-coupled pathway, as documented in several cases (VanderKuur etaf., 1995; He etal., 1995; Chauhan et al., 1995).However, not all cytokine receptor pathways utilize JAK for activating ERKs (Miura et af., 1994), indicating alternative mechanisms for carrying out these processes. Cross-regulation between JAK and ERK pathways also involves STAT regulation by ERK phosphorylation. Serine phosphorylation of STAT1 and STAT3 is necessary for full transcriptional activity, most likely stabilizing STAT dimers and STAT-DNA complex formation (Wen et af., 1995; X. Zhang et al., 1995). Phosphorylation of STAT1 or STAT3 at Ser,,, is important for this effect. These sites lie within the consensus sequence for ERK phosphorylation (Pro-X-Ser(P)-Pro), thus allowing positive feedback of STAT signaling by MKWERK. 9. SIGNALING THROUGH PHOSPHATIDYLINOSITOL 3’ KINASE

Evidence for an involvement of phosphatidylinositol3’ kinase (PI3’K) on MKKERK activation is based primarily on the suppression of signaling by pharmacological inhibitors or dominant negative mutants of PI3’K. Although PU’K signaling is likely to be more important in stress-activated kinase pathways, the PI3’K inhibitors wortmannin or Ly294002 (Powis et d., 1994; Vlahos et al., 1994) inhibit ERK activation in response to insulin, serum, vasopressin, platelet- activating factor, growth hormone, GPVsubunit expression, and T-cell receptor activation (Cross et af., 1994; Welsh et al.,

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1994; Ferby et al., 1994; Nishioka et al., 1995; Urich et al., 1995; Kilgour et al., 1996; Von Willebrand et al., 1996). Supporting evidence shows inhibition of ERK following the expression of dominant negative mutant PI3’K and ERK activation by constitutively active mutant PI3’K (Hu, Q. et al., 1995b; Von Willebrand et al., 1996). However, various studies have yielded conflicting results. In some cases, wortmannin or Ly294002 blocks ERK activation at doses uncorrelated with their inhibition of PI3’K, suggesting nonspecific mechanisms of action (Frevert and Kahn, 1997). Regulation of ERK by PI3’K might be mediated through Ras, which binds directly to the p l 1 0 catalytic subunit, resulting in elevation of PI3’K activity (Rodriguez-Viciana et al., 1996).

B. Stress-Activated Protein Kinase Pathways 1. STRESS-ACTIVATED MAPKs: JNWSAPK AND p38 MAPK

NH,-terminal cJun kinase (JNK)/stress-activated protein kinase (SAPK) and p38 MAP kinase are implicated in responses to cellular stress, inflammation, and apoptosis. Members of both families are ubiquitously expressed and are activated in response to lipopolysaccharides and proinflammatory cytokines interleukin-1 or tumor necrosis factor (TNF-a), ionizing or ultraviolet (UV) radiation, inhibitors of translation such as cycloheximide or anisomycin, cancer chemotherapeutics, tumor promoters, heat shock or hyperosmotic stress (DCrijard et al., 1994; Kyriakis et al., 1994; Han et al., 1994; Raingeaud et al., 1995; Y. Chen et al., 1996a; Kuroki et al., 1996; Osborn and Chambers, 1996).These are distinct from cell growth and transformation responses controlled by MKKERK, although the stress-activated kinases can be activated by growth stimuli under some conditions (Fig. 1). JNK/SAPK was first identified through purification as a cycloheximideactivated MAP2 kinase (Kyriakis and Avruch, 1990) as well as a protein kinase that copurified with and phosphorylated c-Jun (Adler et al., 1992; Hibi et al., 1993). Subsequent cloning efforts revealed three genes encoding JNWSAPK isoforms: JNK1, JNK2, and JNK3 (corresponding to SAPKa, p, and y) (DCrijard et al., 1994; Kyriakis et al., 1994). Alternative mRNA splicing at the C terminus results in further diversification of the JNK/SAPKs into as many as 10 isotypes, including four forms of JNK1, four forms of JNK2, and two forms of JNK3 with molecular masses ranging from 45 to 57 kDa (Gupta et ul., 1996). p38 MAPK was first purified as a protein phosphorylated in response to lipopolysaccaride (Han et al., 1994) and as a “reactivating kinase” able to phosphorylate and activate phosphatase-treated MAPKAP kinase-2 (Rouse et al., 1994). Both studies led to cloning of a protein kinase sharing 52% amino acid identity with the S . cerevisiue HOG1 protein kinase that responds

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to osmotic stress (Brewster et al., 1993; Han et al., 1994). The functional similarity of JNWSAPK and p38 MAPK was established by complementation of yeast hog1A strains (Han et al., 1994; Galcheva-Gargova et al., 1994). Four forms of p38 MAPK (a,p, y, and 6, also known respectively as RWSAPK2a, SAPK2b, ERK6ISAPK3, and SAPK4), have been identified (Jiang et al., 1996; Z. Li et al., 1996; Goedert et al., 1997). p38 MAPK a and p are inhibited by novel cytokine-suppressive anti-inflammatory drugs (CSAIDs) (Lee et al., 1994), are sometimes referred to as CSAID-binding proteins (CSBP1 and CSBP2). An alternatively spliced variant of p38 MAPK, called Mxi2, is a C-terminal truncation of p38 MAPKa, discovered by its interaction with Myc and its interacting partner, Max (Zervos et al., 1995). The overall sequence identity among JNWSAPK, p38 MAPK, and ERKs is 4 0 4 5 % . All three enzymes share a common mechanism for activation through phosphorylation of a Thr-X-Tyr motif found in the activation lip (DCrijard et al., 1994; Kyriakis et al., 1994; Gupta et al., 1996).JNWSAPK is activated by the phosphorylation of Thr-Pro-Tyr, whereas p38 MAPK is phosphorylated at a Thr-Gly-Tyr motif. The length of the activation lip differs among ERK, JNWSAPK, and p38 MAPK, which contain 25,21, and 1 9 amino acid residues, respectively, which are located between the Asp-Phe-Gly and Ala-Pro-Glu conserved sequences in subdomains 7 and 8. The sequences in the N-terminal half of the activation lip also differ among the three MAPKs. However, these differences do not account for the specific recognition by upstream protein kinases because substitution of the ERK2 activation lip by p38 MAPK or JNWSAPK sequences does not affect ERK2 phosphorylation by MKKl (Robinson et al., 1996a). Sequences immediately C-terminal to the P+ 1 recognition site are similar in all three MAPKs, consistent with their common recognition of Sermhr-Pro motifs. Crystallographic studies of the inactive form of p38 MAPKa show similarities in the overall domain structure, but significant differences in the folding of the activation lip compared to that of inactive ERK2 (Wilson et al., 1996; Z. Wang et al., 1997). Presumably, conformational changes induced by phosphorylation of p38 MAPK lead to an activation lip structure similar to that of active ERK2.

2. STRESS-ACTIVATED MAP KINASE KINASES 3, 4 , 6 , AND 7 Polymerase chain reaction (PCR) strategies were used to identify the upstream activators of JNWSAPK and p38 MAPK from mammalian libraries, using yeast PBS2 (Brewster et al., 1993), Xenopus XMEK2 (Yashar et al., 1993), or Drosophila hemipterous (Glise et al., 1995) as templates (Sanchez et al., 1994; DCrijard et al., 1995; Raingeaud et al., 1996). To date, four distinct MKKs have been identified (Fig. 1).MKK3 selectively phosphorylates p38 MAPK (DCrijard et al., 1995; Raingeaud et al., 1996). MKK6 is closely

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related to MKK3 (80% sequence identity) and also phosphorylates p38 MAPK, although its basal activity is approximately 300-fold higher than that of MKK3 (Raingeaud et al., 1996; Han et al., 1996; Moriguchi et al., 1996b; Stein et al., 1996).Both MKK3 and MKK6 have been found in two splice variant forms (Han et al., 1996; Moriguchi et al., 1996a).MKK4 and MKK7 (also called JNKKl/SEKl and JNKK2, respectively) are regulators of JNWSAPK, although MKK4 also phosphorylates p38 MAPK in vitro (Sgnchez et al., 1994; Dirijard et al., 1995; Lin et al., 1995; Tournier et al., 1997). MKKs 3, 4, 6, and 7 range from 35 to 45 kDa and lack the proline rich insertion between subdomains 9 and 10 that is found in MKKl and MKK2, although MKK4 retains a consensus site for ERK phosphorylation at its N terminus and is a substrate for phosphorylation by ERK (Dirijard et al., 1995). MKKs 3,4, 6, and 7 are responsive to inflammatory cytokines, ultraviolet or y irradiation, translation inhibitors, and osmotic stress, as expected from the behavior of JNWSAPK and p38 MAPKs. In some cells, MKK4 may be preferentially activated by translation inhibitors, whereas MKK3 and MKK6 may be preferentially activated by hyperosmotic shock, although this appears to vary substantially according to cell type (Moriguchi et al., 1995b, 1996a; Cuenda et al., 1996; Meier et al., 1996; Zanke et al., 1996a). The activation lip of these enzymes all contain serine residues at the position homologous to Ser,,, in hMKK1 and threonines at the position occupied by Ser,,,. Activation of these MKKs occurs through dual phosphorylation at these sites because mutagenesis at both positions is needed to obliterate phosphorylation by upstream kinases (Yan et al., 1994; Zanke et al., 1996a). MKK5 was cloned as a MKK homolog with 45% identity to MKK1, identified with two alternatively spliced forms of 50 and 40 kDa (English et al., 1995; Zhou et al., 1995). Yeast two hybrid screens identified an ERK homolog, ERK5, a 90-kDa protein identified in separate studies as big MAP kinase 1 (BMKl), that contains a Thr-Glu-Tyr motif within its activation lip (Zhou et al., 1995; Lee et al., 1995). Both ERK5 and MKK5 contain sequences outside their consensus kinase core, suggesting the presence of cytoskeletal-binding motifs, and MKKS is recovered in the particulate fraction of cell extracts. Despite the interaction between these enzymes, no evidence exists that ERK5 can serve as a substrate for MKKS or any other MKK. To date, ERK5 has no known function; however, it is activated by hydrogen peroxide or sorbitol in smooth muscle cells (Abe et al., 1996), suggesting its regulation by oxidant or osmotic stress.

3. STRESS-ACTIVATED MAP KINASE KINASE KINASES

a. MEKK MEK kinases (MEKKs) are mammalian homolog of yeast kinases STEl 1 and Byr2, first discovered through PCR strategies by Lange-Carter et al.

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(1993). At least nine mammalian MEKKs have been identified so far, including MEKKs 1, 2, 3, and 4, MAPKKKS, Tpl-2, TAK1, ASK1, and NIK. MEKK1, originally identified as a 5’-truncated mouse clone encoding a 73kDa polypeptide (Lange-Carter et al., 1993), was later identified in rat as a full-length 195-kDa polypeptide consisting of a C-terminal catalytic domain and several N-terminal domains (proline-, cysteine-rich and pleckstrin homology domains) likely to be involved in protein-protein association (Xu et al., 1996). MEKKl phosphorylates MKK1, MKK2, and MKK4 in vitro, although expression studies indicate that JNWSAPK pathways regulated through MKK4 are preferentially activated over ERK pathways in mammalian cells (Yan et al., 1994; Minden et al., 1994a; Xu et al., 1995; Zanke et al., 1996a). Interestingly, MEKKl is a poor activator of p38 MAPK in vivo, despite the recognition of p38 MAPK by MKK4 in vitro; thus it has been proposed that the formation of complexes through interaction domains of various protein kinases are important determinants of specificity (Zanke et al., 1996a). Expressed MEKKl can be activated by nerve growth factor or phorbol ester through Ras-coupled pathways in PC12 cells (Lange-Carter and Johnson, 1994), and endogeneous proteins immunocross-reactive with MEKK can be activated by a peptide chemoattractant in neutrophils (Avdi et al., 1996). Because MEKKl is active when expressed in mammalian cells, the mechanisms that govern its regulation are unclear. The ability of MEKKl to recognize its substrates for phosphorylation might involve cellular targeting, consistent with the observation that the full-length enzyme is enriched within membrane fractions of cell extracts (Xu et al., 1996). MEKK2 and MEKK3 predict 70- and 71-kDa polypeptides, respectively, with 94% sequence identity to each other within their kinase domains (vs 50% identity to MEKK1) and greater divergence in their N-terminal domains (Blank et al., 1996). In vitro, MEKK2 phosphorylates MKKs 1 and 4, while MEKK3 phosphorylates MKKs 1,3, and 4 (Ellinger-Ziegelbauer et al., 1997; Blank et al., 1996; Deacon and Blank, 1997), although on expression in mammalian cells, both stimulate JNWSAPK and ERK, but not p38 MAPK. MEKK4 and MAPKKK5 are respectively 180 and 150 kDa polypeptides with 55% and 40% identity to MEKKs 1,2, or 3, and stimulate JNW SAPK, but not ERK or p38 MAPK following cell expression (X. Wang et al., 1996; Gerwins et al., 1997). Tpl-2 is an oncogene discovered as a locus of provirus insertion in Moloney murine leukemia virus-induced rat T-cell lymphomas (Patriotis et al., 1993,1994) and is >90% identical to the human and mouse Cot oncogene (Miyoshi et al., 1991). Tpl-2 encodes a 51- kDa polypeptide with 32% sequence identity to MEKKl within its kinase domain and is activated by deletion of 43 amino acids at its C-terminal tail (Patriotis et al., 1994). In vitro, Tpl-2 phosphorylates and activates MKKl and MKK4 (Salmer6n et

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al., 1996) and activates ERK and JNWSAPK on expression in mammalian cells. A protein kinase of 64 kDa with 30% sequence identity to the catalytic domain of MEKKl was revealed in screens for mammalian regulators of yeast STE7. This enzyme was named TGF-@-activatedkinase (TAKl) because it is rapidly activated and transcriptionally induced in response to TGF-@and bone morphogenic protein 4 (BMP4) and mediates transcriptional activation of TGF-@-responsiveenhancers (Yamaguchi et al., 1995b). In vitro, TAKl phosphorylates MKK4 and MKK6 but not MKK1, raising the possibility that it mediates TGF-P signaling through JNWSAPK pathways (Yamaguchi et al., 1995b; Moriguchi et al., 1996b). Two-hybrid yeast screens identified TAB, a novel protein that interacts with and enhances kinase activity of TAKl in intact cells (Shibuya et al., 1996).Thus, stress activated kinase cascades are implicated in transcriptional responses to TGF-p signaling. Apoptosis signal-regulating kinase (ASK1) has 30% sequence similarity to MEKKl and STEll in its kinase domain (Ichijo et al., 1997) and is more closely related to MEKKs SSK2 and SSK22 in the yeast HOG1 pathway (Maeda et al., 1995; Section VI,A,3). ASKl activates MKK3 and MKK4, but not MKKl in vitro, and activates the JNWSAPK and p38 MAPK pathways, but not ERK, following mammalian cell expression. Endogeneous ASKl is activated by TNF-a in several cell lines and its overexpression leads to apoptosis (Ichijo et al., 1997). Another MEKK homolog implicated in the cell death response is NF-&-inducing kinase (NIK), which was identified through its association with TNF receptor-associated factor 2 (TRAF2) adaptor (Malinin et al., 1997). Catalytically inactive NIK mutants block TNF-a or interleukin-1 signaling to NF-KB,and the expression of wild-type NIK inhibits cytotoxic responses to TNFa, suggesting a role for this enzyme in NF-KB-mediated resistance to cell death.

b. Mixed Lineage Kinases A second class of enzymes, distinct from MEKKs, also activate JNWSAPK and p38 MAPK pathways. Mixed lineage kinases (MLKs) contain leucine zipper domains for protein-protein interactions outside the conserved kinase core. Members of this family include MLK-2 (Dorow et al., 1993; Katoh et al., 1995; Hirai et al., 1997), MLK-3/SPRWPTK-l (Ezoe et al., 1994; Gallo et al., 1994; Ing et al., 1994) and DLWZPWMUK (Holzman et al., 1994; Reddy and Pleasure, 1995; Hirai et al., 1996). MLKl (Dorow et al., 1993) is a related enzyme that is not as thoroughly characterized (Fig. 1). MLK2 and MLK3/SPRK are 105- and 93-kDa polypeptides, each with a catalytic domain flanked by an SH3 domain on its N-terminal side and by two leucine zipper domains on its C-terminal side. Between the leucine zippers and a proline/glycine-rich C-terminal tail lies a Cdc42Rac-interactive

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binding (CRIB) motif (Ile-Ser-X-Pro-X,-,His-X-X-His)that enables interaction of these enzymes with Racl-GTP or Cdc42-GTP (Burbelo et al., 1995; Teramoto et al., 1996b). In vitro, MLK-2 and MLK3/SPRK phosphorylate and activate MKK4. Cell expression of either enzyme triggers both JNWSAPK and p38 MAPK, and coimmunoprecipitation of MLK3/SPRK with MKK4 or MKK6 has been observed (Rana et al., 1996; Tibbles et al., 1996), indicating a specific role for these enzymes in stress signaling pathways. DLWMUK is a 100-kDa polypeptide that migrates as a 130-kDa polypeptide on SDS-PAGE. It contains a catalytic domain flanked on its C-terminal side by a dual leucine zipper motif and proline/glycine rich regions at both N and C termini, although only an incomplete CRIB domain is present (Fan et al., 1996). DLWMUK also activates JNWSAPK and p38 MAPK when expressed in mammalian cells, and catalytically inactive DLWMUK can inhibit Ras-dependent JNWSAPK activation (Hirai et al., 1996; Fan et al., 1996). Furthermore, the activation of JNWSAPK is blocked by the expression of a catalytically inactive MEKKl mutant, indicating that DLK/MUK may directly or indirectly regulate MEKKl activity (Fan et al., 1996). Neither MLK3/SPRK nor DLWMUK show significant signaling through the MKW ERK pathway, although MLK-2 activates ERK to an extent comparable to p3 8 MAPK. However, detailed comparisons of the substrate specificities of these enzymes in vitro are still awaited. 4. UPSTREAM REGULATORS OF JNWSAPK AND p38 MAPK PATHWAYS

a . Rac, Rho, and Cdc42-Regulated Kinases In analogy to Ras-GTP regulation of MKWERK, members of the Rho family of small GTPases, including RhoA, Racl , Rac2, and Cdc42, are positive regulators of JNWSAPK and p38 MAPK pathways (Coso et al., 1995; Minden et d., 1995)(Fig. 1).These GTPases are implicated in regulating cytoskeletal reorganization. Rac and Cdc42 regulate cell motility through the formation of lamellipodia and filopodia at the membrane leading edge, whereas Rho is involved in the formation and maintenance of actin stress fibers and focal adhesion plaques as well as the formation of the actin contractile ring at cell division (Nobes and Hall, 1995). Mechanisms linking these proteins to the activation of various MKKs are still unclear but may involve the action of several protein kinases that couple Rac and Cdc42 to stress-activated kinase pathways. p21Ras-activated kinases (PAKs) are 60- to 70-kDa enzymes that are closely related to yeast STE20 (Leberer et af., 1992) within their C-terminal kinase domains; they interact with GTP-Rac and GTP-Cdc42, but not RhoA, through N-terminal CRIB domains. Four mammalian enzymes have been de-

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scribed so far, including PAK1, PAK2, PAK3/@PAK,and yPAK (Manser et al., 1994,1995; Martin et al., 1995; Bagrodia et al., 1995a; Teo et al., 1995). PAK activity and autophosphorylation are stimulated by GTP-Rac or GTPCdc42 in vitro and in vivo, whereas the expression of PAK in mammalian cells leads to activation of JNWSAPK and p38 MAPK (Bagrodia et al., 1995b; Zhang, S. et al., 1995; Frost et al., 1996). ERK is not a major target for activation by Rac or PAK; however, a potential mechanism for crossregulation is indicated by the ability of PAK to synergize with Raf-1 in stimulating MKK and ERK activity (Frost et al., 1996). This may be regulated by the phosphorylation of Ser,,, on MKKl by PAK because mutagenesis of this residue to Ala significantly reduces the synergy (Frost et al., 1997). The mechanisms by which PAK activates JNK are not clearly defined, but may involve signaling through MEKKs or MLKs. Dominant negative MLK3 has been shown to inhibit Cdc42 and Rac signaling to JNWSAPKs (Teramoto et al., 1996b). Alternatively, PAK3 interacts with phospholipase Cy, and PAKs 1 and 3 interact with the adapter protein, Nck, through SH3 domain interactions with N-terminal proline-rich motifs in PAK (Bagrodia, 1996a; Galisteo et al., 1996; Bokoch et al., 1996). PAKl binding to Nck is enhanced by PDGF, leading to elevated Nck phosphorylation, whereas PAKl activity is enhanced in response to EGF on coexpression with Nck. Thus, Nck might mediate signaling upstream or downstream of PAK. Although RhoA also regulates JNWSAPK and p38 MAPK pathways, this does not appear to involve PAK (Teramoto et al., 1996a). Instead, RhoA interacts with protein kinase N (PKN),a 120-kDa protein kinase C-related enzyme with N-terminal leucine zipper motifs (Mukai and Ono, 1994; Amano et al., 1996; Watanabe et al., 1996). Rho-PKN interactions lead to the activation of PKN in vitro and in vivo (Watanabe et al., 1996; Amano et al., 1996), and PKN translocates from cytosolic to nuclear pools following cell stress (Mukai et al., 1996), suggesting that this kinase mediates Rho-dependent activation of JNWSAPK or p38 MAPK. Rac, Rho, and Cdc42 also interact with RhoA-binding kinases a and @ (ROKs), 150- to 160-kDa protein kinases with lengthy coiled coil domains, related to human myotonic dystrophy kinase (Leung et al., 1995,1996; Ishizaki et al., 1996). These induce actin polymerization on cell expression (Leung et al., 1996) and are thus implicated in Rho-dependent cytoskeletal rearrangements. However, experiments introducing point mutations into Rac, Cdc42, and Rho effector domains have shown that the effects of these GTPases on PAK, JNWSAPK, and p38 MAPK activation are not correlated with their effects on ROK activation, lamellipodia and filopodia extension, or actin stress fiber formation (Lamarche et al., 1996; Joneson et al., 1996). This suggests that stress-activated kinase pathways are uncoupled from cytoskeletal reorganization events occurring in response to Rac, Cdc42, or Rho.

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b. Germinal Center Kinases Other protein kinases implicated in stress-activated signaling pathways are members of the germinal center kinase (GCK) family, including GCWRab8ip, HPK1, MST1, MST2, and SOKl (Katz et al., 1994; Creasy and Chernoff, 1995a,b; Ren et al., 1996; Hu, M. et al., 1996; Pombo et al., 1996), which are mammalian homologs of yeast STE20 and Spsl (Friesen et al., 1994). These enzymes are characterized by N-terminal catalytic domains followed by proline-rich regions, leucine-rich regions, and potential PEST sequences (but no Rac interaction motifs). Although none have been found to directly phosphorylate MKKs, on mammalian cell expression these enzymes activate stress-activated kinases, indicating an indirect regulatory role. The 97-kDa GCK is expressed in several tissues, but in lymphoid follicles is restricted to germinal centers, suggesting a role in B-cell development (Katz et al., 1994). It is stimulated in response to TNF-a, and its expression leads to MKK4 and JNWSAPK activation, but not ERK or p38 MAPK activation (Pombo et al., 1995). A closely related mouse homolog, Rab8ip, is found to coimmunoprecipitate and colocalize with Rab8, implicating this enzyme in Golgi vesicle targeting or fusion (Ran et al., 1996)(Fig.1). Hematopoetic progenitor kinase 1 (HPK1) is a 97-kDa enzyme that is found predominantly in blood cells and, when overexpressed, selectively activates JNK/SAPK (Kiefer et al., 1996; Hu, M. et al., 1996). The activation of JNWSAPK by HPKl is inhibited by dominant negative mutants of MEKK1, MLK-3, or MKK4. HPKl coprecipitates with and phosphorylates both MEKKl and MLK-3, suggesting a mode of regulation involving multicomponent complex formation. Mammalian STE20-like kinases (MSTl, MST2) and STE20/oxidant stress response kinase (SOK-1)also belong to the GCK family (Creasy and Chernoff, 1995a,b; Pombo et al., 1996). SOKl is activated by cytokines and oxidants, but does not appear to regulate JNK/ SAPK or p38 MAPK.

5. RECEPTORS AND SECOND MESSENGERS INVOLVED IN CELL STRESS Although JNK/SAPK and p38 MAPK pathways are stimulated by many different types of physical stressors, little is known about the mechanisms for sensing these stimuli. In yeast, HOG1 activation by hyperosmolarity involves at least two osmoreceptors, SLNl and SHOl (Maeda etal., 1995; see Section VI,A,3) which may be activated through mechanical membrane distortions. Physical membrane events are also possible in JNWSAPK activation, based on findings that UV light or hyperosmolarity induces clustering of EGF, TNF-a, or interleukin-1 receptors that might then signal in response to dimerization (Rosette and Karin, 1996). Alternative models invoke DNA damage as common signals induced by UV or y irradiation, chemical muta-

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gens, and cancer chemotherapeutic agents (e.g., cisplatinum, adriamycin, vinblastine, etoposide). The degree of JNWSAPK activation has been correlated with the number of DNA lesions induced by UV-B or W - C irradiation (Adler et al., 1995), although conflicting results have been obtained regarding the ability of UV to induce JNWSAPK activation in enucleated cells (Devary et al., 1993; Adler et al., 1995). Reactive oxygen intermediates (ROIs)such as hydroxyl-free radicals are generated by many agents that also activate JNWSAPK and p3 8 MAPK, including GTPase-deficient mutant Rac(Vall2) (Sulciner et al., 1996), suggesting that ROIs provide common signals to JNWSAPK pathways in response to irradiation, anticancer drugs, or cytotoxic cytokines. This model is supported by the potent activation of JNWSAPK by oxidants (e.g., H,O,) (Lo et al., 1996) and by the inhibitory effect of radical scavengers (e.g., N-acetyl-L-cysteine) on JNWSAPK activation by cell stress (Tao et a/., 1996). However, several studies on the effects of oxidants and antioxidants on JNK/SAPK are also conflicting (Yu, R. et al., 1996; Gomez del Arc0 et al., 1996), and so far a causal role of ROIs in signaling through the kinase pathways has not been definitively established. Ligand-receptor-mediated pathways implicated in JNWSAPK and p38 MAPK activation include those involved in inflammatory and/or apoptotic responses, triggered by receptors for TNF-a, IL-1, and Fas ligand as well as the CD14 receptor for lipopolysaccharide. Rac or Cdc42 are activated in response to all of these stimuli and may be common signaling intermediates, although the mechanisms regulating their respective guanine nucleotide exchange factors are unknown. Receptor-mediated signaling may also involve several heterotrimeric Ga proteins that have been shown to stimulate JNWSAPK preferentially over ERK. Activation of JNWSAPK by GTPasedeficient mutant GmlZor GmI3occurs through mechanisms dependent on Rac and Cdc42 and can be blocked by dominant negative mutant MEKKl (Prasad et al., 1995; Collins et al., 1996; Voyno-Yasenetskaya et al., 1996). In addition, GP./ subunits mediate JNWSAPK signaling through Gmi-coupled muscarinic receptors (Coso et al., 1996). Ras is implicated in some of these studies, based on inhibitory effects of dominant negative mutant Ras (Prasad et al., 1995; Adler et al., 1996); however, in the majority of cases, stress-activated pathways are Ras independent. Abelson tyrosine kinase (Abl) is implicated in regulating cell growth, but is also activated by compounds that induce DNA damage, including ionizing radiation (Kharbanda et al., 1995a,b). Both v-Abl and Bcr-Abl activate JNWSAPK (Renshaw et al., 1996; Raitano et al., 1995).Furthermore, Able’cells generated by homologous recombination are unable to activate JNWSAPK in response to cisplatinum or l-P-D-arabinofuranosylcytosine (AraC), although cells still respond to W light and alkylating agents, implying the existence of convergent stress-signaling pathways (Kharbanda et al., 1995b; Pandey et al., 1996). These findings indicate an upstream regu-

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latory role for c-Abl, most likely mediated through Rac, because dominant negative mutant Rac inhibits v-Abl-induced JNWSAPK activation (Renshaw et al., 1996). Another tyrosine kinase implicated in JNWSAPK activation is Pyk2, which is activated in response to TNF-a, UV irradiation, and hyperosmolarity, and activates JNWSAPK on overexpression (Tokiwa et al., 1996). A calcium-dependent tyrosine kinase, partially purified and found to be related to Pyk2, was stimulated by thapsigargin and angiotensin I1 under conditions resulting in substantial JNK activation and minimal ERK activation (Yu, H. et al., 1996). Lipid second messengers are implicated in stress-activated kinase signaling. In response to TNF-a, interleukin-1, Fas ligand, or ionizing radiation, ceramide levels increase as a result of elevated sphingomyelinase activity. Activation of JNWSAPK is also observed on direct treatment of cells with cellpermeable ceramide analogs (Westwick et al., 1995; Pyne et al., 1996; Welsh, 1996; Coroneos et al., 1996). Lipid products of PI3’K may also be involved in signaling to JNWSAPK kinases. Elevation of Rac-GTP and activation of JNWSAPK are inhibited by wortmannin under some conditions (Hawkins et al., 1995; Ishizuka et al., 1996), and a constitutively active mutant of PI3’K stimulates JNWSAPK (Klippel et al., 1996). Thus, in addition to other Rac or Rho-like morphological responses (Reif et al., 1996), JNK/SAPK may be regulated by products of PI3’K through activation of Rac. Further evidence for a role of PI3’K in JNWSAPK signaling is provided by studies in cells derived from patients with ataxia telangiectasia (A-T), a deficiency in a PI3’K-like enzyme, where ionizing radiation (but not UV or anisomycin treatment) failed to activate JNWSAPK (Shafman et al., 1995). However, ionizing radiation induces an association between JNWSAPK and the p85 . subunit of PI3’K, mediated through Grb2 (Kharbanda et al., 1 9 9 5 ~ )Under the latter conditions, wortmannin stimulated JNWSAPK activity, suggesting inhibitory effects of PI3’K on JNK/SAPK activation. 6 . JNWSAPK AND p38 MAPK PATHWAYS IN APOPTOSIS

Many agents that activate JNWSAPK are cytotoxic, suggesting that this kinase pathway positively regulates cell death. In blood cells, cytokine-stimulated apoptosis is mediated through ceramide synthesis and can be induced by ceramide uptake into cells and antagonized by sphingosine 1-phosphate (Obeid et al., 1993; Cuvillier et al., 1996). A causal role for JNWSAPK in cell death is suggested by the finding that ceramide-induced apoptosis in U937 cells and UVC or y radiation-induced apoptosis in T cells can be inhibited by the expression of dominant negative mutant MKK4 (Verjeijet al., 1996; Chen, Y. et al., 1996a). Furthermore, ERK is activated by sphingosine 1-phosphate and the cytoprotective effect of this metabolite requires ERK

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activity because it is suppressed by PD98059 (Cuvillier et al., 1996; Pyne et al., 1996). Similar results have been reported in model systems for neuronal cell apoptosis. PC12 cells differentiated with nerve growth factor (NGF)undergo apoptosis following NGF withdrawal. Cell death is accompanied by the activation of JNWSAPK and p38 MAPK and appears to be dependent on these pathways, because expression of dominant negative mutant MKK3 or MKK4 promotes cell survival (Xia et al., 1995). PC12 cell survival is also enhanced by the expression of constitutively active MKKl (Xia et al., 1995). Thus, in neuronal and blood cells, relative levels of signaling through ERK vs JNWSAPK or p38 MAPK appear to regulate the choice between cell survival vs death. Consistent with this model, a role of JNWSAPK in epithelial cell apoptosis induced by integrin-extracellular matrix disruption (anoikis) is blocked by dominant negative mutant MKK4 (Frisch et al., 1996). Although JNWSAPK activation is correlated with apoptosis induced by ceramides and UV and y irradiation, the same may not be true for TNF-aor Fas-induced apoptosis. Thus, JNWSAPK activation was inhibited by dominant negative MEKKl or MKK4 in response to TNF-a or Fas, respectively, with no effect on ligand-induced cell death (Liu et al., 1996; Lenczowski et d., 1997). Correspondingly, apoptosis induced by TNF-a or Fas was inhibited by mutagenesis of the FasKNFR1 receptor interacting proteins, RIP or FADD, or with interleukin-1 converting enzyme (ICE) protease inhibitors, with no reduction in JNK activation. Although the p38 MAPK inhibitor, SB203580, had no effect on TNF-a- or Fas-mediated apoptosis (Juo et al., 1996; Bayaert et al., 1996),expression of active MKK3 or MKK6 augmented the Fas-dependent cell death response (Juo et al., 1996; Huang et al., 1997), indicating a potential involvement of MKK3/6 through a p38 MAPK-independent mechanism. Furthermore, whereas JNWSAPK and p3 8 MAPK are involved in some but not all apoptotic responses, activation of JNWSAPK during anoikis and Fas-dependent activation of p38MAPK in T cells can be inhibited by the CrmA protease inhibitor (Frisch et al., 1996; Juo et al., 1996), providing evidence for a positive feedback between stressactivated kinases and ICE proteases.

111. REGULATION OF MAPK PATHWAYS BY PROTEIN PHOSPHATASES Protein phosphatases are catagorized into two general groups, the protein phosphatases (PPs) and the protein tyrosine phosphatases (PTPs). Attenuation of signaling in the various MAPK pathways is controlled by members of both these families (Table I). The PPs specifically hydrolyze serinehhreo-

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Table I Regulatory Phosphatases in MAPK Pathways Phosphatasen Mammalian PPS PP2A

Target

Localization

Reference

ERK and MKK

Cytosol (mainly)

Anderson et al. (1990), Gomez and Cohen (1991), Sontag et al. (1993), Alessi et al. (1995), Cohen (1989)

ERK, JNK, p38

Nucleus

PAC 1

ERK and p38

Nucleus

MKP-2 (hVH-2, TYP- 1)

ERK, JNK, p38

Nucleus

MKP-3 (RVH-6, PYST1)

ERK

Cytosol

hVH-3 (B23)

ERK

Nucleus

M316

JNK and p38

Cell type dependent

hVH-5 VHR

ERK ERK

ERK, JNK, p38

Sun et al. (1993),Alessi et al. (1993), Noguchi etal. (1993),Zheng and Guan (1993),Brondello et al. (1995), Chu et al. (1996) Rohan etal. (1993), Ward et al. (1994), Chu et al. (1996) Guan and Butch (1995), Misra-Press et al. (1995), King et al. (1995), Chu et al. (1996),Brondello et al. (1997) Mourey et al. (1996), Muda etal. (1996b), Groom et al. (1996), Muda et al. (1996a) Kwak and Dixon (1995), Ishibashi et al. (1994) Theodosiou et al. (1996), Muda et al. (1996a) Martell et al. (1995) Ishibashi et al. (1992), Ishibashi et al. (1994) Groom et al. (1996 j, Muda et al. (1996b) Muda et al. (1997)

DSPs MKP-1 (CL100, ERP,3CH134, hVH-1)

MKP-X (PYST2) MKP-4 Saccharomyces cerevisiae

PPS PTC 1,2 PTPs PTP2,3

(HOG1 and PBS2)?

Maeda et al. (1994)

HOG1

Maeda et al. (1994),Jacoby et al. (1997),WurglerMurphy et al. (1997)

DSPs MSG5

FUS3 and MPKl

Doi et al. (1994), Watanabe et al. (1995) (continues)

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Table I (continued) ~

Phosphatasea

Target

Localization

Reference

Schizosaccharomyces pombe PPS Ptc1,2,3

(Spcl and Wisl)?

