Oncogene (1998) 17, 1415 ± 1438  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Rho family proteins and Ras transformation: the RHOad less traveled gets congested

Irene M Zohn1, Sharon L Campbell2,3, Roya Khosravi-Far4, Kent L Rossman2 and Channing J Der1,3,5

1Department of Pharmacology, 2Department of Biochemistry and Biophysics, 3Lineberger Comprehensive Cancer Center, 4Department of Biology, MIT, Cambridge, Massachusetts 02139; 5Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, North Carolina 27599, USA

The Rho family of small GTPases has attracted diverse downstream e€ectors to cause its diverse considerable research interest over the past 5 years. actions on cell proliferation, di€erentiation and During this time, we have witnessed a remarkable apoptosis. Among these Raf-independent signaling increase in our knowledge of the biochemistry and pathways are those that connect Ras with speci®c biology of these Ras-related proteins. Thus, Rho family members of the Rho family of small GTPases. Since proteins have begun to rival, if not overshadow, interest Rho family proteins are regulators of actin organiza- in their more celebrated cousins, the Ras oncogene tion, gene expression, and cell cycle progression, it is proteins. The fascination in Rho family proteins is fueled likely that Rho family proteins will contribute primarily by two major observations. First, like Ras, signi®cantly to the actions of oncogenic Ras. The Rho family proteins serve as guanine nucleotide- Rho family of proteins have been the subject of a regulated binary switches that control signaling pathways number of excellent reviews (Symons, 1996; Narumiya, that in turn regulate diverse cellular processes. Rho 1996; Van Aelst and D'Souza-Schorey, 1997). There- family proteins are key components in cellular processes fore, in this review we will emphasize the involvement that control the organization of the actin cytoskeleton, of Rho family proteins in the regulation of cell activate kinase cascades, regulate gene expression, proliferation and Ras transformation. Despite our regulate membrane tracking, promote growth trans- wealth of knowledge on these small GTPases, we are formation and induce apoptosis. Second, at least ®ve Rho clearly in the early days of comprehending the complex family proteins have been implicated as critical involvement of these small GTPases in regulating regulators of oncogenic Ras transformation. Thus, it is normal and neoplastic cell biology. More excitement suspected that Rho family proteins contribute signi®- is certain to follow as we continue to develop and cantly to the aberrant growth properties of Ras- revise, or discard, our current models and concepts. transformed cells. Rho family proteins are also critical mediators of the transforming actions of other trans- forming proteins and include Dbl family oncogene Rho family proteins are members of the Ras superfamily proteins, G protein-coupled receptors and G protein a of small GTPases subunits. Thus, Rho family proteins may be key components for the transforming actions of diverse The Ras superfamily of small (20 ± 25 kDa) GTPases oncogene proteins. Major aims of Rho family protein (480 mammalian members) can be categorized into at studies are to de®ne the molecular mechanism by which least nine distinct branches. These include the Ras, Rho family proteins regulate such a diverse spectrum of Rab, Rho, Ran, Rheb, Rad/Gem, Rin/Rit and Arf cellular behavior. These e€orts may reveal novel targets families. Rho family proteins constitute one of the three for the development of anti-Ras and anti-cancer drugs. major branches of the Ras superfamily and its members share approximately 30% amino acid identity with the Keywords: Ras superfamily; signal transduction; actin four Ras proteins (Chardin, 1993). Presently, at least 14 cytoskeleton; e€ectors mammalian Rho family proteins have been identi®ed: RhoA, RhoB, RhoC, RhoD, RhoE/Rnd3, Rnd1/Rho6, Rnd2/Rho7, RhoG, Rac1, Rac2, Rac3, Cdc42, TC10 and TTF that share signi®cant (ranging from 50 ± 90%) An important theme that has emerged during the past amino acid identity with each other (Ridley, 1996) 5 years is that Ras transformation is mediated by (Figure 1a). Two mammalian isoforms of Cdc42 have signaling activities that are much more complex than been identi®ed from brain (B) or placenta (P). originally envisioned (Marshall, 1996; Khosravi-Far et Much of our knowledge of Rho family protein al., 1997; Campbell et al., 1998). Considerable function has been derived primarily from the studies biological, biochemical and genetic evidence support of Rac1, RhoA and Cdc42 and each exhibits distinct the importance of the Raf serine/threonine kinase a key cellular functions. A recent report indicates that RhoD e€ector of Ras function. However, the evidence that possesses functions distinct from these three proteins Ras has a life beyond simply activating Raf is strong (Murphy et al., 1996). Sequence comparisons suggest and continues to mount. Instead, our current model that RhoE and TTF may also share functional proposes that Ras employs a spectrum of functionally relationships, whereas TC10 and RhoG are anticipated to be most related in function to Cdc42 and Rac1, respectively (Figure 1b). Recent observations that Rho/ Correspondence: CJ Der Rnd3 or Rnd1 causes a disruption of stress ®bers Rho family of small GTPases and transformation IZohnet al 1416 support a distinct function for RhoE and related members, are likely to occur in the future. Finally, proteins (Nobes et al., 1998; Guasch et al., 1998). although RhoA, RhoB and RhoC are likely to share Furthermore, RhoE and related proteins possess common functions in regulating stress ®ber formation substitutions at positions corresponding to Ras (Ridley and Hall, 1992), they di€er in subcellular residues 12, 13, 59, 61 ± 63 and 66 ± 67 that in¯uence location (Adamson et al., 1992), regulation of expres- both intrinsic and GAP-mediated GTP hydrolysis in sion (Jahner and Hunter, 1991; Fritz et al., 1995) and Ras, and consequently, they exist in a predominantly posttranslational lipid modi®cation (Adamson et al., GTP-bound state. Thus, sequence and functional 1992; Lebowitz et al., 1995). Thus, even closely related di€erences allow the de®nition of at least six distinct members are likely to exhibit unique roles as well. Rho subfamilies: (1) Cdc42 and TC10; (2) Rac1, Rac2, Like Ras, Rho family proteins function as GTP/ Rac3 and RhoG; (3) RhoA, RhoB and RhoC; (4) GDP regulated switches that cycle between active RhoE, Rnd1/Rho6 and Rnd2/Rho7; and (5) RhoD and GTP- and an inactive GDP-bound forms (Figure 2) TTF. However, a preliminary report that a dominant (Ridley, 1996). This cycle is regulated by three distinct negative mutant of RhoG did not impair formation of classes of regulatory proteins. First, Rho guanine membrane ru‚es, ®lopodia or stress ®bers suggests that nucleotide exchange factors (GEFs; also referred to RhoG function may be distinct from both Cdc42 and as Dbl homology proteins) serve as activators and Rac (Roux et al., 1997). Thus, further subdivision of the stimulate the replacement of GDP by GTP (Whitehead Rho family, as well as the identi®cation of additional et al., 1997). To date, over 20 Dbl homology proteins

a Rho family of small GTPases and transformation IZohnet al 1417 have been identi®ed, and the majority were initially Several regulators of Rho GDP/GTP cycling appear discovered as transforming proteins in NIH3T3 focus- to function as multifunctional regulators. For example, formation assays (e.g., Dbl, Vav) (Figure 3). Second, at BCR and ABR each possess distinct catalytic domains least 16 Rho GTPase activating proteins (GAPs) have that serve as both Rho GEFs and GAPs (Chuang et also been identi®ed that serve as negative regulators of al., 1995) (Figure 3). In addition, some Rho GAPs may Rho family protein function by stimulating their also serve as downstream e€ectors (e.g., N-chimaerin intrinsic GTPase activities (Cerione and Zheng, 1996). and the p85 subunit of phosphoinositide-3-OH kinase Some GAPs show preferential stimulation of speci®c (PI3K)) (Kozma et al., 1996; Zheng et al., 1994a). Rho family proteins. Although RasGRFs and SOS have been shown to A third class of Rho regulators are the Rho GDP- function as Ras GEFs, the presence of tandem DH/PH dissociation inhibitory factors (RhoGDIs). The ®rst domains in these molecules suggests that they may RhoGDI was originally identi®ed as an inhibitor of possess a Rho family GEF function which is distinct Rac GDP dissociation (Ueda et al., 1990) and from their Ras GEF function (Nimnual et al., 1998; subsequently, of Cdc42 and Rac as well (Abo et al., Fan et al., 1998). Finally, one Dbl family protein (Trio) 1991; Leonard et al., 1992). This ubiquitously possesses two distinct DH domains and each shows expressed, cytosolic protein, was later shown to distinct GEF activities. interfere with both intrinsic and GAP-stimulated Like Ras, Rho family GTPases share high sequence GTP hydrolysis of Rac and Cdc42 (Chuang et al., similarity, especially in the core GTP binding 1993; Hart et al., 1992). Thus, RhoGDI can perturb sequences, and diverge primarily at their COOH- Rho GDP/GTP cycling via two distinct mechanisms. termini. The x-ray and NMR structures of truncated Finally, RhoGDIs also serve an important role in forms of Rac1-GMPPNP, Cdc42-GDP, G14V RhoA- regulating the association of Rho family proteins with GDP and RhoA-GTPgS have recently been solved membranes (Takai et al., 1995). A cytosolic complex (Hirshberg et al., 1997; Feltham et al., 1997; Ihara et with RhoGDI is disrupted during Rho membrane al., 1998; Rittinger et al., 1997; Wei et al., 1997). Like translocation and activation by GEFs. Two additional Ras these Rho family GTPases share a common a/b RhoGDIs include the hematopoietic cell speci®c D4- fold with the core GTP-binding domain as a GDI/LY-GDI (Lelias et al., 1993; Scherle and Staudt, conserved structural unit. In contrast to Ras, the 1993) and RhoGDIg/RhoGDI-3, which is preferen- Rho subfamily contains an insert of 13 amino acids in tially expressed in brain, pancreas and other tissues a region between b5 and a4 of H-Ras. One exception, (Adra et al., 1997; Zalcman et al., 1996). however, is TTF, which contains a smaller insert b

Figure 1 Sequence comparison of Rho family proteins. (a)To date, at least 14 distinct mammalian Rho family proteins have been described. A multiple sequence alignment of human Rho family proteins and H-Ras was generated using the dynamic algorithm alignment program ClustalW (Thompson et al., 1994). A pro®le alignment of the Rac1 primary sequence containing Rac1 secondary structural elements and the remaining Rho family proteins was initially constructed. The primary sequence of H-Ras was then added before the ®nal multiple sequence alignment was produced. All alignments were carried out using PAM series protein weight matrices. The ClustalW multiple sequence alignment was shaded using Boxshade 3.2 (Thompson et al., 1994). Residues that are conserved in greater than 50% of sequences at a given sequence position are colored blue, while residues that are similar are colored yellow. (b) The sequence alignment described above was used to construct the Rho dendrogram. The branch lengths in the dendrogram are proportional to the estimated divergence along each branch. Rnd3/Rho8 di€ers from RhoE only in having an additional 15 amino acid NH2-terminal extension (Nobes et al., 1998) Rho family of small GTPases and transformation IZohnet al 1418 consisting of 7 amino acids. In Rac1 and RhoA, the forms contacts with the loop between b4 and a3. It is insert consists of two alpha helices followed by an not clear whether these di€erences represent differ- extended loop. The insert represents a highly charged ences between X-ray and NMR methods or whether surface, is mobile and exposed. However, Cdc42 the fact that the structure of Rac1 was solved on a contains only one helix in the insert. Moreover, the F87S mutant that may alter the structure and contacts insert appears to form a compact loop structure that between the insert and the b4/a3 loop. NMR studies indicate that the conformation of the insert is not sensitive to the binding of GTP versus GDP. Like Ras, the conformation of the switch I and switch II domains is sensitive to the binding of GTP versus GDP and both appear to be dynamic structures. However, in contrast to Rac1, helix two in the switch II domain of Cdc42 is absent. Interestingly, NMR chemical shift mapping of Cdc42-GDP and Cdc42- GMPPCP suggest the existence of an additional switch region comprised of b4/a3anda3 (Feltham et al., 1997).

Rho family proteins are regulators of diverse cellular processes

Rho family proteins have been implicated in the regulation of a diverse and extensive spectrum of cellular processes. Most prominent among these are Figure 2 Regulators of Rho family protein GDP/GTP cycling. their e€ects on the organization of the actin Like Ras, Rho family proteins function as GDP/GTP-regulated binary switches. A variety of extracellular stimuli regulate Rho cytoskeleton. Rho proteins also initiate signaling family protein GDP/GTP cycling, through modulation of Rho cascades that cause activation of a variety of GEF, GAP or GDI function. Activated Rho family proteins transcription factors. Hence, Rho-mediated changes exhibit diverse cellular functions that include regulation of actin in the actin organization or in gene expression may organization, gene expression and cell cycle progression regulate many of the cellular processes associated with Rho protein function. These include regulation of cell shape, cell attachment, cell motility and invasion, cell- cell interactions, cell proliferation, di€erentiation and apoptosis. Below, we summarize our current knowl- edge regarding the involvement of Rho proteins in controlling speci®c cellular processes.

