I. INTRODUCTION

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Small GTP-binding proteins (G proteins) are monomeric G proteins with molecular masses of 20-40 kDa. The Ha-Ras and Ki-Ras genes were first discovered as the v-Ha-Ras and v-Ki-Ras oncogenes of sarcoma viruses around 1980 (111, 660). Their cellular oncogenes were then identified in humans, and their mutations were furthermore found in some human carcinomas (146, 252, 499, 561, 626, 661). The mutated forms were subsequently shown to stimulate proliferation and transformation of cultured cells (71, 84, 182, 681). Moreover, the mutated forms were shown to induce cell differentiation in neuronal cells (41, 249, 523). These findings drew the attention of many scientists not only in the cancer research field but also in many other fields. Finally, these Ras proteins were shown to be related to the heterotrimeric G proteins, such as G_s and G_i, and G proteins involved in protein synthesis, such as elongation factor Tu (EF-Tu) (222, 644, 659).

The Rho gene was discovered as a homolog of the Ras gene in Aplysia in 1985 (421); the YPT1 gene, which had been discovered as an open reading frame between the actin and tubulin genes in the yeast Saccharomyces cerevisiae (S. cerevisiae) in 1983 (208), was identified to encode a small G protein in 1986 (641); Arf protein, which was purified as a cofactor for the cholera toxin-catalyzed ADP-ribosylation of G_s in 1984 (326), was identified to encode a small G protein in 1986 (327). The SEC4 gene, which had been isolated as a gene involved in secretion in the yeast in 1980 (527), was identified to encode a small G protein in 1987 (623). These results suggested the presence of a big family of Ras-like small G proteins. Actually, many small G proteins were systematically isolated by molecular biological (100, 101, 105, 551, 573) and biochemical (291, 337, 344, 534, 535, 793, 797) methods.

Now, more than 100 small G proteins have been identified in eukaryotes from yeast to human, and they comprise a superfamily (60, 250, 701). The members of this superfamily are structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families (Table 1 and Fig. 1). In the yeast S. cerevisiae, sequence analysis against complete genomic sequence has revealed that there are 4 Ras family members, 6 Rho family members, 11 Rab family members, 7 Sar1/Arf family members, and 2 Ran family members (210, 383). The functions of many small G proteins have recently been elucidated: the Ras subfamily members (Ras proteins) of the Ras family mainly regulate gene expression, the Rho/Rac/Cdc42 subfamily members (Rho/Rac/Cdc42 proteins) of the Rho family regulate both cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf family members (Rab and Sar1/Arf proteins) regulate intracellular vesicle trafficking, and the Ran family members (Ran) regulate nucleocytoplasmic transport during the G₁, S, and G₂ phases of the cell cycle and microtubule organization during the M phase. Many upstream regulators and downstream effectors of small G proteins have been identified, and modes of activation and actions have gradually been elucidated. In this review, functions of small G proteins and their modes of activation and action are described. However, this review may not cover all detailed information regarding each small G protein; readers may refer to other recent excellent reviews (3, 49, 58, 80, 82, 139, 325, 417, 420, 428, 483, 491, 519, 536, 630, 713, 744, 758).

View larger version (35K): [in this window] [in a new window]   Fig. 1. Dendrogram of the small G protein superfamily. [Modified from Garcia-Ranea and Valencia (210).]

As to nomenclature, the term small GTPases is often used, but "small G proteins" is used here because small G proteins have both GDP/GTP-binding and GTPase activities. In many cases, the GTPase activity is necessary for the termination of the functions of small G proteins, but not essential for them to perform their functions. From this point of view, the term GTPase is misleading. "G proteins" represent heterotrimeric G proteins and "small GTP-binding proteins" should be used, but just for simplicity "small G proteins" is used here. G proteins used here include heterotrimetric G proteins (223), G proteins involved in protein synthesis (338), and small G proteins. Guanine nucleotide exchange factor (GEF) is often used, but guanine nucleotide exchange protein (GEP) is used here, because all GEFs thus far found are proteins and "GEPs" is a more correct term.

