Rho and the Actin Cytoskeleton Alan Hall, et al. Science 279, 509 (1998); DOI: 10.1126/science.279.5350.509

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Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 1998 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS. FRONTIERS IN CELL BIOLOGY Rho GTPases and the Actin Cytoskeleton Alan Hall

The actin cytoskeleton mediates a variety of essential biological functions in all eukaryotic dynamic structure at the tip of the axon, cells. In addition to providing a structural framework around which cell shape and polarity consisting of filopodial and lamellipodial are defined, its dynamic properties provide the driving force for cells to move and to protrusions (see Fig. 2 and compare with divide. Understanding the biochemical mechanisms that control the organization of actin Fig. 1G) that respond to both positive and is thus a major goal of contemporary cell biology, with implications for health and disease. negative external guidance cues. Activation Members of the Rho family of small guanosine triphosphatases have emerged as key of Rac and Cdc42 in a neuroblastoma cell regulators of the actin cytoskeleton, and furthermore, through their interaction with line, N1E-115, has been shown to promote multiple target proteins, they ensure coordinated control of other cellular activities such the formation of lamellipodia and filopo- as gene transcription and adhesion. dia, respectively, along neurite extensions. More informatively, lamellipodia and filop- odia formation induced by a concentration gradient of an external agonist can be spe- The story begins back in the early 1990s present some of the recent evidence that cifically blocked by introducing dominant with the analysis of Rho, then a newly supports and extends this view. negative Rac or Cdc42 into these cells (5). described member of the of Activation of Rho in neuronal cells has small guanosine triphosphatases (GTPases). Not Just Fibroblasts been shown by a number of groups to in- In Swiss 3T3 fibroblasts, it was shown that duce neurite retraction and cell rounding, Rho can be activated by the addition of Although the effects of Rho GTPases on and although this appears strikingly differ- extracellular ligands [for example, lysophos- the organization of the actin cytoskeleton ent from what is seen in fibroblasts after phatidic acid] and that Rho activation leads are perhaps still best characterized in fibro- Rho activation, the underlying biochemical to the assembly of contractile actin-myosin blasts, there is now compelling evidence of cause seems to be the same: the Rho-depen- filaments (stress fibers) and of associated a similar role for these proteins in all eu- dent formation of contractile actin-myosin on May 7, 2008 focal adhesion complexes (Fig. 1, C and D) karyotic cells. Some of the most exciting filaments (5, 6). The difference is that fi- (1). It was concluded that Rho acts as a observations have been in neuronal cells, broblasts, unlike neuronal cells, can main- molecular switch to control a signal trans- where mechanisms of axonal growth and tain a flattened shape through the forma- duction pathway that links membrane re- guidance are being intensively studied. Ax- tion of strong focal adhesion attachment ceptors to the cytoskeleton. Rac, the next onal extension is driven by actin polymer- sites. It has been proposed that the opposing member of the Rho family to be analyzed, ization within the growth cone, a highly effects of Rac or Cdc42 and Rho might be a could be activated by a distinct set of ago- nists (for example, platelet-derived growth factor or insulin), leading to the assembly of www.sciencemag.org a meshwork of actin filaments at the cell periphery to produce lamellipodia and membrane ruffles (Fig. 1E) (2). More re- cently, activation of Cdc42, a third member of the Rho subfamily, was shown to induce actin-rich surface protrusions called filopo- dia (Fig. 1G) (3, 4). As with Rho, the cytoskeletal changes induced by Rac and Downloaded from Cdc42 are also associated with distinct, in- tegrin-based adhesion complexes (Fig. 1, F and H) (3). Moreover, there is significant cross-talk between GTPases of the Ras and Rho subfamilies: Ras can activate Rac (hence Ras induces lamellipodia), Cdc42 can activate Rac [hence filopodia are inti- mately associated with lamellipodia (Fig. 1G)], and Rac can activate Rho (although Fig. 1. Rho, Rac, and Cdc42 control the assembly and organization of the actin cytoskeleton. Quies- in fibroblasts, this is a weak and delayed cent, serum-starved Swiss 3T3 fibroblasts (-) contain very few organized actin filaments (A) or vinculin- response) (2, 3). These observations suggest containing integrin adhesion complexes (B). The effects of Rho, Rac, or Cdc42 activation in these cells that members of the Rho GTPase family are can be observed in several different ways such as with the addition of extracellular growth factors, key regulatory molecules that link surface microinjection of activated GTPases, or microinjection of guanosine diphosphate (GDP)–guanosine receptors to the organization of the actin triphosphate (GTP) exchange factors. Addition of the growth factor lysophosphatidic acid activates Rho, cytoskeleton. The aim of this article is to which leads to stress fiber (C) and focal adhesion formation (D). Microinjection of constitutively active Rac induces lamellipodia (E) and associated adhesion complexes (F). Microinjection of FGD1, an exchange factor for Cdc42, leads to formation of filopodia (G) and the associated adhesion complexes The author is at the Medical Research Council Laboratory (H). Cdc42 activates Rac; hence, filopodia are intimately associated with lamellipodia, as shown in (G). for Molecular Cell Biology, Cancer Research Campaign Oncogene and Signal Transduction Group, University In (A), (C), (E), and (G), actin filaments were visualized with rhodamine phalloidin; in (B), (D), (F), and (H), College London, Gower Street, London WC1E 6BT, UK. the adhesion complexes were visualized with an antibody to vinculin. Scale: 1 cm ϭ 25 ␮m. [Figure E-mail: [email protected] courtesy of Kate Nobes]