Rho proteins are regulators of actin cytoskeletal organization The best characterized function of Rho family proteins involves their regulation of speci®c ®lamentous F-actin organization. Actin ®laments are components of one of the three major cytoskeletal protein networks that determine cell shape, movement and regulate cellular processes: actin ®laments, microtubules and intermedi- ate ®laments. The actin cytoskeleton is a highly dynamic cytoplasmic structure that is reshaped and reformed in response to diverse extracellular signals. In ®broblasts, polymerized actin is assembled into a variety of distinct structures. Lamellipodia are curtain- like extensions that consist of thin protrusive actin sheets. Membrane ru‚es represent lamellipodia that have lifted from the substratum at the leading edge of Figure 3 Dbl family proteins are guanine nucleotide exchange cells. Actin stress ®bers consist of actin bundles that factors and activators of Rho family proteins. Presently, at least 22 mammalian Dbl family proteins have been described (reviewed traverse the cell and promote cell attachment to the in Whitehead et al., 1997); Fam et al., 1997; Schuebel et al., 1996; extracellular matrix via focal adhesions. Focal adhe- Henske et al., 1995; Gebbink et al., 1997; Alam et al., 1997). All sions consist of integrins and cytoplasmic proteins such share a Dbl homology (DH) followed by a pleckstrin homology as vinculin and talin. Filopodia are thin, ®nger-like (PH) domain. Beyond the tamdem DH/PH domains, each Dbl family protein possesses distinct catalytic (Ras GEF, Rho GAP) cytoplasmic extensions that contain tight actin bundles or protein-protein or protein-lipid interaction motifs [Src and may be involved in the recognition of the homology 2 and 3 (SH2 and SH3), cysteine-rich zinc-binding extracellular environment (Nobes and Hall, 1995). domains (CRDs)]. A Rho GEF activity has been de®ned for Speci®c Rho family proteins are regulators of distinct most, but not all, members of this family. Whereas some exhibit changes in these actin-based structures, as well as very speci®c activity, others serve as activators of multiple Rho family proteins. For example, FGD1 is an activator of Cdc42, others (e.g., tight junctions in polarized epithelial cells) while Vav can activate Rac1, RhoA and Cdc42 (Nusrat et al., 1995), in ®broblasts and other cell types. Rho family of small GTPases and transformation IZohnet al 1419 The ®rst clues that Rho family proteins regulate Rho family proteins have also been described in the actin organization were provided by studies using C3 yeasts S. cerevisiae and S. pombe (reviewed in Chant toxin from Clostridium botulinum, an inhibitor of and Stowers, 1995). RhoA, B and C function (Chardin et al., 1989) and Although a Cdc424Rac4Rho cascade has also by microinjection analyses using mutant Rho family been seen in other cell types (Allen et al., 1997), proteins (Paterson et al., 1990; Ridley et al., 1992; variations on this theme, involving separate pathways Ridley and Hall, 1992; Nobes and Hall, 1995). These or feedback loops, have also been observed. For studies demonstrated that speci®c Rho family proteins example, Cdc42 was shown to inhibit, rather than are components of signaling pathways that induce promote, stress ®ber formation in other cell types unique morphological changes involving rearrange- (Kozma et al., 1995; Qiu et al., 1997). Activated Rac ments of F-actin. RhoA, Rac1 and Cdc42 control did not induce actin stress ®ber formation in MDCK distinct changes in the actin cytoskeleton and distinct epithelial cells (Ridley et al., 1995). Inactivation of Rho cellular structures. Furthermore, they can act in caused activation of Cdc42 and Rac in N1E-115 concert in cascades that link their activities. Constitu- neuroblastoma cells (Kozma et al., 1997). These tively activated Cdc42 caused induction of ®lopodia in observations, when taken together with the fact that Swiss 3T3 ®broblasts as well as the activation of Rac each Rho family can be activated by distinct signals, (Nobes and Hall, 1995; Kozma et al., 1995). Activated re¯ect the versatility of Rho family proteins in Rac1 in turn caused the induction of lamellipodia and orchestrating di€erent cellular processes in di€erent membrane ru‚ing, and the activation of Rho (Ridley situations and cell types. et al., 1992). Activated RhoA caused the formation of Finally, the precise links between these GTPase stress ®bers and focal adhesions (Ridley and Hall, cascades have not been established. However, based on 1992). Both Rac and Cdc42 also regulate formation of the involvement of Dbl family-related proteins (CDC24) focal complexes distinct from those caused by RhoA. in the activation of Cdc42 in yeast pathways (Zheng et A second cascade of small GTPases involves the ability al., 1994b), it is logical that Dbl family proteins will of oncogenic Ras to activate Rac and subsequently serve as intermediates between GTPases. GAPs and Rho (Ridley et al., 1992). GTPase cascades that involve GDIs may also facilitate these GTPase cascades.

Figure 4 Rho family proteins are regulators of signaling pathways that regulate the organization of the actin cytoskeleton. Cdc42, Rac1 and RhoA function as downstream components of signaling pathways initiated by ligand-stimulated G protein-coupled serpentine receptors (SRs), receptor tyrosine kinases (RTKs) or integrin receptors (IRs). Cdc42, Rac1 and RhoA each modulate distinct changes in actin organization. At least two GTPase cascades have been identi®ed that regulate actin reorganization. One involves oncogenic Ras activation of Rac1, then Rac1 activation of RhoA. A second involves Cdc42 activation of Rac1 and RhoA. These hierarchies of small GTPases may vary in di€erent cell types Rho family of small GTPases and transformation IZohnet al 1420 Like Ras, Rho family proteins also serve as GDP/ membrane ru‚ing (Kotani et al., 1994; Wennstrom et GTP-regulated relay switches that transmit extra- al., 1994; Nobes and Hall, 1995). cellular ligand-mediated signals that promote changes In general, speci®c Rho family proteins show the in actin structures (Figure 4). It had been observed same e€ects on actin organization in di€erent cell types. previously that lysophosphatidic acid (LPA), bombe- However, some cell type di€erences have been observed. sin and bradykinin each bind to GPCRs to activate For example, Rac and Rho cause distinct membrane the formation of ®lopodia, membrane ru‚es and ru‚ing response in KB human epithelial carcinoma cell stress ®bers. Rho family proteins were subsequently lines (Nishiyama et al., 1994). Whereas Rho causes implicated in these processes (Ridley et al., 1992; neurite retraction, Rac causes neurite extension in NIE- Ridley and Hall, 1992). For example, LPA stimula- 115 neuroblastoma cells. Other members of the Rho tion of actin stress ®bers formation and the family are also regulators of additional actin cytoske- assembly of focal adhesion complexes in Swiss 3T3 letal rearrangements and cellular processes. For cells was blocked by C3 toxin (Ridley and Hall, example, transient expression of activated RhoD in a 1992). variety of cell types caused rearrangements of the actin PDGF stimulation of its receptor, a transmembrane cytoskeleton and cell surface and involved the forma- tyrosine kinase, causes membrane ru‚ing which was tion of long thin F-actin containing membrane blocked by dominant negative Rac (Ridley et al., processes together with the disassembly of stress ®bers 1992). Dominant negative Cdc42 blocked the forma- and focal adhesions. These cytoskeletal changes tion of ®lopodia induced by bradykinin (Nobes and corresponded with regulated endosome motility and Hall, 1995). Finally, insulin- or hepatocyte growth distribution (Murphy et al., 1996). RhoG has been factor-induced membrane ru‚ing in KB human reported to cause a Rac-dependent membrane ru‚ing in epidermoid carcinoma cells was dependent on Rac or Swiss 3T3 cells (Roux et al., 1997). Finally, activated Rho, respectively (Nishiyama et al., 1994). There is Rnd1 and RhoE/Rnd3 has been reported to cause a evidence that PI3K may link PDGF- and insulin- disruption of stress ®bers (Nobes et al., 1998; Guasch et mediated signaling to Rac activation and induction of al., 1998).

Figure 5 Rho family proteins are regulators of mitogen-activated protein kinase cascades. At least three distinct MAPK modules have been identi®ed and involve a cascade of a MAPK kinase kinase (MAPKKK; also MEKKs), a MAPK kinase (MAPKK; also MKKs) and a MAPK. Rac and Cdc42 have been shown to be activators of pathways that lead to the activation of the JNK and p38 MAPK modules (reviewed in Vojtek and Cooper, 1995; Treisman, 1996). Although several kinases have been implicated as Rac/Cdc42 e€ectors (e.g., PAKs, MLKs, MEKKs), the precise link(s) between Rac and Cdc42 and these cascades is presently not known. Ras activates ERKs via a Raf-dependent pathway, and JNKs and p38 MAPKs via a Raf-dependent pathway that involves Rac/Cdc42. Activated MAPKs in turn regulate a variety of substrates that include nuclear transcription factors (reviewed in Treisman, 1996); (Wang and Ron, 1996; Zervos et al., 1995). Note that common transcription factor targets are shared by the di€erent MAPKs Rho family of small GTPases and transformation IZohnet al 1421 Rho family proteins are also associated with are activated by many mitogenic stimuli, whereas JNK processes that involve the actin cytoskeleton in other and p38 are more commonly activated by cellular stress organisms. The regulation of cytoskeletal organization (heat shock, ionizing radiation, etc.) and in¯ammatory by Rho family members is evident from genetics cytokines. studies in yeast and Drosophila. Yeast Cdc42 has These cascades involve kinase modules where a been demonstrated to coordinate polarization of the MAPK kinase kinase (or MEKK) causes activation of actin cytoskeleton during cell division by budding, and a MAPK kinase (or MKK), which in turn activates a involves a cascade of other small GTPases (BUD1/ MAPK. The activated MAPKs have a variety of RSR1 and RHO proteins) (reviewed in Chant and targets and include various Ets domain, bZIP and Stowers, 1995). In Drosophila, Drac1 is required to MADS box containing transcription factors reviewed assemble actin at adherens junctions of the wing disc in Treisman, 1996). Although we have depicted each epithelium, while Dcdc42 is involved in the regulation MAPK module as a separate linear array, there is cross of polarized cell shape during various stages of wing talk between these cascades, as well as branch points disc development (Eaton et al., 1995). Studies have also and feedback loops within each module emphasizing implicated a requirement for Rho family proteins the complex nature of signaling pathways. during cytokinesis in sand dollar (Mabuchi et al., Constitutively activated Rac1 and Cdc42, but not 1993) Xenopus (Kishi et al., 1993; Drechsel et al., RhoA, are activators of JNKs (also called stress- 1996), Dictyostelium (Larochelle et al., 1996) and activated protein kinases, or SAPKs) and p38/HOGs, mammalian cells (Madaule et al., 1998). but not the p42 and p44 ERKs (Coso et al., 1995; Minden et al., 1995; Olson et al., 1995; Bagrodia et al., 1995a; Zhang et al., 1995). However, RhoA can Rho family proteins are regulators of gene expression activate JNK in some cells (Teramoto et al., 1996b) In addition to their involvement in regulation of and RhoG is also an activator of JNK, but not ERK cytoskeletal organization, it has been shown that Rho (Roux et al., 1997). JNK in turn activates the ATF-2 family proteins regulate protein kinase cascades that and Jun nuclear transcription factors. ATF-2 and Jun control the activity of a variety of nuclear transcription can dimerize with other transcription factors to factors (Figure 5). To date, at least three distinct stimulate transcription from promoters containing mitogen-activated protein kinase (MAPK) cascades AP-1 and related DNA sequences (e.g., the c-jun have been identi®ed in mammalian cells and more are promoter) (Figure 6) (Karin, 1995). Although Rac, likely to be discovered (Treisman, 1996). The p42 and RhoA and Cdc42 are not activators of ERKs, it has p44 extracellular signal regulated kinases (ERKs; also been observed that they can indirectly modulate ERK called p42 and p44 MAPKs), Jun NH2-terminal kinases activity in 293 cells (Luttrell et al., 1997). Dominant (JNKs) and p38/HOG kinases are distinct kinase negative Rac2 was found to impair Ras activation of cascades activated by distinct small GTPases. ERKs ERK2, whereas activated RhoA, Rac2 and Cdc42