Shionaki and Russell (1995b)

PTPs PYPlJ-

spc 1

Shiozaki and Russell (1995a), Miller et al. (1995), Degols et al. (1996)

DSPs Dspl

Pmkl ~

Toda etal. (1996) ~

~~~~

aPPs, Ser/Thr protein phosphatases; PTPs, protein tyrosine phospharases; DSPs, dual specificity phosphatases.

nine phosphoesters and include PP1, PP2A, PP2B, and PP2C (Wera and Hemmings, 1995). Hydrolysis of tyrosine phosphoesters is catalyzed by the PTPs, which include a subfamily of dual specificity phosphatases (DSPs) capable of hydrolyzing both phosphotyrosine and phosphoserine/threonine (Fauman and Saper, 1996). Although PPs and PTPs have similar functions, their catalytic mechanisms and structures are different (reviewed by Denu et al., 1996a; Barford, 1996). The crystal structures of PP1 (Goldberg et al., 1995; Egloff et al., 1995), PP2B (Griffith et al., 1995; Kissinger et al., 1995), and PP2C (Das et al., 1997) have been determined, revealing a central P-cx-P-a-P motif containing a dinuclear metal ion center at the active site. Based upon structural data, a single-step catalytic mechanism has been proposed in which metalactivated water directly attacks the phosphorous center of the substrate. In contrast, PTPs do not require metal ions for catalysis, but utilize a catalytic cysteine present in the conserved active site sequence motif: His-Cys-X-X-Gly-X-X-Arg-Ser/Thr.Catalysis occurs in a two-step mechanism in which a phosphoenzyme intermediate forms at the cysteine followed by hydrolysis by water to restore the enzyme (Denu and Dixon, 1995a, 1996b). Comparison of the PTPlB structure (Jia et al., 1995) with that of the dual specificity phosphatase VHR (Yuvaniyama et al., 1996) suggests that the depth of the active site pocket determines substrate specificity. The shallow active site pocket of VHR accommodates phosphorylated serine, threonine, and tyrosine, whereas the deeper active site of PTPl B favors the longer phosphotyrosine side chain (Yuvaniyama et al., 1996).

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A. Dual Specificity Phosphatases Dual specificity phosphatases constitute the largest family of phosphatases capable of regulating MAPKs in mammalian systems. DSPs that modulate MAPK pathways are homologous, especially in their catalytic domains; each is related to the prototypical VH1 dual specificity phosphatase of vaccinia virus (Guan et al., 1991). This group of MAPK phosphatases (MKPs) includes the following members: MKP-1 (also named CL100,3CH134, hVH1, or ERP), MKP-2 (hVH-2 or TYP-l), MKP-3 (rVH-6 or PYSTl), MKP-4, MKP-X (PYST2), PAC1, hVH-3 (B23), M3/6, hVH-5, and VHR (Table I). Although similar in function, members of this family vary in substrate specificity, localization, regulation of gene expression, and tissue distribution. Because MAPKs require both threonine and tyrosine phosphorylation for full activity, dual specificity MKPs are in a unique position to regulate MAPK signal transduction cascades. MKP-1, the first of the MKPs to be characterized, inactivates ERK2 in vitro by concomitant dephosphorylation of phosphothreonine and phosphotyrosine residues (Charles et al., 1993; Alessi et al., 1993; Zheng and Guan, 1993b).ERKs are also effectively inactivated by MKP-1 in vivo (Sun et al., 1993; Duff et al., 1995). Similarly, PAC1, MKP2, and MKP-3 inactivate ERK1/2 both in vitro and in vivo, whereas hVH-3, hVH-5, and VHR do so only in vitro (Table I). The stress-activated kinases JNWSAPK and p38 MAPK are also substrates for MKP-1 and MKP-2, whereas PAC1 recognizes only p38 MAPK (Chu, Y., et al., 1996; Brondello et al., 1997). However, JNWSAPK inactivation requires neither MKP-1 nor MKP-2 in vivo, and ERK is a better MKP-1 substrate than JNWSAPK (Sun et al., 1994; Brondello et al., 1997). MKP-3 is specific for ERKs1 and 2, whereas M3/6 selectively inactivates JNWSAPK and p3 8 MAPK (Muda et al., 1996a; Groom et al., 1996). The substrate specificities of individual MKPs imply their differential ability to regulate various MAPK pathways. Indeed, MKP-3 blocks growth factor stimulation of ERKl in vivo, but not stress-induced activation of JNKUSAPK or p38 MAPK (Groom et al., 1996). Conversely, M3/6 is restricted to JNKUSAPK and p38 MAPK and has no effect on EGF stimulation of ERKl (Muda et al., 1996a).Overexpressed MKP1 is able to block both signaling pathways (Sun et al., 1993; Liu, 1995). In general, MKPs localize within nuclei, providing a mechanism for the inactivation of nuclear MAPK (Table I). MKP-1 is exclusively nuclear in both quiescent and stimulated cells (Brondello et al., 1995; Lewis et al., 1995), as are PAC1, MKP-2, and hVH-3 (Rohan et al., 1993; Guan and Butch, 1995; King et al., 1995; Kwak and Dixon, 1995). In contrast, microinjected M3/6 shows localization dependent on cell type, being nuclear in Swiss 3T3 cells, but cytoplasmic in MDCK and PC12 cells (Theodosiou et al., 1996).MKP3 and MPK-4 are excluded from the nucleus and thus appear to be a regulator of cytoplasmic ERKl and ERK2 (Groom et al., 1996; Mourey et al.,

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1996; Muda et al., 1996b, 1997).Cytoplasmic vs nuclear regulation of MAPK signaling functionally distinguishes MKP-3 and -4 from the other MKPs. Most MKPs are transcriptionally upregulated in response to mitogenic and stress stimuli, although in different ways. MKP-1 was originally identified as an immediate early gene responsive to oxidative stress and heat shock, as well as serum stimulation (Lau and Nathans, 1985; Keyse and Emslie, 1992). UV light and anisomycin treatment also rapidly enhance MKP-1 mRNA levels (Liu et al., 1995; Bokemeyer et al., 1996). PAC1 is a mitogen-induced early response gene that is predominantly expressed in hematopoietic tissues (Rohan et al., 1993). Similarly, induction of hVH-5 by NGF and insulin is characteristic of an immediate early gene (Martell et al., 1995). In contrast, the other MKPs differ from MKP-1 in their magnitude and kinetics of mRNA expression. hVH-3 is induced neither by oxidative stress nor UV light and the time course of its mRNA expression in response to insulin is prolonged (Kwak and Dixon, 1995; Groom et al., 1996). The upregulation of MKP-2 message in response to mitogenic stimuli is delayed and less pronounced compared to MKP-1. In addition, MKP-2 transcription is not induced by oxidative stress or heat shock (King et al., 1995; Guan and Butch, 1995; Misra-Press et al., 1995). VHR is unresponsive to extracellular stimuli, including oxidative stress or growth factor treatment (Kwak and Dixon, 1995). The cytosolic MKP-3 is not an immediate early gene; its message expression is weakly induced by serum and is unaffected by heat shock, oxidative stress, or UV light (Groom et al., 1996). In PC12 cells, NGF causes a robust upregulation of MKP-3, whereas in MM14 muscle cells, bFGF causes a similar induction (Muda et al., 1996b; Mourey et al., 1996). bFGF withdrawal from MM14 cells decreases MKP-3 expression, resulting in a subsequent increase in ERK activity, concomitant with differentiation (Mourey et al., 1996). MKP mRNA expression is regulated by mitogenic and stress-activated MAPK pathways. MKP-1 promoters contain Spl, NFl-like, AP2, AP1, Ebox, and CRE enhancer sites as well as a TATA basal promoter (Noguchi et al., 1993; Kwak et al., 1994). AP1 promoters are known to be regulated by ERK or JNWSAPK phosphorylation of c-Jun and CRE promoters can be regulated by JNUSAPK or p38 MAPK phosphorylation of ATF2 (see Section IV,A). Indeed, MKP-1 appears to be regulated by both ERKlI2 and JNWSAPK in a cell type-specific fashion. In NIH3T3 cells, activation of the JNWSAPK pathway induces MKP-1 gene expression whereas selective stimulation of the ERK pathway inhibits MKP-1 transcription (Bokemeyer et al., 1996). Thus, MKP-1 mediates cross-talk between ERK and JNWSAPK pathways in this system because JNWSAPK induces MKP-1, which in turn inactivates ERK (Bokemeyer et al., 1996). In contrast, in CCL39 fibroblasts, activation of ERK induces MKP-1 and MKP-2 expression, forming a negative feedback loop, whereas agonists that activate JNK have no effect on MKP-

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1 or MKP-2 (Brondello et al., 1997). The incongruous results in these studies necessitate further investigation into the role of MAPKs in MKP-1 regulation. Similar to MKP-1, the PACl promoter contains an AP2 site and an E-box element. PACl is positively regulated by the ERK pathway in mouse hematopoietic cells, constituting another inhibitory feedback loop (Grumont et al., 1996). Activation of yeast pheromone response and stress-activated MAPK pathways is also attenuated by the feedback induction of PTPs and DSPs (see Section VI). Thus, it is apparent that negative feedback loops are a conserved mechanism for MAPK pathway downregulation.

B. Serinenhreonine Phosphatases Dual specificity MKPs are not the exclusive regulators of MAPK pathways, as serinekhreonine protein phosphatases 1 (PPl) and 2A (PP2A) negatively regulate both MKKs and MAPKs. PP1 and PP2A inactivate ERKl and ERK2 and MKKl and MKK2 in vitro by dephosphorylation of regulatory Ser/Thr residues within their activation lip (Anderson et al., 1990; Ahn et al., 1991,1993; G6mez and Cohen, 1991; Nakielny et al., 1992). In addition, treatment of cells with okadaic acid, a potent inhibitor of PP1 and PP2A, activates the ERK pathway (Haystead et af., 1990; Gotoh et af., 1990b; Casillas et af., 1993). Overexpression of SV40 small T antigen, which interacts with and inhibits PP2A, stimulates MKKl and ERK activity while having no effect on Raf-1 activity in CV-1 cells (Sontag et al., 1993).In PC12, PAE, or 3T3-Ll cells, the rapid (15 min) inactivation of ERK following growth factor stimulation occurs independently of MKP-1 expression (Wu et al., 1994; Alessi et al., 1995b). In these cells, PP2A is the major phosphatase acting on ERK and MKKl, functioning coordinately with an unidentified Tyr,,,-specific phosphatase.(Alessi et al., 1995b).PP2A is negatively regulated by phosphorylation of its catalytic subunit (Tyr307)by receptor and nonreceptor tyrosine kinases (Chen, J. et al., 1992, 1994). Furthermore, phorbol ester stimulation of ventricular cardiomyocytes transiently suppresses PP2A activity, correlating with phosphorylation of the PP2A catalytic subunit (Quintaje et al., 1996). This temporary decrease in PP2A activity is accompanied by a transient increase in cytosolic ERK activity, indicating that PP2A is a physiological regulator of ERK.

C. Protein Tyrosine Phosphatases Tyrosine-specific phosphatases also attenuate signaling in MAPK modules. S . cerevisiae and S . pombe utilize PTPs in coordination with PP2C-related PTCs to regulate stress response pathways (Table I). In mammals, selective

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dephosphorylation of Tyrlss on ERK2 by the transmembrane PTP, CD45,is completely inactivating (Anderson et al., 1990). In general, a well-defined PTP that regulates ERK physiologically has yet to be reported in vertebrate systems, although one ERK-inactivating PTP has been purified to near homogeneity from Xenopus eggs (Sarcevic, 1993). Such an enzyme would probably act in conjunction with PP2A, efficiently dephosphorylating Tyrl8? following the rate-limiting removal of Thr,,, phosphoester by PP2A (Alessi et al., 1995b). The advantage of MAPK regulation by dual specificity phosphatases vs tyrosine-specific phosphatases probably resides in substrate specificity. For example, dual specificity VHR preferentially recognizes diphosphorylated substrate in an ordered mechanism that entails rapid removal of phosphotyrosine followed by slow hydrolysis of phosphothreonine (Denu et al., 1995b). Thus, DSPs do not require the initial action of a serinekhreonine phosphatase in order to efficiently dephosphorylate phosphotyrosine.

IV. CELLULAR SUBSTRATES OF MAP KINASES In uitro, the only targets for MKKs that have been identified are MAP kinases, therefore, specificity of downstream signaling in MKWMAPK cascades appears to be controlled at the level of ERK, JNWSAPK, and p38 MAPK. ERK favors substrates containing Pro residues at the P+ 1 position, with secondary preference for Pro at the P-2 position (Alvarez et al., 1991; Davis, 1993). JNWSAPK and p38 MAPK also recognize Pro at P+1, although the sequence determinants that confer differential recognition by ERK, JNK, or p38 MAPK have not been reported. In uitro targets for MAP kinases include nuclear transcription factors, metabolic enzymes, cytoskeletal proteins, and other signaling components (Table 11). Many of these also appear to be physiological targets, based on peptide map comparisons between in uitro vs in uiuo phosphorylation sites. Important reagents have become available that enable further correlation between kinase activation and substrate phosphorylation in response to various stimuli. Constitutively active mutants of Raf and MKKs have been expressed or microinjected into cells to activate signaling through various MAP kinase pathways. A particularily useful construct fuses the N-terminally truncated Raf-1 catalytic domain to the steroid-binding domain of the estrogen receptor (McCarthy et al., 1995). This chimera can be activated within minutes of adding estradiol to cells and has been used to measure effects of acute signaling through the Raf/MKK/ERK pathway (Pumiglia and Decker, 1997; Bianchini et al., 1997). Active MKKl and MKK2 mutants have been designed with activities ranging from 10-fold over basal to those comparable

OD

N

Table 11 Substrates for MAP Kinases and MAPKAP Kinases Substrate

Function

A. ERK 112 substrates 1. Protein kinases Rsk-1, Rsk-2 Rsk-3 MAPKAP kinase 2

MAPKAP kinase 3 Mnkl, Mnk2 2. Transcription factors ~62~~~Elk-2 SAP-la, SAP-2 ER8 1 Pointed P2 (Drosophila) Yan (Drosophila) ERF PPAR-71 Estrogen receptor C-FOS Fra-1, Fra-2 c-Jun JUN (Drosophila) C-MYC N-Myc HSF-1 GATA-2 TAL-1/SCL NF-IL6

Sequence

LLMT,,PCYT,,ANFV PQPPT,,PALP NSLTT,,,PCY AIS,,,PGMK VF’QT,,,PLHTSR

Reference

Sutherland et al. (1993a) Y. ZHao et al. (1995) Ben-Levy et al. (1995)

Ludwig et al. (1996) Waskiewicz et al. (1997) SRF activation SRF activation Ets activation Ets repression Ets repression Ets repression Suppression adipogenesis Receptor activation Fos stabilization

STLS,,,PIAPRS,,,PAK STLS,,,PVAPLS,,,PAR

Inhibition DNA binding AP-1 activation Myc activation Myc repression Heat shock response Hematopoietic transcription TAL-1 activation Interleukin-6 signaling

TPPLS,,,PIDME VINS,,PD, TVNT,,PD LPPTS,,PSRR PLS,,PS PPSPPQS,,,PR

PLT15,PG GGPLT,,,PRRV PAS,,,P PPPQLS,,,PF DSLSS,,PTLL

MVQLS,,,PPAL SSPPGT,,,PSP

Cavigelli et al. (1995) Price et al. (1995) Janknecht (1996) Brunner et al. (1994a) Rebay and Rubin (1995) Sgouras et al. (1995) Hu et al. (1996) Kato et al. (1995) R. Chen et al. (1996) Gruda et al. (1994) Alvarez et al. (1991) Peveraii et al. ( 1996) Alvarez et al. (1991) Manabe et al. (1996) B. Chu et al. (1996) Towatari et al. (1995) Wadman et al. (1994) Nakajima et al. (1993)

NTF-1IElf-1 (Drosophila) Tristetraprolin AMLl P53 3. Signaling components SOSl

EGF receptor Phospholipase A2 HSPDE4B2B Rab4 Inhibitor-2 PTP-2C 4. Cytoskeletal proteins MAP-2 Caldesmon Dystrophin Synapsin I Tau 5. Other targets Tyrosine hydroxylase Connexin-43 Stathmin Oncoprotein M Topoisomerase I1 Q

Torso response element Early response gene Acute myeloid leukemia Checkpoint control p2lRas activation

EGF signaling PLA, activation CAMPphosphodiesterase Glucose transport PP1 activation PTPase inhibition Microtubule association Smooth muscle contraction Cytoskeleton organization Neurotransmitter release Microtubule association Neurotransmitter synthesis Gap junction communication Microtubule polymerization DNA supercoiling

PLS,,,PS QIQPS,,,PPWS GSIAS,,,PSVH TET,,PGP, PAT,,PW GPRS ,,AS AESS,,,,PS HLDS,,,,PPA PRYS,,,,IS VEPLT,,,PSGEA SYPLS,,,PLSD 1pQs48,ps,,9ppL

QLRS,,,PRTT PST,PY

NKS,,,PAPK VTS,,PTK PVAS,,PAAPS6, PGS PPAS,,,PSPQ YSS199PGS202P

AIMS,,PRFK PLS,,,PSK PLS,,9PMS,,,PP ILS,,PRS, PLS,,PP LILS,,PRSK LPS,,,,PRG

Liaw et al. (1995) Taylor et al. (1995) Tanka et al. (1996) Milne et al. (1994) Corbalan-Garcia et al. (1996)

Northwood et al. (1991) Lin et al. (1993) Lenhard et al. (1996) Cormont et al. (1994) Q. Wang et al. (1995) Peraldi et al. (1994) Ray and Sturgill (1987) Adam and Hathaway (1993) Shemanko et al. (1995) Jovanovic et al. (1996) Drewes et al. (1992) Haycock et al. (1992) Warn-Cramer et al. (1996) Leighton et al. (1993) Marklund et al. (1993) Wells and Hickson (1995) (continues)

Table 11.

(continued)

Substrate

Function

Sequence

B. J W S A P K substrates MAPKAP kinase 3 ~62'~~Elk-l P53 Neurofilament H c-Ju~ C. p38 MAPK substrates MAPKAP kinase 2 MAPKAP kinase 3 ATF2 CHOP(GADD 153) Phospholipase A2 Max D. RSK1,2 substrates GSK-3 (Y GSK-3 /3 Glycogen targeting subunit L1 Tyrosine hydroxylase Nur77 C-FOS SRF CREB CBP E. MAPKAP b a s e 2 substrates hsp27 Tyrosine hydroxylase LSP-1 F. MAPKAP kinase 3 substrate Hsp27

SRF activation

Neurofilamentpolymerization AP-1 activation

STLS,,,PIAPRS,,,PAK VTET,,PGP APAT,,PW KSPXE repeats LLTS,,PDVGLLKLAS,,PEL LTT,,,PCY,

ATF2 activation CEBP activation

PQT,,,PL

DQT,9PT,1PT

RTSQS,,PHS,,PDSS

Myc activation

PP1 activation Neural cell adhesion DOPA synthesis Transcriptional regulation Fos stabilization SRF activation CREB activation Transcriptional coactivation

RARTSS,,FAEP RPRTTS,FAES RRGS,,ESS KGGKYS,,,,VK RRQS,,LIE AHRKGS,,,SSN RSLS,,,EM RRPS1,,YR

Reference Ludwig et al. (1996) Cavigelli et al. (1995) Milne et al. (1995) Giasson et al. (1996) Pulverer et al. (1991) Engel et al. (1995a) Ludwig et al. (1996) Gupta et al. (1995) Wang and Ron (1996) Kramer et al. (1996) Zervos et al. (1995) Sutherland and Cohen (1994) Stamhlic and Woodgett (1994) Dent et al. (1990) Wong et al. (1996) Sutherland et al. (1993b) Chen et al. (1992) R. Chen et al. (1996) Chen et al. (1992) Xing et al. (1996) Nakajima et al. (1996)

Actin polymerization DOPA synthesis Actin binding

RQLS,,SSGV RRAVS,,ELD, RRQS,,LIE KSNS,,VKK, AVA!5252TKT

Stokoe et al. (1992b) Sutherland et al. (1993b) Huang et al. (1997)

Actin polymerization

RQLS,,SSGV

Clifton et al. (1996)

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to phosphorylated enzyme (Brunet et al., 199410; Huang and Erikson, 1994; Mansour et al., 1994; Cowley et al., 1994; Mansour et al., 1996b). Constitutively active mutants of MKK3 and MKK6 have also been developed by the substitution of regulatory phosphorylation sites with acidic amino acids (Xia et al., 1995; Raingeaud et al., 1996). Finally, a constitutively active mutant of ERK mutation (“sevenmaker,” D319N) was identified by its gain-offunction phenotype in Drosophila eye development (Brunner et al., 1994b). Although the mutant ERK appears to be regulated normally by phosphorylation, its elevated activity is conferred by its greater resistance to dephosphorylation and inactivation by several protein phosphatases (Bott et al., 1994; Chu, Y. et al., 1996; Oellers and Hafen, 1996). This mutant has been used with variable success in mammalian systems due to its low level of activity (three- to fourfold). Several inhibitors have been useful for defining the involvement of various MKKs or MAPKs in signaling responses. Catalytically inactive mutants of MKKs 1-4 and 6, ERKs 1 and 2, JNK/SAPK, and p38 MAPK have been reported to work as dominant negative regulators of signaling, presumably through nonproductive interactions with upstream activators or downstream targets (Alawi et al., 1993; Frost et al., 1994; Zanke et al., 1996; Raingeaud et al., 1996). Inhibition of signaling has also been achieved using full-length antisense transcripts or antisense oligonucleotides directed toward Raf-1 or ERK (Pa& et al., 1993; Sale et al., 1995; Muthalif et al., 1996). PD98059 is a cell-permeable inhibitor selective for MKKl and MKK2, which blocks MKK activation at concentrations ranging from 10 to 100 p M (Dudley et al., 1995; Alessi et al., 1995a). In vitro, the IC,, for PD98059 ranges from 2 p M (for MKK1) to 50 p M (for MKK2). Antiinflammatory drugs, SB203580 and SB202190, are inhibitors of p38 MAPK a and p (but not y or S ) , with IC,, -1 pM. (Lee et al., 1994; Bayaert et al., 1996). Using these reagents, the involvement of different MKKs or MAPKs in various cellular responses can be established by correlating dose-response curves for signaling inhibition vs kinase inhibition.

A. Protein Kinase Substrates for MAPKs Five protein kinases can be activated by ERK phosphorylation and thus mediate MKWERK signaling, referred to as MAPK-activated protein (MAPKAP) kinases 1, 2, and 3 and Mnks 1 and 2. MAPKAP kinase-1 (Y and p were first described as ribosomal S6 kinases, pp90 Rsk-1 and Rsk-2 (Sturgill et al., 1988), which are activated through phosphorylation at two threonine residues within the activation lip (Sutherland et al., 1993a; Grove et al,, 1993). However, although ribosomal protein S6 was the substrate first used to identify Rsk activity, S6 appears to be a physiological target not of Rsk,

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but of p70 S6 kinase (Chung et af., 1992).Another homolog, Rsk-3, is phosphorylated but not activated by ERK2, suggesting that ERK may be needed but is not sufficient for activation (Zhao, Y. et af., 1995).MAPKAP kinase-1 isoforms each have two consensus kinase catalytic domains, both which are functional, with ERK phosphorylation elevating the activity of the C-terminal domain (Fisher and Blenis, 1996). These enzymes target several substrates for phosphorylation, with specificity for Arg at position P-3 (Stokoe et al., 1993). The glycogen-associated targeting subunit of PP1 is phosphorylated by ISPK, a protein kinase with properties similar to MAPKAP kinase-1, conferring activation of PP1 in response to insulin (Lavionne et af., 1991; Begum, 1995). Glycogen synthase kinase-3, a protein kinase involved in glycogen synthesis as well as cell fate determination, is inactivated in vitro and in intact cells on phosphorylation by MAPKAP kinase-la (Stambolic and Woodgett, 1994; Sutherland and Cohen, 1994; Eldar-Finkelman et al., 1995). Some substrates for MAPKAP kinase-1 are involved in transcription, including CAMP response element-binding protein (CREB)(Ginty et al., 1994; Xing et al., 1996),CREBbinding protein (CBP) (Nakajima et af., 1996), c-fos, Nur77, and serum response factor (Chen, R. et al., 1992,1996). Thus activation of RsUMAPKAP kinase-1 by ERK provides a means of gene regulation. Genetic lesions in MAPKAP kinase-1 p are implicated in Coffin-Lowry syndrome (Trivier et al., 1996), indicating a role for Rsk in human developmental diseases. MAPKAP kinase-2 is a 40-kDa enzyme, which in addition to a single catalytic domain contains a C-terminal autoinhibitory domain (Zu et al., 1995) and an N-terminal proline-rich region that interacts with SH3 domaincontaining proteins (Engel et af., 1993; Plath et af., 1994).Activation occurs through phosphorylation within the catalytic as well as the autoinhibitory domain (Ben-Levy et al., 1995; Engel et al., 1995a). MAPKAP kinase-2 is phosphorylated by both p38 MAPK and ERK, although p38 MAPK is probably the more important physiological regulator because many growth factors that strongly activate ERK do not activate MAPKAP kinase-2 (Stokoe et al., 1992a; Rouse et al., 1994). Once activated, MAPKAP kinase-2 recognizes substrate specificity determinants of Arg at P-3 and hydrophobic residues at P-5 (Stokoe et al., 1993). MAPKAP kinase-3 (also called chromosome 3p kinase, 3pK) is a 42-kDa enzyme identified in a genomic locus frequently disrupted in small cell lung cancers (Sithanandam et al., 1996). In vitro, MAPKAP kinase-3 can be activated by ERK, p38 MAPK, and JNWSAPK (Ludwig et al., 1996; McLaughlin et al., 1996) and is thus an example of a substrate targeted by all three pathways. Both MAPKAP k'inase2 and MAPKAP kinase-3 are activated by cellular stresses and inflammatory cytokines, and both enzymes phosphorylate heat shock protein 2 7 (hsp27) (Engel et al., 1995b, McLaughlin et al., 1996; Clifton et al., 1996). hsp27 behaves as an actin capping protein, thus MAPKAP kinases 2 and 3 are potential regulators of cytoskeletal assembly.

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M n k l and 2 were identified in screens for ERK interacting proteins (Waskiewicz et al., 1997; Fukunaga and Hunter, 1997). Both enzymes can be directly activated by ERK or p3 8 MAPK phosphorylation. Once activated, M n k l and 2 phosphorylate eukaryotic initiation factor 4E (Waskiewicz et al., 1997) suggesting a mechanism for translational control by these two MAPK pathways.

B. Nuclear Transcription Factors MAP kinases phosphorylate several proteins belonging to the Ets family of helix-turn-helix transcription factors. The p62 ternary complex factor ( ~ 6 2 ~ ~ ~ Aregulates 4 k - l ) serum response element (SRE)transcription through its interaction with serum response factor (SRF) (Shaw et al., 1989; Graham and Gilman, 1991). Transactivation is potentiated by phosphorylation of ~ 6 2 ~ ~ ~ / Eby l kERK, - 1 JNK/SAPK, or p38 MAPK (Marias et al., 1993; Janknecht et al., 1993; Gille et al., 1992, 1995a,b; Cavigelli et al., 1995; Whitmarsh et al., 1995; Price et a/., 1996). Ternary complex factors Sapl and Net/Erp/Sap2, which also interact with SRF and SRE, are substrates for ERK and p38 MAPK; Sapl is also a substrate for JNK/SAPK (Price et al., 1995, 1996; Janknecht et al., 1995). Transactivators ERM/ER81 and most likely Etsl and Ets2 are phosphorylated by ERK (Janknecht, 1996; Janknecht et al., 1996; Conrad et al., 1994). Ets family members that function as transrepressors are also directly regulated through phosphorylation by ERK. An Ets2 repressor factor (ERF), similar to p62TCF/Elk-l in its DNA-binding domain, is phosphorylated and inhibited in response to vRaf in mammalian cells (Sgouras et al., 1995). In vitro, ERF is phosphorylated by both ERK2 and cdc2 and is most likely regulated by phosphorylation at ThrS26, as substitution of this residue with Glu leads to loss of repression. Genetic screens for downstream transcriptional targets of ERK in C. elegans and Drosophila have so far identified mainly Ets family members as physiological substrates controlling developmental pathways (see Section V,B). MAP kinases also target bZIP transcription factors for phosphorylation and activation. Both JNWSAPK and p38 MAPK phosphorylate activating transcription factors ATFa and ATF2 (Gupta et al., 1995,1996; Bocco et al., 1996), members of the ATF/CREB family that heterodimerize with c-Jun, NF-KB, and CREB. Phosphorylation enhances ATF2 transcriptional activity, whereas mutation of phosphorylation sites or expression of dominant negative mutant JNK/SAPK inhibits transcription (Gupta et al., 1995).Dominant negative ERK mutants block transactivation by AP-1, showing that ERK activation is necessary for AP-1 transcriptional function (Frost et al., 1994). Although ERK was originally found to phosphorylate c-Jun at Ser,,

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and Ser,, within its activation domain (Pulverer et al., 1991), later studies showed that ERK also phosphorylates residues at the C terminus of c-Jun, which may lead to negative regulation through inhibition of DNA binding (Alvarez et al., 1991; Chou et al., 1992; Minden et al., 199413).JNWSAPK is the more likely physiological c-Jun kinase in mammalian systems because it forms a tight complex with c-Jun and phosphorylates it more efficiently (Hibi et al., 1993; Minden et al., 1994b; Dirijard et al., 1994).In agreement, in vivo studies show a stronger correlation between c-Jun phosphorylation and JNWSAPK activation than ERK activation (Smeal et al., 1994; Westwick et al., 1994). However, it is still possible that ERK activates AP-1 through direct phosphorylation of c-Jun under some conditions. For example, regulation of photoreceptor differentiation in Drosophila involves c-Jun phosphorylation at Ser,, and Ser9,, dependent on the activity of Rolled (Treier et al., 1995; Peverali et al., 1996). Drosophila JNWSAPK is not required for photoreceptor differentiation, indicating that ERK is the physiological regulator of c-Jun in this system. In vitro, Fos, Fral, and Fra2 are potential targets for direct phosphorylation by ERK, as well as MAPKAP kinase-1, and sites phosphorylated on these transcription factors in vitro are similar to those observed in vivo (Chen et al., 1993; Gruda et al., 1994). In intact cells, phosphorylation of Fos in response to ERK stimulation leads to enhanced Fos protein stability (Okazaki and Sagata, 1995a). A serum-stimulated Fos kinase (FRK) distinct from ERK was shown to activate AP-1 transactivation and may represent an equally important regulator of Fos (Deng and Karin, 1994). c-Myc is phosphorylated by ERK at Ser6,, the primary site for phosphorylation in vivo (Alvarez et al., 1991; Davis, 1993). This residue may be important for transcriptional regulation (Seth et al., 1992), although conclusive evidence for transactivation through ERK is not available. In cell extracts, ERK binds tightly to the N terminus of c-Myc (Gupta and Davis, 1994), an interaction that is disrupted by Myc phosphorylation, suggesting functional interactions between enzyme and substrate. Another member of the Myc family, the N-Myc repressor, is phosphorylated by ERK at Ser,,. This phosphorylation event may be required for N-Myc function because mutation of Ser,, results in loss of repression (Manabe et al., 1996). ERK is also involved in steroid hormone receptor signaling, as demonstrated by its regulation of receptors for estrogen and peroxisome proliferator-activated receptor-y (PPAR-y). Estradiol activates ERK, most likely mediated through tyrosine phosphorylation of Shc (Migliacio et al., 1996). Transactivation of estrogen-response element/luciferase reporters by EGF is dependent on Ras and MKK and can be inhibited by MAP kinase phosphatase-1 expression, suggesting that transcriptional activity is mediated through ERK (Kato et al., 1995; Bunone et al., 1996). Correspondingly, estrogen receptor is phosphorylated by ERK at Ser,,, in vitro; this residue is

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necessary for optimal transcription because its mutation leads to decreased response. In uiuo, however, Serlla can be phosphorylated in the absence of estradiol, suggesting that ERK either mediates steroid-independent transactivation or coregulates steroid-dependent activation (Arnold et al., 1995; Bunone et al., 1996). The analogous residue (Serl12)on the related PPAR-.I, a transcriptional regulator controlling adipocyte differentiation, is phosphorylated in response to EGF, phorbol esters, serum, or insulin (Hu et al., 1996). Phosphorylation at Ser, 12 by ERK blocks transcriptional activity of PPAR-y, providing a means by which growth factors may block adipogenesis through MKKERK signaling.

C. Signaling Components Cytosolic substrates for MAP kinases include signaling components that may be involved in feedback regulation or cross-regulation of other pathways. A likely target for feedback inhibition by ERK is the Ras guanine nucleotide exchange factor, Sosl. In uitro, Sosl is highly phosphorylated by ERK, leading to a characteristic retardation in gel mobility that is also observed in factor-stimulated cells (Cherniack et al., 1994; Buday et al., 1995). Peptide mapping studies have identified at least five residues on hSosl that are phosphorylated by ERK, four of which are phosphorylated in uivo following growth factor stimulation (Corbalan-Garcia et al., 1996). Interestingly, not all of these sites conform to the consensus Sermhr-Pro recognition motif for the MAP kinases (Table 11), suggesting some deviation in ERK substrate specificity. Disruption of the Sosl-Grb2 complex is sensitive to PD98059, indicating a functional role for MKKl in Sos regulation (Dong et al., 1996; Holt et al., 1996). Part of this effect is insensitive to MKP-1 and may thus represent a unique example of ERK-independent MKKl signaling. Phosphorylation of Sosl following cell stimulation correlates with the destabilization of interactions among Sosl-Shc, Sosl-EGFR, and Sosl-Grb2 (Rozakis-Adcock et al., 1 9 9 9 , thus interfering with Ras/Raf activation. Thus, through this mechanism, ERK may serve as its own negative regulator. Negative feedback regulation by ERK has also been proposed to occur through ERK phosphorylation of EGF receptor at Thr,6, (Morrison et al., 1996). However, the effect of phosphorylation at this site is ambiguous, as its mutation to Cys had no effect on receptor autophosphorylation or downregulation of EGF signaling. MAP kinases also phosphorylate enzymes involved in eicosanoid biosynthetic pathways. Cytosolic phospholipase A, (cPLA,) is the rate- limiting enzyme in pathways of agonist-stimulated arachidonic acid release. In vitro, ERK phosphorylates cPLA, on Ser,,,.-, resulting in elevated activity (Lin et al., 1993; Nernenoff et al., 1993; Gordon et al., 1996). Temporal correla-

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tions between the agonist stimulation of ERK and Ser,,, phosphorylation versus the activation of cPLA, and its translocation to the plasma membrane support the importance of ERK as a physiological regulator of cPLA, (Durstin et al., 1994; Sa et al., 1995). Furthermore, in Madin-Darby canine kidney cells, activation of arachidonic acid release in response to stimulation of or,-adrenergic receptors by epinephrine is inhibited by PD98059 (Xing and Insel, 1996). However, cPLA, can be regulated by other kinase cascades as well, depending on the cell type. For example, thrombin stimulation of cPLA, in platelets is insensitive to PD98059, but is completely blocked by SB203580, implicating a pathway mediated by p38 MAPK (Kramer et al., 1996). Another potential target in eicosanoid signaling is cyclooxygenase2 (COX-2) which is transcriptionally upregulated in response to all three MAPK pathways (A. Sorokin, personal communication). In addition, membrane translocation and activation of 5-lipoxygenase in response to the Ca2+ionophore have been shown to be inhibited by PD98059 (Lepley and Fitzpatrick, 1996), implicating ERK in the regulation of leukotriene synthesis.

D. Cytoskeletal Proteins Earlier studies demonstrated that addition of active ERK to cell-free extracts of interphase Xenopus oocytes resulted in microtubule polymerization resembling an M-phase array (Gotoh et al., 1991), suggesting a role of MAPK in meiotic spindle formation. Association of ERK with microtubule networks, determined by indirect immunofluorescence in mammalian cells has also been reported (Reszka et al., 1995), although this is may be due to high levels of ERK expression in cells. Several cytoskeletal substrates for MAP kinases are implicated in neuronal function. ERK was first described as a protein kinase that phosphorylates microtubule-associated proteins 1 and 2 (Hoshi et al., 1988; Ray and Sturgill, 1987), although the physiological significance of these phosphorylation events has not been resolved. The microtubule-binding protein, Tau, is highly phosphorylated by ERK, as well as by other proline-directed (GSK3, cdk5) and nonproline-directed (CAMP-dependent kinase, protein kinase C, casein kinase I, CaM kinase 11)protein kinases (Hosoi et al., 1995; Singh et al., 1996). ERK phosphorylation of Tau resembles an abnormal phosphorylation pattern associated with Alzheimer’s disease and inhibits microtubule-Tau interactions (Drewes et al., 1992). However, cotransfection of Tau with Raf-1 in COS cells does not result in Tau phosphorylation (Latimer et al., 1995), suggesting that ERKs may not be as important as other proline-directed kinases. The neuronal intermediate filament, neurofilament-H, is phosphorylated by JNWSAPK at repeated Lys-Ser(P)-Pro-X-Glu motifs

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within the C-terminal tail domain (Giasson et al., 1996). Hyperphosphorylation of neurofilament H occurs in response to stress activation of JNWSAPK in PC12 cells and dorsal root ganglia and is a characteristic feature of several neurodegenerative diseases. Synapsin I, an actin-binding protein involved in cross-linking synaptic vesicles with cytoskeleton, is phosphorylated concomitantly with ERK activation in response to neurotrophins (Matsubara et al., 1996; Jovanovic et al., 1996). Phosphorylation by ERK at Ser,-, Ser,-, and Ser,,, of synapsin I reduces its F-actin bundling activity in vitro, whereas other proline-directed kinases had no effect.

V. RESPONSES TO MAPK PATHWAYS: GROWTH AND DIFFERENTIATION A. Regulation of Cell Growth and Transformation The rapid activation of Raf-1, MKK1/2, and ERK1/2 observed in response to mitogen stimulation implies that this pathway is necessary for cell cycle progression through G1. This is supported by evidence showing elevated DNA synthesis or cell growth under conditions of ERK activation such as expression of constitutively active mutant MKKl (Brunet et al., 1994; Seger et al., 1994). Correspondingly, mitogen-induced DNA synthesis is blocked by antisense RNA-mediated ERK downregulation (Pa& et al., 1993), dominant negative ERK mutants (Frost et al., 1994; Troppmair et al., 1994), or treatment of cells with the MKK inhibitor, PD98059 (Dudley et al., 1995; Seufferlein et al., 1996). In many cells, ERK activation occurs within minutes following factor treatment and declines after 1-2 hr. However, the kinetics can be variable under different conditions, raising the possibility that different responses may depend on the time frame of activation. For example, responses of CCL39 fibroblasts to thrombin, which is mitogenic, vs thrombin proteolytic peptide, which is nonmitogenic, respectively, occur with sustained vs transient kinetics of ERK activation (Vouret-Craviari et al., 1993), suggesting that persistent activation of ERK is required for cells to pass G1. Likely targets for regulation by ERK during G1 are immediate early genes such as Fos and Egr-1, which can be induced through ~ 6 2 ~ ~ ~ Ephoslk-l phorylation and activation of serum response factor promoter elements (Kortenjann et al., 1994; Hipskind et al., 1994a,b; Beno et al., 1995). Cyclin D1 is transcriptionally induced by MKKERK, and provides a link between the ERK pathway and S-phase entry through regulation of cyclidcdk activity (Lavioe et al., 1996).Phosphorylation of eIF4E by Mnk may also promote translation of key proteins needed for S phase entry (Waskiewicz et al., 1997). Although the best documentation of Raf/MKKERK activation occurs in

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early G1, a few reports exist demonstrating Raf and ERK activation during mitosis in synchronized mammalian cells (Laird et al., 1995; Edelmann et al., 1996). Antibodies that specifically stain activated ERK show colocalization of ERK with spindle poles and condensed chromatin in mitotic cells (P. Shapiro and N. Ahn, unpublished results). In oocytes, ERK associates with microtubule organizing centers and is involved in meiotic spindle formation (Verlhac et al., 1993).Furthermore, activation of ERK by v-Mos or constitutively active MKKl leads to partial chromosome condensation and formation of novel germinal vesicle microtubule arrays (Choi et al., 1996b). These data suggest a role for ERK in microtubule rearrangements during M phase. Transfection of 3T3 cells with constitutively active mutant MKKl and MKK2 (Mansour et al., 1994a, 1996b; Brunet et al., 1994b; Cowley et al., 1994) demonstrated that MKKERK induces hallmarks of cellular transformation, such as high saturation density, anchorage independent growth, cell rounding, and solid tumor formation in mice. In addition, cell transformation induced by v-ras or v-mos can be blocked by dominant interfering mutants of MKKl or ERK 1 or 2 (Troppmair et al., 1994; Okazaki and Sagata, 1995b). Thus, sustained ERK activation most likely accounts for many of the cellular responses observed with oncogenic Ras, Raf-1, and Src. In somatic cells, elevated expression of v-Mos leads to irreversible growth arrest, apoptosis, and increased ploidy, effects that are blocked by p53 (Fukasawa et al., 1995, 1997). This has led to the suggestion that constitutive MKK/ ERK activation in the absence of p53 may enhance chromosome instability during cell transformation. Although transformation is clearly dependent on RaWMKKIERK activation, the degree of tumorigenicity observed with activated forms of Raf or MKK1/2 is low compared to that observed with oncogenic ras. Interestingly, foci formation in NIH3T3 cells induced by v-rafcan be synergistically enhanced to levels observed with v-ras by coexpression of GTPase-deficient mutants of Racl or RhoA (Qiu et al., 1995; Khosravi-Far et al., 1995).Furthermore, dominant negative mutants of Rac or Rho blocked transformation in response to v-ras. Similar effects are observed with PI3’K, acting upstream of Rac (Rodriguez-Viciana et al., 1997).Thus, activation of MKK/ ERK is necessary but not sufficient to account for effects of Ras transformation, which instead requires the coordinated activation of other Ras effectors to control cytoskeletal reorganization.

B. Regulation of Cell Differentiation and Development In contrast to the role of MKK1/2 and ERK1/2 in regulating cell growth, MAPK pathways also play an important role in regulating embryonic development and cell differentiation. This is best demonstrated in whole animal

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studies, where activation of MKK and ERK is essential for vulval development in C. elegans, photoreceptor cell specification and anterior-posterior body patterning in Drosophila, mesoderm induction during Xenopus laevis embryo development, and positive T-cell selection. In addition, MAPK cascades have been shown to regulate the differentiation of mammalian cell lines that undergo processes of commitment along neuronal, blood cell, or fat cell lineages. Such examples illustrate the ability of different cells to utilize identical pathways to control seemingly opposite responses of cell proliferation vs cell growth arrest and expression of lineage-specific genes. 1. WORM DEVELOPMENT