www.sciencemag.org ⅐ SCIENCE ⅐ VOL. 279 ⅐ 23 JANUARY 1998 509 general feature of these GTPases (5–7); in molecular structures appear to go hand in effects of GTPase activation in different the case of neurons, Rac and Cdc42 might hand (1). Further analysis has revealed that cell types may be difficult to predict. be under the control of chemoattractants, Rho activity is required to maintain focal Rho, Rac, and Cdc42 have been report- whereas Rho could be activated by che- adhesions in attached cells such that within ed to regulate the c-Jun NH2-terminal ki- morepellants, leading to either localized 15 min of inactivating cellular Rho, inte- nase (JNK) and the p38 mitogen-activated protrusion or retraction of the growth cone. grin clusters can no longer be seen at the protein (MAP) kinase cascades and thereby Activation of Cdc42 and Rac in macro- cell surface (12). It is not clear whether the regulate gene transcription in a more direct phages has similar effects on the actin cy- GTPase promotes assembly of the adhesion way than through their effects on adhesion toskeleton as it does in fibroblasts and neu- complex directly, by modification of one or complexes (17). Although these results rons, that is, it induces the formation of more of its constituents, or indirectly, have been largely obtained with overex- filopodial and lamellipodial protrusions (8). through cross-linking of actin filaments (to pressed proteins and transfected tissue cul- Moreover, filopodia and lamellipodia in- which many of the constituents bind). In ture cells, there is now compelling evidence duced by the macrophage chemoattractant any case, the results have important impli- from Drosophila genetics that regulation of colony-stimulating factor–1 (CSF-1) are cations. Integrin complexes are the source kinase pathways does in fact represent a blocked by dominant negative Cdc42 and of adhesion-dependent signals required for physiological function of these GTPases. Rac, respectively, although it has yet to be cell cycle progression and survival; because The ability of Rho GTPases to coordinately established whether CSF-1–induced che- their assembly is controlled by Rho, then so regulate the actin cytoskeleton and MAP motaxis is blocked by either or both. Acti- must be their signaling activity (13). It was kinase pathways is an emerging theme and vation of Rho, on the other hand, induces a somewhat more surprising to find that the will be discussed below. contractile actin-myosin filament network actin structures induced by Rac and Cdc42 Although the activation of MAP kinase but no focal adhesions; as a consequence, are associated with integrin adhesion com- pathways and the stimulation of integrin macrophage cells round up in a similar way plexes (Fig. 1, F and H) (3). These com- complex assembly offer ample opportunities to neuronal cells (7, 8). plexes contain many of the same constitu- for Rho, Rac, and Cdc42 to affect gene Distinctive effects of Rho, Rac, and ents as classical focal adhesions, but in the transcription, there is evidence that there Cdc42 activation on the organization of the case of those induced by Rac at least, they may be yet other mechanisms. In an appar- actin cytoskeleton have been observed in are morphologically quite distinct. The role ently JNK- and p38-independent manner, many other cell types, including epithelial of these integrin complexes is not clear— the GTPases have been reported to stimu- cells, endothelial cells, and astrocytes, as they do not seem to be required for the late transcription from the cyclin D pro- on May 7, 2008 well as in circulating cells such as lympho- formation of lamellipodia, but they may be moter and to activate the serum response cytes, mast cells, and platelets (9–11). The required for cell movement or perhaps for transcription factor (SRF) (18, 19). Rho specific response of different cell types can signaling (14). GTPases trigger progression of the G1 phase be modified by other parameters, in partic- Cadherin-based adherens junctions, found of the cell cycle when introduced into qui- ular, the cell’s ability to assemble integrin- between polarized epithelial cells for exam- escent fibroblasts, and their activities are based cell-matrix or cadherin-based cell- ple, are also intimately associated with the essential for serum-induced G1 progression cell adhesion complexes. This then leads to actin cytoskeleton, and a recent and excit- and for Ras-induced cell transformation another interesting chapter in the Rho- ing revelation has been that Rho (Fig. 3) (20, 21). The signals responsible for these