	II. GENERAL PROPERTIES

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A comparison of the amino acid sequences of Ras proteins from various species has revealed that they are conserved in primary structures and are 30-55% homologous to each other. Among Ras proteins, each protein shares relatively high (50-55%) amino acid identity, whereas Rab and Rho/Rac/Cdc42 proteins share ~30% amino acid identity with Ras proteins (250, 742). Nevertheless, like other G proteins, all small G proteins have consensus amino acid sequences responsible for specific interaction with GDP and GTP and for GTPase activity, which hydrolyzes bound GTP to GDP and P_i (61, 701, 742) (Fig. 2A). Moreover, they have a region interacting with downstream effectors. In addition, small G proteins belonging to Ras, Rho/Rac/Cdc42, and Rab proteins have sequences at their COOH termini that undergo posttranslational modifications with lipid, such as farnesyl, geranylgeranyl, palmitoyl, and methyl moieties, and proteolysis (89, 228, 427, 701, 811) (Fig. 3). Arf proteins have an NH₂-terminal Gly residue that is modified with myristic acid (493). Sar1 and Ran do not have such sequences to direct posttranslational modifications.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Structure of small G proteins. A: consensus amino acid sequences responsible for specific interaction with GDP and GTP and for GTPase activity. B: crystallographic structure of small G proteins. The crystallographic structure of Ha-Ras is representatively shown. A, Ala; D, Asp; E, Glu; G, Gly; K, Lys; N, Asn; S, Ser; X, any amino acid. [Modified from Pai et al. (560).]

View larger version (25K): [in this window] [in a new window]   Fig. 3. The COOH-terminal structures and posttranslational modifications of small G proteins. The COOH-terminal regions of small G proteins are classified into at least four groups (1-4). The Cys-A-A-X structure is furthermore subclassified into two groups (1a and 1b). A, aliphatic acid; X, any amino acid; P, palmitoyl; F, farnesyl; GG, geranylgeranyl.

Crystallographic and NMR analyses of some small G proteins, including Ha-Ras, N-Ras, Rap2A, RhoA, Rac1, Rab3A, Rab7, Arf1, and Ran, have revealed that all GDP/GTP-binding domains have a common topology (219) (Fig. 2B). By comparison of the structure of Ha-Ras in the GTP-bound conformation and the GDP-bound conformation, two highly flexible regions surrounding the -phosphate of GTP have been established (471, 560): the switch I region within loop L2 and ₂ (the effector region) and the switch II region within loop L4 and helix ₂. A two-state model for the movement of the effector loop in the GTP-bound form of Ha-Ras has been established; the flexibility of the loop can conveniently be monitored by a large shift of Tyr-32 relative to the phosphate groups, because the hydroxyl group of Tyr-32 forms hydrogen bond with the -phosphate of GTP. Binding of c-Raf-1, an effector of Ras proteins (see below), stabilizes the effector loop in the active conformation (459).

The COOH-terminal regions are classified into at least four groups: 1) Cys-A-A-X (A, aliphatic acid; X, any amino acid); 2) Cys-A-A-Leu/Phe; 3) Cys-X-Cys; and 4) Cys-Cys (89, 228, 427, 701) (Fig. 3). The Cys-A-A-X structure is furthermore subclassified into two groups: one has an additional Cys residue upstream of the Cys residue of the Cys-A-A-X structure (1a), and the other has a polybasic region (1b). In the case of the Cys-A-A-X structure, Ha-Ras and Ki-Ras are first farnesylated at the Cys residue followed by the proteolytic removal of the A-A-X portion and the carboxylmethylation of the exposed Cys residue (90, 201, 247, 256, 313). Ha-Ras has an additional Cys residue that is further palmitoylated (256). The Cys-A-A-Leu structure of Rap1 is first geranylgeranylated followed by the same modifications (336). Both Cys residues of the Cys-X-Cys structure of Rab3A are geranylgeranylated, and the COOH-terminal Cys residue is carboxylmethylated (177). Both Cys residues of the Cys-Cys structure of Rab1 are geranylgeranylated, but the COOH-terminal Cys residue is not carboxylmethylated (672). The lipid modifications of these small G proteins are necessary for their binding to membranes and regulators and for their activation of downstream effectors as described below (89, 228, 257, 427, 701, 702, 811).