An ERK module is central to the process of vulval development in the C. elegans hermaphrodite. During vulval formation, a set of vulval precursor cells (VPCs) receive an inductive signal from the gonadal anchor cell (AC), directing those VPCs nearest the AC to adopt the vulval fate (1"or 2"). The pathways determining vulval fate have been analyzed genetically utilizing Vulvaless (Vul) and Multivulva (Muv) mutants (reviewed by Sundaram and Han, 1996). Characterization of these mutants has revealed a Ras-regulated MKWERK pathway analogous to that found in mammalian cells. The inductive signal is encoded by the lin-3 gene, which is homologous to mammalian EGF (Hill and Sternberg, 1992). This signal is likely received at the VPC through the let-23 receptor tyrosine kinase (RTK), a member of the EGF receptor subfamily (Aroian et al., 1990, 1994). High dosages of lin-3 result in 1"VPCs, whereas lower doses induce 2" VPCs, indicating that VPC fate is modulated by the strength of signaling through the pathway (Katz et al., 1995). LET-23 appears to be localized to VPC cell junctions by LIN-2 and LIN-7, membrane-associated guanylate kinase (MAGUK)-like proteins (Hoskins et al., 1996; Simske et al., 1996). This localization may serve to position LET-23 toward the AC, the presumptive source of LET-3. SEM-5 is an adaptor protein homologous to mammalian Grb2 (Clark et al., 1992), presumably binds activated LET-23 (Stern et al., 1993), and associates with an as yet unidentified guanine-nucleotide exchange factor. Further downstream is the let-60 gene, which encodes the C. elegans Ras protein (Han and Sternberg, 1990; Beitel et al., 1990). LET-60 mediates the activation of the LIN45 Raf homolog (Han et al., 1993), which in turn activates a downstream MKK referred to as MEK-2 (Wu et al., 1995; Kornfeld et al., 1995a). MEK2 functions upstream of the ERK homolog SUR-UMPK-1 (Wu and Han, 1994; Lackner et al., 1994). Downstream of SUR-UMPK-1 are putative transcription factors, LIN-1 and LIN-31 (Miller et al., 1993; Beitel et al., 1995),whose corresponding promoter elements have yet to be identified. lin1 is an Ets family member, containing multiple ERK consensus phosphorylation sites, which negatively regulates vulval development, and lin-3 1 is re-

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lated to the HNF-3/fork head family of DNA-binding transcription factors. A reasonable model analogous to the behavior of mammalian ERK is that these transcription factors may be direct substrates for SUR-l/MPK-1 phosphorylation, activated through let-60 signaling. A novel Raf-related kinase, KSR, has been identified genetically as an enhancer of let-60 ras signaling in the vulva1 development system (Sundaram and Han, 1995; Kornfeld et al., 1995b).KSR appears to function either downstream or in parallel to let-60. In support of the genetics, biochemical analysis of human KSR indicates that this component enhances signaling through accelerating MKK and ERK activation (Therrien et al., 1996). 2. FRUIT FLY DEVELOPMENT

a . T h e ERK Pathway in Eye Development A variety of developmental processes in Drosophila are regulated by receptor tyrosine kinases. The torso receptor tyrosine kinase organizes the formation of the anterior and posterior regions of the embryo, whereas the Drosophila EGF receptor (DER)executes multiple developmental functions, including establishment of dorsal-ventral polarity of the egg (Sprenger et al., 1989; Price et al., 1989). Similarly, cell fate determination of the R7 photoreceptor cell in eye development is regulated by the sevenless RTK (Sev) (Hafen et al., 1987).These receptor tyrosine kinases signal through common ERK pathway component genes, although they are differentially expressed and activated by distinct ligands (reviewed by Perrimon, 1994). The sevenless eye development pathway is well characterized and will be the focus of this discussion. The Drosophila compound eye is composed of approximately 800 ommatidia (unit eyes), each of which contain eight photoreceptor cells (Rl-R8). Photoreceptor differentiation occurs in an ordered sequence, first generating R8, followed by R2IR.5 and R3/R4, R1 and R6, and finally R7 (reviewed by Yamamoto, 1994). Differentiation of the R7 photoreceptor, studied extensively using both genetic and molecular approaches, has established the ERK pathway as the mediator of the developmental signal. Signaling is initiated at the R7/R8 cell interface, where the sevenless receptor tyrosine kinase of R7 interacts with its ligand, bride of sevenless (Boss), a transmembrane protein expressed on the surface of the R8 photoreceptor (Kramer et al., 1991). Upon dimerization and tyrosine autophosphorylation of Sev, the SH2-SH3 adaptor protein Drk(Grb2) binds Sev and recruits Drosophila Sos (Olivier et al., 1993; Simon et al., 1993), resulting in the activation of Rasl (Simon et al., 1991; Bonfini et al., 1992). Rasl activation is attenuated by the Ras GTPase, Gap1 (Gaul et al., 1992). Another potential adaptor protein, daughter of sevenless (Dos), functions upstream of Rasl. Dos contains an N-terminal pleckstrin homology domain and is a substrate of the PTPase,

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corkscrew (Csw) (Raabe etal., 1996; Herbst etal., 1996).Although the function of dos is unclear, genetic relationships and biochemical data are consistent with a model in which dephosphorylation of Dos by Csw activates the signaling pathway downstream of Sev, leading to Rasl activation. Following Rasl, Drosophila Raf-1 activates the Drosophila MKK1, D-MeWDsorl kinase (Dickson et al., 1992; Tsuda et al., 1993; Hsu and Perrimon, 1994; Lu et al., 1994).The Drosophila ERK homolog, Rolled, is then activated by D-Mek (Brunner et al., 1994b; Biggs et al., 1994). As discovered in C. eleguns, KSR was identified in genetic screens as an enhancer of signaling, functioning downstream or in parallel to Rasl (Therrien et al., 1995). Interestingly, the protein phosphatase PP2A has appeared in genetic screens, both positively and negatively regulating the pathway downstream of Rasl; however, substrates for Drosophila PP2A are unknown (Wassarman et al., 1996b).Thus, the fundamental ERK signaling pathway is conserved between invertebrates and mammals. Several transcription factors regulated by the sevenless signaling cascade have been identified as Ets family members. yan and pointed (pnt)have negative and positive regulatory roles, respectively, in R7 photoreceptor differentiation (Lai and Rubin, 1992; O’Neill et al., 1994; Brunner et al., 1994a). The repressor activity of yan is abrogated on activation of the sevenless signaling pathway. The yan protein has multiple ERK consensus phosphorylation sites, and ERK phosphorylation of Yan appears to decrease its stability and alter its subcellular localization (Rebay and Rubin, 1995). The pointed gene encodes two related proteins, pntPl and pntP2, which are products of alternative splicing. pntPl is a constitutively active transcription factor, whereas pntP2 is transcriptionally active only when phosphorylated at its single ERK phosphorylation site (O’Neill et al., 1994; Brunner et al., 1994a). Thus, yan and pointed have opposing transcriptional activities and are differentially regulated by activation of the sevenless signaling pathway. Drosophila Jun (DJun) is also required for Ras-induced R7 photoreceptor differentiation (Bohmann et al., 1994). DJun and Pointed synergistically activate transcription from an AP-1Ets promoter element, indicating functional cooperation between these two transcription factors (Treier et al., 1995). Further, yan antagonizes the enhanced Pointed/DJun transcriptional effect. DJun is a MAPK substrate, and its phosphorylation correlates with photoreceptor differentiation (Peverali et al., 1996). Downstream target genes upregulated by signaling through the sevenless pathway include phyllopod and seven in absentia (sina), which encode nuclear proteins (Carthew and Rubin, 1990; Dickson et al., 1995; Chang et al., 1995). The prosper0 gene, a putative transcription factor involved in nervous system differentiation, is also upregulated in response to sevenless activation, and this induction appears to be mediated by yan and pointed (Doe et al., 1991; Kauffmann et al., 1996). Intriguingly, Phyllopod is also able to upregulate

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prosper0 in coordination with Sina. Phyllopod forms a complex with Sina both in vitro and in vivo, and it is likely that the PhyllopodSina complex has transcriptional activity.

b. T h e JNWSAPK Pathway in Epithelial Cell Migration A second Drosophila MAPK pathway is important for dorsal closure and the insect immune response, representing a unique example of the role of the JNWSAPK pathway in development. Dorsal closure occurs at midembryogenesis and entails the dorsalward spreading of epidermal cells over the amnioserosa membrane until fusion occurs at the dorsal midline (Young et al., 1993). Interestingly, this developmental process is regulated by the JNW SAPK pathway (see Section 11,B). The loss of function hemipterous gene is defective in dorsal closure and encodes a Drosophila JNK kinase (Hep) that is homologous to mammalian MKK3 and MKK4 (Glise et al., 1995). Hep is able to phosphorylate and activate a JNWSAPK homolog, DJNK, encoded by the basket gene (Sluss et al., 1996). Embryos lacking DJNK or that express dominant negative Drosophila cdc42, an upstream GTPase, are also deficient in dorsal closure (Sluss et al., 1996; Riesgo-Escovar et al., 1996). Thus, JNWSAPK pathway components appear to be conserved between mammals and flies. A Drosophila MEKK, Pk92B, has been cloned (Wassarman et al., 1996a).Although its role in development has not been reported, it is reasonable to expect a function upstream of Hep. In addition, DJNK is activated on exposure of cultured Drosophila cells to endotoxic lipopolysaccharide. DJNK activation coincides with a marked induction of antibacterial immune response genes, indicating that the JNWSAPK pathway also mediates an immune response (Sluss et al., 1996). Both developmental and immune responses are likely to be dependent on activation of the DJun transcription factor. Like the ERK homolog, rolled, DJNK efficiently phosphorylates DJun, yet the two pathways appear to be distinct as DJNK is not required in eye development (Sluss et al., 1996; Riesgo-Escovar et al., 1996). Interestingly, the Hep-DJNK pathway upregulates the expression of the puckered gene, which encodes a MAPK phosphatase (Riesgo-Escovar et al., 1996). Hence, regulation through a negative feedback loop is another conserved feature of the pathway.

3. MESODERM INDUCTION IN Xeuopus EMBRYOS

A role for MKWERK in vertebrate development has been documented in the early process of mesoderm induction. MKK and ERK act as cytostatic factors following the fertilization of Xenopus oocytes when their activities drop (Haccard et al., 1993; Kosako et al., 1994); however, their activities appear again following the blastula stage (MacNicol et al., 1995). In Xenopus embryos, signals derived from the vegetal hemisphere, including activin and

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basic fibroblast growth factor (bFGF), diffuse into the animal pole, leading to changes in gene expression and morphogenesis. Treatment of explanted animal poles with various factors show that signaling through bFGF is a key regulator of mesodermal induction, synergistic with activin signaling (reviewed by Gotoh and Nishida, 1996). The importance of Ras and Raf was indicated by inhibition of mesoderm induction on microinjection of dominant negative mutants of these enzymes into animal pole explants treated with bFGF (Whitman and Melton, 1992; MacNicol et al., 1993; Gotoh et al., 1995b). bFGF, but not activin, leads to the stimulation of ERK (Graves et al., 1994), and the involvement of MKKERK in bFGF signaling was confirmed by the induction of mesoderm on injection of constitutively active MKK, ERK (sevenmaker), or STEll mutants into explants (Labonne and Whitman, 1994; Gotoh et al., 1995; Umbhauer et al., 1995). The resulting mesodermal tissue was capable of undergoing further morphogenesis and cardiac actin expression by the dorsalizing factor, noggin. Likewise, the bFGF-induced mesodermal development was inhibited by microinjection of MAP kinase phosphatase-1 . Thus, signaling through MKKERK appears to be sufficient to mimic the effects of bFGF and most likely targets early regulators of mesoderm development, such as the Xenopus Brachyury (Xbra) immediate early gene. 4. NEURONAL DIFFERENTIATION

One approach to understanding the processes of differentiation at a mechanistic level is by manipulating cell lines that differentiate in culture. The rat pheochromocytoma (PC12) line is a widely used model for neuronal differentiation, in which nerve growth factor (NGF) or bFGF cause cell cycle arrest in G1, cytoskeletal rearrangements leading to neurite outgrowth, and expression of neuronal markers. Inhibition of MKKlI2 with PD98059 suppresses NGF-induced neuronal differentiation (Pang et al., 1995), and expression or microinjection of constitutively active MKKl induces neurite outgrowth in the absence of NGF (Cowley et al., 1994; Fukuda et al., 1995). The inhibition of cell proliferation in response to NGF is mediated through the induction of p21(Cipl/Wafl) and suppression of cyclidcdk activity (Pumiglia and Decker, 1997). This is blocked by PD98059, indicating that MKK/ERK induces G1 cell cycle arrest through control of p21(Cipl/Wafl) transcription. Under certain conditions, positive correlations between ERK activation and differentiation are not observed. For example, activation of ERK by mutant NGF or PDGF receptors does not necessarily lead to cell differentiation (Peng et al., 1995; Vaillancourt et al., 1995), and induction of cell differentiation by bone morphogenetic protein or activin A does not require ERK activation (Iwasaki et d.,1996). Therefore, the sufficiency of MKK/ERK activation in regulating PC12 cell differentiation is debatable.

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Nevertheless, MKWERK appears to play an essential role in controlling neuronal differentiation in response to NGF or bFGF signaling. An interesting feature of PC12 cells is that ERK is activated in response to both proliferative and differentiative stimuli. Several laboratories have reported short-term activation of ERK, which is downregulated within a few hours following treatment of cells with proliferation factors vs prolonged activation of ERK on treatment with differentiation factors (Gotoh et al., 1990a; Qiu and Green, 1992; Traverse et al., 1992).This prolonged activation can last up to 10 days and appears to be necessary to maintain the differentiated state and cell survival. The observed variation in kinetics of ERK activation has suggested that sustained activation of ERK may be necessary to trigger a late event necessary for differentiation, but not proliferation, of PC12 cells. A similar model has been proposed to explain proliferative responses in CCL39 cells (Vouret-Craviari et al., 1993; see Cell Growth above). In both cases, nuclear translocation of ERK occurs only in response to prolonged activation (Traverse et al., 1992; Vouret-Craviari et al., 1993), suggesting that the late event responsible for the differentiation of PC12 cells or mitogenesis of CCL39 cells involves the phosphorylation of nuclear targets.

5. BLOOD CELL DIFFERENTIATION MAPK activation occurs as an acute response in lymphoid cells to T-cell receptor or B-cell receptor stimulation (Whitehurst et al., 1992; Izquierdo et al., 1993; Tordai et al., 1994; Nel et al., 1995). In mature T cells that respond to TCR stimulation by transcription of interleukin-2, proliferation, or apoptosis, MAPKs are stimulated through a pathway involving coupling of the TCR-CD3 complex to Ras and Raf, mediated by Lck and ZAP70 tyrosine kinase interactions with TCR. ERK and JNK/SAPK activation appear to be involved in the transcriptional induction of interleukin-2, and TCR signaling is blocked by dominant negative mutant MEKKl in these pathways (Faris et al., 1996). Inhibition of IL-2 transcription also correlates with suppressed ERK and JNK stimulation following T-cell anergy produced in the absence of receptor costimulation (Li et al., 1996). Immature CD4-/CD8- T cells respond to TCR stimulation by positive selection pathways, controlling T-cell differentiation into CD4+/CD8+cells, or negative selection, controlling cell death. The involvement of MKWERK in TCR-regulated pathways was investigated by targeting a dominant negative mutant MKKl for expression in T cells under control of the Ick proximal promoter (Alberola-lla et al., 1995). Mice expressing this mutant showed lowered levels of CD4'8- or CD4-8' cells, indicating a requirement for MKKl during maturation of double-positive to single-positive thymocytes. No effects on negative T-cell selection or TCR-induced proliferation or apop-

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tosis were observed, suggesting an important role of alternative signaling pathways, such as those involving stress-activated kinases (Sen et al., 1996). Results consistent with these studies were observed in fetal thymic tissue utilizing retroviral transfection of MKKl into organ cultures (Crompton et al., 1996). Both double-positive and single-positive T cells were increased following transfection of constitutively active MKKl into tissue from TCRadeficient mice, and transfection of dominant negative MKKl blocked differentiation. No evidence for an effect of MKKl on T-cell proliferation was observed, in agreement with the whole animal study. A role for MKKERK in the differentiation of other blood cell types has also been examined by expression of constitutively active MKK mutants. A late event in blood cell differentiation is the commitment of nonlymphoid cells into megakaryocyte versus erythroid lineages. The human erythroleukemia cell lines, K562 and CMK, retain the capacity to differentiate into megakaryocytes in response to phorbol ester. In either cell type, expression of constitutive active MKKl leads to inhibition of cell growth, induction of characteristic megakaryocytic morphology, including increased cell size and multinucleation, and cell surface expression of integrin aIIbP3, an adhesion receptor specifically expressed in platelets (Whalen et al., 1997; Vik et al., 1997). In addition, the expression of globin genes in K562 cells is blocked by elevated MKWERK activity and is upregulated by the PD98059 inhibitor (Whalen et al., 1997), thus MKWERK promotes megakaryocyte differentiation at the same time it suppresses erythroid differentiation. These results illustrate an example of how MKKERK regulates lineage commitment in pluripotent cells at the level of marker gene expression.

6. ADIPOCYTE DIFFERENTIATION Adipocyte differentiation from fibroblasts involves a series of transcriptional events leading to growth arrest and induction of specific genes required for fat synthesis and degradation (reviewed by MacDougald and Lane, 1995). The 3T3-Ll cell line is a useful model that proliferates with a fibroblast morphology, but can be induced to differentiate into adipocytes by continuous exposure to insulin-like growth factor 1, glucocorticoid, fatty acids, and agents that elevate CAMP. Earlier studies showed induction of differentiation by expression of v-ras or v-rafin 3T3-Ll fibroblasts (Benito et al., 1991; Porras et al., 1994). The evidence implicating ERK in this process is conflicting, however. In one study, ERK antisense oligonucleotides, used to inhibit ERK activation in response to insulin, successfully blocked insulin-induced differentiation of 3T3-Ll fibroblasts (Sale et al., 1995). In another study, differentiation could be activated by v-raf, as scored by the accumulation of fat droplets and transcription of the aP2 lipid-binding protein, but activation of ERK or Rsk was not observed under these conditions,

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suggesting that differentiation through Ras and Raf occurs through pathways separate from those involving MKWERK (Porras et al., 1994). More recent studies have shown that the expression of constitutively active MKKl inhibits insulin-induced adipocyte differentiation, whereas the PD98059 MKK inhibitor suppresses ERK activation, with no effect on differentiation (Font de Mora et al., 1997). It thus appears that the primary effect of MKK/ERK is to negatively regulate adipogenesis and that v-raf may positively regulate differentiation through an alternative pathway. Among the important tissue-specific regulators of adipogenesis are the transcriptional activators CCAAT/enhancer-binding protein (C/EBPa, p, 6) and peroxisomal proliferator activated receptor y2 (PPAR-y); the latter mediates differentiation in response to thiazolidinediones and 15-deoxy-Af2>l4prostaglandin 52 (Wu, Z. et al., 1996). PPAR-y levels increase concomitantly with CEBPa, and together they synergize in promoting differentiation (Lin and Lane, 1992; Tontonoz et al., 1994; Brun et al., 1996). The inhibition of adipocyte differentiation by MKWERK can be explained by the finding that ERK2 phosphorylates PPAR-y2, most likely at Ser,,2. Mutation of Ser, 12 to Ala did not affect ligand-induced transcription, but relieved repression of transcription in response to phorbol ester (Hu et al., 1996), suggesting that ERK phosphorylation inhibits adipocyte differentiation by suppressing PPARq2 transactivation. The C/EBP family member, CHOP/ Gaddl53, inhibits adipocyte differentiation through heterodimerization with other C/EBP family members. In separate studies, phosphorylation of CHOP/Gaddl53 by p38 MAPK was found to be necessary for a full inhibitory effect (Wang and Ron, 1996), providing a mechanism by which ERK and p38 MAPK cooperate to antagonize differentiation in this system.

VI. YEAST MAPK PATHWAYS The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have provided an important complement to metazoan systems in the dissection of MAPK pathways. Through the use of genetic and molecular approaches, five distinct MAPK pathways have been revealed in S . cerevisiae and three in S. pombe. There are striking similarities between yeast and metazoan MAPK signaling, especially at the level of the MAPK module, phosphatase regulation, and transcriptional control. However, the yeasts lack homologs of Raf and appear to signal exclusively through MEKK-like kinases. In addition, the yeasts have thus far been shown to use heterotrimeric G protein-coupled seven transmembrane domain receptors and, in one case, a two-component system, thus receptor tyrosine kinases appear to be utilized exclusively in multicellular systems. Nevertheless, yeast models have

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led to important insights into the dual function of small GTPases in cytoskeletal organization and signaling, as well as mechanisms of heterotrimeric G protein signaling to MAPK modules.

A. Saccharornyces cerevisiae (Budding Yeast) 1. PHEROMONE RESPONSE PATHWAY

Mating in S . cerevisiae occurs between haploid cells of opposite mating types (aor a), and is characterized by the induction of mating-specific genes, cell cycle arrest, and cell morphology changes. This process occurs through activation of the pheromone response pathway, which employs a MAPK module composed of STE11, STE7, and FUS3/KSS1 (Fig. 2). The pheromone response is initiated when a or a peptide mating pheromone binds the appropriate seven transmembrane domain cell surface receptor (STE2 or STE3, respectively), which activates an associated heterotrimeric G protein through guanine nucleotide exchange, releasing Ga-GTP from GPY.The GPr dimer (STE4 and STE18) activates the downstream MAPK cascade through an incompletely understood mechanism, resulting in the activation of STE20 serinehhreonine kinase. The small GTPase, CDC42, and its GTP exchange factor, CDC24, are required in the pheromone response pathway. CDC42 binds to and activates STE20 (Simon et al., 1995; Zhao, Z. et al., 1995), as observed with mammalian Cdc42 and PAK. Although STE20 is required for mating, this interaction functions to localize STE20 to sites of bud emergence (Peter et al., 1996; Leberer et al., 1997) and may not be a necessary event in MAPK signaling. Once activated, STE20 phosphorylates and presumably activates STEll (MEKK) (Wu et al., 1995), although it is not clear how phosphorylation leads to activation. In analogy to the mammalian kinase cascades, STEll phosphorylates and activates STE7 (MKK) (Neiman and Herskowitz, 1994), which in turn phosphorylates and activates FUS3 and KSSl (MAPKs) (Gartner et al., 1992; Errede et al., 1993; Ma et al., 1995). FUS3 has the dual task of controlling the transcription of genes involved in mating and inducing cell cycle arrest. STE12, a transcription factor, and FAR1, a cell cycle regulator, are both substrates for FUS3 (Elion et al., 1993; Peter et al., 1993). Phosphorylation of STE12 is thought to activate transcription of mating specific genes, such as FUSland FARl (Trueheart et al., 1987; Oehlen et al., 1996), and phosphorylation of FARl arrests the cell cycle at G1 by associating with and inhibiting the cyclin-dependent protein kinase, CDC28-CLN2 (Peter et al., 1993; Tyers and Futcher, 1993; Peter and Herskowitz, 1994). Hence, as in mammalian systems, external signals received at the membrane are transduced to the nucleus via a MAPK cascade. STE5 is a signaling molecule that appears to serve as a scaffold for other

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components in the cascade (Choi et al., 1994; Printen and Sprague, 1994; Marcus et al., 1994). STE11, STE7, and FUS3/KSS1 all bind independently to different regions of STES, thus enabling association into a multikinase complex. Only the active form of STEl1 is present in the STES complex, suggesting a mechanism whereby STES binding facilitates STEl1 activation. Indeed, the N terminus of STEll (amino acids 1-435), which is required for STE5 interaction, also functions as a negative regulatory domain (Cairns et al., 1992). STES complexes also bind hypophosphorylated STE7, suggesting that STES may recruit active STEll to inactive STE7 and FUS3/KSS1. Activated STE7 and FUS3KSS1 may then be released, allowing FUS3/KSS1 to phosphorylate downstream targets (Choi et al., 1994). In absence of STES (ste5A),complexes among STE11, STE7, and FUS3KSS1 are still able to form, suggesting that other mechanisms exist to account for the role of STES in mating. However, although STEll and STE7 interact with FUS3/KSSl, STEll associates only marginally with STE7 (Choi et al., 1994; Printen and Sprague, 1994; Bardwell et al., 1996). Hence, facilitation of multikinase complexes by STES is probably important for signal transmission. Protein-protein interactions are implicated with other components that play a vital role in the pheromone response pathway. STE4 ( GP)binds to the N terminus of STES, potentially mediating signaling from heterotrimeric G proteins to the MAPK module (Whiteway et al., 1995). STE4 also binds the N terminus of the guanine nucleotide exchange factor CDC24 (Zhao, Z . et al., 1995).This suggests a model in which the STE4-STES complex activates CDC42 and recruits STE20 (via STE4), bringing it in proximity to its substrate, STEll (via STES). The SH3 domain-containing BEMl protein, involved in cell polarity, is required for efficient signaling in the pheromone response pathway. In a situation analogous to STE4, BEMl directly binds CDC24 (Peterson et al., 1994) and interacts with both STE20 and STES, as well as actin (Leeuw et al., 1995). In addition to binding the STES complex, BEMl associates with FARl (Lyons et al., 1996). Conceivably, BEMl could serve to localize the MAPK module, including its upstream activating components as well as the downstream FARl substrate, at the shmoo tip via its association with the actin cytoskeleton. The involvement of BEMl/CDC24/ CDC42 in the pheromone response pathway demonstrates that components necessary for cytoskeletal reorganization are also fundamental in signal transduction, reminiscent of Rac/Rho function in mammalian systems. In addition to facilitation of signaling through complex formation, STES might prevent cross-talk between the pheromone response pathway and the other S. cerevisiae MAPK pathways. In support of this, a STE7 gain of function mutant can complement a mutation in the cell wall integrity pathway (bcklA) only when it is overproduced or when STES is missing (Yashar et al., 1995). Desensitization of the pheromone response pathway involves dephosphory-

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lation and inactivation of FUS3. The dual specificity phosphatase MSGS (Doi et al., 1994) appears to regulate this process because loss of the MSGS gene results in diminished desensitization. Thus, a phosphatase homologous to the VHI-like class of dual specificity phosphatases, which in humans includes MKP-1 and PAC1 (Table I), is involved in inactivating yeast MAPK. Mating pheromone treatment enhances MSGS expression by fivefold within 30 min. This supports a negative feedback mechanism in which activation of FUS3 causes the transcriptional upregulation of MSGS, as is seen with mammalian MKP-1 and MKP-2 on serum stimulation (Brondello et al., 1997).

2. INVASIVE/PSEUDOHYPHAL GROWTH Invasive growth, characterized by filament formation and agar penetration, occurs in haploid yeast (aor a mating type) when nutritionally starved for nitrogen (Roberts and Fink, 1994). This pathway shares many components with the pheromone response pathway, including STE20 (PAK), STEl1 (MEKK), STE7 (MKK), and STE12 (transcription factor). Invasiveness does not require pheromone receptor, G proteins (STE4, STE18), STES, or FAR1, leaving the signal input component unknown. Deletion of both FUSS and KSSl does not impair invasive growth; however, loss of KSSl alone shows impaired agar penetration, suggesting that KSS 1 regulates the invasive response. Indeed, KSSl in its inactive state negatively regulates invasive growth, yet on activation by STE7, KSSl promotes invasiveness (Cook et al., 1997). Interestingly, mating specific genes are not induced, despite the presence of STE12 in the invasive growth pathway (see below). Two homologous proteins, DIGl and DIG2 (RST1 and RST2), have been identified that suppress invasive growth (Cook et al., 1996; Tedford et al., 1997). Both DIGl and DIG2 coprecipitate with KSSl and FUS3, indicating that they interact in v i m , and DIG112 bind STE12. DIGl colocalizes with KSSl in the nucleus, and both DIGs are phosphorylated by KSS1. These data suggest a model in which DIGs inhibit transcription from filamentous growth response elements (FREs), perhaps through binding of STE12. However, on pathway stimulation induced by starvation, KSSl might phosphorylate DIG112, thereby relieving transcriptional repression and allowing invasive growth. Pseudohyphal growth occurs in diploid ( d a ) cells and, like invasive growth, entails the formation of agar-penetrating filamentous growth in response to nitrogen starvation (Gimeno et al., 1992; Kron et al., 1994). STE20, STEl1, STE7, and STE12 are essential to pseudohyphal growth (Liu et al., 1993); and, as is the case for the invasive pathway, KSSl is the MAPK targeted by STE7 (Cook et al., 1997). Upstream components have also been further characterized in this system. Constitutive activation of U S 2 (through

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mutant RAS2""'19) enhances pseudohyphal growth (Gimeno et al., 1992). This may be mediated through CDC42, which has also been shown to regulate filamentous growth through binding of STE20, and functions downstream of RAS2 (Mosch et al., 1996; Peter et al., 1996; Leberer et al., 1997). However, RAS2 does not function in the pheromone response pathway and thus mediates signaling from a different source into the same MAPK module. Similarly, the 14-3-3 homologs BMHl and BMH2 are essential for pseudohyphal growth signaling, but not pheromone signaling (Robertset al., 1997). BMHl and BMH2 may function in the pathway via interaction with STE20. Activation of the pseudohyphal growth pathway does not activate mating specific genes, analogous to the invasive response (Mosch et al., 1996). Rather, STEl2 and the transcription factor TECl cooperatively bind to FREs to promote transcription required for both invasive and pseudohyphal development (Gavrias et al., 1996; Madhani and Fink, 1997). Other possible targets of the pseudohyphal growth MAPK module are MSSlO (a transcription factor) and MUCl (homologous to mucin-like membrane proteins), both of which are essential for pseudohyphal differentiation (Lambrechts et al., 1996).

3. HIGH OSMOLARITY RESPONSE (HOG PATHWAY) In S. cereuisiae, increases in external osmolarity are compensated for by increased glycerol synthesis and decreased glycerol permeability (Luyten et al., 1995). This high osmolarity glycerol response is rare among eukaryotic signaling pathways in that it combines a two-component response regulatory system (reviewed by Appleby et al., 1996) with a MAPK module composed of SSK2/SSK22 (redundant MEKKs), PBS2 (MKK), and HOGl(MAPK) (Fig. 2). Two distinct cell surface receptors, SLNl and SHOl, act as osmosensors that regulate the HOG cascade. SLNl was initially identified in a screen for synthetic lethal mutations requiring the ubiquitin protein-degradation pathway (Ota and Varshavsky, 1993). Both PBS2 and HOG1 were identified as osmoregulation-defective mutants (Brewster et al., 1993), and SSK2 and SSK22 were discovered in a screen for extragenic suppressor mutants of the lethal SLNl null mutation (Maeda et al., 1995). Strikingly, SLNl is a transmembrane protein with homology to bacterial two-component receptors, containing osmotic sensor, histidine kinase transmitter, and phosphate receiver domains. The remainder of this two-component system includes the response regulators YPDl and SSKl (Maeda etal., 1994; Posas et al., 1996), activating the HOG pathway at the level of SSK2 and SSK22 (Maeda et al., 1995). Under conditions of low osmolarity, SLNl autophosphorylates at His,,, in its transmitter domain. The phosphoryl group is first transferred to Asp,,,, in the SLNl receiver domain, from which it is transferred to His,, on YPD1. Finally, the

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phosphoryl group is passed to Asp,,, in the C-terminal receiver domain of SSKl (Posas et al., 1996).SSKl is phosphorylated and inactive when the external osmolarity is low. Under conditions of high osmolarity, the SLNl histidine kinase is inactivated and the resulting unphosphorylated SSKl activates the downstream SSK2/SSK22, most likely by binding the N-terminal inhibitory domain of SSK2 or SSK22 and relieving autoinhibitory constraints (Maeda et al., 1994). Upon activation, SSK2 or SSK22 phosphorylates and activates PBS2, which in turn phosphorylates and activates H O G l . Thus, histidine kinase activity negatively regulates HOG1, and slnld mutant strains are lethal due to hyperactivation of the HOG pathway. The second osmosensor, SHOl, is a putative transmembrane protein needed for viability in slnld strains. SHOl associates with PBS2 through the binding of its SH3 domain to a proline-rich domain in PBS2 (Maeda et al., 1995). This interaction activates PBS2 through phosphorylation; however, neither SSK2 nor SSK22 is involved. Instead, SHOl binding recruits PBS2 to a multikinase complex with STEll and HOG1, although no crossregulation of HOG by pheromone is apparent (Posas and Saito, 1997). Once activated, H O G l completes the signaling response by inducing the transcription of genes necessary for maintaining osmotic balance. Among these are glycerol-3-phosphate dehydrogenase (GPD1) (Albertyn et al., 1994) and glycerol-3-phosphatase (GPP2) (Norbeck et al., 1996), both essential for glycerol synthesis. Other HOG1-regulated genes include a cytosolic catalase T, CTTl (Schuller et al., 1994), and a small heat shock protein, HSP12 (Varela et al., 1995), both of which are under the control of stress response elements (STREs). The zinc finger transcription factors MSN2 and MSN4 are important regulators of STRE-dependent promoters and are required for activation of C TTl and HSP12 genes (Schmitt and McEntee, 1996; Martinez-Pastor et al., 1996). Strains deficient in MSN2 and MSN4 are less sensitive to high osmolarity than hogld strains but are still viable; therefore, not only do these transcription factors function in HOG signaling, other factors are likely to be involved. The HOG pathway can be downregulated by protein tyrosine phosphatases PTP2 (Ota and Varshavsky, 1992; Guan et al., 1992; James et al., 1992) and PTP3 (Jacoby et al., 1997; Wurgler-Murphy et al., 1997), as well as PTC1, PTC2, and PTC3, which are homologous to the mammalian serinehhreonine phosphatase, PP2C (Maeda et al., 1993; I. Ota, personal communication). PTPs 2 and 3 and PTCs 1-3 are able to suppress lethality in slnld strains when overexpressed, indicating that these phosphatases suppress hyperactivation of the HOG pathway (Maeda et al., 1994; I. Ota, personal communication). PTP2 and PTP3 target HOG1, whereas the substrate specificities of PTCs 1, 2, and 3 have yet to be determined in this system (Table I). It has been suggested that the PTCs regulate both PBS2 and H O G l activity, although this has not been directly demonstrated. Both

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PTP2 and PTP3 are transcriptionally induced in response to hyperosmolarity in a HOG1-dependent manner (Jacoby et al., 1997), thus downregulation of the pathway can be controlled through a negative feedback mechanism. 4. CELL WALL INTEGRITY (PKC PATHWAY)

A MAPK cascade modulates part of a pathway regulating cell wall integrity. S. cerevisiae has a single PKC gene (PKC1) that is closely related to the mammalian PKC a, p, and y isoforms (Levin et al., 1990). Mutations that inactivate PKCl result in cell lysis, caused by deficiencies in cell wall construction (Levin and Bartlett-Heubusch, 1992; Paravicini et al., 1992). Downstream of PKCl is an MEKK (BCKUSLKl), which, when mutated, also results in cell lysis (Lee and Levin, 1992; Costigan et al., 1992). BCKl has a PKC consensus phosphorylation site at Ser,134, and PKCl selectively phosphorylates BCKl in vitro (Levin et al., 1994), suggesting that PKCl activates BCKl through phosphorylation. In addition, two functionally redundant MKKs in this pathway (MKK1 and MKK2) suppress a BCKl deletion mutant when overexpressed (Irie et al., 1993). MKKl and MKK2 are upstream of and activate a MAPK, MPKUSLT2 (Torres et al., 1991; Lee et al., 1993), which, like FUS3, is negatively regulated by the dual specificity phosphatase MSG5 (Watanabe et al., 1995). Although the PKCl pathway does not appear to involve a STE5-like protein, MKKl interacts with PKC1, whereas MKKl and MKK2 bind both BCKl and Mpkl (Soler et al., 1995; Paravicini and Friedli, 1996). Thus, the kinase components form a multienzyme complex through association with MKK1/2. Although each component in this pathway (BCK1, MKK1/2, and MPK1) yields a cell lysis defect when deleted, none is as severe as the loss of PKC1. This implies that the MAPK module functions only in one branch of a bifurcated pathway regulated by PKCl (Errede and Levin, 1993). Upstream regulation of PKCl involves a number of stimulatory inputs. Tyrosine phosphorylation and activation of MPKl increase in response to hypotonic shock (Davenport et al., 1995) or mild heat shock (Kamada et al., 1995). In addition, MPKl is activated during periods of polarized cell growth (budding) in a CDC28-dependent manner (Mazzoni et al., 1993; Zarzov et al., 1996; Marini et al., 1996). Each of these events disrupts the structural integrity of the plasma membrane, suggesting a mechanism whereby plasma membrane stretch activates mechanosensitive ion channels, resulting in an influx of Ca2+(Kamada et al., 1995). Increased intracellular Ca2+ might activate PKCl directly or indirectly through the stimulation of phospholipase C (PLC) and diacylglycerol synthesis. In the case of polarized cell growth, CDC28 upregulates diacylglycerol production, possibly through activation of a phosphatidylcholine-

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specific PLC (Marini et al., 1996). Although PKCl is not activated by phospholipids, Ca2+,or DAG in uitro, it is conceivable that the proper conditions have not been found (Watanabe etal., 1994).For example, the phospholipiddependent regulation of PKCl is facilitated by RHOl-GTP, a homolog of mammalian RhoA, which binds PKCl at its pseudosubstrate site and C1 domain (Nonaka et al., 1995). When complexed with RHO1-GTP, PKCl is strongly activated by phosphatidylserine (Kamada et al., 1996),which serves as the exclusive cofactor in the R H O l P K C l complex, as neither Ca2+ nor DAG is stimulatory. A R H O l loss of function mutation is complemented by overexpression of MPKl or a gain of function MKK1, and R H O l is required for MPKl activation during heat shock (Kamada et al., 1996).These results suggest that PKCl is a target for RHOl and that RHO1-dependent stimulation of PKCl activates the downstream MAPK cascade (Nonaka et al., 1995). Other evidence suggests that PKCl might be regulated by phosphoinositides. A yeast phosphatidylinositol4-kinase (STT4) has been shown genetically to function upstream of PKCl and BCKl (Yoshida et al., 1994). However, a strain bearing an STT4 deletion is incompletely suppressed by overexpression of PKCl or a gain of function BCKl mutant, implying that a second branch point likely exists between STT4 and PKCl . Targets of the MAPK cascade in the PKCl pathway are just beginning to be defined. Two high-mobility group (HMG)-like proteins (NHP6A and NHP6B) function downstream of MPK1, although they are not likely to be direct targets (Costigan et al., 1994).A transcription factor (RLM1)with homology to the MADS box family is a probable target of MPKl induction (Watanabe et al., 1995). Five genes involved in cell wall biosynthesis are upregulated by MPKl, including those encoding enzymes needed in the synthesis of (1-3)-P-glucan, (1-6)-P-glucan, chitin, and N- and 0-linked mannoproteins (Igual et al., 1996).Upstream regulatory promoter sequences and MPK1-specific transcription factors that regulate these genes have yet to be determined.