Rac-Cdc42 story, namely, their ability to and Rac are required for their assembly in effects are clearly of great interest, but what www.sciencemag.org regulate other cellular activities coordinate- keratinocytes (15). These observations raise are they? Activation of G1 progression by ly with actin. some interesting issues. First, components Rac correlates well with its ability to stim- of adherens junctions participate in signal ulate lamellipodia, but not its ability to Not Just Actin transduction pathways that affect gene regulate JNK, suggesting that actin fila- transcription; thus, Rho and Rac may influ- ments or integrin adhesion complexes The observation that both stress fibers and ence these pathways (16). Second, previous might be the source of a triggering signal focal adhesion complexes are assembled work has shown that activation of Rac con- (22, 23). Another suggestion that has been when Rho is activated in fibroblasts did not tributes to scattering of Madin-Darby ca- made is that reactive oxygen species (ROS) Downloaded from come as much of a surprise; the two macro- nine kidney epithelial cells treated with may be important. Rac is known to regulate hepatocyte growth factor, whereas in the the nicotinamide adenine dinucleotide keratinocyte experiment referred to above, phosphate (reduced) oxidase enzyme com- Rac promotes cell-cell adhesion (9, 15). plex in professional phagocytes to generate These apparently contradictory responses superoxide and ROS, but until recently it may well be explained by differences in the had been assumed that this was a highly activity or availability of cadherins or other specialized function of Rac in these cells. junctional components, suggesting that the There is now evidence to suggest that Rac-

Fig. 3. Rho is required for the establish- C3 injected Cadherin ment of cadherin-based adherens junc- tions. Primary human keratinocytes were plated and allowed to assemble cell-cell contacts over a period of 3 hours. A group Fig. 2. Filopodial and lamellipodial activity in neu- of cells was injected with an inhibitor of Rho ronal growth cones. A migrating growth cone at (C3) and visualized 30 min later with an the end of an axon isolated from a chicken dorsal injection marker (red) and an antibody to root ganglion was visualized with phase contrast E-cadherin (green). Scale: 1 cm ϭ 60 ␮m. microscopy. Scale: 1 cm ϭ 20 ␮m. [Figure cour- [Figure courtesy of Vania Braga] tesy of Dennis Bray]