Figure 6 Rho family proteins are regulators of genes that control cell growth, di€erentiation and apoptosis. Rho family proteins regulate the activity of genes that regulate cell proliferation and apoptosis (reviewed in Treisman, 1996). RhoA, Rac1 and Cdc42 activate SRF (Hill and Treisman, 1995), which forms a complex with ternary complex factors (e.g., Elk-1) at the serum response DNA element found in the promoter sequences of c-fos and other early response genes. RhoA and Rac/Cdc42 are believed to activate SRF through distinct pathways. Rac1/Cdc42 (and RhoA in some cells) activate JNK, which in turn can activate the Jun, ATF-2 and Elk-1 nuclear transcription factors. Activated c-Jun:ATF-2 heterodimers stimulate AP-1 like DNA elements in various promoters that regulate c-jun and other genes. Rac1 activates NF-kB by as yet unknown pathways that involve production of superoxide and phosphorylation and inactivation of IkB (Perona et al., 1997). Among these NF-kB-responsive genes are those that have an anti-apoptotic function (Baichwal and Baeuerle, 1997). Transduced and mutated versions of cellular Fos, Jun and NF-kB sequences were identi®ed as potent oncogene proteins responsible for the highly oncogenic properties of retroviruses (Bishop, 1991). Thus, these Rho family mediated changes in gene expression are likely to contribute to the growth promoting actions of these small GTPases Rho family of small GTPases and transformation IZohnet al 1422 synergistically enhanced Raf activation of ERK2. How Baltimore, 1996). In one study, Finkel and colleagues Rho family proteins may modulate ERK2 activation is found that activated Rac1, but not Cdc42, strongly not presently known. Furthermore, this crosstalk may stimulated NF-kB in HeLa cells (Sulciner et al., 1996). not occur in all cell types, since we have not observed Interleukin 1 b-stimulated NF-kB was also dependent this in NIH3T3 cells (Khosravi-Far et al., 1995). on Rac function. However, a second study found that Oncogenic Ras activates ERKs via Raf and JNK and Rac1, RhoA and Cdc42 stimulated NF-kBbya p38 via a Raf-independent pathway(s) (Minden et al., mechanism that involves phosphorylation of IkBto 1994; Olson et al., 1995; Clark et al., 1997) that are promote NF-kB translocation to the nucleus (Perona et likely to involve Rac/Cdc42. al., 1997). Consistent with this second study, we have RhoA, Rac1 and Cdc42 have been shown to found that a variety of Dbl family proteins that are not activate the transcription factor SRF (serum response activators of Rac (e.g., Dbl and Dbs) also activate NF- factor) by as yet unde®ned signaling pathways kB (Westwick et al., 1998). (Whitmarsh et al., 1995). SRF cooperates with Ras activation of JNK and NF-kB has been shown ternary complex factors (TCFs; Elk-1 and SAP1/2) to be mediated, in part, by activation of Rac (Minden and the serum response DNA elements found in et al., 1995; Sulciner et al., 1996). Rac1(12V) activation certain promoters such as the c-fos promoter and of NF-kB was found to be independent of JNK many other growth factor-regulated promoters activation. Instead, Rac1 stimulated increased produc- (Marais et al., 1993). TCFs are activated by the tion of superoxides and other reactive oxygen species Raf4MEK4MAPK pathway (Figure 6). Interest- (ROS), and inhibitors of their production also ingly, Rac and Cdc42 activation of SRF is not inhibited NFkB activation (Baeuerle and Baltimore, dependent on Rho indicating that Rho family 1996). Inhibition of either ROS production (Irani et al., proteins utilize distinct pathways to activate SRF 1997) or NF-kB activation (Finco et al., 1997) has been (Hill et al., 1995). Thus, the Cdc424Rac4Rho shown to block oncogenic Ras transformation. cascade that regulates actin structure is clearly Furthermore, since NFkB inhibition caused Ras- distinct from pathways that regulate SRF. Again, transformed NIH3T3 cells to undergo apoptosis cross-talk at the level of transcription factors is also (Mayo et al., 1997), the role of NFkB activation in seen. For example, Elk-1 can also be activated by Ras, and possibly Rac, transformation may be to cause JNK and p38. upregulation of as yet to be identi®ed anti-apoptotic Rho family proteins have also been implicated in the genes. An anti-apoptotic role for NFkB has been regulation of NF-kB. NF-kB recognizes DNA elements described for other extracellular stimuli (reviewed in found in a wide variety of promoters (Baeuerle and Baichwal and Baeuerle, 1997). Whether this is the mechanism by which superoxides promote Ras transformation remains to be determined.