5. SPORE FORMATION Sporulation occurs in diploid yeast during conditions of nitrogen starvation in the presence of a nonfermentable carbon source. A MAPK gene, SMK1, has been identified as a regulator of spore formation (Krisak et al., 1994). A STE20-like serinekhreonine protein kinase, called SPS1, is also required in sporulation (Friesen et al., 1994). Yeast strains mutant in either of these genes are defective in spore wall biosynthesis. Both SMKl and SPSl transcripts are expressed in a sporulation-specific manner and are required for the normal expression of late sporulation-specific genes. SPSl and SMKl probably represent components in a MAPK module specific to the sporula-

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tion process. However, the homologs of MEKK and MKK in this pathway have not yet been identified.

B. Schizosaccharomyces pom6e (Fission Yeast) 1. PHEROMONE RESPONSE PATHWAY

Sexual differentiation in S. pombe is similar in many respects to S. cuevisiae; however, there are fundamental disparities. In particular, fission yeast require a mating pheromone as well as nutritional starvation in order to mate. These are respectively mediated by the pheromone response pathway and the stress response MAPK pathway (discussed later). Hence, sexual differentiation involves two distinct MAPK signaling pathways. Another difference is that G protein signaling is transmitted through the Ga subunit in S. pombe. Furthermore, Ras is required for the pheromone response, whereas it has no such role in S. cerevisiae. Mating in S. pombe occurs between cells of opposite mating type (P or M). Initiation of the pheromone response takes place when P or M pheromones associate with their receptors (Mam2 or Mam3, respectively), stimulating the release of GTP-bound Ga. The Ga subunit, encoded by the gpal gene, is required for both mating and sporulation (Obara et al., 1991), although effectors that interact with GOiare not known at present. An essential constituent of the pheromone pathway is Rasl (Nadin-Davis et al., 1986),which regulates a MAPK module composed of Byr2 (MEKK), Byrl (MKK), and Spkl (MAPK) (Nadin-Davis and Nasim, 1988, 1990; Wang et al., 1991; Toda et al., 1991). Each component of this MAPK cascade is necessary for the execution of sexual differentiation. Genetic evidence indicates that Ga and Rasl coordinately stimulate the kinase module, converging at B y d (Xu et al., 1994). Rasl directly binds to the N terminus of Byr2 in a GTP-dependent manner, although this interaction has not been reported to activate Byr2 (Van Aelst et af., 1993; Masuda et al., 1995). Rasl also indirectly activates the MAPK module through the STE20 homolog, Shklmakl. Two-hybrid interaction experiments have shown that Rasl-GTP forms a complex with Scdl (CDC24 homolog), Scd2 (BEM1 homolog), and the small GTPase Cdc42, important for both mating and morphogenesis (Chang et al., 1994). Shkl binds to Cdc42 both in vitro and in vivo, and thus is likely to be a Cdc42 effector (Marcus et al., 1995; Ottilie et al., 1995). Shkl restores mating in ste20A strains of S. cerevisiae, and recombinant Shkl is able to activate both ERK2 and JNUSAPK in Xenoptrs oocyte extracts (Polverino et al., 1995),supporting its involvement in MAPK signaling. Another protein essential for sexual differentiation, Ste4, binds the N-terminal regulatory domain of Byr2 at a site distinct from the Ras-1 bind+

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ing domain (Okazaki et al., 1991; Barr et al., 1996). Ste4 acts upstream of Byr2, and Byr2 requires Ste4 binding for complete function. Therefore, full activation of the MAPK cascade involves multiple inputs at the point of Byr2. Signaling downstream of Byr2 is conducted through the Byr2-Byrl-Spkl pathway, which is structurally and functionally homologous to the S. cerevzsiae STEl lSTE7-FUS3KSSl module (Neiman et al., 1993). Consistent with homology and complementation data, tyrosine phosphorylation of Spkl is dependent on Byrl (Gotoh et al., 1993).The matl-Pm gene, which controls entry into meiosis, is positively regulated by signaling through the pheromone response pathway (Aono etal., 1994).The transcription factor, Stell, is a regulator of numerous mating-specificgenes, including matl-Pm; however, it has not been shown to be a substrate of Spkl (Sugimoto et al., 1991).

2. STRESSEEXUAL RESPONSE PATHWAY In S. pombe, multiple forms of environmental stress activate a MAPK cascade with components Wikl/Wakl/Wis4 (MEKK), Wisl (MKK) and Spcl/ Styl/Phhl (MAPK) (Warbrick and Fantes, 1991; Millar et al., 1995; Shiozaki and Russell, 1995a; Kato et al., 1996; Samejima et al., 1997; Shieh et al., 1997; Shiozaki et al., 1997). Spcl is phosphorylated and activated by Wisl in response to hyperosmolarity, starvation, oxidative stress, and heat shock (Millar etal., 1995; Shiozaki and Russell, 1995a; Degols et al., 1996). Like the HOG pathway, the Wikl-Wisl-Spcl module is regulated by an upstream two-component system (Shieh et al., 1997). Mcs4 is homologous to S. cerevisiae SSKl and functions upstream of Wikl, which activates Wisl by phosphorylation. Wisl can also be activated by Win1 in response to osmotic stress independent of Mcs4/Wikl, while Spcl is activated by heat and oxidative stress independent of Wisl, perhaps involving Pypl phosphatase inactivation (Samejima et al., 1997). This MAPK pathway is regulated by several phosphatases from both the PTPs and PP2C families, in a situation analogous to the HOG pathway. The PTPs are Pypl and Pyp2, which are negative regulators of mitosis (Ottilie et al., 1991, 1992; Millar et al., 1992; Hannig et al., 1994). Pypl and Pyp2 both form complexes with Spcl and efficiently inactivate it by direct dephosphorylation of Tyr173 (Shiozaki and Russell, 1995a; Millar et al., 1995). Interestingly, Spcl activation induces expression of Pyp2, forming a negative feedback loop (Millar et al., 1995; Degols et al., 1996; Wilkinson et al., 1996). The PP2C-related phosphatases, Ptcl, Ptc2, and Ptc3, are also implicated in downregulating the Wisl/Spcl pathway, and mutants in both Spcl and Wisl suppress the lethality of a AptclAptc3 double mutant (Shiozaki et al., 1994; Shiozaki and Russell, 1995b). However, the particular substrate(s) for Ptc has yet to be determined. Dual specificity phosphatases that act on this pathway have not been reported at this time.

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Activation of Spcl is required for the transcription of genes involved in stress responses and sexual development. Examples of stress-responsive genes induced by the Spcl pathway are glycerol-3-phosphate dehydrogenase (gpdl+),trehalose-6-phosphate synthase (tpsl+),and catalase (A) (Aiba et al., 1995; Degols et al., 1996; Shiozaki and Russell, 1996; Wilkinson et al., 1996). Spcl is also essential for expression of the Stel 1transcription factor, which regulates sexual differentiation genes in response to nitrogen starvation (Kato et al., 1996; Shiozaki and Russell, 1996). This implicates the Wisl/Spcl pathway as an essential element in sexual differentiation. Atfl, a transcription factor homologous to mammalian ATF-2 (Takeda et al., 1995), is a likely effector of transcriptional regulation by Spcl. In vitro, Atfl and Spcl physically associate and Spcl phosphorylates Atfl . Furthermore, stress-induced expression of gpdl+, tpsl+, catalase, pyp2+, and stel 1 requires Atfl (Shiozaki and Russell, 1996; Wilkinson et al., 1996). However, Atfl deletion mutants are not defective in mitosis, indicating a branch point in the Wisl/Spcl pathway between mitotic and stress responses. In S. pombe, the stress response pathway may indirectly stimulate the pheromone response pathway, an example of cross-regulation between MAPK cascades. Transcription of the Byr2 regulator, Ste4, is dependent on Stell (Okazaki et al., 1991). Thus, regulation of the pheromone response pathway through Ste4 may occur following Stel 1 expression in response to the stress pathway. This may explain the dual requirement for both pheromone factor and nutritional starvation in the mating response. +

3. CELL WALL INTEGRITY PATHWAY A third MAPK isolated in S . pombe, called Pmklhmpl, is a potential regulator of cell wall integrity (Toda et al., 1996; Zaitsevskaya-Carter and Cooper, 1997). p m k l d strains have multiple phenotypes including cell wall weakness, unusual cell shape, defective cytokinesis, and altered cation sensitivity. Pmkl shares 65% identity with S. cerevisiae Mpkl, and Mpkl overexpression is able to rescue p m k l d phenotypes. However, despite the similarities between these two MAPKs, Pmkl is not activated by either hypoosmolarity or heat shock nor does it appear as though Pmkl regulates cell wall integrity in a linear pathway with PKC. S . pombe has two known PKCs, p c k l + and pck2+ (Mazzei et al., 1993; Toda et al., 1993), encoding homologs of the 6, E, and q isoforms of mammalian PKC. Strains lacking Pck2 have a cell wall defect (Shiozaki and Russell, 1995b), that is more severe than the Pmkl null strain. However, genetic data argue that Pmkl and Pckl/2 do not function independently in the regulation of cell wall integrity, but rather in a coordinate manner. One possibility is that Pckl/2 regulate a bifurcated pathway with Pmkl functioning in one of the branches.

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VII. INTRACELLULAR TARGETING AND SPATIAL REGULATION OF MAPK PATHWAY COMPONENTS A. Signaling Complexes An important principle that has emerged from studies on kinase cascades is that protein kinases and their targets interact in a way that confers specificity in signaling. Part of this specificity can be attributed to recognition motifs within the active site of each protein kinases. For example, a systematic comparison of different chimeras between ERK2 and p38 MAPK showed that helix C within the N-terminal lobe of the conserved kinase structure is an important determinant for recognition by growth factor vs stress signaling (Brunet and PouyssCgur, 1996). Presumably, this domain forms part of the recognition surface between MKKs and MAPKs, as observed in the recognition surface between cyclin A and cdk2 (Jeffrey et al., 1995). Domains outside the active site may extend the interaction surface between various enzymes and their substrates. For example, stable complexes between STE7 and FUS3 involve interactions between FUS3 and the STE7 N terminus (Bardwell et al., 1996), and a proline-rich domain between subdomains 9 and 10 of MKKl is important for association between MKKl and Raf-1 (Papin et al., 1996). Extensive regulatory domains containing conserved interaction motifs are present within several protein kinases, such as Raf-1, MEKKs, PAKs, and multilineage kinases, providing mechanisms to confer multiple selective interactions that augment active site recognition. Complexes formed through such extensive protein-protein interactions would be expected to facilitate signal transmission and quite possibly provide an additional insulating function, preventing cross-regulation between homologous enzymes in growtWdifferentiation vs stress regulatory pathways. The best example for this is in S. cerevisiae, where complex formation of STE5 with STE4, BEM1, STE11, STE7, and/or FUS3 provides a mechanism to facilitate the interaction between several pathway components. However, STE5 appears to be unique to the pheromone response pathway. Homologs have not been found in other systems, and STES does not interact with S. pornbe MEKK or MKK (Byr2, Byrl) nor does it bind to mammalian MEKK, MKK, or Raf (Marcus et al., 1994).Thus, in other cell types, conserved binding motifs within the kinases themselves might provide the scaffolding for interactions that facilitate signaling. Most of the evidence for stable complexes formed between protein kinases and their substrates is derived from biochemical coprecipitation experiments as well as from two-hybrid interaction screens. Raf-1 and B-Raf form stable complexes with MKKl and ERKs, mediating the interaction of these kinases with Ras (Moodie et al., 1993; Jelinek et al., 1996; Papin et al., 1995, 1996) and potential interactions with other proteins such as 14-3-3 or KSR.

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Raf-1 also interacts with GPr subunits and tyrosine kinases Src and Fyn (Cleghon et al., 1994; Pumiglia et al., 1995). Evidence also exists for stable interactions between Ras with MEKK (Russell et al., 1995), Rac-GTP or Cdc42-GTP with PAK20 or MLK-3 (Bagrodia et al., 1995; Teramoto et al., 1996), MLK-3 with JNKUSAPKl or MKK3 (Tibbles et al., 1996), and JNKWSAPKl with JNWSAPK (Zanke et al., 1996). Interactions between MAPKs and their downstream substrates have also been detected biochemically, best exemplified by JNWSAPK interactions with c-Jun, which are stable enough to enable the detection and purification of JNWSAPK (Hibi et al., 1993). These interactions are mediated through interactions between a JNK docking site removed from the phosphorylation site on c-Jun and a specificity determination site on JNK/SAPK outside the kinase catalytic cleft (Kallunki et al., 1994,1996). Other substrates that interact with JNWSAPK include transcription factors ATF and Elk-1 (Bocco et al., 1996; Gupta et al., 1996), and p38 MAPK has been found complexed with MAPKAP kinase 2 and 3pk (McLaughlin et al., 1996; Krump et al., 1997). Substrates that copurify with ERK include MAPKAP kinase-1, cMyc, and c-Jun (Scimeca et al., 1992; Hsiao et al., 1994; Gupta and Davis, 1994; Bernstein et al., 1994).

B. Nuclear Translocation of MAPK and MKK Signaling from an extracellular ligand at the plasma membrane requires the migration of at least one of the components in the signaling cascade into the nucleus, where an important end result is transcriptional activation. In some cases, MAPKs fulfill this function, enabling regulation of gene expression through phosphorylation of nuclear transcription factors. The localization of ERKl and ERK2 is predominantly cytoplasmic in quiescent cells (Chen, R. et al., 1992; Lenormand et al., 1993). However, upon serum or growth factor stimulation, a fraction of cytoplasmic ERK translocates into nuclei (Chen, R. et al., 1992; Traverse et al., 1992; Lenormand et al., 1993; Gonzalez et al., 1993). Translocation occurs rapidly (within 5-30 min, depending on cell type) and typically endures for several hours. Similarly, JNKUSAPK is both cytoplasmic and nuclear in unstimulated cells, but upon UV irradiation migrates into the nucleus (Cavigelli et al., 1995). Although y radiation also stimulates JNKUSAPK activity, it does not appear to cause nuclear translocation (Chen, Y. et al., 1996a).Although a study of endogenous p38 MAPK translocation has yet to be reported, overexpressed p38 MAPK localizes at the cell periphery, in the cytoplasm, and in the nucleus; however, UV irradiation does not lead to its redistribution between cytoplasm and nucleus (Raingeaud et al., 1995). ERK3, unlike the other MAPKs, is constitutively nuclear (Cheng et al., 1996).

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Mechanisms for MAPK translocation have yet to be determined. Neither ERKl nor ERK2 contain nuclear localization or nuclear export signals, and ERKs are below the size limit for passive diffusion through nuclear pores, suggesting that nuclear export may be passive. One possibility is that ERK is retained in the cytoplasm through its interaction with a cytoplasmic localization factor. One candidate for this factor is MKK1, based on data showing a specific interaction of ERK with the N terminus of MKK, cytoplasmic retention of ERK upon expression of MKK, and reduced coimmunoprecipitation of MKK and ERK on cell stimulation (Fukuda et al., 1997). Interestingly, ERK nuclear translocation does not require its activation as nonphosphorylatable mutants ERK-Thr,,,Ala, Tyr,,,Phe or catalytically inactive ERK still translocate in response to serum (Gonzalez et al., 1993; Lenorrnand et al., 1993). However, the rapid uptake of ERK and its nuclear retention against a gradient following stimulation suggest that active import is also likely. Unlike the ERKs, MKKl and MKK2 are cytoplasmic before and after growth factor stimulation (Zheng and Guan, 1994b; Moriguchi et al., 1995a). MKK has a short N-terminal, leucine-rich stretch of amino acids (amino acids 32-44) that acts as a strong and autonomous nuclear export signal (Fukuda et al., 1996). Ovalbumin, covalently coupled to a synthetic peptide containing the MKK export sequence, is directed out of the nucleus immediately after microinjection. Thus, a mechanism is in place to export MKK to the cytoplasm, implying that nuclear import of wild-type MKK may occur under physiological conditions. One study has demonstrated serumdependent nuclear uptake of MKK mutants lacking the export sequence (Jaaro et al., 1997). This raises the question of whether MKK import is required for signaling. For example, MKK and ERK activation may be elevated over several hours following cell stimulation under certain conditions. The existence of high levels of nuclear phosphatases, including dual specificity phosphatases, which target ERK, necessitates a mechanism for maintaining elevated ERK activity in nuclei. Therefore, one possibility is that MKK nuclear uptake may serve as a means for maintaining active nuclear ERK.

VIII. FUTURE DIRECTIONS Eukaryotic MAP kinase cascades provide excellent examples of signal transduction mechanisms that embody key principles common to many, if not all, signaling pathways. These include regulation of reversible posttranslational modification by phosphorylation, sequential activation of multiple signaling components, and involvement of second messengers. Studies

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have revealed additional features of these pathways important for signal transmission, such as stable protein complex formation, translocation between intracellular organelles, cross-regulation, and the involvement of homologous kinase modules in parallel pathways. Many fundamental questions remain for future studies to investigate the mechanisms by which these pathways are regulated as well as the cellular responses that they control. For example, in several cases, the identification of pathway components is incomplete; in particular, many enzymes that mediate the activation of stress-activated kinases by upstream effectors, as well as the sensors for these stress signals, have yet to be identified. We also have little understanding of how these kinase cascades can control such diverse processes as cell transformation, stress responses, and cell differentiation, and why different types of cells respond to the same pathway in different ways. The substrates found so far for MAPKs are likely to be a small fraction of the total number of targets that exist. We have few clear examples of how phosphorylation regulates their function, and we have yet to identify other substrates for upstream kinases that enable these pathways to bifurcate. Finally, little is known of how the events in signaling and cross-regulation are coordinated in order to modulate and tune the cellular responses to these pathways. These kinase cascades are clearly not fabricated from assorted enzymes jumbled together, but require processes of ordered assembly and hierarchal activation to enable the efficient facilitation of signal transduction. The combined use of genetics, molecular biology, and biochemical approaches, together with newly acquired genomic sequences, now set the stage for further exciting research into this highly complex problem.

ACKNOWLEDGMENTS This work was supported by Grants GM48521 (N.G.A.)and GM18151 (P.S.S.) from the National Institutes of Health. We thank Katheryn Resing, Irene Ota, Loree Kim, and Donna Louie for many helpful discussions.

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Zaitsevskaya-Carter,T., and Cooper, J. A. (1997).E M B O ] . 16,1318-1331. Zanke, B. W., Rubie, E. A., Winnett, E., Chan, J., Randall, S., Parsons, M., Boudreau, K., McInnis, M., Yan, M., Templeton, D. J., and Woodgett, J. R. (1996a). 1.Biol. Chem. 271, 29876-29881. Zanke, B. W., Boudreau, K., Rubie, E., Tibbles, L. A., Zon, L. I., Kyriakis, J. M., Liu, F. F., and Woodgett, J. R. (1996b). Curr. Biol. 6, 606-613. Zarzov, P., Mazzoni, C., and Mann, C. (1996).E M B O J. 15, 83-91. Zervos, A., Faccio, L., Gatto, J. P., Kyriakis, J. M., and Brent, R. (1995). Proc. Nutl. Acud. Sci., USA. 92,10531-10534. Zha, J., Harada, H., Yang, E., Jockel, J. and Korsrneyer, S. J. (1996).Cell 87, 619-628. Zhang, F., Strand, A., Robbins, D., Cobb, M. H., and Goldsmith, E. J. (1994). Nature 367, 704-71 1. Zhang, J., Zhang, F., Ebert, D., Cobb, M. H., and Goldsmith, E. J. (1995).Structure 3,299-307. Zhang, S., Han, J., Sells, M. A., Chernoff, J., Knaus, U. G., Ulevitch, R. J., and Bokoch, G. M. (1995).j. Biol. Chem. 270,2393423936. Zhang, X., Blenis, J., Li, H. C., Schindler, C., and Chen-Kiang, S. (1995). Science 267, 1990-1 994. Zhang, X. F. Settleman,J., Kyriakis, J. M., Takeuchi-Suzuki,E., Elledge, S. J., Marshall, M. S., Bruder, J. T., Rapp, U. R., and Avruch, J. (1993).Nature 364, 308-313. Zhao, Y., Bjerbaek, C., Weremowicz, S., Morton, C. C., and Moller, D. E. (1995).Mol. Cell. Biol. 15,4353-4363. Zhao, Z.-S., Leung, T., Manser, E., and Lirn, L. (1995).Mol. Cell. Biol. 15,5246-5257. Zheng, C.-F., and Guan, K.-L. (1993b3.1. Biol. Chem. 268, 16116-16119. Zheng, C.-F., and Guan, K.-L. (1993a).]. Biol. Chem. 268, 11435-11439. Zheng, C.-F., and Guan, K.-L. (1994a).E M B O J . 13, 1123-1131. Zheng, C.-F., and Guan, K.-L. (1994b).]. Biol. Chem. 269, 19947-19952. Zhou, G., Bao, Z. Q., and Dixon, J. E. (1995).1. Biol. 270,12665-12669. Zu, Y. L., Ai, Y. and Huang, C. K. (1995).]. Biol. Chem. 270,202-206.

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FHIT in Human Cancer Cabdella Sozzi,"Kay Huebner,f and Carlo M. Crocet *Division of Experimental Oncology A, lstituto Nazionale Tumori, 201 33 Milan, Italy, and tKimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 191 07

I. Introduction A. Chromosomal Rearrangements and Loss of Heterozygosity of the Short Arm of Chromosome 3 in Tumors B. Fragile Sites and FRA3B 11. Cloning and Structural Features of the FHIT Gene 111. The Fhit Protein and Its Biochemical Properties IV. FHIT Abnormalities in Human Cancer A. Tumors of the Gastrointestinal Tract B. Tumors of the Aerodigestive Tract C. Tumors and Preinvasive Lesions of the Breast D. Other Tumors V. Conclusions and Perspectives References

1. INTRODUCTION

A. Chromosomal Rearrangements and Loss

of Heterozygosity of the Short Arm of Chromosome 3 in Tumors Chromosomal deletions and loss of heterozygosity (LOH) involving the short arm of chromosome 3 (3p)occur frequently in carcinomas of the lung, head and neck, kidney, breast, and other epithelial neoplasms (Devilee et al., 1989; Hibi et al., 1992; Lothe et al., 1989; Ogawa et al., 1991; Yang-Feng et al., 1993; Kohno et al., 1993; Maestro et al., 1993). However, the pattern of 3p losses in tumors is complex and involves several distinct regions: 3p12, 3 ~ 1 4 ~ 3 ~and 2 1 3p24-25. , Deletions of 3p are probably early events in cancerogenesis as they have been reported in preneoplastic lesions of the lung (Sozzi et a!., 1991; Sundaresan et al., 1992; Hung et al., 1995a), in benign proliferative breast diseases (Teixeira et al., 1996; Panagopoulos et al., 1996), and in oral leucoplakia (Ma0 et al., 199613; Roz et al., 1996). These findings, together with the identification of homozygous deletions in cancer cell lines, have led to the hypothesis that these regions are the sites of tumor Advances in CANCER RESEARCH 0065-230W98 $2S.00

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suppressor genes (TSG),whose inactivation is achieved by the loss of one allele and the presence of inactivating mutations or lack of expression in the remaining allele. These genetic changes ultimately result in the loss of the wild-type protein expression or function. Tumor suppressor activity has been demonstrated for the entire chromosome 3 in lung adenocarcinoma (Satoh et al., 1993), renal cell carcinoma (Shimizu et al., 1990; Yoshida et al., 1994; Sanchez et al., 1994), ovarian carcinoma (Rimessi et al., 1994), and mouse fibrosarcoma (Killary et al., 1992).The tumor suppressor activity was subsequently associated with portions of 3p, indicating the existence of two regions displaying such an activity. The region 3p22-p21 suppressed the tumorigenic properties of a mouse fibrosarcoma cell line (Killary et al., 1992), and the introduction of region 3~14-12into renal carcinoma cells with a translocation t(3;8) (p14.2;q24) resulted in partial suppression of tumor growth in nude mice (Sanchez et al., 1994). The region within 3p22-p21 has been narrowed down to an 80-kb clone from 31321.3 as demonstrated by suppression of tumor growth in vivo (Todd et al., 1996). However, no solid candidate for the tumor suppressor gene(s) in these regions has yet been identified. So far the only definite 3p-linked TSG is the Von Hippel-Lindau gene (VHL),located at 3p25. Von Hippel-Lindau disease is a familial cancer syndrome, dominantly inherited, that predisposes affected individuals to a variety of tumors, among them renal cell carcinomas. The VHL gene was shown to be mutated in the germline DNA of VHL disease families (Latif et al., 1993). The VHL gene is also inactivated in a considerable fraction of sporadic renal cancer (Gnarra et al., 1994) but is only rarely mutated in other tumors, such as lung lesions (Sekido et al., 1994). Region 3p14 is frequently lost in primary carcinomas (Rabbitts et al., 1989; Brauch et al., 1990; Hibi et al., 1992; Lubinski et al., 1994; Hung et al., 1995b; Druck et al., 1995; Pandis et al., 1996; Teixeira et al., 1996; Mao et al., 1996b; Roz et al., 1996) and is the site of homozygous deletions in a range of cancer-derived cell lines (Lisitsyn and Wigler, 1995; Kastury et al., 1996; Boldog et al., 1997). It also contains the 3p break of a hereditary renal-carcinoma-associated translocation, t(3;8) (p14.2;q24), which has been shown to segregate in a family with an early onset of bilateral and multifocal clear cell renal carcinoma (Cohen et al., 1979). FRA3B, another cytogenetic landmark in chromosome region 3 ~ 1 4 . 2 ,is the most active of the common constitutive aphidicolin-inducible fragile sites in the human genome (Glover et al., 1984).

B. Fragile Sites and FRA3B Fragile sites are specific regions of chromosomes that reveal cytogenetically detectable breaks when cells are exposed to certain chemical reagents or cul-

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ture conditions and are divided into two classes, common and rare (for review see Sutherland, 1991). Whereas common fragile sites are expressed in all individuals, although at different levels, rare fragile sites are present in a small proportion of the population and are heritable. Several folate-sensitive, heritable, X-linked, and autosomal fragile sites have been localized to unstable CCG or CGG repeats (Yu et al., 1991; Fu et al., 1991; Kremer et al., 1991) and were shown to be implicated directly in human diseases. The best known examples are the fragile X syndrome (FRAXA) and the X-linked mild mental retardation locus (FRAXE)characterized by the dramatically increased size of simple trinucleotide repeats, (CCG)n. This amplification of repeats is associated with abnormal methylation of the adjacent CpG islands and loss of expression of the FMRl and FMR2 genes (Verkerk et al., 1991; Pieretti et al., 1991; Knight et al., 1993; Mulley et al., 1995). A direct link between a fragile site and “in vivo” chromosome breakage has been demonstrated with the association between a chromosome deletion syndrome and a fragile site (Jones et al., 1995). In fact, the fragile site FRAllB at l l q 2 3 has been localized to the (CCG)n repeat of the CBL2 protooncogene. Jacobsen syndrome, recognized by specific dysmorphic features and moderate mental retardation, is characterized by a deletion of the long arm of chromosome 11 (llq23-qter). A proportion of Jacobsen syndrome patients have been shown to inherit a chromosome carrying a CBL2 expansion, which was truncated close to FRAllB. In addition, a CpG island adjacent to FRAllB was found to be methylated when its length exceeded certain limits. The rare dystamycin A-sensitive fragile site, FRAl6B located at 16q22.1, has also been isolated by positional cloning and is an expanded 33 bp ATrich minisatellite repeat (Yu et al., 1997). Therefore, the unstable repeat sequence mutation, so far associated with the increase in copy number of trinucleotide repeats, as mentioned earlier, is also a property of repeats of various compositions and also with various lengths of repeat motif. Common fragile sites represent a cytogenetic puzzle. The biologic role, if any, and the molecular basis for chromosomal fragility at common fragile sites are not known. The apparent association among common fragile site localization, cancer breakpoints, and genes involved in tumorigenesis (Yunis and Soreng, 1984; Le Beau, 1986) led to the hypothesis that fragile site breakage was involved in the chromosomal rearrangements and allele losses observed in malignant diseases. Common fragile sites are highly conserved during evolution (Schmid et al., 1985), are induced by agents that perturb DNA replication, are located in the euchromatic and late replicating genome regions, and are induced by agents known to attack chromatin DNase I hypersensitive sites, i.e., sequences associated with expressed genes (Yunis et al., 1987; Musio and Sbrana, 1996). In fact, common fragile site Xp22.1 is expressed only on the active X chromosome. Thus, common fragile sites could be associated with transcriptional activity. Consequently, the potential role of a fragile site located at the 5’ end of a tumor suppressor gene could

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be the loss of transcription of such a hypothetical gene as a result of fragile site expression. It is of interest that the rate of expression of common fragile sites can vary from person to person; occasionally, individuals are encountered who have high levels of expression of one of these fragile sites (Sutherland and Richards, 1995). The most active of the inducible common fragile sites of the human genome is FRA3B, contained in the 3p14.2 chromosomal band. FRA3B expression is observed after exposure of cultured cells to diverse mutagens and carcinogens, including benzo[a]pyrene diol epoxide, the ultimate carcinogen of benzo[a]pyrene (a major constituent of tobacco smoke) (Yunis et al., 1987) and ethanol (Kuwano and Kajii, 1987). A significantly increased frequency of FRA3B expression has been reported in peripheral lymphocytes of smokers (Kao-Shan et al., 1987). Aphidicolin induction of breakage and rearrangement of FRA3B in somatic cell hybrids resulted in the generation of hybrid clones with human/ hamster translocations involving breakpoints at FRA3B (Glover and Stein, 1988; Paradee et al., 1995). Subsequent studies of the position of hybrid clone breakpoints (Wilke et al., 1994; Boldog et al., 1994) have shown that the genomic region involved was up to 100 kb, suggesting that FRA3B may represent a region of fragility rather than a single site. It has been shown that an area of frequent breaks within FRA3B coincides with the spontaneous HPV16 integration site, offering direct evidence for the coincidence of viral integration sites and fragile sites (Wilke et al., 1996).

11. CLONING AND STRUCTURAL FEATURES

OF THE FHIT GENE The observations summarized in Section I,A indicate that the 3p14.2 region probably harbors one of the long sought tumor suppressor genes on the short arm of chromosome 3. The isolation of a YAC contig covering the t(3;8) break and FRA3B (Wilke et al., 1994; Boldog et al., 1994; Michaelis et af., 1995; Kastury et al., 1996) and the development of STS markers allowed the definition of homozygous deletions in a range of cancer-derived cell lines (Kastury et al., 1996). Ohta et al. (1996) developed a cosmid contig covering the common homozygously deleted region and identified and characterized a gene that is partially deleted in uncultured tumors of the aerodigestive tract and other organs. This gene has been designated the fragile histidine triad gene or the FHZT gene. The cosmid contig was assembled from cosmids subcloned from the library prepared from YAC 648D4, the shorter YAC clone covering the commonly deleted region (Fig. 1).Individual cosmids were used in exon-trapping

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telomere Exons 10 9

145

a

8

~1

n 6 m n

2 3 BA

z

8

7

6

IHPVl61

n 5

1 t3;8 1 I

3

4

centrornere

n 2

1

Y648D4 Y850A6 YAC75OF1

Fig. 1 Organization of the FHITgene. Approximate location of the YAC clones, the relevant STSs, plasmid and HPV16 integration sites, and the t(3;8) translocation breakpoint are indicated.

experiments aimed at identifying genes within the deleted region. An oligonucleotide primer designed from the initial trapped exon was used in primer extension to obtain a 5’ extended product of the cDNA by a rapid amplification of cDNA ends (RACE) reaction. The longest product from the RACE reaction detected a ubiquitously expressed 1.1-kb mRNA by Northern blot analysis of mRNA from various normal tissues. Because cDNA sequences 5’ and 3‘ of the first trapped exon (exon 5 in Fig. 1)were not within the cosmid contig, cosmid libraries from YACs 850A6 and 750F1, which extend centromeric and telomeric to the fragile region deletions, respectively, were screened with the 5’ and 3’ cDNA probes flanking exon 5. Cosmids containing the remaining exons were then used to derive intron sequences using cDNA primers, and the structure of the gene was determined as shown in Fig. 1. The 1.1-kb FHITcDNA consists of 10 small exons and is distributed over a genomic locus of about a megabase. Only exon 5 falls within the region of homozygous deletion originally observed in tumor-derived cell lines. The coding region of the open reading frame begins in exon 5 and ends in exon 9. Interestingly, the first three exons (El, E2, and E3 of Fig. 1)of the gene are centromeric to the t(3;8) break. Thus this gene has become a strong candidate for involvement in initiation of the clear cell renal carcinoma of the t(3;8) family because one copy of the gene is disrupted by the translocation. In addition, the location of several markers, such as BE758-6 (Lisitsyn and Wigler, 1995) and D3S1300, previously found deleted at high frequency in tumors and cell lines (Kastury et al., 1996), within the FHIT gene locus, close to the first coding exon 5, suggested that FHITis the target of these deletions. Analysis of FHIT expression in normal and tumor tissues by Northern blot revealed a low level of expression in all normal human tissues tested, whereas varying levels of FHIT transcripts, from barely detectable to almost normal levels, were found in tumor-derived cell lines (Ohta et al., 1996). Reverse transcription-polymerase chain reaction (PCR) was used to

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detect abnormalities in FHZT transcripts in primary gastrointestinal tumors; a pattern of products ranging from one apparently normal-sized amplified transcript to numerous aberrant bands without a normal sized band was observed. Sequence analysis of the usually shorter aberrant products revealed an absence of various regions between exons 4 and 9, whereas normal tissue mRNA from the same organ did not exhibit alteration in the amplified sequences (Ohta et al., 1996). Because the aberrant transcripts frequently lacked exon 5 , which begins the open reading frame of FHIT, or exon 8, the highly conserved histidine triad-containing domain, it is likely that these aberrant products could not encode functional proteins. Insertions of various lengths of DNA, either between or replacing exons, were also observed (Ohta et al., 1996).

111. THE Fhit PROTEIN AND ITS BIOCHEMICAL PROPERTIES The FHlTgene encodes a polypeptide of 16.8 kD that is composed of 147 amino acids that show 52% identity and 69% similarity to a core region of 109 amino acids of the diadenosine S’,S’’’-Pl,P4-tetraphosphate (Ap,A) hydrolase from the fission yeast Schizosaccaromyces pombe (Robinson et al., 1993; Huang et al., 1995; Ohta et al., 1996). The latter enzyme is a 182 amino acid protein that catalyzes the in vitvo hydrolysis of dinucleoside polyphosphates, with Ap,A as the preferred substrate. Both the Fhit protein and the S. pombe Ap,A hydrolase are related by sequence to the HIT proteins, a group of molecules of unknown functions characterized by four conserved histidines, three of which make up a histidine triad (HIT) sequence, H X H X H, where X is most frequently valine (Huang et al., 1995; Ohta et al., 1996). Subsequent biochemical studies (Barnes et al., 1996) clearly demonstrated that the human Fhit protein could be classified as an Ap,A hydrolase on the basis of its in uitro enzymatic activity. Ap,A is the preferred substrate among ApnAs, and AMP is always one of the reaction products. By site-directed mutagenesis, each of the four conserved histidine residues of FHZT was changed to an asparagine. Each change resulted in a decrease in Ap,A hydrolase activity, demonstrating that all four conserved histidines are required for full activity, but the central histidine of the triad (H96) is absolutely essential for Ap,A hydrolase activity (Barnes et al., 1996). Consequently, alterations of exon 8 could be critical for the biological activity of the Fhit protein. The crystal structure of histidine triad nucleotide-binding protein (HINT) showed that histidine triad proteins (HIT) are nucleotide-binding proteins (Brenner et al., 1997). HINT-nucleotide complexes demonstrated that the

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most conserved residues in the superfamily mediate nucleotide binding and that the HIT motif forms part of the phosphate binding loop. Thus, FHIT is an enzyme of the HIT family, a family of genes involved in nucleotide metabolism. FHIT substrates Ap,A and Ap,A have been considered alarmones in bacterial systems, which are produced in times of cellular stress (Kitzler et al., 1992). Accumulation of Ap,A in human cultured cells is induced by interferon (Vartanian et al., 1996), and Ap,A is reported as a consequence of contact inhibition of growth and toxic stress (Segal and Le Pecq, 1986). FHIT-substrate or substrate-enzyme complexes may thus be involved in signaling responses to cellular stress, resulting in cell cycle arrest.

IV. FHIT ABNORMALITIES IN HUMAN CANCER For a summary of FHIT abnormalities, see Table I.

A. Tumors of the Gastrointestinal Tract The initial finding of a high incidence (46%) of LOH at 3p14.2 in gastric cancer and the definition of a small region of homozygous deletion (60 kb) in a gastric carcinoma cell line (KatoIII) (Kastury et al., 1996) contributed to the positional cloning of the 1-Mb FHIT gene, which includes FRA3B (Ohta et al., 1996). In the latter study, abnormal expression of FHIT was reported in 5 of 10 esophageal cancers, in 5 of 9 stomach cancers, and in 3 of 8 colon cancers. Subsequently, three other studies investigated FHIT abnormalities in colon and gastric tumors and cell lines (Thiagalingam et al., 1996; Baffa, submitted for publication; Gemma et al., 1997). Because most of the initial results on the FHIT gene were obtained mainly by nested RT-PCR analysis of gene expression, some investigators cautioned that such analysis may produce artifactual aberrant products not directly related to an alteration of the FHIT gene and its function. In fact, Thiagalingam et al. (1996) reported LOH at loci internal to the FHIT gene in only 22% of the colon cancer xenografts examined, and abnormal transcription of the gene was found in a minority ( 4 of 31) of the same tumors. They suggested that FHIT may not be causally related to colon cancer pathogenesis or that it is a bystander casualty of the genomic instability of this fragile site. The development of specific antibodies recognizing the Fhit protein has allowed a correlation of DNA lesions at the FHIT locus with aberrant RT-PCR products and altered Fhit protein in cell lines derived from tumors of the gastrointestinal tract, as well as from other sites (Druck et al., 1997). In this study the authors demonstrated that even when DNA and RNA alterations

Table I Summary of FHIT Abnormalities in Tumors Tumor type

Gastrointestinal Esophageal Colon

Gastric

Aerodigestive Lung

Type of specimena

10T 31 T 8T 9T 32 T 6 CL 40 T

14 SCLC (T) 45 NSCLC (T) 17 SCLC (CL) 24 NSCLC (CL) 1 s SCLC (CL)

FHIT abnormalitiesb

Types of Assay'

Reference

5/10 (50) 7/31 (22) 4/31 (13) 3/8 (37) 5/9 18/32 (56) 4/8 (50) 16/38 (42) 1/40

RT-PCR LOH RT-PCR RT-PCR RT-PCR LOH, SB, RT-PCR SB, RT-PCR, WB LOH SSCP

Otha et al. (1996) Thiagahgametal. (1996) Thmgahgam d al. (1996) Otha et al. (1996) Otha et al. (1996) Baffa et al. (1997) Baffa et al. (1997) Gemma et al. (1997) Gemma et al. (1997)

11/14 (80) 18/45 (40) 63-92% 0117 ( 0 ) 7/24 (29) 100% LOH; 73% RT-PCR

RT-PCR RT-PCR LOH DNAPCR, SB, RT-PCR DNA-PCR, SB, RT-PCR

Sozzi et al. (1996) Sozzi et al. (1996) Yanagisawa et al. (1996) Yanagisawa et al. ( 1996) Fong et a/. (1997)

Head and neck

17 NSCLC (CL) 8 SCLC (T) 108 NSCLC (T) 8 CIS 26 CL

16 CL Breast

Merkel cell carcinoma Pleomocphic adenoma parotid gland

11 Cl 30 T 3 14 T 1T

88% LOH; 77% RT-PCR 100% LOH 45% LOH; 59% RT-PCR 6/8 (75) 15/25 (55) 13/20 (65) 3/22 (12) 13/16 (81) 7/16 (45) 3/11 (27) 9/30 (30) 3/3 (100) 8/14 (57) 1/1

LOH RT-PCR FISH SB LOH RT-PCR RT-PCR RT-PCR DNA-PCR, RT-PCR RT-PCR Cytogenetics, FISH sequence, RT-PCR

Fong et al. ( 1 997) Fong et al. (1997) Fong et al. (1997) Fong et a[. (1997) Virgilio et al. (1996)

Mao et al. ( 1996) Negrini etal. (1996) Negrini et al. (1996) Panagopodoset al. (1996) Sozzi et al. (1996) Geurts et al. (1997)

dT, tumors; CL, cell line; AH, atypical hyperplasia; CIS, carcinoma in situ. *Number of cases. Numbers in parentheses represent percentage of total number of cases.