The farnesyl moiety is derived from farnesyl pyrophosphate, an intermediate product of the mevalonate pathway which produces cholesterol from mevalonate (231). Mevalonate is produced from 3-hydroxy-3-methyglutaryl-CoA by the action of 3-hydroxy-3-methyglutaryl-coenzyme reductase. A specific inhibitor for this enzyme, named pravachol, is used as a very effective drug for arteriosclerosis (171, 231). The geranylgeranyl moiety is derived from geranylgeranyl pyrophosphate, which is an intermediate product for the synthesis of dolichol and ubiquinone (220, 231). The palmitoyl moiety is derived from palmitoyl CoA. The methyl moiety is derived from S-adenosyl-methionine. The enzymes that transfer the prenyl moieties have been isolated and characterized (811). The farnesylation of the Cys-A-A-X structure is catalyzed by farnesyltransferase, the geranylgeranylation of the Cys-A-A-Leu structure is catalyzed by geranylgeranyltrasnferase I, and the prenylation of the Cys-X-Cys and Cys-Cys structures is catalyzed by geranylgeranyltransferase II. Farnesyltransferase and geranylgeranyltransferase I consist of two subunits,  and  subunits, and the -subunits of both enzymes are identical (648). Geranylgeranyltransferase II consists of three subunits, originally termed component A but recently renamed Rab escort protein I (Rep1), and - and -subunits (289, 645, 646, 672). Rep1 binds unprenylated Rab proteins, presents them to the catalytic -subunits, and remains bound to Rab proteins after the geranylgeranyl transfer reaction (20). In cells, Rab GDP dissociation inhibitor (GDI) (see below) may dissociate this product from Rep1, allowing multiple cycles of catalysis. The human Rep1 gene has been identified by positional cloning as that responsible for choroideremia, which is an X-linked form of retinal degeneration (131, 132, 187). Loss of Rep1 activity causes the reduced prenylation of Ram/Rab27, which is expressed at high levels in the retinal cell layers, and the degeneration of this protein in the progression of this disease (647). Palmitoyltransferase that palmitoylates Ha-Ras has been purified (406), but it is not yet known whether this enzyme is the one that functions in vivo. Methyltransferases that transfer the methyl moieties to small G proteins having Cys-A-A-X, Cys-A-A-Leu, and Cys-X-Cys structures have not been well characterized. A protease, Rce1, that removes the A-A-X and A-A-Leu portions of Ha-Ras, N-Ras, Ki-Ras, and Rap1B, has recently been identified (345, 557).

According to the structures of small G proteins, they have two interconvertible forms: GDP-bound inactive and GTP-bound active forms (60, 250, 701) (Fig. 4). An upstream signal stimulates the dissociation of GDP from the GDP-bound form, which is followed by the binding of GTP, eventually leading to the conformational change of the downstream effector-binding region so that this region interacts with the downstream effector(s). This interaction causes the change of the functions of the downstream effector(s). The GTP-bound form is converted by the action of the intrinsic GTPase activity to the GDP-bound form, which then releases the bound downstream effector(s). In this way, one cycle of activation and inactivation is achieved, and small G proteins serve as molecular switches that transduce an upstream signal to a downstream effector(s).

View larger version (27K): [in this window] [in a new window]   Fig. 4. Regulation of small G protein activity.