Rho family proteins regulate cell proliferation Some early indications that Rho family proteins may regulate cell growth include observations that expres- sion of speci®c Rho family proteins is regulated by growth factor stimulation. RhoB was identi®ed as an immediate-early response gene induced by receptor tyrosine kinase activation (EGF and PDGF stimula- tion), by nonreceptor tyrosine kinases (viral Src and Fps), UV irradiation, and by DNA damaging agents (cisplatin and N-methyl-N-nitrosurea) (Jahner and Hunter, 1991; Fritz et al., 1995). RhoB was also transiently induced by EGF or TGFa in human mammary epithelial cells and was overexpressed in human breast cancer tissues and cell lines (de Cremoux et al., 1994; Linares-Cruz et al., 1994). RhoG gene expression was also found to be growth-inducible (Vincent et al., 1992). The hematopoietic cell-speci®c Rac2 was induced in T cells by phytohemagglutinin A growth stimulation (Reibel et al., 1991). Rho family proteins have also been implicated in cell cycle regulation. RhoA, Rac1 and Cdc42 were shown Figure 7 Oncogenic Ras causes transformation by activation of to be requirement for cell cycle progression through the Raf-independent pathways that cause activation of speci®c Rho G1 phase of the cell cycle (Yamamoto et al., 1993; family proteins. The ability of dominant negative mutants of Olson et al., 1995). For example, C3 toxin treatment RhoA, RhoB, Rac1, Cdc42 and RhoG to block oncogenic Ras and inhibition of Rho function caused Swiss 3T3 cells transforming activity demonstrates the requirement for speci®c Rho family proteins in mediating, in part, Ras transformation. to accumulate in the G1 phase of the cell cycle and Prevailing evidence suggests that each Rho family protein may growth inhibition (Yamamoto et al., 1993). Further- function in distinct pathways and that the Cdc424Rac4Rho more, microinjection of constitutively activated mu- hierarchy seen for actin reorganization in Swiss 3T3 cells may not tants of Ras, RhoA, Rac1 and Cdc42 caused G1 occur for Ras transformation of rodent ®broblasts. Rho family proteins contribute to multiple aspects of the transformed and progression and stimulation of DNA synthesis in tumorigenic phenotype of Ras-transformed cells quiescent Swiss 3T3 ®broblasts, whereas the dominant Rho family of small GTPases and transformation IZohnet al 1423 negative mutant counterparts blocked serum-stimu- observations suggested that increased formation of the lated DNA synthesis (Olson et al., 1995). The active GTP-bound protein, together with accelerated mechanism by which Ras and Rho proteins regulate GDP/GTP cycling, may both be required to promote cell cycle progression is not yet understood but it is Cdc42 transformation. Enhanced GDP/GTP exchange, thought to involve stimulation of D-type cyclins which rather than impairment in GAP-stimulated GTP are stimulated by Ras and AP1 proteins (Albanese et hydrolysis, has also been shown to activate the al., 1995). We recently showed that Rac1 and RhoA transforming potential of Ras proteins (Walter et al., stimulated transcription of cyclin D1 (Westwick et al., 1986; Feig and Cooper, 1988; Der et al., 1988; 1997). Rho proteins have also been implicated in the Reinstein et al., 1991). degradation of the cyclin-dependent kinase inhibitor A growth-regulatory function for other Rho family p27Kip1, to facilitate entry into S phase (Hirai A et al., proteins is also likely. For example, activated 1997). Thus, it is possible that Rho family proteins are RhoG(12V) showed only limited growth transforma- mediators of Ras-induced cell cycle progression. tion of NIH3T3 cells (Roux et al., 1997). However, it The involvement of BCR, a bifunctional Rho GAP showed strong synergistic focus-forming activity when and GEF, as the translocation partner of the Abl co-expressed with activated mutants of RhoA, Cdc42 tyrosine kinase in Philadelphia Chromosome-positive or Rac1. Whether the aberrant upregulation of other chronic myelogenous leukemias implicated deregulated Rho family proteins also promote growth transforma- Rho protein function in human leukemias. However, tion remains to be determined. Additionally, whether whether the BCR sequences present in the p185 or Rho family proteins can cause growth transformation p210 BCR-Abl chimeric proteins contribute to their of epithelial and other cell types has not been function as oncoproteins has not been established. addressed. Also, although transcripts of the reciprocal Abl-BCR fusion have been detected, whether it encodes a Rho family proteins promote cell motility and invasion functional protein has not been described. Finally, one Rho family protein (TTF) was rearranged by a Rac1, RhoA and Cdc42 function have also been shown t(3 : 4) chromosome translocation in large cell non- to promote cell motility and invasion. Rho protein Hodgkin's lymphomas (Dallery et al., 1995). What function was found to be required for crawling function the hematopoietic cell-speci®c TTF protein movements of NIH3T3 cells on a ¯at surface may have, and whether its loss of function is important (Takaishi et al., 1993) and for hepatocyte growth for lymphoma development, has not been addressed. factor induced motility of mouse karatinocytes Direct evidence for the importance of Rho family (Takaishi et al., 1994) or MDCK epithelial cells proteins in the regulation of cell growth came from (Ridley et al., 1995). Similarly, C3 treatment showed studies to determine if constitutively activated mutants that Rho is required for LPA-induced invasion of of various Rho family proteins could cause transforma- hepatoma cells through a mesothelial cell monolayer tion of rodent ®broblasts. The ®rst evaluation of this (Yoshioka et al., 1995; Wang and Ron, 1996). C3 possibility did identify a transforming activity for RhoA treatment inhibited the invasive of three T cell (Avraham and Weinberg, 1989). However, transforma- lymphoma cell lines through a monolayer of tion of NIH3T3 cells was associated with wild type, C3H10T1/2 cells (BW-O-Lil, BW-19 and CCRF- rather than activated, RhoA protein. Lacal and CEM). Invasion in this in vitro assay was shown to colleagues showed that both wild type and constitu- correlate with the ability or a cell line to induce tively activated Aplysia Rho caused tumorigenic experimental metastasis formation in mice (Verschue- transformation of NIH3T3 cells (Perona et al., 1993). ren et al., 1997). Collard and colleagues showed that Expression of activated mutants of human Rac1, activated Rac1(12V) and the Tiam1 Rac GEF, but not RhoA or RhoB alone have been shown to cause activated RhoA(14V), induced invasion of T lympho- tumorigenic transformation of NIH3T3 and Rat-1 ma cells in the same ®broblast invasion assay (Habets rodent ®broblasts (Qiu et al., 1995a, b; Khosravi-Far et al., 1994; Michiels et al., 1995). Tiam1 also et al., 1995; Prendergast et al., 1995). Constitutively promoted metastasis in nude mice. Finally, aberrant activated Cdc42 has been shown to cause partial Rac1 or Cdc42 function increased the motility and transformation of Rat-1 cells (Qiu et al., 1997). invasion in vitro of T47D human breast epithelial cells Cdc42(12V)-expressing cells formed colonies in soft (Keely et al., 1997). Both the increased motility and agar and tumors in nude mice, but did not show invasion were blocked by inhibitors of PI3K and reduced requirement for serum or loss of density- constitutively activated PI3K alone was sucient to dependent growth in culture. We have found that cause these alterations. Thus, PI3K may be a key Cdc42(12V) is growth inhibitory in NIH3T3 cells, but e€ector in mediating Rac- and Cdc42-mediated can cooperate with activated Raf to cause focus- invasion. When taken together with the involvement formation in NIH3T3 assays (Whitehead et al., 1998). of Rho proteins in mediating cell attachment and cell- Interestingly, Cdc42, but not RhoA or Rac1, has been cell interactions, Rho regulation of motility and shown to cause G1 arrest by a p38-dependent invasion supports the possibility that aberrant func- mechanism in NIH3T3 cells (Molna r et al., 1997). tion of Rho family proteins promote the invasive and A recent report also found that overexpression of metastatic properties of human tumor cells. constitutively activated, GAP-insentive Cdc42(61L) was growth inhibitory in NIH3T3 cells. In contrast, Dbl family proteins: Rho family GEFs and transforming they showed that a mutant of Cdc42 (F28L), which proteins exhibited enhanced GDP/GTP cycling could cause growth transformation of NIH3T3 cells (growth in Finally, the transforming activities associated with low serum and soft agar) (Lin et al., 1997) These many Dbl family proteins also provide evidence that Rho family of small GTPases and transformation IZohnet al 1424 upregulation of Rho family protein function can cause stratum adhesion, and that oncogenic Ras caused growth deregulation (reviewed in Whitehead et al., increased membrane ru‚ing and motility (Bar-Sagi 1997). Dbl family proteins are presumed to cause and Feramisco, 1986) suggested an involvement of Rho transformation by causing constitutive upregulation of family proteins. In this section, we summarize the Rho protein function. Consistent with this, Dbl family evidence for a Ras connection with Rho family proteins cause the same changes in actin organization proteins and discuss our current understanding of and gene expression as seen with Rho family proteins how they may contribute to oncogenic Ras-mediated (Westwick et al., 1997b). transformation. All Dbl family proteins share a domain of One of the ®rst suggestions of a link between Ras approximately 180 amino acids that has been and Rho proteins was made by the observation that a designated the Dbl homology (DH) domain. In p120 GAP-associated protein, p190, is a GAP for addition, all family members possess a second, shared RhoA, and to some degree for Cdc42 and Rac domain designated the pleckstrin homology (PH) (Settleman et al., 1992). Thus, p120 GAP may serve domain of approximately 100 amino acids (Figure 3). as an e€ector that facilitates Ras regulation of Rho The PH domain is invariably located immediately family protein function. However, at present, whether COOH-terminal to the DH domain and this invariant the association of a p120 GAP : p190 Rho GAP topography suggests a functional interdependence complex with activated Ras leads to upregulation or between these two structural modules. The DH downregulation of Rho family protein function has not domain provides the GEF activity and each Dbl been established. family protein may activate a speci®c or several Rho Genetic analysis in S. pombe Ras (Ras1) also family proteins. Whether the DH domains of some identi®ed a link with Rho family proteins and de®ned family members actually serve as Rho GEFs remains two distinct Ras1 e€ector-mediated activities. One to be determined (e.g., SOS, RasGRF, BCR). involves Ras1 interaction with Byr2 which is a MEK PH domains are also found in many non-Dbl family kinase homolog (Wang et al., 1991; Van Aelst et al., proteins that comprise a large, diverse group of 1993; Masuda et al., 1995). The other involves Ras1 proteins that are involved in cell signaling or interaction with the Scd1 Dbl family protein (Chang et cytoskeletal functions that requires association with al., 1994). Scd1 is a putative exchange factor and cell membranes (Lemmon et al., 1996). PH domains activator of the S. pombe Cdc42 homolog. Interest- can function as protein-protein or protein-lipid ingly, Scd1 was found to bind directly to activated Ras interaction motifs and it is generally believed that and possess properties of a Ras e€ector, thus directly they serve to recruit proteins to the cell surface by lipid linking Ras activation to activation of Rho family binding. Thus, it is speculated that the PH domain may proteins. Whether a speci®c mammalian homolog(s) of serve two roles in Dbl family protein function. First, it Scd1 exists and functions as a Ras e€ector remains to may promote membrane translocation. This function is be determined. Finally, this model is an oversimplifica- supported by the observation of Whitehead and tion since Scd1 is likely to promote a multiprotein colleagues that addition of a plasma membrane complex formation, with the SH3 domain-containing targeting sequence restored the loss of PH function Scd2 protein and GTP-complexed Ras1 (Chang et al., and recreated a transforming version of Lfc (White- 1994). Such a role for an e€ector emphasizes a theme head et al., 1996). Second, the PH domain may serve as where e€ectors of small GTPases mediate their actions an intramolecular regulator of the DH domain. Thus, by promoting the formation of signaling complexes upstream signaling pathways that cause activation of rather than activating simply linear cascades. beta-gamma subunits of G proteins or upregulation of Further evidence for the involvement of Rho family phosphatidylinositol lipids may regulate Dbl family members in mediating oncogenic Ras function came protein function via interaction with the PH domain. from microinjection studies in serum-starved Swiss 3T3 How Dbl family proteins link extracellular signals with cells by Hall and colleagues (Ridley et al., 1992; Ridley Rho family proteins remains a poorly understood area. and Hall, 1992). H-Ras(12V) induction of membrane Three recent studies showing that speci®c tyrosine ru‚ing and stress ®bers were blocked by dominant kinases cause phosphorylation of Vav to stimulate its negative Rac1(17N) and C3, respectively. These GEF activity de®ne one mechanism where extracellular observations established the ability of oncogenic Ras signals cause, via a Dbl family protein, activation of to transiently stimulate a Rac4Rho cascade and Rho family proteins (Crespo et al., 1997; Han et al., prompted investigations into the possible involvement 1997; Teramoto et al., 1997). of RhoA and Rac1 in Ras transformation. Conse- quently, the morphologic transformation seen in Ras- transformed cells may be caused, in part, by Ras activation of a GTPase cascade: an involvement of deregulation of Rho and Rac function. Rho family proteins in transformation Presently, the function of ®ve Rho family proteins have been shown to be important for oncogenic Ras While it is clear that Ras must utilize e€ectors other transforming activity. Three experimental approaches than Raf, the precise nature of these Raf-independent have been applied to support key contributions of pathways remains to be fully delineated (Marshall, RhoA, RhoB, Rac1, Cdc42 and RhoG to Ras 1996; Khosravi-Far et al., 1997). However, several lines transformation (Qiu et al., 1995a; Khosravi-Far et of evidence have implicated speci®c Rho family al., 1995; Prendergast et al., 1995; Qiu et al., 1995b, proteins as key downstream mediators of Ras 1997; Roux et al., 1997; Lebowitz et al., 1997). First, transformation. In retrospect, the early observations co-expression of dominant negative mutants [equiva- that Ras-transformed cells undergo morphologic lent to the Ras(17N) mutant] of each Rho family alterations, loss of stress ®bers and decreased sub- protein caused a signi®cant, although incomplete, Rho family of small GTPases and transformation IZohnet al 1425 reduction in oncogenic Ras focus-forming activity cells do possess enhanced membrane ru‚ing and when analysed in rodent ®broblasts. Second, co- pinocytosis, consistent with persistent Rac activation. expression of constitutively activated versions of each However, the loss, rather than enhancement, of stress Rho family GTPase with activated Raf-1 mutants ®bers and focal adhesions, are not consistent with showed cooperative and synergistic focus-forming persistent Rho activation. activity. Finally, constitutively activated mutants Several observations suggest that each Rho family caused growth transformation of NIH3T3 or Rat1 protein contributes a distinct aspect of Ras transfor- rodent ®broblasts. Taken together, these observations mation. First, constitutively activated mutants of each support a model where oncogenic Ras causes consti- Rho family protein caused a distinct transformed tutive activation of speci®c Rho family proteins which phenotype in NIH3T3 cells. Second, co-expression of in turn activate a spectrum of functions that contribute di€erent combinations of activated mutants showed to full Ras transforming activity. However, it should be cooperative focus-forming activity, suggesting that emphasized that a demonstration that Ras-transformed they activate distinct pathways (Roux et al., 1997). cells exhibit constitutively elevated GTP-bound levels Interestingly, the greatest cooperative focus-forming of a speci®c Rho family protein has not been activity was seen with co-expression of activated determined. Thus, which, if any, of the known Rho Cdc42(12V) and Rac1(12V). Third, the characteriza- family proteins are clearly targeted by oncogenic Ras tion of Rat1 ®broblasts coexpressing H-Ras(12V) and remains to be established. either dominant negative Rac1 or Cdc42 argued that The evaluation of e€ector domain mutant of each Rho family protein played distinct roles in Ras oncogenic H-Ras also supports the contribution of transformation (Qiu et al., 1997). Dominant negative Rho family proteins to Ras transformation (White et Cdc42(17N) blocked the soft agar growth and cause al., 1995; Joneson et al., 1996b; Khosravi-Far et al., morphologic reversion of Ras-transformed Rat-1 1996). Speci®cally, two mutants with single amino acid cells. However, low serum growth was retained. In substitutions (37G and 40C) lost the ability to bind and contrast, Rac1(17N) reverted low serum growth and activate Raf and the ERK pathway. Despite this colony formation in soft agar, but not morphologic defect, both mutant proteins retained the ability to transformation. Hence, it was proposed that Cdc42 cause full transformation of NIH3T3 cells (Khosravi- and Rac play distinct roles in Ras transformation, Far et al., 1996). However, the transformed phenotype with Cdc42 mediating morphological transformation caused by Ras(12V, 37G) or Ras(12V, 40C) was and Rac1 promoting serum-independent growth. distinct from that caused by activated Ras or Raf, Finally, distinct contributions of each Rho family and instead, was indistinguishable from the trans- protein to Ras transformation is also suggested by formed phenotype caused by activated Rac or Rho the observation that dominant negative RhoA and proteins. These observations lend further support for a Cdc42, but not Rac1, blocked Raf transforming Raf-independent connection between Ras and Rho activity. These ®ndings also suggest that the family proteins that contributes to cellular transforma- Cdc424Rac4Rho cascade for actin reorganization tion. may not apply for their association with oncogenic The evaluation of Ras-transformed rodent fibro- Ras transformation. Finally, since dominant negative blasts that stably co-expressed either dominant Rac1 impaired RhoG-mediated transformation, it was activated or negative Rho family proteins has proposed that Rac1 functioned downstream of provided some clues regarding the contribution these RhoG. small GTPases to the Ras transformed phenotype. For example, coexpression of activated Rac1 or RhoA caused further morphologic transformation and de- A search for the missing link that connects Ras with Rho creased attachment to the plastic substratum of Ras- family proteins transformed NIH3T3 cells (Khosravi-Far et al., 1995). These cells also formed colonies in soft agar that Although how Ras may connect with Rho family suggested increased motility and invasive properties. In proteins is not clearly understood at present, the contrast, co-expression of dominant negative Rac1 or prevailing evidence support the linkage via a Raf- RhoA caused a morphologic reversion of Ras- independent pathway. First, dominant negative mu- transformed NIH3T3 cells (Khosravi-Far et al., tants of Rac1, RhoA, Cdc42 and RhoG showed 1995). Taken together, these observations support the complete, or preferential, inhibition of Ras versus Raf possibility that Ras causes morphologic transforma- focus-forming activity. Second, the potent cooperative tion, in part, by upregulation of Rac1 or RhoA focus-forming activity seen when activated Raf and function. However, the observations that Rac1- and Rho family proteins are co-expressed suggests activa- RhoA-transformed NIH3T3 cells do not undergo any tion of distinct signaling pathways. Third, activated signi®cant morphologic transformation, and retain Raf does not cause direct activation of JNK, indicating well-organized stress ®bers and focal adhesions, argue that Rac, Cdc42 and RhoG are not downstream that aberrant Rac or Rho function is not responsible components of this pathway. for that altered morphology and loss of stress ®bers. The studies presented above clearly establish a Instead, since Raf- and MEK-transformed NIH3T3 critical role for Rho proteins in mediating Ras- cells also undergo morphologic transformation, it induced cytoskeletal alterations, cellular transforma- suggests that the Raf4MEK4ERK pathway pro- tion, gene expression and cell cycle progression. motes morphologic transformation. However, the components that transmit the signal How the transient changes in actin organization from activated Ras to Rho proteins remain to be caused by Rho family proteins relate to long term determined. Evidence that support the involvement of changes is also not clear. Ras-transformed NIH3T3 known Ras-binding proteins as candidate e€ectors that Rho family of small GTPases and transformation IZohnet al 1426 link Ras with Rho proteins is summarized in this G protein-coupled receptor transformation and Rho section. family proteins At least ®ve candidate e€ectors of Ras display properties that support their possible role in linking In addition to the involvement of Rho family proteins Ras to Rho family proteins. First, similar to S. in Ras and Db1 family protein transformation, speci®c pombe Ras (Chang et al., 1994), a mammalian Rho Rho family proteins also contribute to the transform- GEF (Whitehead et al., 1997) that is analogous to ing actions of other oncogene proteins. For example, a yeast Scd1 may function as an e€ector and directly requirement for Rho family protein function has been link Ras with speci®c Rho family proteins. However, determined for viral Abl (Renshaw et al., 1996). although over 20 candidate mammalian Rho GEFs, Dominant negative Rac1(17N) was shown to block v- or Dbl family proteins, have been identi®ed, to date Abl activation of JNK and cause partial reversion of none have been implicated as Ras-GTP binding transformation of NIH3T3 cells (P-3T3 subclone). proteins (Whitehead et al., 1997). Nevertheless, by Serum-independent growth, but not morphologic analogy to Ras proteins, it is anticipated that a Rho transformation or anchorage-independent growth were GEF will still be an important intermediate between inhibited. Polyoma virus middle T oncoprotein focus- Ras and Rho proteins. Second, Ras interaction with formation on F111 ®broblasts was inhibited completely a p120 GAP:p190 Rho GAP complex may lead to by co-expression of dominant negative Rac1(17N) or activation or inactivation of Rho family proteins Cdc42)(17N) (Urich et al., 1997). Thus, it will not be (Settleman et al., 1992). Overexpression of p190 GAP surprising if Rho family proteins will be required for has been shown to cause inhibition of stress ®ber the transforming activity of other oncoproteins. formation (Ridley et al., 1993). However, it is not In addition to mediating a spectrum of normal known whether Ras association in¯uences p190 Rho physiological responses that include neurotransmission, GAP function. Indirect evidence for such a role is metabolism, growth and di€erentiation (van Biesen et provided by the recent observation that dominant al., 1996), there is also emerging and growing evidence negative mutants of p120 GAP, block Ras activation for the involvement of aberrant GPCR function in of JNKs but not MAPKs (Clark et al., 1997). Third, cellular transformation and oncogenesis (Dhanasekar- three groups recently identi®ed independently a Ral- an et al., 1995). As described above, Rho family binding protein (RalBP1/RLIP1/RIP1) that interacted proteins are components of a number of GPCR with Ral in a GTP-dependent manner, required an receptor signaling pathways. Therefore, it is not intact Ral e€ector domain, and contains a Rho GAP surprising that Rho family proteins have been domain (Cantor et al., 1995; Jullien-Flores et al., implicated as essential mediators of transformation by