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of the FHIT locus are not detected, the Fhit protein may be absent, reduced, or possibly altered. Consequently, the best way to assess the involvement of the FHIT gene in human cancer could be the detection of the presence of the Fhit protein by Western blot and immunohistochemistry. The types of alterations, mainly deletions and/or rearrangements, and the rarity of point mutations observed in the FHZT locus (Gemma et al., 1997) suggest that the mechanisms of FHIT inactivation may be different than those described for other, now classic, tumor suppressor loci, possibly due to the fact that FHIT encompasses the FRA3B region. In fact, the two regions of FRA3B that have already been isolated as flanking sites to an HPV16 or pSV2neo insertion (Wilke et al., 1996; Rassool et al., 1991) occur within intron 4 and 5 of FHZT, respectively, surrounding the frequently deleted exon 5. As chromosome breakage and integration of exogenous DNA at chromosome band 3p14.2 is a frequent event in aphidiColin(apc)-treated somatic cell hybrids, cancer cells, and apc-treated lymphocytes, a fluorescence in situ hybridization (FISH)approach using cosmids covering specific regions of the FHITgene was used to demonstrate that most of the apc-induced gaps at FRA3B fall within the FHIT gene, with the highest frequency of gaps falling in intron 5 (Zimonjic et al., 1997). Gaps were also observed in intron 4, the site of the integration of HPV16, and in intron 3, the location of the t(3;S) breakpoint. These observations mirror findings in tumor cells where loss and rearrangements of exons 3 , 4 , and 5 were reported (Druck et al., 1997), suggesting that these cancer-specific deletions originated from breaks in fragile sites, although the molecular bases for the fragility of this region have not yet been identified. A more recent study (Baffa et al., submitted for publication) used RT-PCR, Southern analysis, LOH, and protein studies to analyze eight gastric cancerderived cell lines and 32 primary adenocarcinomas of the stomach in order to define the role of the FHIT gene in gastric cancer development. Four of the eight cell lines showed deletions or rearrangements within the FHITgene, together with an absence of the wild-type transcript and Fhit protein. Among the primary gastric carcinomas, a total of 18 of 32 (56%) exhibited rearrangement of the FHIT gene and/or aberrant RTPCR products. In analyzing evidence for changes in the FHZT gene and its expression relative to clinicopathological parameters of gastric carcinoma, a significant correlation was observed between the intestinal subtype of gastric carcinoma and the occurrence of FHIT alterations. It is of interest that gastric carcinomas of the intestinal type are thought to develop from carcinogenic exposure (Correa et al., 1973). Thus, this study strengthens the hypothesis that abnormal FHZT transcripts are a consequence of carcinogen-mediated DNA damage, resulting ultimately in the absence of the Fhit protein. An analysis of matched cancer and normal tissues from 40 cases with primary gastric cancer using PCR single-strand conformation polymorphism (SSCP) and direct sequenc-

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ing revealed the presence of a missense mutation in exon 6 in a signet ring cell adenocarcinoma (Gemma et al., 1997). In addition, allelic deletion of FHIT occurred in 42% of the tumors analyzed. The occurrence of point mutation, although occasional, as well as the frequent allelic deletions observed in these tumors, points to the FHIT gene as a target of inactivation in 40-50% of gastric cancer.

B. Tumors of the Aerodigestive Tract Lung cancer is a major cause of mortality worldwide, and the overall survival rate has not improved significantly since the mid-1970s. The understanding of the molecular pathogenesis of this disease may help to prevent it and to provide new and more sensitive means to diagnose and treat lung cancer patients. The most common genetic alteration in small cell (SCLC) and non-small cell lung cancer (NSCLC) is loss of genetic material on the short arm of chromosome 3 (3p). A cytogenetically visible deletion, de1(3)(p14p23),was first reported in 1982 in SCLC cell lines and then in primary tumors of both small cell and non-small cell types (Whang-Peng et al., 1982; Testa et al., 1994). Refined molecular analyses with polymorphic markers indicated LOH at loci on 3p at three distinct regions located at 3 ~ 2 5p21.3-21.2, , as well as more proximal deletions in the 3~12-14region (Naylor et al., 1987; Yokota et al., 1987; Brauch et al., 1987; Rabbitts et al., 1989; Mori et al., 1989; Hibi et al., 1992).LOH of loci on 3p appears to be an early event in lung cancer, as it has been reported in early precancerous lesions such as bronchial dysplasia, metaplasia, and hyperplasia (Sozzi et al., 1991; Sundaresan et al., 1992; Hung et al., 1995a). These observations led to the hypothesis that these regions could be the sites of a critical tumor suppressor gene(s).However, no solid candidate for a tumor suppressor gene(s) on 3p had been identified. To determine the role of the FHIT gene in lung cancer, more than 100 primary lung tumors were analyzed for abnormalities of FHIT expression by reverse transcriptase-PCR analyses and sequencing, LOH, and Southern blot, as well as by immunocytochemistry (Sozzi et al., 1996b, 1997). Abnormalities in products amplified from FHIT transcripts were found in 80% of SCLC tumors and in 42% of NSCLC. Sequence analysis of the aberrant products showed that the absence of exons 4 or 5 through 8 was the most common abnormality. Most of the tumors also displayed a normal size amplified product, possibly due to normal tissue mixed with the tumor. LOH at three microsatellite markers internal to the FHIT gene, located either in the fragile region (D3S1300, D3S4103) or in the more telomeric distal 3' end of the gene (D3S1234),was detected in 63% of the tumor specimens. Tumors exhibiting aberrant FHIT amplification products also lost one FHIT allele, suggesting a loss of function of the FHIT gene. In or-

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der to correlate specific FHIT locus DNA lesions with their effects on transcription products and protein expression, 11 lung cancer cell lines of different type were studied by Southern blot analysis, reverse transcriptionPCR, Western blot analysis, and immunocytochemistry. Three cell lines, after hybridization with the cDNA and specific cosmid probes covering large intronic regions of FHZT, showed abnormal restriction patterns, consisting in deletions or rearrangements involving exon 3, exon 4, and intron 5, as well as a more distal region surrounding exons 6 and 7. In addition, one cell line showed a homozygous deletion in intron 5. The RT-PCR analysis of RNAs revealed a complete absence of the normal FHIT transcript in three cell lines: one showed lack of exon 4 only, whereas in the other two only multiple abnormal transcripts lacking exons 3 or 4 through 8 and 9 were present. In six cell lines, FHZT mRNA products of both normal and abnormal sizes were found, whereas two cell lines showed the wild-type transcript only. Cloning and sequencing of the apparently normal-sized transcripts in several cell lines revealed a mixture of different transcripts lacking crucial coding exons, such as exons 5 through 8, and often exhibiting insertions of sequences of various lengths, either between or replacing exons. The inserted sequences derived from human DNA show identical size and sequence in some cell lines and primary tumors, suggesting that they could derive from FHIT introns. Thus, potential proteins resulting from the aberrant transcript would lack relevant portions of the wild-type protein; the insertion of new nucleotides would result in either adding new amino acids or altering the reading frame of the gene. This multiplicity of genetic lesions may be explained by the fact that the FHZT gene encompasses the fragile 3B region, which by definition is highly susceptible to breakage induced by carcinogens such as those in tobacco smoke. Western blot analysis, using a rabbit polyclonal antibody against the GST-FHIT fusion protein revealed that indeed 9 of the 11 lung cancer cell lines did not produce Fhit protein. Immunocytochemistry studies using the same antibody showed that the two cell lines positive for Fhit by Western blot displayed clear cytoplasmatic immunostaining. These data indicate that due to the complexity of FHIT rearrangements, immunocytochemistry may be the best way to assess the level of involvement of FHIT in lung tumors as the Fhit protein was absent or greatly reduced in tumor cell lines for which DNA or RNA alterations of the FHIT locus were not observed. Other investigators have confirmed that FHZT is altered in a significant proportion of NSCLC cell lines. Yanagisawa et al. (1996) observed either lack of expression or aberrant splicing in 7 out of 24 (29%)cell lines, often accompanied by intragenic homozygous deletions. Very recently, a thorough analysis of molecular abnormalities of the FHZT gene in primary lung tumors, cell lines and preneoplastic bronchial lesions confirmed that FHIT and FRA3B abnormalities are associated with lung cancer pathogenesis (Fong et

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al., 1997). The results showed that the FHZTIFRA3B region undergoes allele loss in the vast majority of lung cancers (occurring first at the stage of carcinoma in situ), which may exhibit homozygous deletions (including intragenic deletions), and very frequently expresses aberrant FHIT transcripts, although in most cases an intact wild-type transcript was coexpressed, as assessed by RT-PCR analyses. FHIT mRNA was undetectable or barely detectable by Northern blot analysis in tumor-derived cell lines, suggesting that most cells in the population were expressing very little wild-type or altered FHZT transcripts. Head and neck cancers (HNSCC)represent 3% of all cancer in Western countries and are associated with a high mortality rate, with a 5-years survival of 40%. Tobacco and alcohol have been recognized as etiological factors in these carcinomas, and the reported increase in the incidence of HNSCC by epidemiological studies is probably due to changes in consumption of these agents. Several regions of LOH have been identified in HNSCC, with a 45-50% rate of loss on the short arm of chromosome 3 (Nawroz et al., 1994; Ishwad etal., 1996), not only in oral carcinomas but also in oral dysplastic lesions at the precancerous stage (Roz et a\., 1996). In particular, it has been reported that LOH at the 3p14 region is a frequent event in oral premalignant lesions and is associited with oral cancer development (Ma0 etal., 1996b).FHlTgene alterations have been investigated in head and neck cancer cell lines in two studies (Virgilio et al., 1996; Mao et al., 1996a). Twenty-six oral cancer cell lines were examined for deletions by Southern blot analysis, for allelic losses of specific FHIT exons by interphase nuclei FISH, and for integrity of FHIT transcripts by RT-PCR analysis (Virgilio et al., 1996). Three cell lines exhibited homozygous deletions within the FHZT gene, the presence of aberrant transcripts was found in 55%, and 65% presented more than one subpopulation of cells with losses of different portions of FHZT alleles by FISH. Most of the cell lines that showed multiple FHZT transcripts also displayed two or three cell populations, with diverse regions of deletion of FHZT, explaining the amplification of both normal and aberrant transcripts by RT-PCR. This study suggested that an absence of FHZT does not provide a growth advantage in tissue culture for HNSCC. In analysis of 16 HNSCC cell lines from 11 patients, Mao et al. (1996a) reported LOH at 3p14.2 in 81% of cell lines and abnormal FHZTexpression in 45% of the patients. However, in this study, the genomic rearrangements observed by Southern blotting were not clear enough to permit a correlation between the presence of abnormal transcripts and DNA lesions. Additional studies are necessary to determine if disruption of FHIT contributes to malignant transformation of precancerous oral lesions presenting LOH at 3 ~ 1 4 . 2 . Taken together, the genetic lesions within the FHZT gene may be explained by the location of a fragile region within the gene, rendering the gene highly susceptible to breakage induced by carcinogens (Glover and Stein, 1988).

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Tumors associated with carcinogen exposure, such as cancers of the aerodigestive tract, could be particularly susceptible to alterations of the FHZT gene. Because of its etiology, lung cancer is likely to be strongly and directly associated with the effects of agents that interfere with DNA replication, such as chemicals in tobacco smoke. Accordingly, tumors exhibiting the highest frequency of FHIT abnormalities, such as lung, head and neck, and gastric cancer of the intestinal type, have been recognized to be caused by etiological agents such as tobacco and alcohol. Accordingly, a molecular analysis of microsatellite alterations within the FHZT gene and FRA3B in lung tumors from heavy smokers and in tumors developed in never smokers was undertaken to seek genetic damage attributable to tobacco smoking (Sozzi et al., 1997a). LOH at D3S1300 and D3S4103 microsatellite markers, located in the epicenter of the fragile region encompassing exon 5 and intron 5 of the FHIT gene (Fig. 1) and at D3S1234 in the more distal 3' end of the gene, was analyzed in tumor tissue. LOH affecting at least one locus of the FHZT gene was observed in 41 out of 51 tumors in smokers (80%) but in only 9 out of 40 tumors in nonsmokers (22%). The comparison between the frequency of losses in smokers and nonsmokers was statistically significant (80% vs 22%, p = 0.0001). All the tumors with loss of one FHIT marker had lost all the informative markers, suggesting that the tumor cells had lost an entire FHZTallele. These findings suggest that FHIT is a preferential target of carcinogens in tobacco smoke at a molecular level and indicate the possibility of using LOH a t FHIT and FRA3B as early molecular indicators of damage related to tobacco smoke in screening high-risk individuals, such as those belonging to the heavy smokers category.

C. Tumors and Preinvasive Lesions of the Breast Breast cancer is the most frequent neoplasm among women in most Western countries (Wingo et al., 1996). However, little is known regarding the genetic changes associated with the various stages of mammary carcinogenesis, precluding the use of genetic changes in clinical applications such as early diagnosis and determination of clinical behavior. Despite the complexity of the tumor karyotype in breast carcinomas, one of the few abnormalities that has been repeatedly detected, first by cytogenetic analysis and then by LOH studies, is an interstitial deletion of the short arm of chromosome 3 (Pandis et al., 1993; Buchhagen et al., 1994; Chen et al., 1994; Pandis et al., 1995). The minimal common deleted segment was 3p14 (Buchhagen et al., 1994; Pandis et al., 1995). 3p deletions were also found in benign hyperproliferative breast disease (Dietrich et al., 1995; Peterson et al., 1996) and in in situ ductal carcinoma (Teixeira et al., 1996),

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indicating that 3p deletions are probably early events in mammary carcinogenesis. To determine whether the FHIT gene might be a target of breast cancerspecific 3p alterations, Negrini et al. (1996) performed an analysis of cell lines and primary tumors for alteration in transcription of the FHlT gene. Lack of expression of FHIT mRNA was found in 10% of the breast cancer samples as detected by RT-PCR analysis. Aberrant and normal-sized transcripts were present in four samples; sequence analysis revealed that aberrant products were generated by the fusion of exon 3 with exons 7, 8, or 9, resulting in an absence of in-frame ATG start codons. Whereas sequencing of the normal-sized FHIT product revealed that most were wild type, an interesting finding was that in several cases the normal-sized band was actually a mixture of normal and aberrant transcripts lacking various coding exons. The simultaneous presence of normal and aberrant transcripts within the same tumor cell population suggests a dominant negative effect of the abnormal Fhit protein, an overall significant reduction of the Fhit native protein, or heterogeneity in the cell population, with some cells expressing wildtype FHIT. With the exception of one cell line (MDA-MB-436) where exon 5 is homozygously deleted, analysis of the DNA coding portions of the FHIT gene in the remaining cell lines showing abnormal FHIT transcripts did not reveal homozygous deletions of exons. Overall, these results suggested that alterations in the FHIT gene may play an important role in at least 30% of breast cancer, a relevant proportion if compared to the low frequency of abnormalities reported for other suppressor gene in the same tumor type. Further confirmation of involvement of the FHIT locus in breast neoplasia came from the analysis of three benign proliferative breast diseases with cytogenetic rearrangements of chromosome band 3 ~ 1 4 . 2(Panagopoulos et a/., 1996). RT-PCR analysis showed that the FHIT gene was either not expressed or that its expression was dramatically reduced to a level not detectable by RT-PCR in the samples with atypical hyperplasia. In addition, genomic analysis of exons 3 and 5 of FHIT provided evidence that DNA segments were homozygously deleted in the majority of the cells. These data are in agreement with the histopathological features and the cytogenetic findings in the three samples analyzed; none contained normal parenchyma and all had chromosomal aberrations involving 3p. Of interest, in addition to chromosomal deletions, translocations and inversions of 3p were also detected in these specimens, indicating that FHIT disruption is achieved by various inter- and intrachromosomal rearrangements. Although these FHIT alterations were observed in samples from two women with familial predisposition to breast cancer, it is likely that they would be detected in sporadic breast lesions with 3p14 aberrations and that

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the finding of homozygous deletions in the cases of epithelial hyperplasia in this study may be due to the selection of samples with known cytogenetic alterations of 3p14.

D. Other Tumors FHZT gene abnormalities have so far been described in two other tumor types. Sozzi et al. (1996a) have examined primary Merkel cell carcinomas (MCCs) for the presence of FHIT abnormalities by RT-PCR and sequencing. MCC is a rare neuroendocrine carcinoma of the skin that shares several features with small cell lung carcinoma, including morphological and immunophenotypical characteristics, as well as some aspects of natural history (Cattoretti et al., 1989). In fact, both of these small cell tumor types have neuroendocrine features and express specific neuropeptides and intermediate filament protein (Gould et al., 1985). Clinically, both SCLC and MCC show a high incidence of recurrences and nodal metastases, although MCC behaves less aggressively (Pilotti et al., 1982; Pilotti et al., 1988; Mercer et al., 1990). Little is known about the genetic changes associated with MCC, with the exception of several cytogenetic studies that reported the occurrence of chromosomal rearrangements involving various chromosomes (Kusyk and Romsdahl, 1986; Sandbrink et al., 1988; Sozzi et al., 1988; Shabtai et al., 1989; Smadja et al., 1991). However, a study by Leonard et al. (1996)of 26 MCC tumors revealed LOH at 3~13-21.1in 69% of the cases, and rearrangement of chromosome 3 was detected in a cell line derived from one tumor by FISH. Because of the similarities of SCLC and MCC and the high frequency of LOH in the 3p region where FHIT is localized, an analysis of FHZT gene abnormalities was conducted in 14 cases of MCC (Sozzi et al., 1996a). Eight of 14 tumors (57%)displayed abnormal FHIT products by RT-PCR analysis. Abnormal transcripts of 360 or 250 bp or both occurred in six of the eight samples, whereas in two specimens a product of 500 and 450 bp was found, respectively. In all cases a normal-sized transcript was observed, which is believed to be caused by normal stromal contamination [as suggested by the presence of partial allelic losses (Leonard et al., 1996)], as well as by histopathological analyses of this tumor type (Pilotti et al., 1982). The aberrant products lacked a variable number of exons of the FHZT gene, including exon 5 , which contains the initial methionine codon, and exon 8 where the HIT domain of the gene resides. It is unlikely that the aberrant transcripts encode functional proteins. In 6 of 8 tumors showing FHZT abnormalities, the abnormal transcripts were fusions of exons 3-7 and exons 3-9. These aberrant products corresponded to the type I and I1 abnormal

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transcripts consistently found in SCLCs (Sozzi et al., 1996a).The occurrence in MCC of abnormal FHIT products, coupled with a high frequency (69%) of LOH reported in region 3p13-p21.1 (Leonard et al., 1996), also suggests that FHZT gene inactivation could be achieved by the loss of one allele and deletion within the remaining one, as shown for the other tumor types investigated so far. Although SCLCs are linked to carcinogenic exposure of tobacco smoke, ultraviolet light exposure may be involved in the etiopathogenesis of some MCC tumors (Gomez et al., 1983; Silva et al., 1984; Mercer et al., 1990), which frequently coexist with basal or squamous cell carcinoma of the skin. Thus, the recurrent pattern of FHIT abnormalities in small cell carcinomas of the lung and of the skin suggests that the FHZT gene could be damaged by chemical or physical agents that preferentially disrupt the fragile region. Another mechanism of disruption of the FHIT gene has been described in a case of pleomorphic adenoma of the parotid gland (Geurts et al., 1997). These tumors, the most frequent are benign tumors of the salivary glands, carry chromosome aberrations in 50430% of the cases. Whereas t(3;8)(p21;q12)is the most frequently observed translocation, many different chromosome segments have been found as translocation partners of both 8q12 and 12q13-15. The HMGIC gene is the target for 12q13-15 translocations, but no fusion partner genes had been identified in this tumor type until recently. Using 3' RACE experiments, ectopic sequences fused to HMGIC were isolated in a tumor presenting a complex rearrangement involving chromosome 3 ~ 1 4 . 2 ,chromosome 12q13-15, and chromosome 1Oq. The ectopic sequences fused to HMGIC were revealed to derive from the FHZT gene. By FISH analysis using HMGIC cosmids, it was demonstrated that both the 5' and the 3' ends of the HMGZC gene were inserted into the 3p14.2 region. Comparative evaluation of the HMGIC fusion transcript and FHIT showed that the first three exons of the HMGIC gene, encoding the AT hook domains, were fused to exons 9 and 10 of the FHZT gene, which encode the last 31 carboxyl-terminal amino acids of this gene. RT-PCR analyses demonstrated the expression of both HMGICIFHIT fusion transcripts and its reciprocal counterpart. The breakpoint observed in this tumor was in intron 8 of FHIT, downstream of the HIT domain, resulting in the exchange of 31 carboxyl-terminal amino acids of the Fhit protein for 26 amino acids of the acidic tail of HMGIC. The putative mRNA destabilizing motif, AUUUA, present in the 3' UTR of the HMGIC gene, also became linked to the hybrid FHITIHMGIC transcript. This could result in a decrease in the levels of FHIT, which presumably occurs in most of the tumor types studied so far. In addition to the t(3;8)(p14.2;q24)of hereditary renal cell carcinoma (Ohta et al., 1996), this represents the second example in which FHIT is disrupted by a tumor-specific chromosome translocation.

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V. CONCLUSIONS AND PERSPECTIVES The discovery of FHIT gene abnormalities in tumors is the first molecular evidence linking the instability of fragile sites to cancer (Pennisi, 1996). Because of the frequent abnormalities in the FHZTgene in a variety of cancer-derived cell lines, as well as in primary tumors of the digestive tract, lung, and breast, three of the most common neoplasms in humans, abnormalities in this gene are becoming one of the most frequent genetic changes occurring in the tumorigenic process. Furthermore, indirect evidence suggests that this gene is also involved in other types of human neoplasms, e g , in cervical carcinoma, where one of the most frequently lost regions is 3p14 and where an insertion site for the human papillomavirus type 1 6 was found to be very close to the FRA3B fragile site (Wilke et al., 1996). It is now known that this insertion site is within the FHIT gene between exons 4 and 5, suggesting that human papillomavirus type 16 disrupts the FHIT gene by the induction of deletion during the integration process. In fact, deletions were detected in seven of eight cervical carcinoma lines, and of five of these cervical cell lines analyzed by RT-PCR amplification, absence of FHIT expression was observed in two and reduced expression in one (Boldog et al., 1997). These results led the authors to conclude that homozygous deletions in this region sometimes had no apparent effect on FHIT mRNA, suggesting either a different target gene or unselected genomic instability. However, protein data collected in cell lines and primary tumors of different types (renal, nasopharyngeal, cervical, gastric, lung) (Druck et al., 1997; Baffa et al., submitted for publication; Sozzi et al., 1997b) suggest that even when DNA or RNA alterations of the FHIT locus have not been observed in tumors and cell lines, the Fhit protein may be absent, reduced, or possibly altered. The complexity of the DNA lesions observed within the FHIT locus suggests that the types of alterations observed differ from those observed in other tumor suppressor loci, possibly because the FHIT gene encompasses the fragile 3B region. In addition, the fact that alterations in the FHIT gene have been observed in different regions of the locus, near exons 3 and 4,in intron 5, and in the more distal exons 8,9, and 10, suggests that the FHITgene is the target of loss and is involved in clonal expansion. In fact, if another single gene were the target, it would need to be spread over the same genomic region as the FHIT gene is. Because of its binding capability and hydrolase activity on diadenosine triand tetraphosphate molecules, which may accumulate in response to cellular stress, lack of the Fhit protein could result in abnormal adaptation of cells to stress. This effect may be an early event in neoplasia of some organs, as preneoplastic cells of various tissues (oral, bronchial, breast) show abnormalities of the FHIT gene. Functional studies aimed at restoration of FHZT function and prevention of tumor growth in vivo will prove that FHlT is a

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tumor suppressor gene. These studies could permit the development of novel therapeutical approaches, such as transfer of the wild-type FHIT gene to precancerous lesions or tumors of cancer patients carrying a disrupted FHIT gene, a gene therapy approach recently attempted for p.53 by gene transfer to tumors of patients with lung cancer (Roth et al., 1996).

ACKNOWLEDGMENTS The authors are grateful to Dr. Marco A. Pierotti for critical reading of the manuscript, Mr. Mario Azzini for professionally preparing the illustration, and Mrs. Anna Grassi for kindly preparing the manuscript.

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Deletion mapping of the short arm of chromosome 3 in Merkel cell carcinoma. Genes Chrom. Cancer 15,102-107. Lisitsyn, N., and Wigler, M. (1995). Representational difference analysis in detection of genetic lesions in cancer. Methods Enzymol. 254,291-304. Lothe, R. A., Fossa, S. D., Stenwig, A. E., Nakamura, Y., White, R., Borresen, A. L., and Brogger, A. (1989).Loss of 3p or 1l p alleles is associated with testicular cancer tumors. Genomics 5,134-138. Lubinski, J., Hadaczek, P., Podolski, J., Toloczko, A., Sikorski, A., McCue, P., Druck, T., and Huebner, K. (1994). Common regions of deletion in chromosome regions 3p12 and 3p14.2 in primary clear cell renal carcinomas. Cancer Res. 54, 3710-3713. Maestro, R., Gasparotto, D., Vukosavljevic,T., Barzan, L., Sulfaro, S., and Boiocchi, M. (1993). Three discrete regions of deletion at 3p in head and neck cancers. Cancer Res. 53, 5775-5779. Mao, L., Fan, Y. H., Lotan, R., and Hong, W. K. (1996a). Frequent abnormalities of FHIT, a candidate tumor suppressor gene, in head and neck cancer cell lines. Cancer Res. 56, 5 128-51 3 1. Mao, L., Lee, J. S., Fan, Y. H., Ro, J. Y., Batsakis, J. G., Lippman, S., Hittelman, W., and Hong, W. K. (1996b). Frequent microsatellite alterations at chromosomes 9p21 and 3p14 in oral premalignant lesions and their value in cancer risk assessment. Nature Med. 2, 682-685. Mercer, D., Brander, P., and Liddell, K. (1990).Merkel cell carcinoma: The clinical course. Ann. Plastic Surg. 25, 136-141. Michaelis, S . C., Bardenheuer, W., Lux, A., Schramm, A., Gockel, A., Siebert, R., Willers, C., Schmidtke, K., Todt, B., van der Hout, A. H. et al. (1995). Characterization and chromosomal assignment of yeast artificial chromosomes containing human 3p13-3p21-specific sequence tagged sites. Cancer Genet. Cytogenet. 81, 1-12. Mori, N., Yokota, J., Oshimura, M., Cavenee, W. K., Mizoguchi, H., Noguchi, M., Shimosato, Y., Sugimura, T., and Terada, M. (1989). Concordant deletions of chromosome 3p and loss of heterozygosity for chromosomes 13 and 17 in small cell lung carcinoma. Cancer Res. 49, 5 130-5 135. Mulley, J. C., Yu, S., Loesch, D. Z . , Hay, D. A., Donnelly, A., Gedeon, A. K., Carbonell, P., Lopez, I., Glover, G., Gabarron, I. et al. (1995). FRAXE and mental retardation. 1. Med. Genet. 32,162-169. Musio, A., and Sbrana, I. (1996). Common and rare fragile sites on human chromosomes. The cytogenetic expression of active and inactive genes? Cancer Genet. Cytogenet. 88,184-1 85. [Letter] Nawroz, H., van der Riet, P., Hruban, R. H., Koch, W., Ruppert, J. M., and Sidransky, D. (1994). Allelotype of head and neck squamous cell carcinoma. Cancer Res. 54, 1152-1155. Naylor, S. L., Johnson, B. E., Minna, J. D., and Sakaguchi, A. Y. (1987). Loss of heterozygosity of chromosome 3p markers in small-cell lung cancer. Nature 329,451454. Negrini, M., Monaco, C., Vorechovsky, I., Ohta, M., Druck, T., Baffa, R., Huebner, K., and Croce, C. M. (1996). The FHIT gene at 3 ~ 1 4 . 2is abnormal in breast carcinomas. Cancer Res. 56,3173-3179. Ogawa, O., Kakehi, Y., Ogawa, K., Koshiba, M., Sugiyama, T., and Yoshida, 0. (1991). Allelic loss at chromosome 3p characterizes clear cell phenotype of renal cell carcinoma. Cancer Res. 51, 949-953. Ohta, M., Inoue, H., Cotticelli, M. G., Kastury, K., Baffa, R., Palazzo, J., Siprashvili, Z . , Mori, M., McCue, P., Druck, T., Croce, C. M., and Huebner, K. (1996). The FHIT gene, spanning the chromosome 3p14.2 fragile site and renal carcinoma-associated t(3:8) breakpoint, is abnormal in digestive tract cancer. Cell 84, 587-597. Panagopoulos, I., Pandis, N., Thelin, S., Petersson, C., Mertens, F., Borg, A., Kristoffersson, U., Mitelman, F., and Aman, P. (1996). The FHIT and PTPRG genes are deleted in benign pro-

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liferative breast disease associated with familial breast cancer and cytogenetic rearrangements of chromosome band 3p14. Cancer Res. 56,48714875. Pandis, N., Idvall, I., Bardi, G., Jin, Y., Gorunova, L., Mertens, F., Olsson, H., Ingvar, C., Beroukas, K., Mitelman, F., and Heim, S. (1996). Correlation between karyotypic pattern and clinicopathologic features in 125 breast cancer cases. Int. I. Cancer 66, 191-196. Pandis, N., Jin, Y., Limon, J., Bardi, G., Idvall, I., Mandahl, N., Mitelman, F., and Heim, S. (1993).Interstitial deletion of the short arm of chromosome 3 as a primary chromosome abnormality in carcinomas of the breast. Genes Chrom. Cancer 6, 151-155. Pandis, N., Jin, Y., Gorunova, L., Petersson, C., Bardi, G., Idvall, I., Johansson, B., Ingvar, C., Mandahl, N., Mitelman, F. et al. (1995). Chromosome analysis of 97 primary breast carcinomas: Identification of eight karyotypic subgroups. Genes Chrom. Cancer 12, 173-185. Paradee, J. D., Mullins, C., He, Z., Glover, T., Wilke, C., Opalka, B., Schutte, J., and Smith, D. (1995). Precise localization of aphidicolin-induced breakpoints on the short arm of human chromosome 3. Genomics 27,358-361. Pennisi, E. (1996). New gene forges link between fragile site and many cancers. Science 272, 649. Petersson, C., Pandis, N., Mertens, F., Adeyinka, A., Ingvar, C., Ringberg, A,, Idvall, I., Bondeson, L., Borg, A., Olsson, H., Kristoffersson, U., and Mitelman, F, (1996). Chromosome aberrations in prophylactic mastectomies from women belonging to breast cancer families. Genes Chrom. Cancer 16,185-188. Pieretti, M., Zhang, F. P., Fu, Y. H., Warren, S. T., Oostra, B. A., Caskey, C. T., and Nelson, D. L. (1991).Absence of expression of the FMR-1 gene in fragile X syndrome. Cell66,817-822. Pilotti, S., Rilke, F., Bartoli, C., and Grisotti, A. (1988). Clinicopathologic correlations of cutaneous neuroendocrine Merkel cell carcinoma. J. Clin. Oncol. 6, 1863-1873. Pilotti, S., Rilke, F., and Lombardi, L. (1982). Neuroendocrine (Merkel cell) carcinoma of the skin. Am. I. Surg. Pathol. 6,243-254. Rabbitts, P., Douglas, J., Daly, M., Sundaresan, V., Fox, B., Haselton, P., Wells, F., Albertson, D., Waters, J., and Bergh, J. (1989). Frequency and extent of allelic loss in the short arm of chromosome 3 in nonsmall-cell lung cancer. Genes Chrom. Cancer 1,95-105. Rassool, F. V., McKeithan, T. W., Neilly, M. E., van melle, E., Espinosa, R., 111, and Le Beau, M. M. (1991). Preferential integration of marker DNA into the chromosomal fragile site at 3p14: An approach to cloning fragile sites. Proc. Natl. Acad. Sci. USA 88,6657-6661. Rimessi, P., Gualandi, F., Morelli, C., Trabanelli, C., Wu, Q., Possati, L., Montesi, M., Barrett, J. C., and Barbanti-Brodano, G. (1994).Transfer of human chromosome 3 to an ovarian carcinoma cell line identifies three regions on 3p involved in ovarian cancer. Oncogene 9, 3467-3474. Robinson, A. K., de la Pena, C. E., and Barnes, L. D. (1993). Isolation and characterization of diadenosine tetraphosphate (Ap4A) hydrolase from Schizosaccharomyces pombe. Biochem. Biophys. A C ~ U1161, 139-148. Roth, J. A., Nguyen, D., Lawrence, D. D., Kemp, B. L., Carrasco, C. H., Ferson, D. Z . , Hong, W. K., Komaki, R., Lee, J. J., Nesbitt, J. C., Pisters, K. M., Putnam, J. B., Schea, R., Shin, D. M., Walsh, G. L., Dolormente, M. M., Han, C. I., Martin, F. D., Yen, N., Xu, K, Stephens, L. C., McDonnell, T. J., Mukhopadhyay, T., and Cai, D. (1996). Retrovirus-mediated wildtype p53 gene transfer to tumors of patients with lung cancer [see comments]. Nature Med. 2,985-991. Roz, L., Wu, C. L., Porter, S., Scully, C., Speight, P., Read, A., Sloan, P., and Thakker, N. (1996). Allelic imbalance on chromosome 3p in oral dysplastic lesions: An early event in oral carcinogenesis. Cancer Res. 56, 1228-1231. Sanchez, Y., el-Naggar, A., Pathak, S., and Killary, A. M. (1994).A tumor suppressor locus within 3p14-pl2 mediates rapid cell death of renal cell carcinoma in vivo. Proc. Natl. Acad. Sci. USA 91,3383-3387.

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Phosphoinositide 4+and 5+Kinases

and the Cellular Roles of Phosphatidylinositol 4,5Bisphosphate 1. Justin Hsuan,*#+Shane Minogue,* and Maria dos Santos* *Ludwig Instrtute for Cancer Research, University College London Medical School, London W l P 8BT, and tDepartment of Biochemistry and Molecular Biology, University College London, London WClE 6B1; United Kingdom

I. Introduction A. The Enzyme Families B. Empirical Restrictions 11. Receptor-Linked Phosphoinositide Metabolism A. Receptors B. Modular Interaction Domains C. Regulation of Phospholipase C Activity D. Phosphatidylinositol (PtdIns) 4-Kinases and PtdInsP 5-Kinases E. Compartmentation and the Organization of Signaling Complexes F. Cross-talk between Signaling Pathways G. Nuclear Signaling 111. Phosphoinositides and the Cytoskeleton A. Gelsolin B. Profilin C. Vinculin D. The rho Family of Small G Proteins IV. Vesicle Biogenesis and Trafficking A. Endoplasmic Reticulum and cis-Golgi Vesicle Formation B. trans-Golgi Network Vesicle Formation and Protein Sorting C. Plasma Membrane Endocytosis D. Exocytosis V. Roles in Cancer, Summary, and Prospects References

I. INTRODUCTION Phosphatidylinositol (PtdIns) is a widely occurring eukaryotic lipid found as a minor component of many subcellular membranes. This acidic molecule contains a D-myo-inositol head group in vivo and its structure is represented Advances in CANCER RESEARCH 0065-23OW98 $25.00

Copyright 0 1998 by Academic Press

All rights of reproduction in any form reserved.

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in Fig. 1. Although many early investigations into the cellular role of PtdIns measured the apparent turnover or hydrolysis of PtdIns itself, it subsequently became clear that agonist-stimulated, intracellular phospholipase C (PLC) activities actually hydrolyze an intermediate form, namely phosphatidylinositol 4,S-bisphosphate (PtdInsP,). More recently, the biological importance of PtdInsP, has been emphasized by the realization that it is the preferred in vivo substrate for agonist-dependent phosphoinositide (PI, a general term used to describe PtdIns and its phosphorylated forms) 3-kinases and that it possesses a signaling ability of its own. This latter property of PtdInsP, still requires considerably more characterization, but several intracellular events have been shown to exploit noncovalent protein-PtdInsP, interactions to localize proteins to specific membrane sites and to allosterically stimulate enzyme activities. As changes in PtdInsP, availability are proposed to regulate several cellular functions, such as secretion and shape in addition to PI 3-kinase and PLC-dependent growth signaling, the enzymes responsible for the biosynthesis and catabolism of PtdInsP, and its precursor phosphatidylinositol4-phosphate (PtdInsP) are obvious points for regulation. Although little is known at present about PtdInsP and PtdInsP, dephosphorylation, their biosynthesis has been studied for many years. For example, in isolated erythrocyte membranes, a near equilibrium “shuttle” exists among PtdIns, PtdInsP, and PtdInsP2, which may be required for maintaining a discoid morphology (Muller et al., 1986 and references therein), whereas receptor-dependent PtdInsP, hydrolysis by many cell types requires the stimulation of PtdInsP, biosynthesis from PtdIns. The object of this review is to describe and assess many of the reported cellular functions of PtdInsP, and the regulation of its biosynthesis. PI and PtdIns-specific 3-kinases are only discussed in this review where their respective enzymatic products require consideration alongside PtdInsP,, as the cellular functions of these enzymes have been subjected to a large number of reported studies, which have been reviewed elsewhere (Car-

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Table I PtdIns 4-Kinases and PtdInsP 5-Kinases" Enzyme PtdIns 4-kinases ~ 5 (type 5 11)

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Inhibitor (ICs0)

TXIOO, Phos

Phos, mAB 4CSG, adenosine (20-1 00 /IM) Wortmannin (-2 PM), adenosine (>200 pM) mAb 4CSG, adenosine (SO100 mM) Wortmannin

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Subcellular localization

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adenosine (>1 mM) PtdInsP S-kinases p68 (type IalSTM7.I) ~ 6 (type 8 Ib) ~ 9 (Type 0 ID)' pS3 (type IQd

PtdOH PtdOH PtdOH Inactive against Active against PtsInsP in PtdInsP micelles native membranes and liposomes

Cytosol, membrane Cytosol, membrane Cytosol, membrane Plasma membrane, cytosol, cytoskeleton, nucleus

"Only the better characterized isozymes are included (TX100, Triton X-100; Phos, phosphorylation). Isozymes that have yet to be cloned are shown in italics. "The localization of these isoforms appeared to be very similar in COS-7 cells (Nakagawa ef al., 1996a,b). However, it is possible that this arises from improper processing as a consequence of this strongly overexpressing system. 'Type Ip may be identical to one or more of the larger STM7 splice variants (Carvajal et al., 1996). dRecent work (Rameh ef al., 1997) suggests that type I1 enzymes catalyze the 4'-phosphorylation of PtdIns 5-phosphate (PtdInsSP),a pathway not previously known to exist.

penter and Cantley, 1996). A degree of caution is needed in interpreting the results of experiments in which the inhibitor wortmannin has been used, frequently at concentrations between 10 and 100 nM, to define the involvement of PI 3-kinase activity as this has now been shown not to be a specific inhibitor. Wortmannin inhibits PI 3-kinases with an IC,, of 1-5 nM, murine p170 at 40-50 nM, and human PtdIns 3-kinase at 2.5 nM in vitro (Virbasius et al., 1996 and references therein). In vitro studies show that certain PtdIns 4-kinases are inhibited at low micromolar concentrations (Table I), and a purified adrenal medulla PtdIns 4-kinase is inhibited with an ICSq of approximately 50 nM (Downing et al., 1996). Conversely, studies in which roles for PtdInsP and PtdInsP, are deduced often ignore the possibility that these polyphosphoinositides are required only as a substrate for PI 3-kinase activities and that the products PtdIns 3,4-bisphosphate, PtdIns 3,4,5-

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trisphosphate (PtdInsP,), or PtdIns 3,4-bisphosphate produced by dephosphorylation of PtdInsP, may in fact be the active lipid in vivo.