Thus the rate-limiting step of the GDP/GTP exchange reaction is the dissociation of GDP from the GDP-bound form. This reaction is extremely slow and therefore stimulated by a regulator, named GEP (also called GEF or guanine nucleotide releasing factor), of which activity is often regulated by an upstream signal. GEP first interacts with the GDP-bound form and releases bound GDP to form a binary complex of a small G protein and GEP. Then, GEP in this complex is replaced by GTP to form the GTP-bound form. Most GEPs, such as Son of Sevenless (SOS), a Ras GEP, and Rab3 GEP, are specific for each member or subfamily of small G proteins (56, 75, 762), but some GEPs, such as Dbl, a GEP active on Rho/Rac/Cdc42 proteins, show wider substrate specificity (259, 788). The GDP/GTP exchange reactions of Rho/Rac/Cdc42 and Rab proteins are furthermore regulated by another type of regulator, named Rho GDI and Rab GDI, respectively (28, 206, 453, 629, 732). This type of regulator inhibits both the basal and GEP-stimulated dissociation of GDP from the GDP-bound form and keeps the small G protein in the GDP-bound form. Rho GDI and Rab GDI show wider substrate specificity than GEPs and GTPase-activating proteins (GAPs) and are active on all Rho/Rac/Cdc42 and Rab proteins, respectively (18, 275, 391, 627, 629, 732, 737). Thus the activation of Rho/Rac/Cdc42 and Rab proteins is regulated by positive and negative regulators. Recently, Ran GDI, p10/NTF2, has also been reported (125, 485, 563, 789), but GDIs have not been identified for other small G proteins. The GTPase activity of each small G protein is variable but relatively very slow and is stimulated by GAPs. Most GAPs, such as Ras GAP and Rab3 GAP, are specific for each member or subfamily of small G proteins (56, 205, 727), but some GAPs, such as p190, a GAP active on Rho/Rac/Cdc42 proteins, show wider substrate specificity (656).

Small G proteins as well as heterotrimeric G proteins are present only in eukaryotes from yeast to human, although G proteins involved in protein synthesis such as elongation factors exist in both prokaryotes and eukaryotes. Most small G proteins are widely distributed in mammalian cells, and most cells have the Ras, Rho, Rab, Sar1/Arf, and Ran families, although expression levels of their members may vary from one type to another. A few members show tissue-specific expression; for instance, Rab3A is expressed in cells having a regulated secretion pathway, such as neurons, neuroendocrine cells, and exocrine cells (140, 183, 476, 477, 625). Rab17 is detected in epithelial cells (413). Most small G proteins are localized either in the cytosol or on membranes. Ran is localized either in the cytosol or in the nucleus. Each small G protein is localized to a specific membrane. Ras proteins are localized at the cytoplasmic face of the plasma membrane. This localization is mediated by the posttranslational modifications with lipid. The farnesyl moiety of Ha-Ras and Ki-Ras alone is not sufficient for their binding to the membrane (256). In the case of Ha-Ras, both the farnesyl and palmitoyl moieties are necessary, whereas in the case of Ki-Ras, both the farnesyl moiety and the neighboring clustered polybasic amino acids are necessary. The farnesyl and palmitoyl moieties may interact with the acyl moieties of the phospholipids, whereas the polybasic amino acids may interact with the polar head groups of the acidic phospholipids. The methyl moiety also contributes markedly to efficient membrane association (255). Rap1 is geranylgeranylated and has clustered polybasic amino acids. Most Rab proteins have either a Cys-X-Cys or Cys-Cys structure of which Cys residues are both geranylgeranylated. These small G proteins are localized at the cytoplasmic faces of distinct membrane compartments. It has not been experimentally clarified how Rap1 and Rab proteins exactly interact with the membranes, but it is likely that both the prenyl moiety and the polybasic region or two prenyl moieties are necessary. In contrast, Arf proteins have one myristoyl moiety and Sar1 has no lipid moiety, but they interact with the cytoplasmic faces of membranes. Arf proteins interact with membrane lipids by its myristoylated and amphipathic NH₂-terminal helix (21, 47). In the case of Sar1, it may interact with the phospholipid through only peptide region. Small G proteins, such as Rho/Rac/Cdc42 and Rab proteins, located on the plasma membrane and the cytosol are translocated between these two sites. Ran is also translocated between the cytosol and the nucleus through the nuclear pore complexes (NPCs).