1995; Park and Weinberg, 1996). Thus, this protein some heterotrimeric G proteins (G12 and G13) and G may serve as a link between Ras and Rho proteins protein-coupled receptors (Mas and XGR). The (Feig et al., 1996). Ras association with RalGDS may following section summarizes the importance of Rho lead to the activation of Ral. Activated Ral then family proteins in their transforming actions. associates with RalBP1/RLIP1/RIP1 and alters the A number of G protein-coupled receptors (GPCRs; activity of certain Rho family members. This is an also serpentine receptors, SRs) have been identi®ed as intriguing model that remains to be validated. This oncogenes in ®broblast transformation assays. A Ral-binding protein showed strongest GAP activity determination that the mas oncogene, detected in an towards Cdc42, and to a lesser degree with Rac, but NIH3T3 transfection/nude mouse tumorigenicity assay, not RhoA. Ral association and regulation of RalBP1 encoded a novel G protein coupled receptor provided GAP activity also has yet to be demonstrated in vivo. the ®rst indication that G protein-mediated signaling Fourth, Ras interaction and activation of MEKK1 pathways could promote cellular transformation may explain how Ras mediates activation of JNK (Young et al., 1988). Subsequently, other G protein- and p38, although this pathway would not be coupled receptors have been identi®ed as having

expected to involve a Rho family protein. Finally, transforming properties and include the M1-, M2-and

while PI3K has been shown to be a candidate M3-muscarinic acetylcholine receptors (Gutkind et al., e€ector of Ras by interacting with Ras in a GTP- 1991), the 5HT1b serotonin receptor (Julius et al., dependent manner, it is also thought to be required 1989), and the a1b-adrenergic receptor (Allen et al., for activation of Rac proteins (Hu et al., 1995). 1991). More recently, the thrombin receptor (White- Furthermore, constitutive activated mutants of PI3K head et al., 1995) and the XGR novel G protein- activate JNK (Klippel et al., 1996), although a coupled receptor (R Kay, unpublished observation) second study did not see this activation (Marte et were also added to this list by virtue of their detection al., 1997). Despite these ®ve possibilities, the exact as transforming proteins in cDNA library screens for mechanism by which signals from activated Ras transforming proteins in NIH3T3 transformation proteins are transmitted to cause activation of Rho assays. We recently found that Mas causes transforma- family of proteins still remains to be characterized. A tion by activation of Rac (Zohn et al., 1997), whereas direct demonstration that speci®c Rho family proteins XGR causes transformation by activation of Rho are constitutively activated in Ras-transformed cells (Zohn et al., in preparation), and it is likely that the also remains to be shown. Finally, Marshall and transforming activity of other GPCRs will also involve colleagues showed recently that Rho signaling speci®c Rho family proteins. antagonized Ras-induced increases in p21/Waf1/Cip1 What G proteins mediate the transforming actions levels (Olson et al., 1998). This suggests that RhoA of these GPCRs? At present, this is not known. does not promote a Ras-mediated signaling event, However, observations that constitutively activated but instead, negatively modulates a Ras-mediated mutants of a number of G protein a subunits can signaling function. cause transformation of rodent ®broblasts suggests Rho family of small GTPases and transformation IZohnet al 1427 candidates for this role. In particular, constitutively family GTPase e€ectors. Finally, we conclude with a activated mutants of Ga12 and the related Ga13 have discussion of how a spectrum of structurally and exhibited the strongest transforming potential and can functionally diverse proteins all share the common cause tumorigenic transformation of rodent ®broblasts property of serving as e€ectors of a particular Rho (Xu et al., 1993; Vara Prasad et al., 1994; Chan et al., family protein. 1993; Jiang et al., 1993; Voyno-Yasenetskaya et al., We have categorized Rho family protein binding

1994). Similarly, Gaq has been shown to transform partners into two general groups based on their binding NIH3T3 cells (Kalinec et al., 1992; De Vivo et al., speci®cities: (1) those that bind Rac and/or Cdc42, but

1992). However, Gaq can also be cytotoxic (Kalinec et not Rho and (2) those that bind Rho. However, al., 1992; Wu et al., 1992), and induces apoptosis exceptions exist and include proteins that bind Rho (Althoefer et al., 1997). Since the signaling and and Rac/Cdc42. It should be emphasized that many of transforming activity of Ga12 and Ga13 have been these analyses have been limited to the analyses of shown to involve Rac or Rho function, they represent interaction with Rac1, RhoA or Cdc42. Therefore, logical connectors between transforming GPCRs and possible e€ector interactions with other Rho family

Rho family proteins. For example, Ga12 transforming members may complicate these simple classi®cations. activity was shown to be inhibited by dominant Many of the binding analyses have been performed negative RhoA and Rac1, but not Cdc42 (Tolkacheva in vitro or by using yeast two-hybrid binding analyses. et al., 1997). Microinjection analyses of activated Ga12 Thus, whether they represent physiologically-relevant and Ga13 in Swiss 3T3 cells caused the induction of interactions remain to be determined for many of these stress ®bers (Buhl et al., 1995; Hooley et al., 1996) and proteins. What properties are required to validate an both have been shown to activate JNK (Prasad et al., e€ector? First, the ability of a particular small GTPase 1995; Collins et al., 1996). Recently, the demonstration to bind to an isolated domain in vitro may be that Ga13 can interaction with and activate the Lsc/ misleading. Therefore, can interaction with the full p115 RhoGEF Dbl family protein, a Rho GEF, de®nes length protein needs to be established in vivo? Second, at least one possible link between GPCRs and Rho does association lead to regulation, positive or (Hart et al., 1998). negative, of e€ector function? Third, does overexpres- In summary, the transforming actions of diverse sion of e€ector function mimic any aspect of the oncoproteins, ranging from Ras to G protein-coupled function of its corresponding GTPase provides? receptors, require the function of speci®c Rho family proteins. Thus, it is likely that speci®c Rho family A plethora of candidate e€ectors of Rac and Cdc42 proteins will play key supportive roles in human carcinogenesis. Whether mutational activation, or Unlike the situation with candidate e€ectors of Ras, at abberant overexpression, of speci®c Rho family least three clear consensus binding sequences for Rho proteins alone occur in human cancers will be an family binding proteins have been identi®ed for at least important goal of future studies. some of these proteins. Hall and colleagues have described a minimal region of 16 amino acids required for binding of Cdc42 and/or Rac, designated the Rho family proteins mediate their actions through Cdc42/Rac Interactive Binding (CRIB) motif (Burbelo interaction with multiple e€ectors et al., 1995) (Figure 8a). Using this motif in a search of the GenBank data base, they identi®ed more than 25 Clearly, Rho family protein-mediated changes in actin proteins from a wide variety of eukaryotic species that cytoskeletal organization, gene expression and regula- contain the CRIB motif. This motif is found in known tin of cell cycle progression are all likely to contribute Cdc42/Rac-binding proteins, such as the three p21 to the aberrant growth phenotype of Ras-transformed (Cdc42/Rac) activated kinase (PAKs) isoforms and the cells. However, what aspects of their function, two ACK nonreceptor tyrosine kinases. Other mam- mediated by which e€ectors, contribute to Ras malian CRIB motif-containing proteins include WASP, transformation are presently not understood. One several mixed lineage kinases (MLK2, MLK3), and an approach for addressing this question has been to uncharacterized human protein MSE55. Further determine what promotes the transforming activity of analyses demonstrated that all showed GTP-depen- speci®c Rho family proteins. This process is compli- dent interaction with Cdc42 and/or Rac1. Therefore, cated by the multitude of functions attributed to each the CRIB motif may de®ne a GTP-dependent Rho family protein and the fact that, like Ras, each interaction site found in a subset of Rac and/or mediates its actions through a plethora of e€ector Cdc42 e€ector molecules. targets. As with Ras, it is suspected that no one speci®c Generally, Rac and Cdc42 share a number of e€ector will be important for the growth-promoting common binding partners which do not bind RhoA, actions of a speci®c Rho family protein. which may be related to the higher sequence homology The roster of candidate e€ectors for Rac1, RhoA between Rac and Cdc42 compared to RhoA. However, and Cdc42 is extensive and continues to expand. some Rac-speci®c- (e.g., p67phox and POR-1) and some Whereas the discovery of binding partners has been Rac/Cdc42-binding proteins have been identi®ed that rapid, progress in de®ning their contributions to Rho lack the CRIB motif (e.g., pp70 S6 kinase, the p85 family protein function has been very limited. In the subunit of PI3K, IQGAPs). Clearly, Rac-binding following section, we provide a brief overview of Rho motifs other than the CRIB motif will be found. family binding proteins and possible functions. We also Among the Rac/Cdc42-binding proteins that have summarize observations from recent studies using been identi®ed, the three mammalian PAK serine/ e€ector domain mutants of Rac1 and Cdc42 that threonine kinase isoforms (rat a-PAK/human hPAK-1, begin to de®ne the precise function of speci®c Rho rat p65 b-PAK/mouse mPAK3 and p62 g-PAK/human Rho family of small GTPases and transformation IZohnet al 1428 h-PAK2) have attracted the most interest (Manser et SRF or for transformation of NIH3T3 cells (Westwick al., 1994, 1995; Teo et al., 1995; Bagrodia et al., 1995b; et al., 1997a) and well as for induction of lamellipodia Martin et al., 1995). By virtue of their striking (Lamarche et al., 1996; Joneson et al., 1996a; Westwick homology with the S. cerevisiae protein Ste20, which et al., 1997). Instead, a correlation was found that is implicated in G protein-associated pheromone implicated PAK as a possible e€ector for mediating

signaling to a MAPK cascade, PAKs have been Rac stimulation of transcription from the cyclin D1 considered likely candidates for e€ectors that promote promoter (Westwick et al., 1997). However, Lamarche Rac/Cdc42 activation of the JNK MAPK cascade et al. (1996) found that PAK was dispensable for Rac- (Simon et al., 1995; Zhao et al., 1995). Support for this mediated progression through G1. Thus, Rac upregu-

possibility was provided by experiments using Xenopus lation of cyclin D1 alone may not be sucient for its oocyte extracts, where Ste20, a related protein from S. regulation of cell cycle progression. Furthermore, the pombe (Shk1) and PAK were shown to activate JNK/ Rac-induced pathways leading to the regulation of