A. The Enzyme Families Two strictly sequential enzyme activities are responsible for the biosynthesis of PtdInsP,, namely PtdIns 4-kinase (EC 2.7.1.67) and PtdInsP 5-kinase (EC 2.7.1.68) activities (see also Table I, note d), whose names derive from the respective position of phosphorylation on the inositol ring (Fig. 1).Splice variation and different genes lead to the existence of multiple isozymes for each activity (Table I), and with the increasingly rapid advances in genome sequencing it is probable that further forms will be identified in the near future. Many investigations of changes in PI phosphorylation compare either total cell extracts of the various polyphosphoinositides following metabolic labeling with tritiated inositol or assays of PI phosphorylation using cell lysates. An appreciation of the range of isoforms and the different subcellular sites in which each isoform operates is missed by such approaches as are subtle changes in the activity of these isoforms, particularly as the basal activity of PtdIns 4-kinases and PtdIns5P 4-kinases is considerably elevated in detergent lysates compared with native membranes. Different metabolic compartments are discussed further throughout the course of this review and include different cellular membranes such as the plasma membrane, the endoplasmic reticulum (ER) and secretory vesicles, discrete sites within individual membranes such as caveolae and coated buds, and phosphoinositides bound to intracellular proteins such as PtdIns transfer protein (PITP) and cytoskeletal proteins. Further problems in identifying the type of isozyme involved in different studies have arisen from the limitations in analytical reagents. For example, the only monoclonal antibody (mAb) available for the analysis of PtdIns 4kinases, 4CSG, inhibits type I1 but not type I11 PtdIns 4-kinase purified from bovine brain (Endemann et al., 1991). However, p97, which is probably a splice variant of p230 type I11 PtdIns 4-kinase, is also inhibited by 4C5G and was therefore proposed to be a type I1 enzyme (Wong and Cantley, 1994). Inhibition by 4CSG alone cannot therefore be used to identify PtdIns 4-kinase isozymes. In Saccharomyces cerevzsiae there are only two PtdIns 4-kinase genes, which encode Stt4p, thought to lie upstream of PtdInsP, hydrolysis and protein kinase C (PKC)activation (Yoshida et al., 1994a),and Piklp, whose cellular role remains unclear (Flanagan et al., 1993; Garcia-Bustos et al., 1994). Although overexpression from multicopy plasmids may cause enzymes from one pathway to affect another, comparisons of yeast and mammalian signaling enzymes have proven useful in many cases. The domain organization

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Cellular Functions of PtdInsP,

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Fig. 2 PtdIns 4-kinases. (A) The degree of similarity between kinase domains of cloned PtdIns 4-kinases is shown as a dendrogram [p230, rat 230 kDa type 111 (Nakagawa et al., 1996b); p97, human p97 PtdIns 4-kinase (Wong and Cantley, 1994); Stt4p, S. cerevisiae Stt4 protein (Yoshida ei al., 1994a); p92, rat 92KDa PtdIns 4-kinase (Nakagawa et al., 1996a); F35H12.4, C. elegans open reading frame encoding a putative PtdIns 4-kinase (g1109864); Sc Piklp, S. cerevisiae Pikl PtdIns 4-kinase (Flanagan et al., 1993; Garcia-Bustos et al., 1994); Sp Piklp, S. pombe Pikl homolog (g1220291); and PIK4, D. discoidium putative PtdIns 4-kinase (Zhou et a[., 199S)l. (B) Schematic of PtdIns 4-kinases showing regions of homology, sites proposed for modular domain interactions, and the number of amino acid residues [LKHl, lipid kinase homology region 1 and putative kinase domain; LKH2, lipid kinase homology region 2 (also known as the lipid kinase unique region); LKH3, lipid kinase homology region 31.

of Stt4p is most similar to the type I11 group represented in Fig. 2, whereas Piklp is most similar to the cloned p92 isozyme. However, none of the cloned mammalian isozymes appears to correspond with the 55-kDa type I1 isozyme, which is believed to lie upstream of agonist-dependent PtdInsP, hydrolysis. This isozyme could be encoded by an as yet unidentified gene or by a splice variant of p230/p97 or p92. Yeast PLC is most similar to PLCG isozymes in mammalian cells, for which the upstream kinases have not been

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C05E7.5

Fabl p STM7ltype la type Ib Skittles Mss4p type II

B

TCP-1

C05E7.5

++

Fablp

STM7Aype la type Ib Skittles Mss4p type II

PUTATIVE KINASE DOMAIN

-

1189

INSERT R f Z C N

2278 539

546

462 779

+

405

PROLINE-FKH

Fig. 3 PtdIns4P 5-kinase family. (A) Similar to Figure 2A, a dendrogram is shown of the cloned PtdInsP 5-kinase family members, excluding the splice variants of STM7 [CO5E7.5, C. elegans open reading frame encoding a putative PtdIns4P 5-kinase (g1065686);Fablp, S . cerevisiae Fabl protein (Yamamoto et al., 1995); STM7/type Ia, human STM7.1 PtdIns4P S-kinase (Carvajal et al., 1995) and mouse type Ia PtdIns4P S-kinase (Ishihara e t a / . , 1996); type Ib, mouse type Ib PtdIns4P S-kinase (Ishihara e t a / . , 1996); Skittles, D. rnelanogaster skittles locus encoding a putative PtdIns4P S-kinase (g1657964); Mss4p, S . cerevisiae Mss4 protein (Yoshida et al., 1994b);and type 11, human PtdInsSP 4-kinase (Boronenkov and Anderson, 1995, Divecha et al., 199S)l. (B) Similar schematic to Fig. 2B showing PtdInsP S-kinase homology regions, sites proposed for modular domain interactions, and the number of amino acid residues (TCP-1, chaperonin TCP-1 homology domain).

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identified and whose regulation appears to be quite different to the regulation of PLCP and PLCy (Section 111,C). It is therefore tempting to suggest that Stt4p/type I11 enzymes lie upstream of PLCG activities and that the Piklp/p92 group comprises the type I1 PtdIns 4-kinases. Although early purification studies suggested the existence of only type I and I1 enzymes in mammals, sequence analysis of the known PtdInsP S k i nases in different species indicates that extra complexity exists (Fig. 3 ) . Type I enzymes have been cloned from Drosophila melanogaster, mouse, and humans, and two genes and several splice variants have now been shown to encode at least five mammalian isozymes. The STM7/Ia isoform has been implicated in causing Friedreich's ataxia (Carvajal et al., 1995,1996). The only cloned type I1 enzyme, p53, is the nearest relative of the type I enzymes along with Mss4p, one of two yeast PtdInsP 5-kinase homologs. Mss4p is believed to lie on the Stt4p/PLC/PKC pathway (Yoshida et al., 1994b). A third class of PtdInsP 5-kinase homologs is defined by the second yeast isozyme, Fablp (Yamamoto et al., 1995), and an open reading frame termed CO5E7.5 in the Caenorhabditis elegans genome. These two sequences have an as yet undescribed region of homology, which is not present in other PtdInsP 5-kinases (Fig. 3). Although the role of this region remains to be studied, database searches suggest that the amino acid sequence of this region is similar to part of the polypeptide binding, apical domain of archaebacterial thermosomes and eukaryotic CCT/TCP-l cytosolic chaperonins (reviewed by Kim et al., 1994).

B. Empirical Restrictions The 55-kDa type I1 PtdIns 4-kinase forms detergent-insoluble aggregates with many other proteins and has proven to be extremely difficult to purify in quantity despite attempts by numerous groups, which explains why this important isoform remains poorly characterized and its cDNA uncloned. It has recently become apparent that preparations of lipids from biological sources may not be sufficiently pure to determine the substrate specificities of different kinases. In particular, PtdInsP preparations have been found to contain significant amounts of PtdInsSP, which is extremely difficult to distinguish from PtdInsP by all but the most sophisticated chromatographic techniques. It is hoped that the availability of synthetic lipids of precisely defined composition will avoid confusion in the future. In addition to the limited number of useful antibodies and inhibitors for the study of PtdInsP, biosynthesis, the experimental systems used for analysis pose certain problems. The questionable results achieved using detergent lysates have already been mentioned, but molecular interactions may also be affected by the loss of cellular architecture and the analysis of strongly anionic amphipathic molecules. For example, just as the anionic detergent sodi-

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um dodecyl sulfate is known to bind very strongly and with little specificity to many proteins, polyphosphoinositides in micelles, in liposomes, and in membrane preparations may be expected to show a wide range of nonphysiological interactions. In particular, the demonstration of proteins binding to PtdInsP, or PtdInsP, micelles in vitro is a poor indicator of binding in vivo. The composition of liposomes and micelles, the presence of detergents, and the concentration of various divalent cations can also have large effects on protein binding and enzyme activities. For these reasons it is probable that several reported effects of polyphosphoinositides will prove to be artifactual. As the range of potential interactions and intracellular roles for PtdInsP, is vast, its localization and biosynthesis are necessarily restricted and regulated in different subcellular compartments. How this is achieved within cells will now be considered.

11. RECEPTOR-LINKED PHOSPHOINOSITIDE

METABOLISM The first cellular function of PtdInsP, to be considered here has been studied the most, namely the widespread receptor-dependent hydrolysis of PtdInsP,. Following a brief description of different receptor classes, the modular domains that mediate specific protein-protein and protein-phosphoinositide interactions during signal transduction are summarized. A considerable number of studies have investigated the stimulation of different PLC enzymes, and only more recently has the agonist-dependent biosynthesis of PtdInsP, been suggested to provide further regulation of signaling. These topics are discussed before finally assessing the compartmentation of PtdInsP, involved in signaling and evidence for cross-talk between different receptor classes. PtdInsP, is also the main cellular substrate for receptor-dependent PI 3-kinases and the mechanisms by which increased flux through these two pathways occurs share many properties. As the hydrolysis of PtdInsP, has been far better characterized it is a more suitable paradigm for this review.

A. Receptors Two major classes of cell surface receptor are known to regulate the metabolism of plasma membrane PtdIns: the heterotrimeric GTP phosphodiesterase (GTPase or G protein)-coupled receptors (GPCRs) and the protein tyrosine kinase-linked receptors. GPCRs are responsible for transducing signals in response to neurotransmitters and many hormones, as well as odourants and light. Members of this large family of receptors are integral membrane proteins characterized by seven transmembrane segments. The

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coupled G protein is an inactive heterotrimer of a,P, and y subunits associated with the cytoplasmic region of the receptor (Hamm and Gilchrist, 1996). When the receptor is activated by its cognate ligand, the a subunit undergoes a conformational change that lowers its affinity for GDP and promotes exchange for GTP. In its active GTP-bound state the heterotrimer dissociates into an a subunit and a tightly associated Py dimer, both of which have the potential to stimulate PLCP activity (Section 11,C).The subunits remain dissociated until the GTP bound to the a subunit is hydrolyzed, a period determined by the rate of hydrolysis that varies depending on the G a subunit and the action of specific GTPase-activating proteins (GAPS).Once the bound GTP has been hydrolyzed to GDP, the subunits reassociate with the receptor in the inactive state (reviewed by Neer, 1995). Growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), and fibroblast growth factor (FGF) bind to cognate transmembrane receptors that possess intrinsic protein tyrosine kinase activity. Ligand binding generally promotes receptor dimerization followed by receptor autophosphorylation, which probably occurs in trans (reviewed by Ullrich and Schlessinger, 1990). Activation of receptor tyrosine kinases leads to the phosphorylation of multiple tyrosine residues, which generates numerous sites for the interaction of proteins containing phosphotyrosine-binding domains, such as the PLCy isozymes (reviewed by Lee and Rhee, 1995; Pawson, 1995; Hsuan and Tan, 1997). As discussed in Section II,C, tyrosine phosphorylation of PLCy is a key event that, along with other regulated molecular interactions, enables the assembly of multienzyme signaling complexes. Dimerization of the EGF receptor is sufficient to activate its intrinsic tyrosine kinase activity, but phosphorylation of a tyrosine residue within the kinase domain of the PDGF receptor is required for its activation. A subgroup of protein tyrosine kinase-linked receptors, which includes the T-cell receptor, IgM, high-affinity IgE, hepatocyte growth factor, cytokine receptors, and integrins, lacks protein kinase activity but can induce tyrosine phosphorylation of specific proteins by interaction with nonreceptor tyrosine kinases. In an analogous fashion to receptor tyrosine kinases, many nonreceptor tyrosine kinases are thought to link ligand binding to PLCy phosphorylation (reviewed by Weiss and Littman, 1994; Sjaastad and Nelson, 1997).

B. Modular Interaction Domains Carefully coordinated, noncovalent molecular interactions are an important characteristic of receptor signaling and, in many cases, permit the assembly and disassembly of complexes containing multiple signaling molecules. These interactions frequently require the presence of noncatalytic, modular domains capable of specific binding to target sites. Although the

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formation of PI signaling complexes in response to GPCR activation has not been widely reported, the rapid recruitment of cytosolic PLCy by an interaction with receptor phosphotyrosine residues has been well studied. Agonist-stimulated changes in the subcellular localization of signaling molecules may have widely important regulatory implications. For example, translocation may juxtapose an enzyme with its substrate, cofactor, or activator. Some substrates are present at very low concentrations in cells, but recruitment of an enzyme to a subcellular compartment in which the local concentration of substrate is much higher or the formation of a complex with sites of substrate biosynthesis can result in more favorable kinetics as signaling ceases to be substrate limited. In other cases, molecular interactions may stimulate enzymatic activity via allosteric mechanisms, e.g., in the stimulation of PI 3-kinase/p85 holoenzymes by binding to PDGF receptor phosphopeptides (Shoelson et al., 1993). A set of protein modules have been identified, and their occurrence in species as diverse as Saccharomyces, Dictyostelium, Drosophila, and humans indicates an ancient ancestry. These modular domains are generally small enough to be studied by nuclear magnetic resonance spectrometry and many three-dimensional structures and their binding specificities have now been elucidated (Pawson, 1995). Modular domains continue to be identified in a growing number of signaling molecules, and the LKH3 region specific to the Piklp family of PtdIns 4-kinases (Fig. 2) may represent a novel type of domain. Although many different types of modular domain have been identified, only those domains relevant to the subsequent discussion of PI signaling are described here. Src homology type 2 (SH2) domains are defined by approximately 100 amino acid residues and allow signaling proteins to bind phosphotyrosine residues with high affinities ( K d 1-10 nM; reviewed by Pawson, 1995). SH2 domains from PI 3-kinase p85 subunits have been shown to bind PtdInsP, in vitro. As this binding was apparently competitive with phosphotyrosine binding (Rameh et al., 1995), the authors suggested that polyphosphoinositides may be able to regulate SH2-phosphoprotein interactions or provide a way of recruiting proteins to specific membranes. It will be interesting to see whether such experiments can be repeated under more physiological conditions, i.e., using full-length molecules and membranes rather than phospholipid micelles, and in the presence of physiological concentrations of divalent cations such as Mg2+. As in the eponymous c-src protein, SH2 domains are frequently found alongside src homology type 3 (SH3) domains, which contain only 50-75 amino acid residues and mediate interactions with proline-rich sequences. In some cases, SH3 domains may have a negative regulatory role as mutations of the SH3 domains of the c-abl and c-src protooncogenes enhance their oncogenic activity (Jackson and Baltimore, 1989; Hirai and Varmus, 1990; Seidel-Dugan et al., 1992). However, the large G-protein dynamin, which is involved in receptor internalization (Section IV,C), can bind and be stirnu-

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lated by isolated SH3 domains from c-src and GRB2 (Gout et al., 1993). Evidence also shows that some SH3 domains, including that of PLCy, localize proteins to the cytoskeleton (Bar-Sagi et al., 1993). SH3 domains have also been identified in a number of cytoskeletal proteins, including spectrin, which is discussed in Section IV,D. Pleckstrin homology (PH) domains were originally identified in the PKC substrate pleckstrin and have since been found in a large number of molecules, including PLC isoforms (Parker et al., 1994) and PtdIns 4-kinases (Fig. 2). The identification of PH domains is complicated by considerable variations in sequence and their function in many proteins is not well understood. Since the three-dimensional structures of several PH domains have been solved it has become clear that, despite the lack of sequence identity between PH domains, they all share a common protein fold and can interact with protein or PI ligands. A subset of PH domains may mediate interactions with Gpy subunits (Touhara et al., 1994), whereas other PH domains bind PtdInsP, or PtdInsP, (Harlan et al., 1994). These domains may therefore play a role in the localization of signaling proteins, such as the PLCS isozymes, to polyphosphoinositide-containingmembranes. The importance of correct signaling localization is exemplified by a naturally occurring point mutation of the PH domain of Bruton’s tyrosine kinase, the human gene identified as defective in X-linked agammaglobulinemia, which causes transformation of NIH 3T3 fibroblasts (Li, T. et al., 1995), possibly by inappropriately targeting this protein tyrosine kinase to the plasma membrane. Several PH domains stimulate enzyme activities, including the amino-terminal PH domain of PLCG1 (Lomasney et al., 1996); the activation of PARK, which may require the synergistic action of GPy and PtdInsP, (Pitcher et al., 1995); and the PH domain-dependent activation of dynamin GTPase by PtdInsP, (Salim et al., 1996; Lin and Gilman, 1996). Evidence also shows that some PH domains, such as that of PLCSI, bind inositol phosphates (reviewed by Irvine and Cullen, 1996). C2 or CalB domains can bind inositol phosphates (reviewed by Irvine and Cullen, 1996), phospholipids, and proteins (Li, C. et al., 1995; Chapman et al., 1996) and are found in many proteins, including PI 3-kinases (Virbasius et al., 1996; Molz et al., 1996) and all types of PI-specific PLC (Ponting and Parker, 1996). Some of these interactions have been shown to be Ca2+ dependent, but the properties of C2 domains in PI 3-kinases and PLC enzymes remain to be defined.

C. Regulation of Phospholipase C Activity PtdInsP, is the preferred substrate both for phosphorylation by PI 3-kinases and for hydrolysis by multiple eukaryotic PI-specific PLC enzymes. PLC activities generate two highly important second messengers: diacyl-

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glycerol, an important activator of PKC (reviewed by Nishizuka, 1988),and inositol 1,4,5-trisphosphate (InsP,), which regulates the release of Ca2+ from intracellular stores (reviewed by Berridge, 1993; Clapham, 1995). In addition, both of these products are precursors for other biologically active molecules, including phosphatidic acid (PtdOH) and inositol 1,3,4,5-tetrakisphosphate, respectively. As a consequence of the widespread importance of PtdInsP, hydrolysis in cellular responses to diverse extracellular stimuli, the molecular mechanisms regulating PLC activity have been studied intensively. To date, three classes of mammalian PtdInsP,-specific PLC have been cloned, which can be divided into PLCp1, p2, p3, and p4 isozymes; PLCyl and y2 isozymes; and PLC61,62,63, and 64 isozymes (Lee and Rhee, 1995). In addition to their catalytic domains, several structural features are common to all types of PtdInsP,-specific PLC, including an amino-terminal PH domain (Parker et al., 1994) and a C2 domain (Ponting and Parker, 1996). All types hydrolyze PtdIns, PtdInsP, and PtdInsP, in vitro, although PtdInsP, is the preferred substrate at physiological concentrations of Ca2+ (reviewed by Katan, 1997). 3-Phosphoinositides are not substrates for any known PLC activity, which is consistent with the idea that these lipids act through quite separate pathways. The regulation of PLCp isoforms by Gqasubunits is well established and appears to involve a unique carboxy-terminal region close to the catalytic domain (reviewed by Lee and Rhee, 1995). However, the sensitivities of the four p isoforms to G,a subunits differ, just as different PLCp isoforms are differentially stimulated by Gi,o Py subunits, probably via an interaction with the amino-terminal PH domain (Table 11). PLCpl also has the unusual property of acting as a GAP for Gqlll (Berstein et al., 1992). This phenomenon suggests a negative feedback loop in which PLCpl rapidly terminates its own activation. Rapid deactivation at the level of the G protein signal may provide a fast response of the type required for certain biological processes such as PLC-dependent phototransduction, which occurs, for example, in Drosophila and Limulus species. A mechanism linking G protein-mediated activation of CAMP-dependent protein kinase (PKA) and inhibition of PLCp2 has been established, in which the direct phosphorylation of PLCp2 by PKA prevents the interaction between PLCp2 and Gpy subunits (Liu and Simon, 1996). This finding helps explain why treatment of cells with cAMP or compounds that are capable of stimulating cAMP production leads to inhibition of PtdInsP, hydrolysis. In addition, Gia subunits are able to negatively regulate adenylate cyclases, thus allowing cross-talk between G protein-linked pathways. Although PLCy phosphorylation by PKA has been reported, this idea does not appear to effect its activity in vitro (Granja et al., 1991). PLCy isozymes are primarily regulated by tyrosine kinase-linked receptors

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Table I I Mammalian PI-Specific PLC Isozymes Type

Modular domain

PLCP PH (Pl-P3>P4>P2) C2 (identified in PLCP2) PLCy

PLCG

PH + “split” PH Two SH2 SH3 c2 PH EF c2

Activation by G protein subunits Stimulated by Gqa Stimulated by Gpy (P2-@3 >> Pl, but P4 is not stimulated in vitro) May bind G,a in vifro

Not significantly stimulated by GPy in vitro

via phosphorylation and the recruitment of cytosolic PLCy to activated receptors (reviewed by Lee and Rhee, 1995). Activation of receptor tyrosine kinases by ligand binding results in receptor autophosphorylation and the phosphorylation of many signaling molecules, including PLCy. Expression of mutant forms of PLCy in mammalian cells has been used to investigate the importance of individual tyrosine residues. In vivo, PLCyl becomes phosphorylated on tyrosine residues 771,783, and 1254. Mutation of tyrosine 783 to phenylalanine abolishes activation (Meisenhelder et al., 1989; Kim et al., 1991), whereas full activation requires phosphorylation of tyrosine 783 and 1254. Purified tyrosine-phosphorylated PLCy has increased activity measured in vitro (Wahl et al., 1992), but the mechanism of activation remains enigmatic, as mutation of residues 771,783, and 1254 in other studies has no effect on activity measured in vitro (Kim et al., 1991).As discussed in Section II,F, it has been suggested that tyrosine phosphorylation is required for the EGF-dependent translocation of PLCy to the cytoskeleton in hepatocytes (Yang et al., 1994). Autophosphorylation of the EGF receptor generates five potential SH2binding sites, each of which can bind PLCyl (Soler et al., 1994). In contrast, FGF and NGF receptors possess only one tyrosine residue that is responsible for PLCy binding (Mohammadi et al., 1991; Obermeier et al., 1993). Stimulation of the B-cell and T-cell antigen receptors results in tyrosine phosphorylation of PLCyl (Carter et al., 1991; Dasgupta et al., 1992), whereas PLCy2, an isoform expressed predominantly in hematopoietic cells, is phosphorylated in response to B-cell receptor ligation (Hempel et al., 1992).It is likely that signaling through PLCy is important in the activation of PKC and in the rapid and sustained phases of increased Ca2+ concentra-

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tion observed in response to receptor activation in lymphocytes (Imboden and Stobo, 1985; Inokuchi and Imboden, 1990). The regulation of PLCG isoforms in the context of a signaling pathway remains a major unsolved question in PI metabolism. A weak and probably insignificant activation by GPy subunits has been observed in vitro (reviewed by Lee and Rhee, 1995). More promisingly, p122 rho-GAP is reported to be capable of stimulating PLCG1 but not PLCy activity in vitro (Homma and Emori, 1995) and may be an important part of the regulation of cytoskeletal organization by PtdInsP, (Section 111). The PH domain of PLCG1 has been implicated in the regulation of enzyme activity as it has a relatively high affinity for PtdInsP, and is required for membrane localization (Garcia et al., 1995).It has been suggested that interaction with PtdInsP,-containing membranes may promote processive catalysis (Cifuentes et al., 1993), a hypothesis that is supported by the publication of mutagenesis data confirming that the amino-terminal PH domain of PLCG1 is necessary for the dose-dependent stimulation of enzyme activity by PtdInsP, (Lomasney et al., 1996). Because the PH domain can competitively bind InsP, with high affinity, a mechanism of product inhibition has also been proposed (Lemmon et al., 1995; Lomasney et al., 1996). A Ca2+-binding EF-hand motif has been identified in PLCG isozymes (Bairoch and Cox, 1990), but this has no established regulatory function (Nakashima et al., 1995).

D. Phosphatidylinositol (PtdIns) 4-Kinases and PtdlnsP 5-Kinases Agonist-stimulated hydrolysis of PtdInsP, and its conversion to PtdInsP, by PI 3-kinases place heavy demands on PtdInsP, biosynthesis. Although mass action alone has been suggested to allow sufficient changes in PtdInsP, biosynthesis, several studies suggest that the synthesis of PtdInsP and PtdInsP, is subject to stringent regulation by receptor-derived signals in order to maintain a sustained flux of intermediates through the pathway and, as may be apparent from the numerous cellular functions of PtdInsP, described in this review, to prevent highly biologically active polyphosphatidylinositols from being synthesized inappropriately. This could be achieved by the regulation of PI phosphatase or PI kinase activities, or both. Some progress has been made in our understanding of the control of PI metabolism, and future work is likely to be facilitated greatly by the cloning of the first members of the PtdIns 4-kinase and PtdInsP 5-kinase families (Table I). Little data exist for the direct regulation of PtdIns 4-kinase activity by heterotrimeric G proteins, although some evidence suggests that a pool of PtdInsP is sensitive to cholera toxin (which causes the ADP-ribosylation of

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EGFR

c-erbB2 c-erbB3 c-erbB4

642 L F M R R R H I V R K R T L R R L L Q . . 652 I L I K R R Q Q K I R K Y T M R R L L . .

645 L Y W R G R R I Q N K R A M R R Y L E . . 648 V Y V R R K S I K K K R A L R R F L E . . CONSENSUS 000RRR. I.RKRA0RR0LE CD4 423 C R H R R R Q A E R M M S Q I K R L L . . Fig. 4 Juxtarnernbrane sequences. The regions proposed to bind PtdIns 4-kinase and PtdInsP 5-kinase are shown for human protein sequences. Conserved basic residues are shown in bold type (0,hydrophobic residue).

Gsa subunits). This surprising observation is of added interest because the toxin was able to specifically inhibit an EGF-dependent increase in PtdInsP in intact A431 cells (Pike and Eakes, 1987), indicating that a G protein may be involved in EGF receptor signaling (Section 11,F). Use of the nonhydrolysable GTP analog GTPyS in assays of partially purified rat liver membrane PtdInsP 5-kinase preparations has suggested the existence of a G protein regulated activity (Urumow and Wieland, 1990).However, studies such as this, which rely solely on the use of GTPyS, do not discriminate between the heterotrimeric and small G proteins. Nevertheless, a cholera toxin-sensitive activity was previously reported in the same tissue (Urumow and Wieland, 1988).More recent work has suggested the existence of a pertussis toxin-sensitive PtdInsP 5-kinase that mediates the increased biosynthesis of PtdInsP, in response to GPCR agonists using permeabilized neutrophils (Stephens et af., 1993). Such studies revealed no apparent increase in PtdInsP biosynthesis (Stephens et al., 1993; Cunningham et al., 1995); however, the measurement of total cell PtdInsP biosynthesis may mask agonist-dependent changes (Section 1,A). Ideally, different PtdIns 4-kinase isozymes should be assayed in isolation; however, suitable reagents such as isozyme-specific mAbs do not yet exist and heterotrimeric G protein complexes do not usually survive cell lysis and immunoprecipitation. The membrane-bound, 55-kDa, type I1 PtdIns 4-kinase and PtdInsP 5-kinase activity have been found in receptor immunoprecipitates from EGFstimulated A431 cells (Cochet et af., 1991; Kauffmann-Zeh et al., 1994).The site of interaction of PtdIns 4-kinase and an unknown PtdInsP 5-kinase with the EGF receptor has been mapped to the receptor juxtamembrane region using a series of carboxy-terminally truncated receptor mutants and synthetic peptides (Cochet et af., 1991). The association with receptor mutants was found to be EGF dependent and did not require the receptor tyrosine kinase activity. The juxtarnembrane amino acid sequence is well conserved among members of the EGF receptor family (Fig. 4), suggesting that they may share a common PtdInsP, signaling mechanism. Indeed, type I1 PtdIns 4-kinase can be immunoprecipitated from c-erbB2 overexpressing breast

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cancer cells (Scott et al., 1991).The juxtamembrane region does not contain a tyrosine phosphorylation site and the mechanism of interaction of these activities is unknown. However, PtdIns 4-kinase and PtdInsP 5-kinase activities were able to bind synthetic peptides in an EGF-independent manner (Cochet et al., 1991),and the high proportion of basic residues in this region suggests the potential for nonspecific interactions with acidic phosphoproteins such as the 55-kDa type I1 PtdIns 4-kinase (Kauffmann-Zeh et al., 1994). PtdIns 4-kinase activity can also be stimulated by cytokine receptors as interleukin-1 treatment of human fibroblast membranes caused a more than 10fold increase in PtdIns 4-kinase activity (Ballou et a!., 1991). The sensitivity of this activity to inhibition by Ca2+ is consistent with the properties of the 55-kDa type I1 enzyme (Downing et al., 1996). Furthermore, the cross-linking of CD4 receptors on T cells, which signal through the associated nonreceptor tyrosine kinase p56lCk,caused a 5- to 10-fold increase in CD4-associated PtdIns 4-kinase activity. A similar sequence to that in the EGF receptor family juxtamembrane region was identified in the carboxy terminus of CD4 (Fig. 4) and was suggested to bind the uncharacterized PtdIns 4-kinase (Prasad et al., 1993). Direct binding to this basic site has yet to be verified. Phosphorylation of the type I1 PtdIns 4-kinase may regulate both its recruitment to the EGF receptor and its activity. Increased PtdIns 4-kinase activity can be found in antiphosphotyrosine and receptor immunoprecipitates from EGF-treated A431 cells (Payrastre et al., 1990),and PtdIns 4-kinase activity can be competitively eluted with phenylphosphate (Cochet et al., 1991). Interestingly, treatment of immunoprecipitates with a nonspecific phosphatase reduced PtdIns 4-kinase activity to levels similar to those seen with unstimulated cells (Kauffmann-Zeh et al., 1994).Although a threonine and serine-specific phosphatase had a similar effect, a purified tyrosine phosphatase was able to stimulate activity threefold (Kauffmann-Zeh et al., 1994). However, these experiments were unable to distinguish direct effects of phosphorylation of PtdIns 4-kinase from indirect effects possibly mediated by changes in EGF receptor phosphorylation. Although the association of PtdIns 4-kinase with the EGF receptor was dependent on receptor autophosphorylation, tyrosine phosphorylation of PtdIns 4-kinase inhibits activity. The EGF receptor tyrosine kinase is probably not responsible for PtdIns 4-kinase phosphorylation as purified PtdIns 4-kinase cannot be phosphorylated by an isolated receptor in vitro. In erythrocyte membranes, tyrosine phosphorylation was found to stimulate PtdIns 4-kinase activity (de Neef et al., 1996). Phorbol esters and CAMPhave both been shown to cause modest and often delayed increases in total cellular PtdInsP and PtdIns 4-kinase activity. Whether these changes were directly due to phosphorylation of PtdIns 4-kinase was not investigated in these studies, neither were the isozymes involved identified (reviewed by Pike, 1992).

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To date, as the 55-kDa type I1 PtdIns 4-kinase has neither been cloned nor satisfactorily purified for characterization, a clear understanding of the effects of phosphorylation will require the isolation of the corresponding cDNA. In addition, whether or not other PtdIns 4-kinase isozymes (Table I) play any part in receptor signaling requires investigation. Although increased PtdInsP 5-kinase activity could be immunoprecipitated using an antiphosphotyrosine mAb following treatment of A431 cell membranes or transfected B82L cells with EGF, dephosphorylation of the latter immunopurified enzyme using a phosphotyrosine-specificphosphatase had no effect on its activity (Payrastre et al., 1990; Cochet et al., 1991).

E. Compartmentation and the Organization of Signaling Complexes Exactly how activated cells control the supply of PI signaling precursors is still poorly understood and many questions remain to be answered, including how PtdIns 4-kinase and PtdInsP 5-kinase activities are regulated during signal transduction against high background activities in unstimulated cells. The answer to this question may lie in an explanation of an empirically observed pool of PtdIns, frequently referred to as the agonist-sensitive pool, which is defined as the fraction of the total cellular PI that is hydrolyzed in response to receptor agonists. The agonist-insensitive pool appears to be resistant to hydrolysis, even during prolonged stimulation. Despite many years of investigation, the physical identity of this pool remains enigmatic, and different studies have both supported and denied its existence (reviewed by Monaco and Gershengorn, 1992). It has been suggested that the turnover of PtdInsP, in response to bradykinin and EGF in A431 cells occurs in discrete membranous structures called caveolae (reviewed by Parton, 1996), which are known to contain receptors, PI kinases, and phosphoinositides (Pike and Casey, 1996; Hope and Pike, 1996 and references therein). However, the mechanism by which these detergent-insoluble subcellular compartments may be able to delimit PI metabolism has yet to be defined. A second means of compartmentalizing PI metabolism relies on an acyl chain specificity of enzymes. The two acyl groups of agonist-sensitive PtdIns are stearyl and arachidonyl residues, although many other types of acyl chain exist in cellular PtdInsP2 (Lee et al., 1991). PITPs and diacylglycerol kinase have been shown to discriminate between different PtdIns acyl groups (Tang et al., 1996), although such studies have yet to be extended to the various PI kinases and PLCs. A third mechanism for intramembrane compartmentation has become apparent from the many in vitro biochemical characterizations of PtdIns 4-kinase, PtdInsP 5-kinase, and PLC activities that have revealed distinct preferences for the mode of substrate presentation, such as the presence of PITP,

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carrier lipids, and detergents. These observations may provide clues to the in vivo specificities of these enzymes and, as summarized later, have led to the suggestion that a specific mode of substrate presentation in vivo may delimit an agonist-sensitive pool of substrates (reviewed by Liscovitch and Cantley, 1995; Hsuan and Tan, 1997). While studies that rely on cell lysis and the isolation of stable, agonistdependent signaling complexes provide much information on the protein components involved, they fail to reveal how the supply and demand of lipid precursors is controlled. Furthermore, caveolae are not solubilized by normal detergent buffers. Equally, the use of artificial assay conditions employing excess exogenous substrates in micellar form instead of substrates in their native membrane context and the disruption of the subtle membrane architecture of the living cell with detergents compromise the understanding of signaling. Detailed studies of phospholipid signaling have also been frustrated by a lack of recombinant proteins and suitable experimental methodologies capable of maintaining the subtle subcellular compartmentation, which may be critical to understanding the supply and demand of signaling precursors. In addition, the existence of multiple isozymes hugely complicates the ability to assign a specific function to any individual isoform. Intact cell analyses in which PI metabolism can be rapidly quenched using organic solvent mixtures are the least metabolically disruptive approach, but are of limited use. The development of permeabilized cell preparations has circumvented some of the problems and has allowed the ability of purified and recombinant cytosolic proteins to reconstitute or inhibit signaling to be assayed. A role for the cytosolic PITP was demonstrated by its ability to reconstitute PLC signaling in response to G protein agonists in permeabilized HL60 cells (Thomas et al., 1993; Cunningham et al., 1995) and EGF signaling in permeabilised A431 cells (Kauffmann-Zeh et a/., 1995). PITPs were originally identified as soluble proteins capable of transferring PtdIns or phosphatidylcholine (PtdCho), but not polyphosphoinositides, between membrane bilayers in vitro (reviewed by Wirtz, 1991). Based on the initial identification of the need for PITP in signaling (Thomas et al., 1993), it was suggested that their role is to replenish PtdIns in the plasma membrane by transporting it from its site of synthesis in the ER (Downes and Batty, 1993). However, evidence from receptor signaling and secretion studies suggests that PITP has a more subtle role to play (reviewed by Liscovitch and Cantley, 1995; Hsuan and Tan, 1997). Briefly, permeabilized and cytosol-depleted HL60 cells are capable of producing an InsP, response, albeit over an extended time course, presumably by using the PtdIns that is normally present in the plasma membranes of all cells, but the addition of exogenous PITP to these cells greatly enhances the initial rate of InsP, production (Cunningham et al., 1995). These data are inconsistent with the mass action hy-

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pothesis and suggest that PITP acutely promotes the synthesis of PtdInsP, and its subsequent hydrolysis by PLC. Furthermore, PITP was found to coprecipitate with the EGF receptor, type I1 PtdIns 4-kinase, and PLCy in an agonist-dependent fashion (Kauffmann-Zeh et al., 1995).As a consequence of these and other data (reviewed by Hsuan and Tan, 1997), PITP, type I1 PtdIns 4-kinase, a PtdInsP 5-kinase, and either PLCP or PLCy have been proposed to be integral parts of multienzyme signaling complexes formed during GPCR and receptor tyrosine kinase signaling, respectively. According to this model, PITP acts as a carrier by retaining and sequentially presenting PtdIns, PtdInsP, and PtdInsP, to the enzymes of the signaling complex (Hsuan, 1993; Cunningham et al., 1995; Liscovitch and Cantley, 1995). In this way, signaling PtdIns can be separated from that in the membrane pool. A major challenge is now to investigate whether agonist-dependent complexes of PITP with PtdInsP and PtdInsP, do in fact exist in vivo.

F. Cross-talk between Signaling Pathways The ability of one signaling pathway to pass on regulatory information to another parallel pathway is an emerging theme in signal transduction, and work from several groups suggests that it may be a more general phenomenon than previously envisaged. A variety of signals are capable of stimulating receptor tyrosine kinase activity independently of the latter’s cognate ligand. For example, the GPCR agonist angiotensin I1 is able to promote tyrosine phosphorylation of PDGF receptors in smooth muscle cells, which may account for the similar stimulation of PLC activity by angiotensin I1 and PDGF in vascular tissues (Linseman et al., 1995). In addition, the EGF receptor is rapidly tyrosine phosphorylated in response to the GPCR agonists thrombin, endothelin-1, and lyso-PtdOH in Rat-1 cells (Daub et al., 1996). In GPCR activation, short wave ultraviolet radiation and the influx of Ca2+ ions can rapidly stimulate EGF receptors in several cell types (Sachsenmaier et al., 1994; Rosen and Greenberg, 1996). As yet, however, no clear mechanism has been identified for these phenomena. Autocrine release of growth factors can be ruled out by the use of inhibitory antibodies and is generally presumed to be too slow a process to account for the speed of these responses. The possibility of cytoplasmic tyrosine kinases that respond to Ca2+, such as PYK2, and radiation have also been suggested (Lev et al., 1995; Rosen and Greenberg, 1996). Conversely, evidence shows that PLCy isoforms are not regulated exclusively by protein tyrosine kinase-linked receptors. For example, PLCy 1 is one of the few proteins that can coprecipitate with G p subunits from EGFstimulated hepatocytes (Yang et al., 1993), and the EGF-induced Ca2+ re-