510 SCIENCE ⅐ VOL. 279 ⅐ 23 JANUARY 1998 ⅐ www.sciencemag.org FRONTIERS IN CELL BIOLOGY:ARTICLES induced generation of ROS occurs in other metastatic or invasive phenotype of human few points of current interest (25). cell types (24). ROS have been implicated cancers. FGD1 was identified by positional The Ser-Thr kinase p160ROCK inter- in the activation of a variety of cellular cloning as the locus for the human genetic acts with Rho in a GTP-dependent manner, responses, including those involving the syndrome faciogenital dysplasia and later and when overexpressed or constitutively transcription factor nuclear factor kappa B, shown to be a GEF specific for Cdc42 (27). activated, it has been reported to mimic which might provide an essential signal for This disease is characterized by severe de- Rho. It would seem, then, that this is an G1 progression or cellular transformation. fects in skeletogenesis, suggesting an impor- excellent candidate for mediating Rho-in- Characterization of the underlying bio- tant developmental role for Cdc42 in bone duced changes to the actin cytoskeleton chemical pathways of ROS generation in morphogenesis. (30). Moreover, two substrates of this ki- nonphagocytic cells is needed to sort out The mechanisms by which GEFs are ac- nase, myosin light chain phosphatase and this potentially interesting story. tivated by membrane receptors are still far myosin light chain, are known to regulate from clear. Exchange activity is encoded the assembly of actin-myosin filament bun- Fishing Upstream and within their DH domain, but it is notable dles, and recent work has shown that Rho- Downstream that in all GEFs this is immediately fol- induced stress fiber assembly occurs primar- lowed by a pleckstrin homology (PH) do- ily through bundling of preexisting fila- To drive processes such as directed cell main. It is thought that the PH domain ments rather than de novo actin polymer- movement and axonal extension, the ac- plays a crucial role in membrane localiza- ization (14, 31). Whether p160ROCK is tivity of Rho GTPases must be restricted to tion by interacting with specific lipids, and the only downstream target of Rho required discrete intracellular locations specified it is known that the generation of phospha- to induce stress fibers remains to be seen. ultimately by extracellular cues. The key tidylinositol-3,4,5-trisphosphate (PIP3), by Assembly of stress fibers is blocked by cy- to understanding this aspect of GTPase phosphatidlyinositol 3-kinase (PI 3-kinase) tochalasin D, suggesting that some actin function is likely to lie in their upstream activity, is essential for receptor-mediated polymerization might be required, but work regulation. activation of Rac in mammalian cells, and from our own laboratory suggests that the Upstream activators. About 10 GTPase- thataPI3-kinase homolog, TOR2, controls actin-myosin filaments induced by this ki- activating proteins and three guanine nu- Rho1p activation in Saccharomyces cerevi- nase are not correctly organized nor are cleotide dissociation inhibitors, both poten- siae(28). A major problem in this field has they contractile as they are when induced tial down-regulators of GTPase activity, been the lack of reliable reagents to mea- by Rho (1, 32). have been described, but little is known sure the concentrations and intracellu- Although not direct targets of Rho, the on May 7, 2008 about their mode of action (25). The 15 lar locations of the active forms of the ERM proteins (ezrin, radixin, and moesin) guanine nucleotide exchange factors (GEFs) GTPases. An exciting possibility is that are emerging as key regulators of the actin (the Dbl or DH family) described to date target proteins (see the next section) could cytoskeleton. In vitro binding assays have have attracted more attention, in part be- be used to recognize the GTP-bound forms revealed that their interaction (through cause many were originally identified as of Rho, Rac, or Cdc42 specifically. Another their NH2-termini) with a transmembrane potent oncogenes capable of transforming problem is that, unlike Ras, which is con- protein, CD44, can be regulated by Rho, NIH 3T3 cells to a malignant phenotype stitutively in the membrane, Rho GTPases and their COOH-terminal ends interact (for example, Dbl, Vav, and Lbc) (25). are thought to be at least partially cytosolic with filamentous actin (F-actin) (33). Fur-