	III. RAS PROTEINS AS REGULATORS OF GENE EXPRESSION

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Three Ras proteins are now known, Ha-Ras, Ki-Ras, and N-Ras, which are capable of transforming mammalian cells when activated by point mutations (71, 84, 182, 681). In the yeast S. cerevisiae, there are two members of Ras proteins, Ras1 and Ras2, that are essential for cell viability, and these yeast genes are functionally replaceable by mammalian genes (141, 577). The downstream effector of Ras proteins was first identified to be adenylate cyclase in the yeast S. cerevisiae (66, 721). Mammalian adenylate cyclase is directly regulated by heterotrimeric G proteins, but not by Ras proteins. Subsequently, genetic, cell biological, and biochemical studies in Caenorhabditis elegans, Drosophila, and mammalian cells established the mode of action of Ras proteins; they directly bind to and activate Raf protein kinase (82, 151a, 254a, 743, 759, 767, 815), which then induces gene expression through the mitogen-activated protein (MAP) kinase cascade in response to various extracellular signaling molecules (145, 299, 416). Other studies have clarified that Ras proteins regulate not only cell proliferation but also differentiation (41, 249, 523), morphology (41a, 182, 779), and apoptosis (334). Ras proteins regulate these functions mainly through gene expression, but it has not been established whether the Ras protein-mediated morphological changes are a direct effect or an indirect effect through gene expression. Another characteristic feature of Ras proteins is that the mutations of their genes and their regulator genes cause human cancers (9, 42, 59, 445, 607, 756, 786). Thus Ras proteins are crucially important molecules not only for biology but also for human health.

Ras protein activity is regulated by GEPs and GAPs, and activation is induced by a large variety of extracellular signals, most notably signals that activate receptors with intrinsic or associated tyrosine kinase activity (168, 207, 399, 549, 617) (Fig. 5). Phosphotyrosines serve as docking sites for the adaptor proteins, such as GRB2 and SHC/GRB2 complex, which then recruit SOS, the most characterized Ras GEP, from the cytosol to produce a receptor-adaptor-GEP complex. SOS recruited to the plasma membrane then stimulates a Ras protein located at the cytoplasmic face of the plasma membrane and converts it from the the GDP-bound form to the GTP-bound form. It is believed that GRB2 recruits SOS from the cytosol to the plasma membrane without affecting its GEP activity, but the possibility has not been totally excluded that GRB2 both recruits and activates SOS. Receptors not directly associated with tyrosine kinases, such as T-cell receptors, may activate Ras proteins indirectly through Src-like tyrosine kinases or ligand-independent activation of receptor tyrosine kinases (243, 690, 774). Moreover, heterotrimeric G protein-coupled receptors, such as -adrenergic receptors, muscarinic acetylcholine receptors, and lysophosphatidic acid, have also been shown to activate Ras proteins (266, 285, 294). In addition, an increase of cytoplasmic Ca²⁺ induced by activation of these receptors in neurons also induces activation of another type of GEPs, p140 Ras GRF, that contains an IQ motif regulated by calcium-bound calmodulin (178).

View larger version (29K): [in this window] [in a new window]   Fig. 5. Mode of action of Ras proteins in gene expression. Ras, Ras proteins.

After the GTP-bound forms of Ras proteins accomplish their effects on downstream effector(s), they are converted to the GDP-bound form by the action of Ras GAPs. However, how termination of Ras protein signaling is achieved is not fully understood. Phosphorylation of SOS by the Raf/MAP kinase pathway (see below) may induce the dissociation of SOS from GRB2 (281, 356), and another possible mechanism is that Ras GAPs are activated by their binding to tyrosine-phosphorylated growth factor receptors such as the platelet-derived growth factor (PDGF) receptor (17, 332).