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sponse in these cells can be inhibited by the microinjection of anti-Ga antibodies (Yang et al., 1993). PLCyl has also been reported to be regulated through a pertussis toxin-sensitive pathway in hepatocytes (Johnson et al., 1986)and renal epithelial cells (Teitelbaum et al., 1990), but treatment with the toxin in other cell types had no effect (Liang and Garrison, 1992). Finally, treatment of cardiac myocytes with the purinogenic GPCR agonist ATP leads to tyrosine phosphorylation and membrane association of PLCy as well as a rapid InsP3 response (Puceat and Vassort, 1996). Nevertheless, direct regulation of PLCy by Ga has yet to be demonstrated. Furthermore, although PLCy has two putative PH domains (Table 11), it is unclear whether they can mediate an interaction with GPy subunits. In the light of these data, it is interesting to note that an increasing number of studies have shown that receptors and enzymes involved in PtdInsP, biosynthesis and hydrolysis can associate with the cytoskeleton, as EGF can also induce the translocation of G,a subunits to the cytoskeleton in hepatocytes (Yang e t al., 1994).For example, in fibroblasts and hepatocytes, PLCy translocates to the cytoskeleton upon stimulation by PDGF and EGF, respectively (McBride et al., 1991; Yang e t al., 1994), and treatment of A431 cells stimulated the association of the EGF receptor, PLCy 1,PtdIns 4-kinase, and PtdInsP 5-kinase activities with the cytoskeleton (Payastre et al., 1991) located within membrane ruffles (Diakonova e t al., 1995).Similarly, a G protein-dependent PLC in turkey erythrocytes is associated with the cytoskeleton (Vaziri and Downes, 1992). It is not known whether the substrate for PLCy resides in the cytoskeleton in hepatocytes. Although profilin, gelsolin, a-actinin, and talin are known to bind PtdInsP, (Section 111),whether this is available as a substrate for PLC in vivo is unclear. The binding of enzymes to cytoskeletal fractions may be mediated by a direct interaction between actin and the EGF receptor (den Hartigh et al., 1992), although the PLCySH3 domain microinjected into fibroblasts has also been shown to localize to the actin cytoskeleton (Bar-Sagi et al., 1993). These examples illustrate intriguing developments in our knowledge of signaling pathways and may add an unexpected complexity to the interpretation and understanding of cellular transformation, as mutations within one pathway could have seemingly disproportionate effects if secondary, potentially oncogenic pathways are corrupted. A further example is the potential for cross-talk between different receptor tyrosine kinases, such as the members of the EGF receptor family, which possess different signaling abilities (reviewed by Carraway and Cantley, 1994).

G. Nuclear Signaling Evidence for PI signaling in the nucleus has been reviewed elsewhere (Divecha and Irvine, 1995)and is therefore not considered in detail here. Briefly,

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PITP, PtdIns 4-kinaseYPtdInsP 5-kinase activities, and PLCpl have been implicated in the intranuclear biosynthesis and hydrolysis of PtdInsP,. The reported nuclear localization of the yeast Piklp PtdIns 4-kinase (Garcia-Bustos et al., 1994) is not supported by other data (Flanagan and Thorner, 1992; Flanagan et al., 1993).A novel rat liver PLC isoform, PLC64, has been characterized at the molecular level (Lee and Rhee, 1996; Liu et d., 1996). Liu et al. (1996) reported the highest levels of expression in regenerating rat liver and intestine and a nuclear localization, whereas Lee and Rhee (1996) reported no detectable expression of protein or message in these tissues. Although data from the two groups are contradictory, the nuclear localization and the specific expression of PLC64 during the transition from G to S phase in the cell cycle is intriguing as it suggests an important role for nuclear PI metabolism (Liu et al., 1996). While this pathway may be affected by growth factors, cell cycle, and differentiation in some cell types (Cocco et al., 1996), its biochemical function and regulation remain largely unclear.

111. PHOSPHOINOSITIDES AND THE CYTOSKELETON The actin cytoskeleton is directly or indirectly involved in many vital cell functions, such as cell shape, motility, cytokinesis, endocytosis, mRNA localization, and growth regulation (reviewed by Janmey and Chaponnier, 1995), and has been implicated as both a target and a mediator of signal transduction initiated through receptor tyrosine kinases and extracellular matrix-integrin systems (Ridley, 1994; Prendergast and Gibbs, 1993). In response to many extracellular signals, cells change their shape, their adhesion to matrix components, and their interaction with adjacent cells (reviewed by Zigmond, 1996).Cells possess a complex army of proteins that bind to actin in its filamentous (F-actin) or monomeric globular (G-actin) forms to regulate the assembly/disassembly, architecture, and distribution of actin filaments. Interestingly, the observation that several actin-binding proteins bind polyphosphoinositides is thought to provide a link between signal transduction and the reorganization of the actin cytoskeleton. Furthermore, as described earlier, the EGF receptor, Giasubunits, PLCy, PtdIns 4-kinase, and PtdInsP 5-kinase have all been reported to associate with the cytoskeleton in response to EGF stimulation, and the type I1 PtdInsP 5-kinase has been reported to associate with the cytoskeleton of platelets in response to thrombin treatment (Hinchliffe et a/., 1996). This review focuses on those actinbinding proteins known to bind PtdInsP and/or PtdInsP,, as other actinbinding proteins have been reviewed elsewhere (Sun et al., 1995; Mooseker and Cheney, 1995 and references therein), and then assesses the role of small G proteins in actin reorganization.

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A. Gelsolin Gelsolin is a key regulator of F-actin length that severs F-actin, caps the fast-growing (barbed) ends, and promotes nucleation of polymerization (reviewed by Yin, 1987). Gelsolin belongs to a Ca2+-regulated family of Factin-binding proteins, which includes villin (Janmey and Matsudaira, 1988), severin (Yin et al., 1990), CapG (Southwick, 1995), and scinderin (also called adseverin; Maekawa and Sakai, 1990; Rodriguez Del Castillo et al., 1990). These proteins are known to be regulated by PtdInsP and PtdInsP,, and in all cases the effects of PtdInsP, are antagonistic to those of Ca2+:inhibiting severing, nucleation, and G-actin binding (reviewed by Janmey, 1994).The biochemical properties of scinderin and gelsolin are almost indistinguishable. However, as described later, scinderin is only expressed in secretory cells (Rodriguez Del Castillo et al., 1990),whereas gelsolin appears to be expressed ubiquitously (Stossel et al., 1985), which suggests that these proteins are not functionally redundant. Evidence in support of gelsolin as a tumor supressor has been steadily accruing. Gelsolin was markedly diminished in H-rus-transformed mouse fibroblasts (Mullauer et al., 1993), human fibroblasts, and epithelial cells transformed with SV40 virus (Vandekerckhove et al., 1990), gastric carcinoma cell lines (Moriya et al., 1994), bladder cancer cell lines, and the majority of bladder cancers (Tanaka et ul., 1995). Transfection and mutation studies have confirmed the tumor supressor function of gelsolin for both epithelial and fibroblastic tumor cells (Mullauer et al., 1993; Tanaka et al., 1995). Two reports on mammary cancers indicated that gelsolin was greatly reduced, the protein was undetectable by immunocytochemistry in 12 out of 12 human breast cancers (Chaponnier and Gabbiani, 1989), and gelsolin RNA was reduced four- to fivefold in mouse mammary tumors (Medina et al., 1993). Partial or complete loss of gelsolin expression appears to be one of the most frequently occurring defects in breast cancer across three species (humans, mice, and rats), regardless of tumor etiology (Asch et al., 1996). The exact role of the loss of gelsolin in mammary tumorigenesis remains to be determined. The absence of gelsolin per se apparently does not induce mammary tumorigenesis because an increased incidence of mammary tumors was not observed in gelsolin-null mice (Witke et al., 1995). Because gelsolin is a component of the actin cytoskeleton, its loss might affect the invasive ability of cells. However, Asch et al. (1996)found that whereas some of the tumor cell lines were invasive in in vitro assays, others were not, suggesting that invasiveness is either independent of gelsolin or loss of gelsolin is not sufficient for this ability. Gelsolin interacts with key components of several signal transduction pathways, and its loss might adversely affect their availability or activity. A role for gelsolin in modulating cell motility comes from experiments that reg-

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ulate expression of gelsolin in vivo. Cells lacking gelsolin demonstrate reduced motility, whereas overexpression of gelsolin increases cell movement (Cunningham et al., 1991; Witke et al., 1995). Using signaling-restricted EGF receptor mutants expressed in NR6 fibroblasts (3T3 derivatives that lack endogenous EGF receptor; Pruss and Henchman, 1977), Chen et al. (1996) have shown that downregulation of gelsolin, through treatment with gelsolin antisense oligonucleotides, abrogates EGF-induced motility. Furthermore, these authors demonstrated that EGF receptor-mediated motility is dependent on the mobilization of membrane-associated gelsolin, which in turn is dependent on and downstream of PLCy activation; only those EGF receptor mutants that elicited cell motility caused a PLCy-dependent dissociation of gelsolin from the plasma membrane. Steed et al. (1996) reported that a physical association between gelsolin and PLD allows actin reorganization and PLD signaling to be coordinated in rabbit brain membranes. Interestingly, these authors show that the activation of PLD by PtdInsP, requires gelsolin and is not a direct effect on PLD, which appears to contrast with the previous proposal that PtdInsP, is a cofactor of PLD; however, the latter studies used PLD in rat brain membranes (Liscovitch et al., 1994), U937 cells (Pertile et al., 1995), and recombinant PLDl in Sf9 cell membranes (Hammond et al., 1995), which may have retained gelsolin. Alternatively, these results could reflect the occurrence of distinct PLD isoforms in the cell, which probably associate with separate pathways (Frohman and Morris, 1996 and references therein), including vesicle budding and exocytosis (Section IV). Apart from direct effects of gelsolin on the cytoskeleton, PLD activity has also been implicated in the stimulation of stress fiber formation in porcine aortic endothelial cells (Cross et al., 1996).

B. Profdin Profilins are ubiquitous, small (12-15 kDa) proteins that bind G-actin, polyphosphoinositides, and poly-L-proline. Whether profilin functions in vivo to inhibit or enhance actin polymerization is perplexing because different studies have drawn opposing conclusions (reviewed by Schafer and Cooper, 1995). Although it may be difficult to compare studies done in different systems using different experimental approaches, even studies on the role of profilin within a single in vitro system (Listeria cells moving in a Xenopus extract) have yielded different conclusions (Theriot et al., 1994; Marchand et al., 1995). Adding further complexity to the investigation of its in vivo role, profilin has been linked to at least two signaling pathways (reviewed by Sohn and Goldschmidt-Clermont, 1994). The first is receptor tyrosine kinase-dependent phosphoinositide turnover. Profilin binds PtdInsP, micelles in vitro with

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high affinity and inhibits PLCy, and probably other PtdInsP,-binding proteins, thereby maintaining a low basal rate of hydrolysis. This inhibition can be overcome by phosphorylation of PLCy (Goldschmidt-Clermont et al., 1991) or by the production of PtdIns 3,4-P, or PtdInsP, (Lu etal., 1996) following growth factor stimulation. Profilin-actin interactions are in turn inhibited by PtdInsP,, PtdIns 3,4-P, or PtdInsP, (Lassing and Lindberg, 1988; Lu et al., 1996). The second system is the yeast RAS signaling pathway, in which the involvement of profilin was inferred from its ability to rescue a yeast mutant defective in a component of this cascade, the cyclase associated protein Srv2p (Vojtek et al., 1991). Profilin mutants that are defective in actin binding do not suppress the yeast mutant phenotype (Haarer et al., 1993). Furthermore, an Acanthamoeba profilin isoform with low affinity for PtdlnsP, was found to be less effective in rescuing the yeast cells than an isoform with higher affinity for PtdInsP,, whereas both isoforms have equivalent affinities for actin (Vojtek et al., 1991). Although these results suggest that PtdInsP, binding has an important role in vivo, Machesky et al. (1994) reported that a vaccinia profilin, which has higher affinity in vivo for PtdInsP and PtdInsP, than for actin, was unable to rescue the S . cerevisiae profilin null mutant, showing that, at least in yeast, the affinity for phosphoinositides alone is insufficient for normal profilin function. PtdInsP, is able to dissociate profilin-actin complexes rapidly and efficiently in vitro, but why this should occur is unclear, as its affinity for profilin is nearly equal to that of actin (Sohn and Goldschmidt-Clermont, 1994). Furthermore, the putative binding sites on profilin for actin and PtdInsP, do not overlap (Vinson et al., 1993). Gieselmann et al. (1995) described a new human profilin isoform, which they named profilin 11. The most striking features exhibited by this new isoform were a lower isoelectric point (5.9compared with 8.4 for profilin I) and a fivefold lower affinity than profilin I for G-actin. However, both isoforms are equally effective at binding PtdInsP, and poly-L-proline and whether the different isoforms have physiologically different roles is not known.

C. Wnculin Vinculin is one of the major structural proteins at the cytoplasmic face of both cell-cell and cell-matrix contact sites (Jockusch and Rudiger, 1996). The role of vinculin in cellular adhesion is well documented (reviewed by Jockusch et al., 1995), and some results indicate that the tumorigenic capacities of some cell lines, which correlate inversely with adhesion, could be reduced by transfection with vinculin (Rodriguez Fernandez et al., 1992). Vinculin is a multiligand protein that interacts in vitro with a variety of

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structural and regulatory proteins of contact sites, including actin, a-actinin, and talin, and protein kinases and paxilin, respectively. In addition, it interacts with acidic phospholipids (Jockusch et al., 1995). The protein consists of a highly flexible C-terminal tail that is linked via a short proline-rich motif (a hinge) to a globular N-terminal head (Coutu and Craig, 1988). The flexibility of the hinge and tail regions allows for intramolecular interactions in which the head touches the tail, producing a closed conformation (Johnson and Craig, 1994; Winkler et al., 1996).This structure is functionally important because different ligands bind to different domains: talin and a-actinin bind to the head, whereas F-actin and phosphoinositides bind to the tail. When vinculin adopts a closed conformation, some binding sites become inaccessible (Jockusch and Rudiger, 1996). Understanding the mechanisms that regulate the conformational changes, and thus the binding capacity, of such a versatile molecule is crucial. Gilmore and Burridge (1996)have shown that PtdInsP, inhibits the head-tail interaction in vivo and thereby increases the binding of talin and F-actin to vinculin by threefold, while the binding of paxilin remains unaffected. This effect appeared to be specific to PtdInsP, as PtdInsP had a weak inhibitory effect and PtdIns and InsP, were ineffective. However, PtdInsP, was not tested. Because the cellular level of PtdInsP, can be affected by cell surface receptor activation, it was suggested that PtdInsP, mediates the assembly or disassembly of adhesion sites in response to growth factor- or hormone-dependent signals by modulating vinculin interactions with talin and F-actin. To test this hypothesis, Gilmore and Burridge (1996) analyzed the role of PtdInsP, in the assembly of focal adhesions using BALB/c 3T3 fibroblasts, which lose stress fibers and focal adhesions on serum starvation. Cells injected with monoclonal antibodies against PtdInsP, to prevent PtdInsP, from interacting with cytoskeletal proteins failed to form stress fibers or focal adhesions on serum stimulation. Although it is very likely that the antibody affects PtdInsP,-binding proteins other than vinculin, these results clearly demonstrate that assembly of focal adhesions requires PtdInsP,. Previous studies had established that the formation of focal adhesions and stress fibers in serum-stimulated Swiss 3T3 fibroblasts is dependent on rho activity (Ridley and Hall, 1992). Accordingly, Gilmore and Burridge (1996) proposed a model in which activation of a PtdInsP 5-kinase by rho elevates the cellular PtdInsP, concentration, which in turn induces a conformational change in vinculin, promoting its association with other components of focal adhesions and the attachment of stress fibers to the membrane. Such a model is consistent with previous results showing that adhesion to fibronectin in C3H 10T1/2 fibroblasts stimulates PtdInsP 5-kinase activity (McNamee et al., 1993) and that one or more type I PtdInsP 5-kinases can be stimulated by rho (Chong et al., 1994; Ren et al., 1996). In addition, a PtdIns5P 4-kinase has been shown to migrate to the cytoskeleton in platelets stimulated with thrombin (Hinchliffe et al., 1996),al-

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though the mechanism by which this isozyme is regulated remains to be identified.

D. The rho Family of Small G Proteins Cell transformation by ras and other oncogenes is accompanied by a dramatic alteration in the organization of the actin cytoskeleton, most notably a strong decrease in stress fiber formation. At the molecular level, these changes are reflected in decreased biosynthesis of several proteins associated with the actin cytoskeleton, including cy-actinin, vinculin, and certain tropomyosin isoforms. Interestingly, if the normal level of any of these proteins is restored, morphological transformation is inhibited and tumorigenicity decreases, indicating that the reorganization of the actin cytoskeleton is an essential component of cell transformation (reviewed by Symons, 1995). A major breakthrough has been the discovery that a specific function of the rho family of small G proteins is to regulate the organization of the actin cytoskeleton and cell surface structures in eukaryotic cells (reviewed by Hall, 1994). It is now well established that members of the rho family, rhoA, B, C, and D, racl and 2, and cdc42Hs, are involved in regulating the organization of the actin cytoskeleton. In fibroblasts, it has been shown that rhoA regulates the formation of actin stress fibers, whereas racl regulates lamellipodium formation and membrane ruffling, and cdc42Hs regulates filopodium formation (reviewed by Hall, 1994; Machesky and Hall, 1996). In BHK cells, rhoD causes the loss of stress fibers and modulates endosomal motility (Murphy et al., 1996). rhoA and rhoD also affect the formation and maintenance of focal adhesions, the sites at which stress fibers are linked via integrins to the extracellular matrix (Craig and Johnson, 1996; Murphy et al., 1996), whereas racl and cdc42Hs regulate the formation of smaller “focal complex” structures associated with lamellipodia and filopodia (Machesky and Hall, 1996). As cell motility involves the coordinated extension of lamellipodia and filopodia at the leading edge of the cell, together with contraction of the cell body and detachment of the rear edge, rho, rac, and cdc42Hs could play crucial roles in these events (Lauffenburger and Horwitz, 1996). Indeed, evidence also shows that rho and/or rac is required for motility (Stasia et ul., 1991; Takaishi et al., 1993; Ridley et al., 1995). rho family proteins have also been implicated in the formation of the actin-based contractile ring at cell division (reviewed by Ridley, 1995; Larochelle et al., 1996), and in the budding yeast S. cereuzsiue, cdc42 and rho1 are involved in regulating polarization of the actin cytoskeleton during budding (reviewed by Ridley, 1995). Despite this accumulation of evidence, until recently very little was known

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concerning the signal transduction pathways acting downstream of these proteins leading to actin reorganization. This review only considers the role of lipid kinases. Other rho “targets” have been discussed elsewhere (Chant and Stowers, 1995; Ridley, 1996; Symons, 1996; Machesky and Hall, 1996). Although no PtdInsP 5-kinase isoforms have been isolated using screens for proteins interacting directly with any members of the rho family, a few reports suggest that either rho or rac, but never both in a single study, interact with a PtdInsP 5-kinase. Chong et al. (1994) were the first to show that acylated rhoA in its GTP-bound state stimulated PtdInsP 5-kinase activity up to 25-fold in murine C3HlOTU2 fibroblast lysates, whereas GDP-bound rhoA stimulated only 2- to 5-fold. racl or bacterially expressed rhoA had no effect. To rule out the possibility that rhoA was indirectly inducing PtdInsP 5-kinase activity, these authors went on to investigate whether rhoA physically interacted with a PtdInsP 5-kinase. Interestingly, they reported that the observed binding was independent of whether rhoA was in a GTP- or GDPbound state (Ren et al., 1996), and moreover that binding occurred using bacterially expressed rhoA. Further characterization of the PtdInsP 5-kinase involved showed that it cross-reacted with polyclonal antibodies against type I PtdInsP 5-kinases, but it was not activated by PtdOH, which had previously been proposed to be characteristic of type I PtdInsP 5-kinases (Jenkins et al., 1994). Two other groups have shown that racl, but not rhoA, can stimulate PtdInsP 5-kinase activity. Using permeabilized platelets, Hartwig and colleagues (1995) demonstrated that bacterially expressed, constitutively active mutants of racl (V12racl) but not rhoA (V14rhoA) caused uncapping of actin barbed ends. racl also stimulated PtdInsP, biosynthesis, and raclinduced uncapping of actin filaments was inhibited by PtdInsP,-binding synthetic peptides derived from gelsolin. Identical results were found using recombinant racl and rhoA produced in Sf9 cells, suggesting that posttranslational modifications are not required in this case. These data suggest that the control of capping proteins by racl is mediated by polyphosphoinositide biosynthesis, although the identity of the capping protein regulated by racl remains to be established. Hartwig et al. (1995) also showed that PtdOH was 25-50% as effective as polyphosphoinositides in uncapping actin filament barbed ends in platelets. Because PtdOH activates type I PtdInsP 5-kinases in uitro (Moritz et al., 1992; Ishihara et al., 1996), its effects on actin assembly could be mediated by the induction of PtdInsP, biosynthesis. Another group has shown that bacterially expressed racl, but not rhoA, associates with a PtdInsP 5-kinase (Tolias et al., 1995), both in rat liver homogenates and in Rat1 fibroblast lysates, and PtdInsP 5-kinase activity can be coimmunoprecipitated with endogenous racl from Swiss 3T3 fibroblasts. The binding was again found to be independent of the bound nucleotide, al-

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though a rat liver cytosol PI 3-kinase associated with both racl and cdc42Hs in a GTP-dependent fashion. The PtdInsP 5-kinase bound to racl was found to be stimulated by spermine and PtdOH, again suggesting the involvement of a type I enzyme. These apparently contradictory results might reflect either the involvement of distinct isoforms of PtdInsP 5-kinase responsive to rho and rac or differences in the experimental conditions used. As rho and rac are known to be involved in distinct pathways, whereas PtdInsP, has many potential cellular roles, it is tempting to speculate that cells evolved several PtdInsP 5-kinase isozymes that are attached to specific pathways. In this regard, two type I PtdInsP 5-kinase isozymes, termed Ia and Ib, from a murine pancreatic cell line have been cloned (Ishihara et al., 1996). Additionally, Carvajal et al. (1996)have reported the occurrence of several splicing variants of STM7, a type I PtdInsP 5-kinase, which suggests that the number of isoforms in mammalian cells might be much larger than initially thought. It is now important to investigate which pathways involve these different isozymes and splicing variants. The intricate relationship among gelsolin, profilin, vinculin, PtdInsP,, and actin in cells has been the focus of many in vivo investigations, but several lines of data dispute the premise that PtdInsP, plays a role in regulating the actin cytoskeleton. Thus it was shown that N-formyl peptide-induced actin polymerization in neutrophils correlated with the intracellular level of PtdInsP, rather than with PtdInsP, (Eberle et af., 1990). Furthermore, Cantley et af. (1991) reported that the cellular event most closely correlated with PI 3-kinase activation was actin filament rearrangement. Additionally, cells with mutations that lacked PI 3-kinase-binding sites in the PDGF p receptor failed to undergo actin rearrangement or other motility responses such as membrane ruffling and chemotaxis in response to PDGF (Severinsson et af., 1990; Wennstrom et al., 1994; Kundra et al., 1994). Exposure of fibroblasts to wortmannin, a PI 3-kinase inhibitor, blocked PDGF-mediated actin rearrangement (Wymann and Arcaro, 1994). Moreover, rho, which is known to regulate cytoskeletal reorganization in response to growth factors, was found to activate PI 3-kinase in platelet extracts (Zhang et af., 1993). Finally, rac, which has been implicated in membrane ruffling, was suggested to be a major effector protein for the PI 3-kinase signaling pathway (Hawkins et al., 1995). All these facts promote the notion that the stimulation of PI 3-kinase activity is crucial to actin polymerization. As PtdInsP, is the primary cellular substrate for PI 3-kinases, the important roles suggested for PtdInsP 5-kinase and PtdIns 3-kinase activities are not mutually exclusive. Indeed, many studies have not distinguished PtdInsP, from PtdInsP, signaling. It is interesting to note that Tolias et af. (1995)reported that racl associated with PtdInsP 5-kinase via a mechanism independent of the GTP- or GDP-bound state, whereas the association of PI 3-kinase with both racl and cdc42Hs

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was GTP dependent. Since it is believed that the GTP-bound form is the “on” state of G proteins, it is difficult to ascribe a physiological role for the association of PtdInsP 5-kinase with GDP-bound forms of either rhoA or racl. A possible model is that instead of having a small G protein activating a PtdInsP 5-kinase, activation proceeds in the opposite direction, allowing PtdInsP 5-kinase activities to regulate associated small G proteins. Indeed, it has been reported that in vitro PtdInsP, strongly stimulates GDP dissociation from cdc42Hs and rhoA and, to a smaller extent, from racl (Zheng et al., 1996a). This would place PtdInsP 5-kinases upstream of these small G proteins, arguably in an analogous fashion to the regulation of ARF by PtdInsP, and rab by PtdInsP, (Section IV). Several key points for further study arise from this discussion. The roles of PtdInsP 5-kinase and PI 3-kinase and their corresponding products should be investigated in a single system, using different experimental approaches. The specificity of PtdInsP, monoclonal antibodies was originally determined using PtdInsP, PtdIns, and InsP,, but D3-phosphoinositides have yet to be tested (Fukami et al., 1988). As described in Section I, the use of wortmannin presents similar difficulties in terms of specificity and, again, information obtained from experiments involving this inhibitor should be complemented by results using other experimental approaches.

IV. VESICLE BIOGENESIS AND TRAFFICKING The uptake and release of a wide range of molecules via intracellular vesicles are termed endocytosis and exocytosis, respectively. Such phenomena occur widely in many cell types and may be considered to include such diverse events as membrane cycling, which includes glucose transporter regulation and nondegradative receptor cycling, hormone and neurotransmitter release, and the targeting of lysosomal proteins. Although each of these processes relies on specific and often complicated collections of proteins that are beyond the scope of this review, polyphosphoinositides have been implicated in several aspects of vesicular function. However, it is worth noting at the outset that in virtually none of the following examples has the biochemical function of the polyphosphoinositide been clearly established. Different types of vesicle are generated at several subcellular loci, including the plasma membrane, ER, Golgi, and nucleus. The formation of all such vesicles requires that a defined area of membrane invaginates (termed budding), followed by fission of the bilayer in the plane between the bud and the membrane. After a brief description of these processes, only vesicular functions relevant to phosphoinositide kinases will be considered. Several reviews deal more fully with other aspects of vesicle formation and trafficking (Roth-

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man and Warren, 1994; Bednarek et al., 1996; de Camilli et al., 1996; Schekman and Orci, 1996).

A. Endoplasmic Reticulum and cis-Golgi

Vesicle Formation The processes of budding in the ER and Golgi compartments rely on the recruitment of cytosolic factors to the cytoplasmic leaflet. These factors minimally comprise two types of protein: a small G protein and a coat protein complex. Where secretory vesicles are being formed at the trans-Golgi network (TGN),a member of the ARF class of small G proteins, typically ARF1, and the clathrin-adaptor protein AP-1 complex assemble; within the ER and Golgi cisternae, anterograde and retrograde transport vesicles (COP-I vesicles) are formed by the recruitment of ARF and a nonclathrin containing coat complex (coatomer) that contains the p-COP adapter protein; and on exclusively anterograde ER transport vesicles (COP-I1 vesicles), a member of the ARF-related Sarlp small G protein family and a unique coat protein complex are recruited (reviewed by Schekman and Orci, 1996). The COP-I fission event can be viewed as a luminal membrane fusion, which is thought to occur spontaneously in the presence of fatty-acyl coenzyme A once the distorted membrane juxtaposes back on itself (Rothman and Warren, 1994). The function of coenzyme A is not clear, but it has been suggested to be required for the acylation of an unidentified luminal protein involved in fission (Ostermann et al., 1993). Although ATP is not required and ARF1, GTP, coatomer, and fatty-acyl CoA are sufficient for COP-I vesicle budding in vitro (Ostermann et al., 1993), both ARF and coatomer are able to bind PtdInsP,. However, it is unlikely that PtdlnsP, is a specific high-affinity receptor for ARFl at the ER and Golgi because PtdInsP, is found in many different cell membranes and because high-affinity binding to purified Golgi membranes is trypsin sensitive (Serafini et al., 1991). It is possible therefore that in the absence of exogenous ATP, sufficient PtdInsP, can exist within membrane preparations to allow budding to occur in vitro. Whether or not continued PtdInsP, biosynthesis is needed to sustain budding in vzvo remains to be investigated, but it is clear that PtdInsP and PtdInsP, biosynthesis occur within the ER compartment (Helms et al., 1993), although the 4- and 5-kinase activities are dwarfed by their respective plasma membrane counterparts. Further evidence that PtdInsP, biosynthesis is needed for ARF activity has been suggested from work in which PITP was shown to be required for the formation of vesicles from the trans-Golgi network (Section IV,B). How does PtdInsP, facilitate high-affinity binding of ARF? One answer to this question comes from the identification of an ARF nucleotide exchange

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factor. A shared requirement for the recruitment of Sarlp to COP-11 vesicles and of the recruitment of ARF to COP-I and clathridAP-1-coated vesicles is that they release GDP in exchange for GTP. As with most G proteins, the basal rate of this exchange is low and relies on the action of cognate exchange factors. In yeast, exchange factors for Sarlp and ARF2 have been identified as Secl2p and Geal, and an exchange factor for human ARFl is the ARNO protein (Chardin et al., 1996). The observation that PtdInsP, mixed micelles increase the rate of nucleotide exchange on ARFl fivefold in vitro led to the suggestion that this phosphoinositide plays a role as a cofactor in the exchange reaction (Terui et al., 1994). Furthermore, the identification of a dynamin and PLCG-like PH domain in ARNO, but which is notably absent from Geal (Peyroche et al., 1996), suggested a site for PtdInsP, interaction (Chardin et al., 1996). However, using purified recombinant ARNO, mixed PtdCho/PtdInsP, vesicles had little effect on its ARFl nucleotide exchange activity, which was stimulated only in the presence of PtdChoPtdInsP, vesicles containing phosphatidylserine or phosphatidylglycerol. Deletion of the PH domain from ARNO abolished all stimulation. These data were taken to suggest that the PH domain of ARNO mediates the interaction with PtdInsP, and that a second interaction with an acidic phospholipid is required to stabilize this interaction (Chardin et al., 1996). The authors note, however, that it remains possible that other polyphosphoinositides, such as PtdInsP,, may prove to be more potent cofactors. In addition, the existence in genome databases of as yet uncharacterized homologs of ARNO (Chardin et al., 1996) provides a possible way of functionally differentiating the five human ARF isoforms and the different subcellular sites where ARF is involved in vesicle trafficking and intriguingly in cell adhesion (Kolanus et al., 1996). Although a cytosolic phospholipid-dependent exchange factor has previously been described (Tsai et al., 1994), it remains to be shown whether this is ARNO. If so, then it is clearly possible that the activity of ARNO, and therefore budding, is regulated and localized by PtdInsP, biosynthesis. Upon binding to PtdInsP,, the myristoylated amino-terminal region of ARF is displaced and anchors ARF by insertion into the membrane leaflet. Tighter binding of ARF is defined by resistance to extraction using PtdCho liposomes and is suggested to occur by association with an integral membrane receptor (Serafini et al., 1991; Helms et al., 1993). However, unlike Sarlp, which binds Sed4p and Sedl6p in yeast (reviewed by Bednarek et al., 1996), no integral receptor for ARF has been identified yet. The interaction between ARF and its putative receptor causes binding of coatomer, but the biochemical signal for this event remains to be firmly established. Nevertheless, a number of empirical observations clearly support a role for phospholipids in this process. A common effector of both PtdInsP, and ARFl in HL60 cells has been shown to be PLD1, which hydrolyzes PtdCho to PtdOH and choline (Brown et al., 1993; Cockcroft et al., 1994;

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Liscovitch et al., 1994; Hammond et al., 1995).As coatomer has been shown to be able to bind directly to PtdOH vesicles in the presence of PtdInsP,, it has been proposed that PtdInsP, and PtdOH form part of the initial binding site for coatomer (Ktistakis etal., 1996; Schekman and Orci, 1996). Indeed, the requirement for ARF in coatomer binding is bypassed in cells with elevated PLD activity and consequently increased levels of PtdOH (Ktistakis et al., 1996). Stronger binding of coatomer is thought to take place through an interaction with a membrane receptor, which has yet to be identified but may be composed of the cytosolic tails of transmembrane cargo proteins and possibly V-SNAREproteins (reviewed by Bednarek et al., 1996). A further role for the acidic phospholipids PtdInsP, and PtdOH has been proposed to be to induce the membrane curvature required for budding through electrostatic repulsion (Sheetz and Singer, 1974), but there is as yet little empirical evidence to support this idea. In summary, a coherent hypothesis for COP-I vesicle formation relies on phospholipid-binding sites for the recruitment of cytosolic proteins that are required for budding. This mechanism is quite different than the proteinonly events involved in COP-I1 vesicle budding (Schekman and Orci, 1996). There is little evidence as yet for the type of PtdIns 4-kinase and PtdInsP 5-kinase responsible for PtdInsP, biosynthesis in the ER and Golgi compartments, whether they are regulated, and whether a PITP activity is required to supply substrate to these enzymes. The observation that PtdIns 4kinase and PtdInsP 5-kinase activities are closely linked to the biosynthesis of PtdIns in the ER (Helms et al., 1991) suggests that sufficient binding sites may exist throughout the ER for anterograde transport to the cis-Golgi. However, coatomer formation has been observed to be limited to a specialized region called the CRER (coatomer-rich ER; Orci et al., 1994). This apparent anomaly is not readily explained, but may be due to the localization of receptors or of PtdInsP, and PtdOH biosynthesis within this region. Despite evidence for the involvement of conventional and novel types of PKC activity in the activation of PLD, there is no reported hydrolysis of PtdInsP, by phospholipases in the ER. It is therefore probable that, analogous to PI metabolism in the erythrocyte plasma membrane, a PI shuttle exists between PtdIns, PtdInsP, and PtdInsP,, although the relevant 4-phosphatase activities have as yet received little attention. As rat liver Golgi contains substantial PtdIns 4-kinase but very little PtdInsP 5-kinase activity (Helms et al., 1991 and references therein) and as PtdIns 4-kinases and a PtdInsP, 5-phosphatase have been localized to the Golgi network (Nakagawa et al., 1996a,b; Suchy et al., 1995), the later stages of protein transport may depend on localized and possibly regulated PtdInsP, biosynthesis through the recruitment of a cytosolic, PtdOH-dependent, type I PtdInsP 5-kinase. The importance of regulating the amount of PtdInsP, in the Golgi is revealed by the effect of toxic levels, which presumably arise in the X-linked

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disorder Lowe’s oculocerebrorenal syndrome (OCRL). In OCRL, mutation of a Golgi-localized PtdInsP, 5-phosphatase compromises visual, renal, and neurological functions (Suchy et a!., 1995), but specific alterations in vesicle formation have yet to be reported.

B. trans-Golgi Network Vesicle Formation and Protein Sorting Vesicles arising at the TGN are destined for several intracellular targets, including lysosomes for degradative functions, endosomes for temporary storage, and the plasma membrane for secretion. Different sets of proteins mediate the formation of each type of vesicle and each set contains the ability to sort its appropriate cargo proteins, carry out budding and fission, and direct the vesicle to its appropriate site. TGN vesicles do not include COP-I or COP-11, but COP-I related vesicles with a “lace-like” vesicle coat (Ladinsky et al., 1994) and clathrin-coated vesicles have been described. The latter are formed by ARFl activity and their coats contain the AP-1 adaptor complex and clathrin (Stamnes and Rothman, 1993), but little is known regarding the part played by phosphoinositide kinases in the formation of these vesicles. Although most attention has focused on the role of dynamin in the biogenesis of clathridAP-2 coated vesicles arising at the plasma membrane (Section IV,C), two yeast dynamins, Vpslp and Dnmlp, are required for sorting vacuolar proteins in the TGN and endosomal compartments, respectively (reviewed by de Camilli et al., 1995).However, although dynamin binds PtdInsP, via its PH domain, PtdInsP, is probably not needed for the formation of TGN-coated buds as Vpslp and Dnmlp lack the PH domain. One area of work that may shed light in the future on the role of PtdInsP, concerns the yeast Fablp putative PtdInsP 5-kinase (Fig. 3), as Fablp is required for proper vacuole formation and the primary defect in temperaturesensitive Fablp mutants is a rapid enlargement of the vacuole (Yamamoto et al., 1995). Further evidence for an essential role for phosphoinositides in the formation of post-TGN vesicles has been produced from studies on secretory vesicle formation in a cell-free preparation of the TGN from rat PC12 neuroendocrine cells. This model system was found to require two cytosolic factors present in bovine adrenal medulla termed CASTl and CAST2. CASTl has been identified as PITP, and the activity of the 01 and p-isoforms of PITP was shown to be equivalent (Ohashi et al., 1995).This requirement for PITP may explain the reported localization of the p-isoform of PITP to the Golgi compartment of Swiss 3T3 fibroblasts that was observed using immunofluorescence microscopy of permeabilized, cytosol-depleted cells (de Vries et al., 1995). The role of PITP is presently unclear, but if it is to supply PtdIns for