There is little doubt that the oncogenic (associated with a guanine nucleotide dis- thermore, with a permeabilized cell recon- www.sciencemag.org activity of Dbl-related GEFs is mediated sociation inhibitor) and therefore must stitution assay, it has been shown that ERM through activation of Rho GTPases, but translocate to the plasma membrane (where proteins are essential for both Rho- and whether subsequent changes to the actin they would presumably meet a GEF). How Rac-induced cytoskeletal effects (34). A cytoskeleton play a role is not clear. Inter- they do so is unknown. reasonable interpretation of these experi- estingly, the constitutively activated ver- Finally, Rho and Cdc42 are required ments is that ERM proteins behave as regu- sions of Rho, Rac, and Cdc42 are at best late in the cell cycle for formation of the latable scaffold proteins that anchor actin only weak oncogenes; one explanation for actin-myosin contractile ring (29). It is filaments to the membrane and that this is this apparent contradiction is that perhaps not known whether the GTPases act an essential prerequisite for Rho and Rac Downloaded from the GTPases must cycle between GTP- and through similar biochemical pathways (acting through target proteins) to induce GDP-bound states to exert optimal onco- during G1 and cytokinesis; however, the stress fibers and lamellipodia, respectively genic effects. Another explanation is that upstream regulation of GTPases at the end (Fig. 4). the GEFs play additional roles in signaling, of mitosis must be tied in to the cell cycle Mutational analysis suggests that the in- perhaps by promoting the formation of a machinery rather than to extracellular duction of actin polymerization and of JNK larger molecular complex. So far there signals. activity are mediated by bifurcating path- have been no reports of genetic alterations Downstream targets. To understand the ways triggered by the interaction of Rac directly affecting DH proteins or Rho biochemical mechanisms through which with two distinct target proteins (22, 23). A GTPases in human cancer. Rho GTPases regulate the organization of similar conclusion has been reached for Two members of the DH family deserve the actin cytoskeleton and other associated Cdc42 (22). Although a dozen or so target further comment. The gene encoding activities, there has been an enormous ef- proteins have been identified for Rac and Tiam-1 was originally identified as being fort to identify cellular targets (effectors). Cdc42, one of these, the Ser-Thr kinase capable of conferring an invasive pheno- Yeast two-hybrid selection and affinity pu- p65PAK, has received much of the attention type when introduced into a noninvasive rification have proved to be powerful tech- to date. Its kinase domain is most closely lymphoma cell line. Tiam-1 is now known niques, and at least 20 candidate targets related to yeast Ste20p, a known regulator to act as a Rac-specific GEF, and indeed have been identified so far that represent a of MAP kinase pathways, suggesting that it Rac itself will also induce an invasive phe- wide variety of enzymatic activities and pro- might mediate activation of JNK by Rac notype in these cells (26). These observa- tein-protein interaction domains. The re- and Cdc42. Although some groups have tions have raised the possibility that dereg- search has been reviewed elsewhere, and provided evidence in support of this, others ulated Rac activity may contribute to the the following discussion will focus on just a have failed to find a link, and it is still not

www.sciencemag.org ⅐ SCIENCE ⅐ VOL. 279 ⅐ 23 JANUARY 1998 511 resolved whether p65PAK is a physiological define the biochemical pathways regulated JNK activity is also required for the mor- regulator of JNK (18, 35). by the Rho GTPases, and in this, as in other phogenesis of a variety of other epidermal A role for p65PAK in Rac-induced actin problems of signal transduction, the genetic cell types. In the Drosophila eye, for exam- polymerization has also been proposed, al- analysis of simpler eukaryotes is playing an ple, the development of cell polarity is un- though on the face of it, the data seem increasingly important role. der the control of the frizzled (fz) receptor; contradictory. Three groups reported that in this case there is evidence that Rho acts Rac mutants that do not interact with The Power of Genetics downstream of fz to mediate JNK activation p65PAK still induce lamellipodia, suggesting (40). These results confirm some of the that the kinase is not involved, whereas The genetic analysis of developmental observations made in tissue culture cells another group reported that a kinase-dead pathways in Drosophila and Caenorhabditis and demonstrate the importance of Rho mutant of p65PAK mimics Rac and induces elegans is rapidly making major contribu- GTPases in coordinating actin changes lamellipodia, suggesting that p65PAK is in- tions to our insight into the physiological with the regulation of MAP kinase volved (18, 22, 23, 36). Clearly both can- role of Rho, Rac, and Cdc42. The area is pathways. not be true. Closer examination of the data already too large to be covered here, so just Drosophila RhoL may be the exception supports a conclusion that the interaction a few examples will be given. During em- that proves this rule. This novel member of of Rac with p65PAK is neither the trigger bryonic development, cells undergo a vari- the Rho GTPase family lacks a tyrosine nor is it required for actin polymerization, ety of changes in their shape and polarity residue at codon 40 (conserved in all other but p65PAK can interact with a molecular and some migrate to new sites within the family members) and does not activate JNK complex that does control actin polymer- embryo in response to specific cues. An (41, 42). Nevertheless, RhoL is required ization. Whether p65PAK is essential for emerging theme underlying these morpho- during oogenesis for the morphogenetic actin polymerization remains to be seen. genetic processes is that they often require changes in the follicular cells that surround The idea that GTPase-induced effects are coordinated changes in gene transcription the oocyte (41). Perhaps in this case, MAP mediated by multimolecular complexes and and in the organization of the actin cy- kinase activity is not required to act in not by linear pathways of biochemical cas- toskeleton. One of the clearest examples of concert with changes in the actin cytoskel- cades should not be so surprising because it this is dorsal closure, where two symmetri- eton. No mammalian homolog of RhoL has has clearly been established for Cdc42 in cal sheets of epithelial cells elongate and yet been reported. yeast (see below). migrate over the embryo, eventually to fuse Genetic analysis has demonstrated the Another target of Rac that may play a at the midline. A driving force for this importance of Rho GTPases in directed cell on May 7, 2008 major part in actin polymerization is phos- morphogenetic movement is a change in movement. Inactivation of Rac (but not phatidylinositol-4-phosphate 5-kinase (PIP the actin cytoskeleton at the leading edge Cdc42 or RhoL) in the Drosophila ovary, for 5-kinase), the enzyme that converts PIP of the migrating cells, but in addition, ac- example, prevents the migration of border to phosphatidylinositol-4,5-bisphosphate tivation of the JNK cascade is essential. cells during oogenesis from the anterior tip,