SAPK (Polverino et al., 1995). Additionally, the lamellipodia, activation of JNK, SRF and cyclin D1 intrinsic kinase activity of PAKs was activated by appear to be mediated by distinct e€ectors (Westwick Rac/Cdc42-binding (Manser et al., 1994), and over- et al., 1997). Which, if any, of the known Rac expression or constitutive activation of PAK showed functions are important for Rac transformation, and enhanced activation of JNK or p38 (Zhang et al., 1995; hence, Ras transformation, is presently unresolved. Frost et al., 1996). Furthermore, dominant negative Finally, RhoB activation of SRE was retained in a mutants of PAK inhibited Rac1 activation of JNK/p38 nonprenylated mutant that was impaired in transform- (Zhang et al., 1995) or of the c-Jun-responsive element ing activity, indicating that SRE activation alone is not in the collagenase promoter (Osada et al., 1997). sucient for transformation (Lebowitz et al., 1997). However, other evidence argues that PAKs are not E€ector mutants of Rac and Cdc42 have also e€ectors for Rac/Cdc42 activation of JNK. First, uncoupled JNK activation from regulation of cytoske- Westwick et al., recently identi®ed Rac e€ector domain letal changes and G1 cell cycle progression. Y40C mutants that no longer bound to, or activated, PAK, mutants of Rac and Cdc42 showed impaired binding to yet they retained the ability to activate JNK and p38 PAK and other CRIB motif-containing proteins (e.g., (Westwick et al., 1997). This demonstrated that PAK WASP), but Rac1(40C) did retain interaction with the

was not required for Rac activation, of JNK. Second, non-CRIB motif-containing proteins p67PHOX or p160 Gutkind and colleagues suggested that MLK3 linked ROCK. However, since the Y40C mutants of Rac1 and Rac/Cdc42 to JNK (Teramoto et al., 1996a). MLK3 Cdc42 retained wild type abilities to induce actin (also known as PTK1 or SPRK) is a member of a reorganization, CRIB motif-containing e€ectors are family of related kinases (including MLK1, MLK2/ not likely to be mediators of actin reorganization nor MST, and MUK/DLK/ZPK) of unknown function. are they involved in linking Cdc42 to Rac or Rac to MLK3 RNA is expressed in most tissues and cell lines Rho. Activated PAK can cause changes in actin (Ezoe et al., 1994; Ing et al., 1994). These investigators found that MLK3 associated with activated Rac1 and Cdc42, but not RhoA, in vivo, and co-expression of a MLK3 (and not PAK) caused a synergistic enhance- ment of Rac1 and Cdc42 activation of JNK (Teramoto et al., 1996a). Overexpression of MLK3 (or MLK2) alone caused activation of JNK, but not p38 or ERK (Tibbles et al., 1996; Hirai S-I et al., 1997). Thus, MLK2/3, rather than PAKs, may serve as Rac/Cdc42 e€ectors that lead to activation of JNK. However, Hall and colleagues described evidence that MLK2/MLK3 were not e€ectors for Rac activation of JNK (Nagata et al., 1998). MEKK1 and MEKK4 represent additional candi- dates for the MAPKKK that links Rac/Cdc42 with JNK (Gerwins et al., 1997; Fanger et al., 1997). MEKK1 was found to interact with Rac and Cdc42, but not RhoA, in a GTP-dependent fashion in vitro.In contrast, MEKK4 associated with both GDP- and GTP-bound Rac1 and Cdc42 in vitro. MEKK4, but not MEKK1, contains a modi®ed CRIB motif. Kinase- inactive mutants of MEKK1 and MEKK4 both blocked Cdc42 and Rac activation of JNK, and both were activators of JNK, but not p38, in COS cells. In contrast, a second study evaluating a human homolog of MEKK4 suggested that it was not a mediator of Rac or Cdc42 activation of JNK and furthermore, that it was an activator of primarily p38, rather than JNK (Takekawa et al., 1997). The basis for these discrepancies is presently not clear. Rac e€ector domain mutant studies also eliminated PAK as the e€ector for directing Rac activation of Rho family of small GTPases and transformation IZohnet al 1429 organization, but in a way distinct from those caused involved in regulating Rac- or Cdc42-induced G1 by Rac1 or Cdc42 (Manser et al., 1997; Sells et al., progression. Finally, since a F37A mutant of 1997). Furthermore, since Y40C mutants of both Rac Rac1(61L) showed impaired p160 ROCK binding, and Cdc42 also retained the ability to stimulate DNA and lost the ability to stimulate lamellipodia and synthesis, CRIB motif-containing e€ectors may not be DNA synthesis, it supports a role for ROCK as an

b

c

Figure 8 Consensus binding sequences de®ne di€erent classes of Rac/Cdc42 and Rho binding proteins. (a) The Cdc42/Rac Interactive Binding (CRIB) motif (Burbelo et al., 1995) is shared by some, but not all, Rac and/or CDC42-binding proteins. CRIB sequences of MEKK4 (Gerwins et al., 1997) and Ack-2 (Yang and Cerione, 1997) and those previously reported by Burbelo et al. (1995) were aligned using ClustalW and shaded using Boxshade 3.2 (Thompson et al., 1994). Residues that are conserved in greater than 50% of sequences at a given sequence position are colored blue, while residues that are similar are colored yellow. R, rat; M, mouse; H, human; Sc, S. cerevisiae, Sp, S. pombe;C,C. elegans; B, bovine; D, Drosophila; As, Ascaris. The domain structure of several known RhoA-binding proteins can be grouped into two distinct classes that share sequence similarity in their minimal Rho binding sequences. (b) The class I Rho e€ector binding motif (REM-1) is shared by the PKN/PRK1 and PRK2 serine/threonine kinases and a novel protein that lack kinase domains (Rhophilin). Shown for PKN/PRK2 are: three predicted homologous coiled- coil (CC) domains referred to as homologous regions 1a, 1b and 1c (HR1a, HR1b, HR1c), (2) a Vo/HR2 domain that shows homology between PKN and PRK2 and highly similar to sequences surrounding the NH2-terminal Vo and pseudosubstrate sites of PCKe and PKCZ and (3) the COOH-terminal kinase domain. The Rho binding sequence (shaded region) is located in the NH2- terminal CC domain of PKN/PRK2 and Rhophilin. The multiple sequence alignment of class I REM shows a highly conserved sequence followed by a heptad repeat and are indicated by a solid square (&). The ®rst position of the heptad repeat is not predicted to be present in Rhophilin. Strictly conserved residues are indicated with an asterisk (*) and positions of very high amino acid similarity are indicated by a period (.); (c) Proteins containing the class II REM-2 motif include Rho-kinase, p160 ROCK and ROKa/b. These related kinases possess an NH2-terminal kinase domain, followed by a predicted large central coiled-coil (CC) domain and a pleckstrin homology (PH) domain that encompasses a cysteine-rich domain (CRD). The Rho binding sequence (shaded box) is located COOH-terminus of the central CC domain. The multiple sequence alignment of Rem-2 motifs contain a region of strict sequence conservation and are indicated by an asterisk (*). Citron shows a similar domain structure to Rho-kinase, p160 ROCK and ROKa/b. The NH2-terminal half of the protein contains a large (*875 residues) predicted coiled-coil (CC) domain. Like the other class II proteins, the CC domain is followed by CRD and PH domains. Citron also contains a proline-rich site (PR) that is a putative SH3 domain binding motif. The putative Rho-binding sequence (shaded box; ) is found in the extreme COOH-terminus of the CC domain. Apart from similarities in domain structure, no strong sequence homology was observed between the Citron and the Rem-1 or Rem-2 motif-containing proteins Rho family of small GTPases and transformation IZohnet al 1430 e€ector for these two Rac functions. Further mutagen- phosphorylated phosphoinositides can interact and esis of Rac and Cdc42 may identify mutants that will regulate a wide variety of protein targets, including a de®ne the contribution of other e€ectors to the number of proteins that regulate actin assembly function of Rac and Cdc42. (reviewed in Toker and Cantley, 1997). Since the A role for PAK in Ras and Rac transformation has oncogene responsible for the transforming activity of been suggested by two di€erent groups. First, Field the avian sarcoma virus 16 in chicken embryo and colleagues showed that kinase-de®cient mutants of ®broblasts encodes the p100 catalytic subunit chicken PAK could block oncogenic Ras transformation (Tang PI3K (Chang et al., 1997), upregulation of PI3K et al., 1997). However, since mutation of the CRIB activity may contribute to Ras and Rac growth motif did not abolish this inhibition of Ras transforma- transformation. tion. The inhibition was not likely to be due to Two Rac-binding proteins have been implicated in formation of nonproductive complexes with Rac or mediating Rac regulation of the actin cytoskeletal Cdc42. Interestingly, this antagonism of Ras was seen organization. In one study, Van Aelst and colleagues in Rat1, but not NIH3T3 cells. A second study showed used yeast two-hybrid screening and identi®ed a that expression of the CRIB motif-containing fragment partner of Rac1(POR1) protein that interacted with of PAK could reverse both Ras and Rac1 transforming GTP-complexed Rac1, but not Cdc42, RhoA or H-Ras activity in NIH3T3 cells (Osada et al., 1997). Since this (Van Aelst et al., 1996). The 34 kDa POR1 protein mutant PAK also blocked Ras and Rac activation of a showed no signi®cant sequence identity with any c-Jun-responsive promoter element (TRE), it was known proteins and no conserved motifs besides the proposed that titration of Rac and/or Cdc42 was presence of a putative leucine zipper motif. The responsible for inhibition of Ras transformation. Rac1(35A) e€ector domain mutant that is impaired The 70 kDa S6 kinase (p70S6K) also appears to be an in membrane ru‚ing also showed impaired binding to important e€ector of Rac and Cdc42 function. p70S6K is POR1. Truncated POR1 inhibited Rac1(12V)-induced activated by diverse mitogenic stimuli, including membrane ru‚ing, whereas full length POR1 acted to growth factors, cytokines and activated oncogenes synergistically enhance activated H-Ras(12V) mem- (Chou and Blenis, 1995). Present evidence supports brane ru‚ing activity. However, no synergistic an important role for p70S6K in the progression of cells interaction was seen between Rac1 and POR1.