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phosphorylation, then the TGN preparation used in these studies must contain limiting amounts of available PtdIns. Support for a role for PITP in supplying substrate for phosphorylation comes from the dependence of postTGN vesicle formation on ATP. In addition, two cloned PtdIns 4-kinases, p92 and p230, have been reported to be localized to the Golgi compartment of transfected COS-7 cells (Nakagawa et al., 1996a,b), as described earlier (Table I), and CAST2 has been suggested to contain a cytosolic PtdInsP 5 kinase activity (Ohashi et al., 1995). However, PITP may carry out an analogous role to the yeast Secl4 protein, which is thought to control protein secretion by maintaining the Golgi Ptd1ns:PtdCho ratio. The mechanism by which Secl4p monitors these Golgi lipids is by inhibition of PtdCho biosynthesis by the PtdCho-loaded form of Secl4p (Skinner et al., 1995).Although this may prove to be one function for PITP in vivo, it is unlikely to account for the observed stimulation of vesicle formation in an isolated TGN preparation. Although the role for PtdInsP, biosynthesis may be similar to the promotion of budding previously described for COP1 vesicles (Section IV,A), an Nethylmaleimide-sensitive protein required for the ATP-independent scission of TGN-coated buds can be replaced by Secl4p (J. P. Simon, personal communication). However, it remains to be shown whether PITP behaves similarly to Secl4p and whether the role of PITP in the scission of coated buds is to supply PtdCho, PtdIns, or even PtdInsP, (reviewed by Liscovitch and Cantley, 1995). Although neither PtdIns 4-kinase nor PtdInsP 5-kinases have been shown to play a role in protein sorting, it is becoming increasingly clear that a PtdIns 3-kinase activity provides one of the key signals for sorting proteins destined for the vacuole in yeast and its equivalent in mammalian cells, the lysosome. This subject deserves attention within the context of this review as reagents used to study the role of PtdIns and PI 3-kinases in mammalian cells are now known to inhibit other enzymes, including PtdIns 4-kinases (Section I). Treatment of K562 erytholeukemia cells (Davidson, 1995) and clone 9 hepatocytes (Brown et al., 1995) with wortmannin or LY294002, both of which inhibit PtdIns 3-kinase activity, causes a specific defect in TGN to prelysosomal compartment trafficking, resulting in the exocytosis of the lysosomal precursor protein procathepsin D. While the actual role of PtdIns 3-phosphate in protein sorting remains enigmatic, the phenotype may be due to a perturbation of mannose 6-phosphate receptor function (reviewed by Shepherd et al., 1996). Although the specificity of the inhibitors used is now questionable, the involvement of PtdIns 3-kinase activity in this process is firmly established by work with yeast vacuolar protein-sorting (VPS) genetic mutations. One such gene product identified in this way is the Vps34p PtdIns-specific 3-kinase, which is the only phosphoinositide 3-kinase activity identified in yeast. This enzyme has a human homolog (Volinia et al.,

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1995) and is required in the TGN to direct newly synthesized vacuolar hydrolases away from the default pathway leading to the plasma membrane and toward the vacuole (reviewed by Stack et al., 1995; Seaman et al., 1996). It remains to be shown whether, like secretory vesicle formation, the proper functioning of the human Vps34p homolog depends on a PITP activity.

C. Plasma Membrane Endocytosis Receptor-mediated and synaptic vesicle endocytosis at a cell surface follow a related sequence of events to the formation of intracellular trafficking vesicles. However, budding and fission of the plasma membrane lead to the formation of vesicles coated with the AP-2 adaptor complex and clathrin. Coat formation depends on specific interactions between AP-2 adaptins and a membrane receptor, during which there is little evidence at present for the involvement of phosphoinosositides. As PtdInsP, micelles and InsP, inhibit AP-2 activity in vitro, PtdInsP, has been suggested to maintain a pool of inactive AP-2 at the plasma membrane (Beck and Keen, 1991) that can be recruited by direct interaction with receptors, synaptotagmin, or PtdInsP, (Li, C. et al., 1995; Gaidarov and Keen, 1995). The G protein most clearly implicated in AP-2 (and its neuronal homolog AP-3/AP-l80)-coated vesicle formation is dynamin, whose function appears to be quite different from that of ARFl and Sarlp. The 100-kDa dynamin protein has been shown to form oligomeric rings around the neck of a coated bud and may provide the machinery for the fission of the vesicle and the membrane (reviewed by de Camilli et al., 1995). Although dynamin has a PH domain that specifically binds PtdInsP, (Salim et al., 1996), the absence of a PH domain in the yeast dynamin homolog Vpslp and Dnmlp, which form similar ring structures on intracellular membranes, implies that the PH domain of dynamin is not required for the ability to promote fission. Instead it is likely that the PH domain serves to localize dynamin to the plasma membrane or to allow regulation of internalization by sensing the level of PtdInsP,. Evidence for the former comes from studies with PH domain deletion mutants of the small G protein exchange factors Ras-GRF (Buchsbaum et al., 1996), dbl (Zheng et al., 1996b), and Ifc (Whitehead et al., 1995), in which loss of their respective PH domains leads to loss of transforming ability and mislocalization. While an interaction with PtdInsP, alone would not be sufficient to target dynamin to the plasma membrane, further interactions with AP-2 adaptins (Wang et al., 1995) or receptor signaling proteins (Gout et al., 1993; Lin and Gilman, 1996) could provide the observed specificity (Damke et al., 1994).The ability of PtdInsP, to stimulate the GTPase activity of dynamin (Tuma et al., 1993) could be interpreted by a model in which dynamin, like AP-2, is held in an inactive, GDP-bound form by free PtdInsP,

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in the plasma membrane until it is incorporated into a coated pit where the GTP-bound form may be more stable (Lin and Gilman, 1996). Assembly of the ring itself may then promote the ordered GTP hydrolysis, which causes coated vesicle fission (reviewed by de Camilli et al., 1995). However, in HeLa cells, dynamin is found only in the cytosol or within coated regions of the plasma membrane, and overexpression preferentially increases the amount in the cytosol (Damke et al., 1994). As described earlier for the existence of CRER (Section IV,A), these observations do not seem to be consistent with the existence of an accessible pool of free PtdInsP,, which in this case is in the plasma membrane. However, if PtdInsP, is somehow localized within coated buds, as it appears to be within caveaolae (Pike and Casey, 1996), then there may be a functional interaction with dynamin. In this regard as rho and rac inhibit transferrin receptor endocytosis via clathrin-coated pits in HeLa cells (Lamaze et al., 1996), it will be interesting to see if rho- or racdependent type I PtdInsP 5-kinases prove to be the relevant effectors. Furthermore, a commercially available anti-PtdInsP, monoclonal antibody (Perceptive Biosystems Inc.), if specific, will serve as a useful tool to reveal the distribution of PtdInsP, within different cell membranes. One of the best studied roles of dynamin is in presynaptic vesicle internalization, where specialized coated vesicles form rapidly following neurotransmitter release (reviewed by Sudhof, 1995). In neurons, dynamin may be recruited to coated pits by interactions with AP-2 either directly or via amphiphysin or possibly GRB2 (David et al., 1996). Although the assembly of this putative complex on synaptotagmin does not appear to require phosphoinositides, a role for PtdInsP, was suggested by the identification of a presynaptic type I1 phosphatase termed synaptojanin (McPherson et al., 1996). More recent results suggest, however, that synaptojanin may preferentially dephosphorylate PtdInsP, rather than PtdInsP, in vitro (R. Woscholski, personal communication). If this is the case in vivo, then PtdInsP, may play a role in sorting and recycling synaptic vesicles following the dissociation of dynamin and uncoating, which is arguably akin to its proposed endosomal role in regulating transferrin receptor recycling and glucose transporter movement (reviewed by Shepherd et al., 1996; Seaman et al., 1996). In either case there does not appear to be any PtdInsP 5-kinase associated with synaptic vesicles (Gross et al., 1995), although it remains possible that the antibodies used in these studies do not recognize the relevant isoform or that PtdInsP, is generated at a specific stage in the vesicle cycle. With the exception of a preliminary report that AP-2 binds PtdInsP, (Gaidarov and Keen, 1995), PI 3-kinase activity does not appear to be involved in receptor internalization (Barker et al., 1995; Hansen et al., 1995; Joly et al., 1995), but it has been implicated in fluid phase uptake, transporter and receptor recycling, and the lysosomal targeting of internalized PDGF re-

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ceptors (reviewed by de Camilli et al., 1996; Shepherd et al., 1996; Seaman et al., 1996). Some of these effects may be due to the regulation of vesicle fusion events by small G proteins of the rab family, possibly in an analogous fashion to the regulation of ARF by PtdInsP, (Section IV,A), as there is evidence (1)that racl, which mediates some of the signaling functions of PtdIns 3-kinase, is not involved in the movement of GLUT4 glucose transporter vesicles in adipocytes (Marcusohn et al., 1995); (2)that a constitutively active rab can overcome the inhibition of early endosome fusion caused by wortmannin (Li, G. et al., 1995); and (3) that insulin but not PDGF stimulates PI 3-kinase activity in a low-density microsomal fraction (Kelly and Ruderman, 1993).

D. Exocytosis The various regulated and constitutive events in different cell types in which vesicles move and fuse with their target membranes rely on complex sets of targeting and fusion proteins (reviewed by Burgoyne and Morgan, 1993; Rothman and Warren, 1994; Rothman, 1994; Sudhof, 1995). In only a few examples has polyphosphoinositide biosynthesis been suggested to be of functional importance, and only these instances will be discussed here. In general, while many different cell types exhibit regulated exocytosis, studies of two broad categories of regulated secretion using permeabilized and cytosol-depleted cells have revealed the importance of polyphosphoinositides, namely in the release of neurotransmitters from neurons and neuroendocrine cells and in the release of lysosomal hydrolases from myeloid cells. In each case a transient increase in Ca2+ concentration stimulates secretion, whereas constitutive secretion does not require Ca2+ ions, and additional cytosolic factors such as ATP and GTP, as well as specific proteins, are required in some cell types. Secretion of noradrenaline from PC12 pheocromocytoma cells can be divided into ATP-dependent priming followed by Ca2+-dependent fusion. Three cytosolic factors required for priming (PEP proteins) have been separated by gel filtration chromatography of rat brain cytosol (Hay and Martin, 1992), of which two have so far been identified: PEP3 contains PITP (Hay and Martin, 1993) and PEP1 contains a type I PtdInsP 5-kinase (Hay et al., 1995). These results suggested that priming requires the biosynthesis of PtdInsP,, which may at least partially explain the requirement for ATP, although the role of PtdInsP, itself remains to be identified. It was suggested nonetheless that PtdInsP, rather than a metabolic derivative is the active agent because secretion can be inhibited by anti-PtdInsP, monoclonal antibodies and by recombinant PLCG (Hay et al., 1995). Priming of noradrenalin secretion by adrenal chromaffin cells had previously been shown to re-

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quire ATP and to be inhibited by the Bacillus thuringiensis Ptdlns-specific PLC (Eberhard et al., 1990).However, these studies do not eliminate the possibility that PtdInsP, is the physiological agent, as removal of PtdInsP, would be expected to inhibit PI 3-kinase activity. Indeed wortmannin and LY294002 inhibit histamine secretion by RBL-2H3 basophilic leukemia cells (Yano et al., 1993; Yano et al., 1995), although the subcellular site of PtdInsP, biosynthesis required for secretion was not identified in these studies. The third and as yet unidentified cytosolic fraction, PEP2, contains a protein of about 120 kDa, which may well prove to be another PtdInsP 5-kinase isozyme, a PI 3-kinase, or a recently cloned soluble PtdIns 4-kinase, p92 (Nakagawa et af., 1996a), which has an apparent molecular mass of about 100 kDa and localizes to vesicles in transfected COS-7 cells. The size of PEP2 is also similar to that of a soluble type 111-relatedPtdIns 4-kinase termed p97 (Table I and Fig. 2; Wong and Cantley, 1994) and a soluble, wortmanninsensitive PtdIns 4-kinase that has been purified from the adrenal cortex (Nakanishi et al., 1995; Downing et al., 1996). Whether this latter enzyme is p92 or p97 remains to be tested. Finally, a requirement for PtdIns 4-kinase activity in priming noradrenalin secretion from adrenal chromaffin cells has been established using reversible inhibition by phenylarsine oxide (Wiedemann et al., 1996). The main site of PtdInsP biosynthesis following disinhibition was identified by subcellular fractionation as the chromaffin granules, which were also shown to contain an associated phenylarsine oxide-sensitive PtdIns 4-kinase. As with Golgi preparations and purified synaptic vesicles, no detectable PtdInsP 5-kinase activity was found in purified chromaffin granules, suggesting that cytosolic activity was lost before or during fractionation or that the assay conditions were inappropriate for the isozyme concerned. In addition to the activation of PLD discussed later, PtdInsP, may be required during secretion to increase membrane fusability (reviewed by Bajjalieh and Scheller, 1995) or to alter interactions with proteins containing PtdInsP,-binding PH domains such as spectrin, which is delocalized from a position close to membrane during secretion (Perrin et al., 1992), or other cytoskeletal proteins (Section 111).In this regard, the Ca2+-dependent, actinsevering protein scinderin (Section II1,A) has been identified as a possible target for PtdInsP, in chromaffin cells (Zhang et af., 1996). During secretion, scinderin appears to sever the cortical cytoskeleton that would otherwise inhibit scinderin, the role of PtdInsP, may be to maintain scinderin in an inactive state, which can be overcome when the Ca2+ concentration is elevated in stimulated cells. Calcium ion-dependent secretion by myeloid cells, as well as several other cell types, requires GTP, but the role of both heterotrimeric and small G proteins in these cells is unclear (reviewed by Burgoyne and Morgan, 1991). Nevertheless, two types of small G protein have been implicated: constitu-

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tively active, bacterially expressed racl and rhoA mutants were reported to stimulate secretion from mast cells, while a dominant negative racl mutant and Clostridium botulinum C3 transferase, which specifically ADP-ribosylates and inhibits rho, inhibited secretion (Price et al., 1995).Although some of the observed responses were probably due to the effects of these small G proteins on the cytoskeleton (Section III,D), a secretory response could still be observed in the absence of cytoskeletal effects. Furthermore, microinjection of mast cells with rhoGDI, which maintains rho in an inactive GDPbound state, inhibited GTP-dependent secretion (Mariot et al., 1996). Studies using permeabilized HL60 cells have led to the observation that bacterially expressed ARFl is able to stimulate secretion and PLD activity in parallel; however, enhanced PLD activity alone in the absence of ATP was not sufficient to stimulate secretion (Fensome et al., 1996). The same workers showed that PITP could also stimulate secretion in the presence of GTP and that the effects of ARF and PITP were additive at limiting concentrations. Finally, ARF and PITP were each shown to stimulate the biosynthesis of PtdInsP,, and secretion was inhibited by the PH domain of PLCG and by neomycin (both of which bind PtdInsP,). This led to the proposal that PtdInsP, is the common effector, as in neuroendocrine cells. The dependence of PtdInsP, biosynthesis on GTP in the presence of PITP (although GTP is not required to promote polyphosphoinositide biosynthesis and hydrolysis in permeabilized A431 cells) (Kauffmann-Zeh et al., 1995) suggests that a PtdIns 4-kinase, a PtdInsP 5-kinase, or both require a G protein for activity. Combining this result with the work on mast cells described earlier leads to the suggestion that racl and rhoA may be the relevant endogenous G proteins, particularly as rac and rho have previously been shown to interact with a type I PtdInsP 5-kinase in vitro (Section II1,D) and as rhoA is able to potentiate the activation of PLD in HL60 cell membranes (Ohguchi et al., 1996). The stimulation of PtdInsP, biosynthesis may therefore provide the pathway by which racl and rhoA enhance secretion in the absence of cytoskeletal changes in mast cells. Although the subcellular site of PtdInsP, biosynthesis was not investigated in these studies in myeloid cells, it would be expected to occur on the secretory vesicle if PtdInsP, plays a similar role to that described for secretion by neuroendocrine cells. Based on the observations that type I PtdInsP S-kinase and PLD activities are stimulated by PtdOH and PtdInsP,, respectively (Moritz et a/., 1992; Liscovitch et al., 1994),and that anionic phospholipids promote bilayer fusion in vitro, a simple model has been proposed to explain the synergistic roles of these two enzymes in coated vesicle exocytosis. According to this model, when a vesicle containing GTP-bound ARF and a PtdIns 4-kinase contacts an acceptor membrane containing PLD and a type I PtdInsP 5-kinase, reciprocal activation of the latter enzymes will ensue mediated by PtdOH and PtdInsP, (Liscovitch et al., 1994). The model there-

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fore proposes that a rapid generation of acidic phospholipids at the intermembrane interface catalyzes subsequent membrane fusion when the Ca2+ concentration is raised. The activity of the system is suggested to be held in check by a PtdOH and PtdInsP,-dependent ARF-GAP (Randazzo and Kahn, 1994). Conversion of ARF to its GDP-bound form will inhibit PLD and also promote uncoating. While many studies now support the notion that the generation of high levels of PtdInsP, on the vesicle outer leaflet is a key event during exocytosis, it is also possible that the PLD present during vesicle formation (Section IV,A,B) is maintained on secretory vesicles and that a type I PtdInsP 5-kinase is recruited directly from the cytosol to the vesicle. Furthermore, the recent identification that the PtdInsP,-binding protein centaurin contains an ARF-GAP domain (Hammonds-Odie et al., 1996) suggests that PtdInsP, may be a signal for uncoating in some cell types.

V. ROLES IN CANCER, SUMMARY,AND PROSPECTS The involvement of receptor signaling in cellular transformation is beyond the scope of this review, but their importance is revealed by the existence of many retroviral oncogenes that are believed to have been originally transduced from vertebrate protein tyrosine kinases. For example, the avian verbB oncogene is derived from the transmembrane and cytoplasmic parts of an avian EGF receptor, and v-erbB transformed chick embryo fibroblasts show enhanced polyphosphoinositide biosynthesis (Kato et al., 1987). Similarly, the role of PLCP and PLCy isozymes in cell growth and transformation has been the subject of numerous studies (reviewed by Grunicke et al., 1996), and the use of PI analogs as inhibitors of PLC activities is currently being exploited in cancer chemotherapy (reviewed by Grunicke et al., 1996). Interesting results from Weber and collaborators (1996)have shown that the proliferation and malignancy of tumors correlate with increased PtdIns 4-kinase and PtdInsP 5-kinase activities. In rat hepatomas of slow and intermediate growth rates, PtdIns 4-kinase activity was increased 5.3- to 7.6fold, whereas in a rapidly proliferating hepatoma (3924A), the increase was 28.5-fold over that of normal liver (Rizzo and Weber, 1994). Similar results were obtained for PtdInsP 5-kinase, with 3.3- to 9.7-fold increased activity in hepatomas with slow to moderate growth rates and a 42-fold increase in hepatoma 392412 (Singhal et al., 1994).A comparable pattern of marked elevations of the two kinase activities was observed in human carcinoma cells (ovarian epithelial carcinoma and breast carcinoma MDA-MB-435) relative to normal tissues (Weber et al., 1996). The elevated kinase activities led to an accumulation of IP,, which suggests that indeed a stimulation of signaling is occurring, despite the observation that the PLC activity was never

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found to be proportionally increased (Rizzo and Weber, 1994; Singhal et al., 1994).These coordinate increases in PtdIns and PtdInsP kinase activities suggest that the capacity for signal transduction is heightened in cancer cells and may well be sensitive targets for designing new chemotherapeutic agents. Interestingly, Friedman ( 1993, 1994) observed that the antiproliferative properties of tamoxifen are linked to increased PtdIns and PtdInsP kinase activity. These results were explained on the basis that tamoxifen binds to phosphoinositides and thereby stimulates the respective kinase activities by reducing product inhibition. However, once PtdInsP, is bound to tamoxifen it is no longer available to PLC and would block signaling. Clearly this mechanism is an inadequate explanation of the nonestrogen receptor-associated effects of tamoxifen. For example, Cabot et al. (1995) have shown in human mammary carcinoma MDA-MB-23 1 cells that tamoxifen elicits the production of PtdOH in a dose-dependent manner, which suggests that tamoxifen may stimulate PtdInsP, hydrolysis or affect another phospholipid pathway such as PtdCho hydrolysis by PLD. Further indirect evidence that tamoxifen interferes with PI metabolism can be inferred from results showing that tamoxifen inhibits the attachment of retinal-pigment epithelial cells to fibronectin (Wagner et al., 1995). Metastasis of a malignant tumor is the leading cause of death from many cancers. Unfortunately, the molecular mechanisms underlying the migration of cells from the primary sites are unclear. However, some progress has been achieved from investigations of cell adhesion and motility. The importance of rho and rac proteins in regulating cell surface projections and cytoskeletal organization has already been discussed (Section 111,D). Studies on the role of integrins in cancer have shown that they are important in a number of cellular processes that have an impact on the development of tumors, including the regulation of proliferation and apoptosis, cell motility and invasion, and angiogenesis (reviewed by Varner and Cheresh, 1996). For example, integrin a5pl (fibronectin receptor, VLA-S), which binds only to fibronectin (Hynes, 1992), has been implicated in the growth regulation of tumor cells. In Chinese hamster ovary cells, overexpression of a5pl integrin led to reduced anchorage-independent growth and tumorigenicity (Giancotti and Ruoslahti, 1990), whereas loss of a 5 p l integrin led to enhanced tumorigenicity (Schreiner et al., 1991). Evidence is slowly accumulating to show that certain integrins can increase PI turnover (reviewed by Sjaastad and Nelson, 1997). For example, both a 5 p l integrin in fibroblasts and pancreatic acinar cells and integrin aLp2 (CDlla/CD18, LFA-1) in Tcells have been reported to stimulate PLCy activity, and integrin aIIbp3 (GPIIbIIIa) in platelets has been reported to cause the translocation of PtdInsSP 4-kinase to the cytoskeleton (Hinchliffe et al., 1996). Furthermore the adhesion of C3H10T1/2 fibroblasts to fibronectin has been shown to stimulate the activity of a PtdInsP 5-kinase (McNamee et al., 1993), but the PtdInsP 5-ki-

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nase isoform involved in integrin a5pl and aLP2 signaling has yet to be identified. Although vesicle formation and trafficking have numerous potential roles within cancer biology, including growth factor and cytokine secretion, receptor downregulation, mistargeting of protooncogenes, and the uptake of therapeutic agents, a direct link between PI metabolism and these roles has not been reported. Nevertheless, an understanding of the diverse roles of PI metabolism in different cell types may allow improvements in the effectiveness and specificity of therapeutic regimes. There are clearly numerous potential roles for PtdInsP, in different parts of the cell. The best studied examples of these are described here, whereas others doubtless remain to be identified. In order to regulate each of the various PtdInsP,-binding proteins and PtdInsP,-modifying enzymes, the biosynthesis and availability of this lipid in cells are subject to different forms of control, including subcellular compartmentation and restricted modes of recognition or high-affinity interaction. The proteins responsible for PtdInsP, biosynthesis are also diverse, allowing differential subcellular localization and regulation, but a full understanding of the extent of this diversity in mammalian cells has not yet been achieved. Of particular note is the absence of a cDNA clone for the 55-kDa type I1 PtdIns 4-kinase and the putative PtdIns 5-kinase. Consequent on this diversity, PtdInsP, biosynthesis should be investigated with the minimum disruption of cell structure, e.g., using permeabilization, transfection, or microinjection. The identification of cDNA clones for several mammalian PtdIns 4-kinase and PtdInsP 5-kinase isozymes allows such studies to be performed for the first time. However, cross-talk between pathways may allow compensation, as observed for the PLC knockout in Dictyostelium (van Dijken et al., 1995). This may limit the usefulness of transgenic animals and demands a careful approach when overexpression is involved. The commercially available anti-PtdInsP, mAb and the identification of specific PtdInsP,-binding polypeptides, such as the PH domains of PLCG and dynamin, may allow changes in PtdInsP, distribution and biosynthesis to be identified, and PtdInsP,-dependent events to be specifically inhibited. Finally, the chemical synthesis of phosphoinositide analogs and fluorescent derivatives has been achieved (see, e.g., Hammonds-Odie et al., 1996), which opens exciting avenues into real-time microscopy as long as these compounds can be appropriately introduced into cells.

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Index

A Abl, see Abelson tyrosine kinase Abelson tyrosine kinase (Abl), stress signaling, 73-74 Actin, reorganization, 192-195 Adipocyte, mitogen-activated protein kinase pathway in differentiation, 99-100 Apoptosis P53 control in tumors, 29-30, 37-39 mutation studies, 32-34 transactivation targets, 30-3 1 stress-activated protein kinase pathways, 74-75 Apoptosis signal-regulating kinase (ASKl), phosphorylation substrates, 69 Arfl, Golgi vesicle formation, 196-197 ARNO, Golgi vesicle formation, 197 ASK1, see Apoptosis signal-regulating kinase ATF, phosphorylation, 87

B Bax apoptosis role, 30-31, 38 gene activation by pS3,30-31 Bcl-2, gene inhibition by p53, 34 Blood cell, mitogen-activated protein kinase pathway in differentiation, 98-99 Breast cancer, chromosome 3 mutations, 154-156 Bur2, pheromone response pathway in yeast, 108-109

C Calcium exocytosis regulation, 203 stimulation of MKKERK pathway, 61-62 CD45, mitogen-activated protein kinase pathway regulation, 81 CDC25A, regulation by MYC, 12-13

Cdc42, regulation of stress-activated protein kinases, 70 Cell wall integrity pathway Saccharomyces cerevisiae, 106-107 Schrzosaccharomyces pombe, I10 Chromosome 3, mutation in tumors, 141-142,147-159 COP-I vesicle, phosphoinositides in formation, 196-199 COX-2, see Cyclooxygenase-2 CREB, see Cyclic-AMP response elementbinding protein Cyclic-AMP response element-binding protein (CREB), phosphorylation, 86 Cyclin D1 G1 progression, role, 3-4, 9-10 MYC pathway interactions, 13-14 regulation of cyclin-dependent kinase complex, 3-4 regulation of expression by RAS, 9-10 Cyclin E, G1 progression, role, 4 Cyclin G, gene activation by p53,27 Cyclooxygenase-2 (COX-2), phosphorylation. 90

D Dynamin, Golgi vesicle formation, 201-202

E E2F, protein-protein interactions, 10-1 1, 38 ELF-1, cell cycle regulation, 8-9 Endocytosis, phosphoinositide role, 201-203 Endoplasmic reticulum, phosphoinositides in budding, 196-198 Epithelial cell, stress-activated protein kinase pathway in migration in fruit fly, 96 ERK-1I2, see also Mitogcn-activated protein kinase activation, 53-54

21 7

218 ERK-1/2 (coat.) adipocyte differentiation role, 99-100 blood cell differentiation role, 98-99 cell cycle progression role, 7-8 cell growth and transformation, regulation, 91-92 inhibition studies, 85 mesoderm induction in Xenopus embryos, 96-97 neuronal differentiation role, 97-98 nuclear translocation, 112-113 signaling complexes, protein-protein interactions, 111-112 structure, 54-55 substrates cytoskeletal proteins, 90-91 nuclear transcription factors, 87-89 protein kinases, 85-87 signaling components, 89-90 table, 82-84 Estrogen receptor, phosphorylation, 88-89 ETS transcription factors cell cycle regulation, 8-9 phosphorylation, 87, 93-95 Exocytosis, phosphoinositide role, 203-206 Eye, ERK pathway in fruit fly development, 94-96

F Fas/APO-l receptor, gene activation by pS3, 32-33 FHIT, see Fragile histidine triad gene FOS, G1 progression, role, 5 Fragile histidine triad gene (FHIT) cloning, 144-145 deletions in tumors, 145-146 gene therapy, 159 mutations in cancer breast cancer, 154-156 gastrointestinal tract, 147, 150-151 head and neck cancer, 153-154 lung cancer, 151-154 Merkel cell carcinoma, 156-157 pleornorphic adenoma of parotid gland, 157 table, 148-149 protein product functions, 158 sequence analysis and structure, 146-147 structure, 145

Index

Fragile site definition, 142-143 FRA3B, 142,144,154,158 FRAllB, 143 FRA16B FRAXA, 143 FUS3, pheromone response pathway, 101, 103

G G1 cyclins in progression, 3-4 growth factor receptors in progression, 1-2,s oncogenes in progression, 2 MYC, 11-14 RAS, 5-11 SRC kinases, 14-15 GUS, pS3 control, 27 G2/M, pS3 control, 28 Gastrointestinal tract tumors, chromosome 3 mutations, 147, 150-151 GCK, see Germinal center kinase Gelsolin functions, 188-189 phosphoinositide binding, 188-189 Genomic instability, p53 and tumor progression, 40-42 Germinal center kinase (GCK), regulation of stress-activated protein kinases, 72 Golgi apparatus, phosphoinositides in vesicle formation cis-Golgi, 196-199 trans-Golgi network formation and protein sorting, 199-201 G proteins, see also Rho cross-talk between signaling pathways, 185 phosphatidylinositol4-kinase regulation, 180-1 8 1 phospholipase C activation, 178-1 80 receptor coupling mechanism, 174-175 stimulation of MKKERK pathway, 62-63 stress signaling, 73

H Head and neck cancer, chromosome 3 mutations, 153-154 Hematopoietic progenitor kinase 1 (HPKl), regulation of stress-activated protein kinases, 72

Index HOG pathway mitogen-activated protein kinase pathway, 104-1 06 osmoreceptors, 72,104 HPK1, see Hematopoietic progenitor kinase 1 Hyperosmolarity response, see HOG pathway

I IGF-BP3, see Insulin growth factor I-binding protein 3 INK4s, inhibition of cyclixdcyclin-dependent kinase complex complexes, 4 Insulin growth factor I receptor, gene inhibition by p53,34 Insulin growth factor I-binding protein 3 (IGF-BP3),gene activation by p53,31-32 Invasive growth, mitogen-activated protein kinase pathway in yeast, 103

J

JAK, see Janus kinase Janus kinase (JAK), stimulation of MKKERK pathway, 64 JNKl, see Stress-activated protein kinase JUN, G1 progression, role, 5 c-Jun, phosphorylation, 87-88

K KSR, worm development role, 94

L LET-23, worm development role, 93 Li-Faumeni disease, p53 and tumor progression, 40-41 LOH, see Loss of heterozygosity Loss of heterozygosity (LOH), chromosome 3 in tumors, 141-142,147-159 Lung cancer, chromosome 3 mutations, 151-154

M MAD, cell cycle regulation, 11-12 MAP4, see Microtubule associated protein 4 MAPK, see Mitogen-activated protein kinase MAPKAP kinases phosphorylation, 85-87 substrates, 82-83

219 MAX, heterodimers in cell cycle regulation, 11 MEK kinase (MEKK) substrates, 68-69 types, 67-68 MEKK, see MEK kinase Merkel cell carcinoma, chromosome 3 rnutations, 156-157 Mesoderm, mitogen-activated protein kinase pathway in Xenopus induction, 96-97 Microtubule associated protein 4 (MAP4), gene inhibition by p53,34 Mitogen-activated protein kinase (MAPK), see also specific proteins discovery, 49-50 family members, 50, 53 inhibition studies, 85 nuclear translocation, 112-113 phosphorylative regulation, see also Mitogen-activated protein kinase kinase activation, 53 phosphatases classification, 75,77 dual specificity phosphatases, 78-80 protein tyrosine phosphatases, 80-81 serinekhreonine phosphatases, 80 types, table, 76-77 responses to pathway adipocyte differentiation, 99-100 blood cell differentiation, 98-99 cell growth and transformation, 91-92 fruit fly development, 94-96 mesoderm induction in Xenopus embryos, 96-97 neuronal development, 97-98 Saccharomyces cerevisiae cell wall integrity, 106-107 hyperosmolarity response, 104-106 invasive/pseudohyphaI growth, 103-104 pheromone response pathway, 101-103 sporulation, 107-108 Schizosaccharomyces pombe cell wall integrity, 110 pheromone response pathway, 108-109 stress response pathway, 109-1 10 worm development, 93-94 signaling complexes, protein-protein interactions, 111-1 12

Index Mitogen-activated protein kinase (cont.) signaling in pathway activation calcium, 6 1-62 G proteins, 62-63 JAWSTAT pathway, 64 phosphatidylinositol3’ kinase, 64-65 protein kinase C, 63 stress-activated kinases, 65-66 substrates cytoskeletal proteins, 90-91 nuclear transcription factors, 87-89 protein kinases, 85-87 signaling components, 89-90 specificity, 53 table, 82-84 Mitogen-activated protein kinase kinase (MKK) cell growth and transformation, regulation, 91-92 inhibition studies, 85, 91 nuclear translocation, 112-1 13 phosphorylation in activation, kinases MEK kinases, 67-69 mixed lineage kinases, 69-70 MOS,60-61 Raf, 56-58 types, 53 signaling complexes, protein-protein interactions, 111-112 stress-activated kinases, 66-67 structure, 55-56 substrate specificity, 53 types, 53 Mixed lineage kinases, phosphorylation substrates, 69-70 MKK, see Mitogen-activated protein kinase kinase MKP-I, mitogen-activated protein kinase pathway regulation, 78-80 MKP-2, mitogen-activated protein kinase pathway regulation, 78-80 Mos cytostatic activity, 60-61 mitogen-activated protein kinase kinase activation, 60 phosphorylation and stability, 60 MYC cell cycle progression role, 5 , 8, 11-14 cyclin D1 pathway interactions, 13-14 MAX heterodimers, 11 phosphorylation, 88 target genes, 12-13

N Neurofilament-H, phosphorylation, 90-91 Neuron, mitogen-activated protein kinase pathway in differentiation, 97-98

0 OCRL, see Oculocerebrorenal syndrome Oculocerebrorenal syndrome (OCRL), gene mutations, 199 ODC, see Ornithine decarboxylase Ornithine decarboxylase (ODC), regulation by MYC, 12-13 Osmolarity response, see HOG pathway

P p21, gene activation by p53, 27 p2 1Ras-activated kinase (PAK), regulation of stress-activated protein kinases, 70-71 p38 mitogen-activated protein kinase apoptosis pathways, 74-75 inhibition studies, 85 nuclear translocation, 112 phosphorylation in activation, kinases, 66-67 signaling complexes, protein-protein interactions, 112 stress activators, 65 stress signaling, receptors and second messengers, 72-74 structure, 65-66 substrates cytoskeletal proteins, 90-91 nuclear transcription factors, 87-89 protein kinases, 85-87 signaling components, 89-90 table, 82-84 upstream regulators, 70-72 P53 apoptosis control mutation studies, 32-34 nontransactivator functions, 33-37 rescue factors, 38-39 transactivation targets, 30-33 cell cycle control GUS, 27 G2/M, 28 nontranscriptional control, 28-29 discovery, 25 functional domains, 26

Index

poly-proline region in nontranscriptional control, 28-29, 37 transcriptional transactivation, 26, 30-33 tumor progression apoptosis inhibition, 29-30, 37-39 genomic instability, 40-42 growth control, 39-40 PAK, see p21Ras-activated kinase Peroxisomal proliferator activated receptor y (PPAR-y),adipocyte differentiation role, 100 Pheromone response pathway Saccharom yces cerevisiae, 101-1 03 Schizosaccharomyces pombe, 108-1 09 Phosphatase, see Protein phosphatase Phosphatidylinositol (Ptdlns), structure, 167-1 6 8 Phosphatidylinositol3’ kinase stimulation of MKKERK pathway, 64-65 stress signaling, 74 Phosphatidylinositol4,5-bisphosphate (PtdInsP2) biosynthesis, 168, 170, 180 cancer role, 206-208 cytoskeleton binding proteins gelsolin, 188-1 89 profilin, 189-190 rho, 192-195 vinculin, 190-192 nuclear signaling, 186-187 transport proteins, 183-1 85 Phosphatidylinositol4-kinase cancer role, 206-208 compartmentation, 183-184 domains, 170-171,176-177 phosphorylation in activation, receptors, 180-183 protein-protein interactions in regulation, 181-1 82 purification, 173 types, 169-1 70 vesicle biogenesis and trafficking role, 195-106 Phosphatidylinositol4-phosphate (PtdInsP) biosynthesis, 168 cytoskeleton binding proteins gelsolin, 188-189 profilin, 189-190 rho, 192-195 vinculin, 190-1 92 transport proteins, 183-1 85 Phosphatidylinositol4-phosphate 5-kinase

22 1 cancer role, 206-208 compartmentation, 183-184 domains, 172-1 73 protein-protein interactions in regulation, 18 1-1 82 types, 169 vesicle biogenesis and trafficking role, 195-106 Phospholipase A2 (PLA2), phosphorylation of cytosolic enzyme, 89-90 Phospholipase C (PLC) cancer role, 206-208 compartmentation, 183-185 cross-talk between signaling pathways, 185-1 86 domains, 176-1 77 exocytosis role, 203-204 isozymes, 178 isozymes in yeast, 171, 173 nuclear signaling, 186-187 phosphorylation in activation, receptors, 174-175,178-180 protein-protein interactions, 179-1 80 recruitment, 176 second messenger generation, 177-178 Phospholipase D (PLD) exocytosis role, 204-206 Golgi vesicle formation, 197-198 PKC, see Protein kinase C PLAZ, see Phospholipase A2 PLC, see Phospholipase C PLD, see Phospholipase D Pleckstrin homology domain, phosphoinositide signaling role, 177, 201 Pleomorphic adenoma of parotid gland, chromosome 3 mutations, 157 PP1, mitogen-activated protein kinase pathway regulation, 80 PP2A, mitogen-activated protein kinase pathway regulation, 80-81 PPAR-y, see Peroxisomal proliferator activated receptor y Profilin functions, 189 phosphoinositide binding, 189-190 RAS signaling pathway role, 190 Protein kinase C (PKC) cell wall integrity pathway, 106-107 stimulation of MKKlERK pathway, 63 Protein phosphatase classification, 75, 77 mechanisms, 77

222 Protein phosphatase (cont.) mitogen-activated protein kinase pathway regulators dual specificity phosphatases, 78-80 protein tyrosine phosphatases, 80-81 serinehhreonine phosphatases, 80 types, table, 76-77 Protein sorting, phosphoinositide role, 199-201 Pseudohyphal growth, mitogen-activated protein kinase pathway in yeast, 103-1 04 PtdIns, see Phosphatidylinositol PtdInsP, see Phosphatidylinositol4-phosphate PtdInsP2, see Phosphatidylinositol4,5-bisphosphate Pyk2 calcium signaling in activation, 61 stress signaling, 74

R Rac phosphatidylinositol4-phosphate 5-kinase activation, 193-194 regulation of stress-activated protein kinases, 70 Raf mitogen-activated protein kinase kinase activation, 56, 91-92 regulation 8-Raf, 59-60 cell growth and transformation, 91-92 Raf-1 lipids, 59 oligomerization, 58-59 phosphorylation in activation, 57-58 recruitment, 56-57 RAS cell cycle progression role, 5-1 1 cell growth arrest, 39-40 downstream effectors, 7-9 ETS transcription factors as targets, 8-9 Rasl, pheromone response pathway in yeast, 108 RB, see Retinoblastoma protein Retinoblastoma protein (RB) E2F binding, 10,38 phosphorylation by cyclin-dependent kinases, 3, 10

Index Rho actin reorganization role, 192-195 phosphoinositide binding, 192-1 95 regulation of stress-activated protein kinases, 70-71 RSK phosphorylation, 85-86 substrates, 84-85

S SAPK, see Stress-activated protein kinase SH2, see Src homology domain 2 SH3, see Src homology domain 3 SHO1, HOG pathway role, 105 SLN1, HOG pathway role, 104-105 Sosl, phosphorylation, 89 Spcl, stress response pathway in Schizosaccharomyces pombe, 109-1 10 Sporulation, mitogen-activated protein kinase pathway in yeast, 107-108 SPS1, sporulation pathway in yeast, 107 Src homology domain 2 (SH2),phosphoinositide signaling role, 176, 179 Src homology domain 3 (SH3) p53 binding, 28-29 phosphoinositide signaling role, 176-177 SRC kinases, cell cycle progression role, 14-15 SSK2, HOG pathway role, 104 STAT, stimulation of MKKERK pathway, 64 STEl1, pheromone response pathway, 101-1 02 STE20, invasive/pseudohyphaI growth pathway, 103-104 Stress-activated protein kinase (SAPK) apoptosis pathways, 74-75 inhibition studies, 85 isoforms, 65 nuclear translocation, 112 phosphorylation in activation, kinases, 66-67 signaling complexes, protein-protein interactions, 111-1 12 stress activators, 65 stress signaling, receptors and second messengers, 72-74 structure, 65-66 substrates cytoskeletal proteins, 90-91

223

Index nuclear transcription factors, 87-89 protein kinases, 85-87 signaling components, 89-90 table, 82-84 upstream regulators Cdc42,70 germinal center kinase, 72 hematopoietic progenitor kinase 1, 72 p2lRas-activated kinases, 70-71 Rac, 70 Rho, 70-71 Synapsin I, phosphorylation, 91

Tpl-2, phosphorylation substrates, 68-69 Transforming growth factor p-activated kinase (TAKl), phosphorylation substrates, 69 Tumor chromosome 3 mutations, 141-142, 147-159 pS3 and apoptosis genomic instability, 40-42 growth control, 39-40 overview, 29-30,37-39

T

V

TAK1, see Transforming growth factor p-activated kinase Tau, phosphorylation, 90 T cell, mitogen-activated protein kinase pathway in differentiation, 98-99 TFIIH, helicase gene regulation by p53,35-37

VHL, see Von Hippel-Lindau gene Vinculin functions, 190-191 phosphoinositide binding, 190-192 Von Hippel-Lindau gene (VHL),mutation in cancer, 142

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