(PIP2). In platelets, Rac can stimulate Inactivation of Rac in the Drosophila em- through the nurse cells, to the oocyte (41). PIP2 formation, leading to barbed-end un- bryo disrupts both actin changes and JNK Interestingly, the transcription factor C/EBP capping and severing of actin filaments activation and blocks dorsal closure (39). and the Drosophila fibroblast growth factor (11). This provides a bolus of nucleation sites for actin monomer addition, resulting www.sciencemag.org in rapid actin polymerization and lamelli- podium formation. There is a growing list of actin-associated proteins (for example, gelsolin, vinculin, and ERMs) that inter- Actin Lamellipodia act with PIP2; the analysis of the enzymes that control the synthesis of this lipid polymerization should provide important insights into the Filament Stress mechanisms of F-actin assembly. bunding Fibers Downloaded from The product of the human Wiskott Al - - Actin Filopodia drich syndrome gene, WASP, has been polymerization identified as a Cdc42-specific target (37). Although it has no catalytic activity, the presence of numerous protein-protein inter- action domains has generated much specu- lation as to its likely function. Mutational analysis has revealed that the interaction of Cdc42 with WASP is not the trigger for filopodia formation; however, it remains to be seen whether WASP plays an active role in F-actin assembly, or whether it interacts Fig. 4. ERM proteins are required for GTPase-mediated cytoskeletal changes. It has been proposed with assembled F-actin and contributes to that ERM proteins exist in a closed (inactive) conformation and an open (active) conformation. There is other Cdc42-induced effects such as gene evidence to suggest that this transition can be regulated by Rho, perhaps through the activation of a Ser-Thr kinase or a lipid kinase (through PIP ). In the active conformation, the NH -terminus of ERM transcription (22, 37). Support for the first 2 2 proteins (pink) can interact with transmembrane proteins, such as CD44, whereas the COOH-terminus suggestion has come from yeast where (blue) interacts with F-actin. This membrane–ERM–F-actin unit is an essential prerequisite for the Rho Bee1p, a WASP-related protein, has been GTPases to induce cytoskeletal changes. In the case of Rho, this is likely to be mediated by the bundling shown to be essential for actin polymeriza- and reorganization of preexisting actin-myosin filaments to generate stress fibers, whereas in the case tion (38). of Rac and Cdc42 it is likely that preexisting filaments are uncapped to allow actin polymerization and There is clearly a long way to go to filament growth leading to the formation of lamellipodia and filopodia.