from the G1 to S phase of the cell cycle. Chou and Finally, POR1 may also serve as an e€ector for Blenis (1996) showed that catalytically inactive p70S6K another small GTPase ARF6 and mediate related interacted with GTP-complexed Rac1 and Cdc42, but changes in actin cytoskeletal organization in CHO cells not RhoA, in vitro (Chou and Blenis, 1996). (D'Souza-Schorey et al., 1997). ARF6 also regulates Constitutively activated mutants of Rac1 and Cdc42, endocytic tracking. Thus, together with RhoD, it but not RhoA, stimulated p70S6K activity in vivo. provides further functional links between actin E€ector domain mutations abolished this activity. cytoskeleton reorganization and membrane tracking. Dominant negative Cdc42(17N) or Rac1(17N) In another study, yeast two-hybrid library screening blocked EGF- and PDGF-induced activation of with activated RhoA(63L) isolated a previously pp70S6K. Similar to the Ras:Raf interaction, Cdc42 identi®ed RhoA-binding protein, p160ROCK (Ishizaki et association with p70S6K in vitro alone did not activate al., 1996), that also interacts with activated forms of its kinase activity. Thus, Cdc42/Rac interaction with Rac1 and Cdc42, but not R-Ras (Lamarche et al., p70S6K must facilitate subsequent events that lead to 1996). Since the Rac1(37A) e€ector domain mutant no full kinase activation in vivo. It is presently not known longer interacted with ROCK and failed to induce S6K what aspect of Rac function is mediated by p70 . lamellipodia or G1 progression, it suggested that Rac1 and Cdc42 have been shown to complex the ROCK may mediate these two activities of Rac1. p85 subunit of PI3K in vitro and to co-precipitate with The Wiskott ± Aldrich syndrome (WAS) protein PI3K activity in vitro and in vivo (Zheng et al., 1994a; (WASP) contains the CRIB motif and was identi®ed Tolias et al., 1995). No CRIB motif is present in the as a Cdc42-binding protein that bound weakly to Rac1, p85 subunit. This interaction was found to be GTP- but not RhoA (Symons et al., 1996; AspenstroÈ m et al., dependent and a Cdc42(35A) e€ector domain mutant 1996; Kolluri et al., 1996). The WASP gene is mutated showed impaired p85 binding (Zheng et al., 1994a). in WAS patients. These WAS-a€ected patients possess Cdc42 stimulated the activity of immunoprecipitated an X-linked recessive disorder characterized by PI3K 2 ± 4-fold. Thus, although the sequence homology thrombocytopenia, which is characterized by recurrent of p85 with other Rho GAPs (e.g., BCR, N-chimaerin infections due to T and B cell function and eczema. and p190 Rho GAP) suggested that it acted as a Abnormalities seen in T and B cells suggested defects in negative regulator of Rho function, no GAP function the organization of the actin cytoskeleton (Kirchhausen has been demonstrated for p85. Instead, this GAP- and Rosen, 1996). WASP overexpression caused the related domain may serve to promote PI3K association formation of WASP clusters that were highly enriched with Rac/Cdc42 as an e€ector in regulating reorganiza- in polymerized actin in porcine aortic endothelial cells tion of the actin cytoskeleton (Zheng et al., 1994a). and clustering was inhibited by dominant negative Interestingly, another Rho GAP (N-chimaerin) has Cdc42(17N), but not Rac1(17N) (Symons et al., 1996). also been shown to exhibit properties of a Rac/Cdc42 Additionally, co-expression of WASP inhibited e€ector that mediates actin cytoskeletal events (Kozma Cdc42(12V) and Rac1(12V) induced changes in actin et al., 1996). cytoskeleton organization. Furthermore, a region of PI3K has also been implicated as an e€ector of Ras. homology between WASP and other proteins (VASP However, it is the p110 catalytic subunit that and Mena) that are involved in the organization of actin complexes with Ras-GTP (Rodriguez-Viciana et al., cytoskeleton and control of micro ®lament dynamics 1994, 1996). PI3K functions as a lipid kinase and its has been identi®ed (Gertler et al., 1996). Interestingly, Rho family of small GTPases and transformation IZohnet al 1431 this domain in VASP and Mena has been shown to Citron is a protein of unknown function that was interact directly with components of focal adhesions. identi®ed as a RhoC-binding protein in a yeast two- These results implicate WASP as an e€ector that hybrid library screen, and found to bind to Rac1, but mediates the activity of Cdc42/Rac in F-actin poly- not Cdc42, in vitro (Madaule et al., 1995). The merization (Symons et al., 1996) and reduced WASP interaction of Citron with two functionally distinct expression may contribute to the cytoskeletal abnorm- small GTPases does not necessarily indicate a common alities seen in WAS a€ected males (Kolluri et al., 1996). function for Citron. It is possible that interaction with Genetic evidence supporting this link comes from Rac and Rho may cause distinct subcellular locations analyses of the yeast counterpart of WASP, designated of Citron, or form distinct, signaling complexes, to Bee1p. Bee1p is a component of the cortical actin cause distinct downstream events. Narumiya and cytoskeleton and plays an essential role in the colleagues have recently described a splice variant of organization of actin ®laments (Li, 1997). Bee1 null Citron that contains an N-terminal Ser/Thr protein cells possess altered actin organization and are defective kinase domain that has *50% sequence identity to the in budding and cytokinesis. However, since Bee1 lacks a kinase domains of ROCK, ROK and MDK that are CRIB motif, its functions may be distinct from those of discussed below (Madaule et al., 1998). Designated human WASP. Finally, since a Cdc42 e€ector mutant Citron-K, this widely expressed form may mediate Rho that lacked WASP binding still stimulated lamellipodia regulation of cytokinesis. Messenger RNA encoding formation, WASP is clearly not the e€ector to mediate the previously discovered Citron variant (designated this Cdc42 e€ect (Lamarche et al., 1996). Citron-N) was detected only in brain tissue. Interestingly, WASP is also recognized by the Nck The 120 kDa ACK tyrosine kinase (ACK-1) adaptor protein, via its Src homology 3 domains speci®cally interacts with Cdc42 and not Rac or (Rivero-Lezcano et al., 1995; Quilliam et al., 1996), RhoA. In addition to its kinase domain, ACK also and Nck also complexes with other Rho family binding has an SH3 domain, a CRIB motif, and a proline-rich proteins (PAK1, PAK3 and PRK2) (Bagrodia et al., C-terminus which may represent putative SH3 domain 1995b; Quilliam et al., 1996; Bokoch et al., 1996; binding sites. To date, little is known regarding the role Galisteo et al., 1996). Nck associates with activated of ACK-1 as a Cdc42 e€ector. Recently, a 96 kDa receptor tyrosine kinases (Li et al., 1992; Lee et al., ACK-2 nonreceptor tyrosine kinase that shares the 1993). Therefore, it may serve as an adaptor that links same structural features with ACK and exhibits speci®c extracellular stimuli to Rho family proteins. interaction with GTP-bound Cdc42 was identi®ed IQGAP1, and the closely-related IQGAP2 (62% (Yang and Cerione, 1997). The reduced size of ACK- identity), have been shown to bind Rac1 and Cdc42, 2 is due primarily to the absence of sequences related but not RhoA (Brill et al., 1996; Kuroda et al., 1996). to those present in the N- and C- terminal regions of No CRIB motif is present in these proteins. IQGAP ACK-1. ACK-2 transcripts were detected in a wide was identi®ed originally as a human protein that variety of human tissues, with highest levels seen in harbored potential calmodulin binding IQ motifs brain and skeletal muscle tissue. Co-expression of upstream of a sequence related to the catalytic domain Cdc42(61L) and ACK-2 in COS cells caused increased of Ras GAPs (Weissbach et al., 1994), and was isolated ACK-2 autophosphorylation. Furthermore, the copre- independently by anity chromatography analyses for cipitation of the two proteins supports that possibility GTP-dependent Cdc42-binding proteins (Kuroda et al., that ACK-2 is a physiologically relevant e€ector for 1996). Whereas IQGAP1 RNA was found highly Cdc42. ACK-2 autophosphorylation was stimulated by expressed in lung, kidney and placenta, IQGAP2 was attachment as well as by EGF or bradykinin observed to be expressed predominantly in the liver stimulation. Thus, ACK-2 may mediate the actions of (Brill et al., 1996). Although both proteins show strong Cdc42 in response to diverse extracellular stimuli. homology with the Ras GAP of S. pombe,no The yeast two-hybrid screening of an Epstein ± Barr stimulation of Ras, Rac1 or Cdc42 GTPase activity virus-transformed B-cell library that identi®ed WASP has been detected (Brill et al., 1996; Kuroda et al., also identi®ed a second Cdc42-binding protein 1996). Since IQGAPs harbor a potential actin binding (AspenstroÈ m, 1997). Designated CIP4, this 545 amino domain, and were found to be accumulated at insulin- acid protein lacks a CRIB motif and shows sequence or Rac1-induced membrane ru‚ing areas, it was homology with a number of proteins that serve as postulated that IQGAPs may serve as e€ectors that regulators of actin organization (e.g., RhoGAP). mediate the actin cytoskeletal events of Cdc42 and Binding to Cdc42 was GTP-dependent, and no Rac1 (Kuroda et al., 1996). Consistent with this binding was seen to Rac1-GTP. Overexpression of possibility, it was found that Cdc42 and IQGAP1 CIP4 caused a reduction in stress ®ber formation and formed a stable complex in vivo along with F-actin co-expression of Cdc42(61L) caused a clustering of (Erickson et al., 1997). EGF stimulation also promoted CIP4. Thus, CIP4 may interact with Cdc42 in vivo to formation of Cdc42:IQGAP1 complexes in COS cells, regulate actin organization although it does not appear suggesting that growth factor stimulation may promote to mediate Cdc42 induction of ®lopodia. an IQGAP1-mediated link between Cdc42 and the In summary, there exists multiple candidate e€ectors actin cytoskeleton. Finally, additional modes of of Rac and/or Cdc42 function and more are likely to IQGAP regulation are suggested by evidence that be identi®ed. The existence of Rac/Cdc42-binding calmodulin and calcium caused disruption of proteins that lack the CRIB motif indicates that at Cdc42:IQGAP1 interaction in vitro (Joyal et al., 1997). least two, if not more, Rac- and/or Cdc42-binding Other candidate e€ectors of Rac include ROKa,a motifs will be identi®ed. Currently, it is not clear, previously described RhoA-binding protein (Leung et which, if any, of these Rac-binding proteins are al., 1996b), POR-2, a protein of unknown function physiologically relevant e€ectors. Furthermore, which (Van Aelst et al., 1996), and tubulin (Best et al., 1996). e€ectors mediate Rac/Cdc42 regulation of actin Rho family of small GTPases and transformation IZohnet al 1432 organization, gene expression and regulation of cell al., 1996). Interestingly, LPA resulted in a Rho- proliferation and invasion remain to be characterized. dependent activation of PKN/PRK1 supporting a role With regards to transformation, our recent evaluation for this kinase in mediating Rho function. The of Rac e€ector domain mutants failed to implicate any observation that staurosporine, a potent inhibitor of known Rac1 function with transforming potential PKC (and possibly PKN/PRK1), blocked formation of (Westwick et al., 1997a). One interpretation is that focal adhesions suggests that PKN may indeed mediate multiple pathways promote Rac growth regulation and this action of Rho proteins. Additionally, PKN has that no one pathway alone is necessary. Alternatively, been shown to interact with and phosphorylate the it suggests that some as yet undiscovered Rac function head-rod domain of neuro®lament proteins suggesting is responsible for Rac-mediated transformation. its role in the regulation of neuro®lament protein Regardless of which is the correct scenario, it remains assembly (MoÈ sch et al., 1996). Hence, PKN may be a to be de®ned what Rac function(s) is required to potential mediator of the growth factor-induced promote oncogenic Ras transformation. Finally, stimulation of intermediate ®laments. despite their signi®cant number of shared e€ectors, it PRK2 was independently isolated by two groups is clear that Rac1 and Cdc42 are functionally distinct searching for Nck- or RhoA-interacting proteins. First, proteins. For example, human Cdc42, but not Rac1 or Quilliam et al. (1996) isolated PRK2 using an Rac2, can restore a Cdc42Sc defect in S. cerevisiae expression library screen for proteins that bound to (Shinjo et al., 1990). Furthermore, as described above, the SH3 domains of the Nck SH2/SH3 adaptor protein the transforming functions of Rac1 and Cdc42 are (Quilliam et al., 1996). It was also shown to bind RhoA, quite distinct. but not Rac1 or Cdc42. In vitro and co-expression of PRK2 with activated RhoA caused synergistic enhance- ment of RhoA-dependent activation of SRF expression. A plethora of candidate e€ectors of Rho Vincent and Settleman (1997) puri®ed PRK2 (and A growing number of Rho-binding proteins have also PKN) by anity chromatography with RhoA from been identi®ed. Two distinct Rho-binding sequence rat liver extracts. However, they found that COS cell- motifs have also been described for a subset of Rho- expressed PRK2 interacted with GTP-bound Rac1, but binding proteins (Reid et al., 1996; Fujisawa et al., interacted with, and activated, both RhoA-GTP and 1996). Both Rho e€ector binding motifs are distinct RhoA-GDP. They also showed that a kinase-defective from the CRIB motif and are associated with proteins mutant of PRK2 caused a disruption in actin stress that contain predicted coiled-coil (CC) domains (Cohen ®bers in NIH3T3 cells. Thus, they suggested that PRK2 and Parry, 1994). These CC domains may provide the may be a common e€ector of both Rac and Rho. The basis for the multimerization of these proteins with reason for the di€erences seen in these two studies is not Rho and other signaling molecules in a complex with known. Finally, PKN/PRK1 and PRK2 were also Rho. identi®ed independently as liver protease-activated Two closely related serine/threonine kinases that kinases 1 and 2, respectively (Yu et al., 1997). show strong sequence similarity to the kinase domain Rhophilin, and to a lesser degree Rhotekin, share

of PKCs (PKN/PRK1 and PRK2), and two novel NH2-terminal sequence homology with the PKN/PRK1 proteins lacking kinase domains (Rhotekin and and PRK2 Rem-1 motif. Rhotekin was identi®ed by Rhophilin), appear to share a distinct Rho e€ector yeast two-hybrid screening for RhoC-binding proteins binding motif (designated class I, REM-1) (Figure 8b). (Reid et al., 1996). It also interacted with GTP- PRK1 and PRK2 (58% amino acid identity) were complexed RhoA and RhoB, but not Rac1 or Cdc42. identi®ed originally by a PCR-mediated approach to Rhophilin was isolated by yeast two-hybrid screening identify sequences that encoded novel PKC-related of a mouse embryo cDNA library for RhoA-binding human proteins (Palmer et al., 1995). The encoded proteins (Watanabe et al., 1996). Rhophilin showed 120 ± 130 kDa proteins identi®ed by this approach strong GTP-dependent interaction with RhoA, little

contain an NH2-terminal basic sequence encompassing with RhoC, and less with RhoB. It also shares a