Three GEPs of Ras (SOS, Cdc25, and Ras GRF) have been discovered to date. The first GEP for Ras, Cdc25, has been identified genetically in S. cerevisiae (67, 81, 605). Cdc25 has a GEP domain that is required for its catalytic activity, located in its COOH-terminal region. In addition, Cdc25 has an SH3 domain in the NH₂-terminal region. In higher eukaryotes, in addition to mammalian Cdc25 (mCdc25), two different types of proteins with homology to Cdc25 have been found. The first group includes SOS. SOS was first identified in Drosophila. Genetic studies have shown that SOS is downstream of Sevenless, a receptor tyrosine kinase, which is homologous to the epidermal growth factor (EGF) receptor (609, 669). One human and two murine homologs of SOS have been cloned. SOS has one pleckstrin homology (PH) domain that may interact with a membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP₂), to determine its localization (62, 98). In addition, SOS has a GEP domain that is required for its catalytic activity in the middle portion and a GRB2-binding site in its COOH-terminal region. The second GEP group includes p140 Ras GRF that is primarily expressed in neural tissues (667). p140 Ras GRF has also a PH domain and a GEP domain. In addition, p140 Ras GRF contains an IQ motif that is regulated by calcium-bound calmodulin (178). Ras GRP, a GEP also predominantly expressed in brain, binds Ca²⁺ directly through a structure similar to the calcium binding EF hand; moreover, Ras GRP is regulated by direct binding of diacylglycerol (166). SOS is active on Ha-Ras and Ki-Ras but not on R-Ras, whereas Ras GRF is active on Ha-Ras and N-Ras but not on RalA or Cdc42 (56, 75, 507, 667).

p120 Ras GAP is the first GAP to be characterized at a molecular level (221, 727, 728, 755). p120 Ras GAP is active on Ha-Ras, Ki-Ras, N-Ras, and R-Ras, but not on Rho/Rac/Rab proteins (56, 221, 727). It contains a hydrophobic NH₂ terminus, two Src homology-2 (SH2) domains, one Src homology-3 (SH3) domain, one PH domain, and a region similar to the calcium-dependent lipid binding region of phospholipase A₂. Although it is clear that p120 Ras GAP acts as a negative regulator of Ras proteins, it has long been speculated that it also has other functions. One possible role of p120 Ras GAP is that it is a downstream effector of Ras proteins (463, 804). However, it now seems unlikely that p120 Ras GAP plays a major downstream role of Ras proteins. A number of tyrosine protein kinases have been shown to stimulate tyrosine phosphorylation of p120 Ras GAP in a variety of cells (169, 481), although the stoichiometry of these phosphorylations is generally low, and no alteration in the activity or localization of p120 Ras GAP has been demonstrated upon tyrosine phosphorylation. Protein tyrosine kinases may control the activity of p120 Ras GAP by forming a complex with it. In fact, p120 Ras GAP forms a complex with activated PDGF receptors (332, 339). Two other proteins have been found to associate with p120 Ras GAP. One is p62 Dok, a docking protein that contains multiple tyrosine phosphorylation sites for its binding to the SH2 domains of p120 Ras GAP and Nck (87, 796). The other is p190 Rho GAP; however, the precise role of the interaction between p190 Rho GAP and p120 Ras GAP is not fully understood (486, 656). p120 Ras GAP-deficient mice die during embryonic development, and the ability of endothelial cells to organize a vascular network is severely impaired in addition to extensive neuronal cell death in these animals (268). Another Ras GAP named GAP1m, which shows a high degree of similarity to the Drosophila Gap1 gene, has been also identified (426). In addition to the GAP catalytic domain, GAP1m has two domains with sequences closely related to those of the phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine kinase.

S. cerevisiae contains two Ras GAPs, Ira1 and Ira2 (711, 712), which contain domains homologous to the COOH terminus of p120 Ras GAP. In wild-type cells, Ras proteins are generally inactive, but in the absence of either the IRA gene product, they accumulate in their GTP-bound state, becoming hyperactive and leading to overproduction of cAMP. In yeast, at least, Ras GAPs are therefore not the effectors of Ras proteins; rather, they serve as negative regulators.

Neurofibromin (NF1), the human protein defective in von Recklinghausen neurofibromatosis (a benign tumor), contains a domain homologous to the catalytic domains of p120 Ras GAP, Ira1, and Ira2 (445, 786). Like p120 Ras GAP itself, neurofibromin possesses GAP activity in vitro and can complement the loss of Ira function in the yeast S. cerevisiae, indicating that it may be the mammalian homolog of Ira1 and Ira2 (34). These proteins share regions of significant similarity outside the GAP-related domain and presumably perform similar functions.

