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 eectors to cause its diverse considerable research interest over the past 5 years. actions on cell proliferation, dierentiation 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 tracking, 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 eorts 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; eectors 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 eector 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 dier 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 dierences 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 rues, ®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 eectors (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.