GAXN (a putative pseudosubstrate domain), a REM-1 putative NH2-terminal CC with PKN/PRK1 and Rho-binding sequence, followed by amphipathic helix PRK2. and leucine zipper domains, and a COOH-terminal A family of novel serine/threonine kinases share a kinase domain (Figure 8b). Unlike PKC, neither are distinct, second Rho e€ector binding motif (designated regulated by phorbol esters or calcium. Both PRK1 class II/REM-2) (Figure 8c). ROKa/Rho-kinase and PRK2 show broad tissue expression. (Leung et al., 1996b) and p160ROCK/ROKb/ROCK-II The 71 kDa Rhophillin protein also contains an (Leung et al., 1996a; Ishizaki et al., 1996) share an

NH2-terminal Rem-1 binding motif. However, it lacks NH2-terminal kinase domain that has strong sequence a kinase domain or any other known catalytic identity with the kinase domain of myotonic dystrophy

sequences. Aside from the NH2-terminal Rem-1 kinase (MDK) (Ishizaki et al., 1996), a central putative motif, two proline-rich motifs and putative SH3- CC domain and a COOH-terminal PH domain (Mayer binding domains are present in the COOH-terminus. et al., 1993) that is split by a cysteine-rich, zinc- Thus, Rho-binding proteins with class I binding motifs dependent folding domain. Interestingly, the Rho/Rac1 also typically contain other putative protein-protein or binding protein Citron (Madaule et al., 1995) contains

protein-lipid binding motifs, suggesting that these a related NH2-terminal CC domain that includes the e€ectors may serve to promote targeting and/or Rho-binding domain, and a related kinase domain multi-protein complex formation with Rho proteins. (Madaule et al., 1998). However, the Rho-binding PRK1 was identi®ed independently by anity sequence exhibits weaker sequence homology to the chromatography analysis for RhoA-binding proteins Rem-2 motif present in these kinases. Hence, Citron and designated protein kinase N (PKN) (Watanabe et may possess a Rho-binding element distinct from the Rho family of small GTPases and transformation IZohnet al 1433 class II motif. Both Citron-K and Citron-N also (Chrzanowska-Wodnicka and Burridge, 1996). The contain a COOH-terminal cysteine-rich motif, a PH COOH-terminal sequences of MBS that bind to RhoA domain and a proline-rich, putative SH3-binding motif. share structural similarity (polybasic region followed Thus, like other Ras and Rho family e€ectors, Citron by leucine zipper-like motif) with PKN/PRK1. may also function as a sca€olding protein for Rho. However no detectable Rem-2 motif was observed ROKa was identi®ed in expression screening for (Kimura et al., 1996). RhoA-binding proteins serine/threonine kinase (Leung Another e€ector that may link Rho with the actin et al., 1996b). ROKa showed a cytoplasmic location cytoskeleton involves members of a formin-related and co-expression of RhoA(14V) caused an increased family of proteins that include the S. cerevisiae association of ROKa with the pellet fraction, suggest- Bni1p, the Drosophila Diaphanous and Cappuchino ing that RhoA promoted its translocation to mem- proteins, and the mammalian Bnip1 homolog branes. Injection of DNA encoding full length ROKa p140mDia (Castrillon and Wasserman, 1994; Emmons caused the formation of stress ®bers and focal et al., 1995; Peterson et al., 1995; Marhoul and Adams, adhesions in HeLa cells (Leung et al., 1996a). A 1995). Bni1p and the related Bnr1p regulate the e€ects closely related protein with 64% overall identity to of Rho proteins on actin organization. p140mDia can ROKa and 90% identity to the ROKa kinase domain, interact with mammalian RhoA-GTP and pro®lin. was subsequently isolated (ROKb; also ROCK-II and Furthermore, p140mDia, pro®lin and RhoA are co- p160 ROCK). In vitro binding analyses using puri®ed localized in a Rho-dependent structures such as recombinant proteins showed that both ROKa and membrane ru‚es in motile cells, cleavage furrows in ROKb bound RhoA, RhoB and RhoC, but not Rac1 mitotic cells, and phagocytic cups induced by or Cdc42 (Leung et al., 1996a), although subsequent ®bronectin (Watanabe et al., 1997). Thus, p140mDia yeast two-hybrid analysis also showed interaction with may mediate actin assembly via a pro®lin-mediated Rac1 (Joneson et al., 1996a) and Cdc42 (Lamarche et mechanism. al., 1996). Since Rhotekin and Rhophillin lack known catalytic Narumiya and colleagues identi®ed p160ROCK (a Rho- functions, essentially nothing has been described associated CC domain-containing protein kinase) as a regarding their functions. At least three possible novel RhoA-binding protein serine/threonine kinase mechanisms for the function of these proteins can be that showed 44% identity with the kinase domain of envisioned. They may either be directly involved in MDK (Ishizaki et al., 1996). RhoA was shown to mediating some aspect of Rho function that does not associate with p160ROCK in vivo and this association require a catalytic function by promoting their promoted weak enhancement of its autophosphorylat- translocation to various components of cytoskeleton, ing activity. Finally, Kaibuchi and colleagues identi®ed or they may somehow negatively regulate the function the p164 Rho-kinase by anity chromatography of Rho proteins by forming an inactive complex and analyses for RhoA-GTP binding proteins (Matsui et preventing the accessibility to other downstream al., 1996). Rho-kinase is likely a splice variant of ROKa, e€ectors. Another possible function is that they may since they di€er only at their NH2-termini, with Rho- act as sca€olding proteins that regulate the coordinate kinase containing an additional nine amino acids. Since activities of Rho proteins and their downstream the kinase activities of these proteins showed only e€ectors in a multimeric structure. limited stimulation by association with RhoA in vitro,it Observations that stimulation by G protein-coupled is likely that RhoA binding may facilitate other events receptor agonists that activate Rho (LPA and that lead to full kinase activation. thrombin), or C3 treatment to block Rho function, Another RhoA-GTP binding protein, myosin light of undi€erentiated neoronal cells (N1E-115, NG108-15 chain (MLC) phosphatase, together with Rho kinase, and PC12 cells) have implicated Rho proteins in provides a mechanism for RhoA regulation of stress causing growth cone collapse, retraction of developing ®ber formation (Kimura et al., 1996). The myosin neurites, and transient rounding of the cell body binding subunit (MBS) of MLC phosphatase was (Mackay et al., 1995; Luo et al., 1996). One recently identi®ed as a RhoA-binding protein by anity identi®ed RhoA-binding protein, designated p116RIP, chromatography analysis of bovine brain membrane showed the same activity as C3 or dominant negative extracts. RhoA binding and activation of Rho kinase RhoA when overexpressed in N1E-115 cells. Although stimulates Rho kinase phosphorylation of MBS. This p116RIP showed preferential binding to RhoA-GTP in results in the inactivation of myosin phosphatase yeast two-hybrid binding analyses, in vitro binding activity, leading to activation of myosin through a analyses showed equivalent binding to either GDP- or net increase in MLC phosphorylation by other protein GTP-bound GST-RhoA. p116RIP is a novel protein that kinases. MLC phosphorylation has been shown to contains a PH domain, two putative SH3 domain- induce a conformational change in myosin, which binding sites and a CC region. It was identi®ed in a increases its binding to actin ®laments, causing yeast two-hybrid screen for RhoA-binding proteins, ultimately the formation of stress ®bers (Chrzanows- together with a novel RhoGDI (RIP1-RhoGDI2), a ka-Wodnicka and Burridge, 1996). Rho-stimulated novel Db1 family protein (RhoGEF), and another contraction of ®broblasts was blocked by the MLC RhoA-GTP binding protein (RIP4) (Gebbink et al., kinase inhibitor (KT5926), resulting in decreased MLC 1997). phosphorylation and loss of stress ®bers and focal adhesions. Additionally, Rho kinase can also phos- Structural requirements for Rho family protein:e€ector phorylate MLC at the same sites that are phosphory- interactions lated by MLC kinase (Amano et al., 1996). Finally, current evidence indicates that RhoA causes stress The identi®cation of multiple binding proteins strongly ®bers and focal adhesion through distinct pathways suggests that Rho family proteins utilize multiple Rho family of small GTPases and transformation IZohnet al 1434 e€ector-mediated pathways to cause its diverse array of concept that some small GTPase:e€ector interactions cellular e€ects. Like Ras, the regions of Rho family are not GTP-dependent (e.g., PI5K). Instead, some proteins important for e€ector interaction also appear may be GDP-dependent, others may associate with to be complex. The regions analogous to the core Ras either GDP or GTP, yet activation may still be GTP- e€ector sequence (Ras residues 32 ± 40), and ¯anking dependent. residues are clearly important for all Rho family protein interactions with e€ectors. Mutagenesis and structural analyses (Hirshberg et al., 1997) suggest at Future directions least two additional, distinct, regions are also likely to mediate interaction with e€ectors. Despite the rapid accumulation of information on Rho There is some evidence that the Rho insert domain family proteins, it is clear that we are only beginning to may be important for interaction with at least some appreciate the complex nature of their biology and e€ectors (Freeman et al., 1996). Mutations in the Rac1 biochemistry. Many questions remain. Will the caused a marked decrease in superoxide production diversity of cellular processes that involve Rho family without perturbing p67PHOX binding, indicating that it proteins continue to increase? Yes, the already was not involved directly in e€ector binding (Nisimoto outdated nature of this review veri®es that this will et al., 1997). Instead, it was proposed that it may be the case. The continued unearthing of yet more Dbl interact with other components of the NADPH oxidase family proteins argues that the diversity of cellular in neutrophils that was important for activity. In a processes that activate Rho family proteins will second study, a Cdc42 mutant protein, where the insert continue to increase. More Rho family proteins will sequence was deleted by replacing it with the also be discovered. Thus, the delineation of additional corresponding region from H-Ras, retained the ability Rho-mediated functions promises to be a major theme to activate PAK-3 in vivo and to bind other CRIB for the coming years. Ample evidence from experi- motif-containing proteins in vitro (ACK and WASP) mental cell systems implicate Rho family proteins as (Wu et al., 1997). However, a reduced anity for critical components of oncogenic Ras transformation. interaction from IQGAP1 was seen. Furthermore, Do Rho family proteins play such a role in human while neither GEF or GAP activities were altered, cancer cells? If so, are Rho-mediated pathways RhoGDI inhibition of GDP dissociation was impaired important targets for the development of anti-cancer in the insert mutant of Cdc42. Mutation of the insert drugs? What speci®c aspects of Ras transformation are domains of Rac1 (Joneson and Bar-Sagi, 1998), Cdc42 contributed by Rho family proteins? (Wu et al., 1998), or RhoA (Quilliam LA, unpublished Understanding the mechanisms of Rho family observation) perturb their growth-promoting and/or protein signaling, both upstream and downstream, transforming activites. Thus, de®ning the e€ector remains limited. How extracellular signals cause targets that interact with their respective insert activation of Rho family proteins remains a poorly domains will delineate the signaling pathways that undestood area. Will Db1 family proteins provide this promote cellular transformation. link? The roster of Rho family binding proteins will Studies with chimeric proteins between Rac1 and continue to expand. Which are physiologically relevant RhoA identi®ed a second, COOH-terminal region of e€ectors? What is the role of each e€ector and how is Rac1 (residues 143 ± 175) important for Rac e€ector e€ector utilization is controlled? Our understanding of function. Hall and colleagues showed that both amino Rho family signal transduction will continue to evolve. and carboxyl terminal regions of Rac1 were required Complex interrelated networks, rather than simply

for PAK and p67PHOX binding, as well as for Rac- linear pathways, will likely be de®ned. Cell type induced lamellipodia formation (Diekmann et al., di€erences will continue to add further complexity. In 1995). In agreement with their observations, we also summary, the only thing that seems certain is that observed that the Rac73Rho chimera did not bind PAK evidence will continue to mount, establishing Rho or induce lamellipodia, but instead induced stress ®ber family proteins as key regulators of many cellular formation (Westwick et al., 1997). Furthermore, we processes in normal and neoplastic cells. observed a loss of JNK activation, suggesting that the e€ector required for JNK activation also requires interaction with COOH terminal Rac sequences. Thus, like Ras, the e€ector-interacting sequences of Acknowledgements Rho family proteins are also composed of multiple, We thank Jennifer Parrish for excellent assistance in the distinct sequences. preparation of the text, references and ®gures. Our studies The importance of sequences beyond switch I and II are supported by NIH grants to CJD (CA42978, CA52072, in interaction with e€ectors underlines an emerging CA55008 and CA67771) and SC (CA64569 and CA70308).

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