512 SCIENCE ⅐ VOL. 279 ⅐ 23 JANUARY 1998 ⅐ www.sciencemag.org FRONTIERS IN CELL BIOLOGY:ARTICLES receptor are also required for border cell nately regulate multiple pathways. For activity and the localization of this com- migration, but whether Rac is involved in example, three specific targets for Rho1p plex. Furthermore, GTPases and their tar- this pathway is not known. Mutations in have been identified to date: Bni1p (which gets can assemble into different multimo- the mig-2 locus in C. elegans lead to migra- affects actin assembly), Pkc1p (an upstream lecular complexes and thereby participate tory defects in a variety of cell types, includ- regulator of a MAP kinase pathway required in different cellular processes. For exam- ing neurons, mesodermal cells, and sex for cell wall biosynthesis), and glucan syn- ple, Cdc42p and Ste20p are required in myoblasts (43). Mig-2 encodes another thase (required for cell wall synthesis) (44). both the pheromone-induced activation of member of the Rho GTPase family, having Two targets of mammalian Rho, PKN and a MAP kinase cascade (leading to cell ϳ60% amino acid identity to C. elegans mDia, are related to Pkc1p and Bni1p, re- cycle arrest) and in the starvation-induced Rac and Cdc42. Whether there is a mam- spectively, and it seems likely that the co- activation of a different MAP kinase path- malian homolog is not known; however, ordinated regulation of the actin cytoskele- way (leading to filamentous growth) (Fig. the exciting observation that a constitu- ton and of MAP kinase pathways by Rho 5). Interestingly, the interaction of tively activated mig-2 causes defects in ax- GTPases is conserved in all eukaryotic spe- Cdc42p with Ste20p is not required for onal guidance, but not axon outgrowth, cies (45). Although there is no mammalian the pheromone response, but it is required seems likely to encourage a search for addi- analog of glucan synthase, it is interesting for the starvation response (48). It re- tional members of the Rho GTPase family to note that Rho has been shown to control mains to be seen whether multimolecular in mammalian cells. the assembly of extracellular matrix fibers complexes are used universally and in to create a microenvironment surrounding higher eukaryotes in GTPase signaling Paradigms from Yeast mammalian cells—perhaps this is somehow pathways. analogous to the cell wall function of A final message to emerge from the anal- Turning to yeast, it becomes clear that the Rho1p in yeast (46). ysis of yeast is that GTPase pathways are biochemical complexity of GTPase signal- A second take-home message is that often linked in a hierarchical fashion. Thus, ing pathways is only just beginning to be components of GTPase-mediated path- Cdc42p, which is required for the assembly appreciated, and any notion that pathways ways assemble into multimolecular com- of components at the bud site during cell are linear, with occasional points of inter- plexes held together by scaffold proteins, a division, acts downstream of another action, is unrealistic. Much progress in un- good example of which is Bem1p. This GTPase, Rsr1p (closest mammalian ho- derstanding the role of Rho1p and Cdc42p Src-homology 3 (SH3) domain–contain- molog Rap1), which is required for localiza- (two of the five Rho proteins) in S. cerevi- ing protein interacts with Cdc24p (a GEF tion of the bud site (49). In fact Rsr1p on May 7, 2008 siae has been made and details can be found for Cdc42p), Rsr1p (an upstream GTPase interacts directly with Cdc24p, a GEF for elsewhere; however, some important gener- that also interacts with Cdc24p), Ste20p Cdc42p, and thereby ensures that compo- al lessons about the of GTPase sig- (a target for Cdc42p), actin, and Ste5p nents of the bud are assembled only at the naling have emerged. First, and in agree- (another scaffold protein that is an essen- bud site. Rho appears to act later in this ment with observations made in mammali- tial component of the MAP kinase path- pathway to promote growth of the bud, but an cells, both Rho1p and Cdc42p coordi- way) (Fig. 5) (47). Cdc42p can affect the how its activation is coordinated with Cdc42 is not clear (50).

Conclusions www.sciencemag.org

Rho GTPases act as molecular switches. In response to extracellular signals, they in- duce coordinated changes in the organiza- tion of the actin cytoskeleton and in gene transcription to drive a large variety of bi- ological responses including morphogenesis, chemotaxis, axonal guidance, and cell cycle Downloaded from progression. It can be predicted with some confidence that the biochemical and ge- netic analysis of the signaling pathways controlled by Rho GTPases will lead to a better understanding of these fundamental processes.

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