Small G protein GDP dissociation stimulator (Smg GDS) is a regulator that is entirely distinct from GEPs, GDIs, and GAPs (328, 795). Smg GDS has two biochemical activities on a group of small G proteins including Ki-Ras and the Rho/Rac/Cdc42/Rap1 proteins: one is to stimulate their GDP/GTP exchange reactions and the other is to inhibit their binding to membranes (18, 275, 479, 480, 788). A detailed kinetic study of Smg GDS with Ki-Ras as a substrate has revealed that it interacts with not only GDP-Ki-Ras and the guanine nucleotide-free Ki-Ras but also GTP-Ki-Ras, under the conditions where a Ras GEP, mCdc25, does not form a ternary complex with GTP-Ki-Ras (506). This property of Smg GDS suggests that it stimulates the GDP/GTP exchange reaction only once. The physiological role of Smg GDS remains to be established, although it shows mitogenic and transforming activities in cooperation with Ki-Ras in fibroblasts (198). Studies on Smg GDS-deficient mice have revealed that mice die of heart failure shortly after birth (708). Enhanced apoptosis is observed in at least heart, thymus, and neuron in Smg GDS-deficient mice. This phenotype is apparently similar to those of Ki-Ras-deficient mice (321, 362), providing another line of evidence that Smg GDS plays a role in Ki-Ras-mediated signaling pathway in vivo.

Ras proteins mediate their effects on cell proliferation mainly by activation of a cascade of protein kinases: Raf protein kinase (c-Raf-1, A-Raf, and B-Raf), MEK (MAP kinase kinases 1 and 2), and MAP kinase. Ras proteins activate this protein kinase cascade by directly binding to Raf proteins (743, 759, 767, 815). Raf proteins then phosophorylate and activate MEK (145, 299, 378), which then phosphorylates and activates MAP kinase (134, 450). The activated MAP kinase translocates to the nucleus, where it phosphorylates and stimulates the activity of various transcription factors, including Elk-1 (442). The recent observation that Ras proteins interact with two distinct NH₂-terminal regions of Raf-1 suggests that Ras proteins promote more than just membrane translocation of Raf-1 and instead may also facilitate the subsequent events that lead to Raf-1 activation (495, 513, 759).

The initial step of the Raf-1/MEK/MAP kinase cascade is the activation of Raf-1 by direct interaction with a GTP-Ras protein (GTP-Ras) (743, 759, 767, 815). The interaction with GTP-Ras localizes Raf-1 to the plasma membrane (389, 687). The first described and best-characterized Ras-binding domain (RBD) is contained within residues 51-131 of Raf-1 (121, 513, 759). The association of the RBD with the Ras effector domain is a high-affinity interaction that is mediated primarily by residues Gln-66, Lys-84, and Arg-89 of Raf-1 (55). The interaction between the RBD and GTP-Ras appears to then allow for a second RBD of Raf-1 to contact GTP-Ras (72, 159). This second RBD (residues 139-184 of Raf-1) encompasses the conserved Cys finger motif within the Raf-1 NH₂ terminus and is referred to as the Cys-rich domain (CRD) (495). In terms of the Ras-Raf-1 interaction, the CRD associates with different residues of GTP-Ras than does the RBD (159), and posttranslational modifications of Ras proteins may be important for CRD binding (297, 376). Thus, in the full-length molecule, the CRD is inaccessible for GTP-Ras binding, but either mutational events or RBD binding can unmask the CRD and allow it to interact with GTP-Ras. Thus, for the Ras-Raf-1 interaction to result in Raf-1 activation, binding to both the RBD and the CRD appears to be required.

Ha-Ras is localized at the cytoplasmic surface of the plasma membrane, while mutant forms of Ha-Ras, which lack posttranslational lipid modification, are cytosolic and lack biological activity. These findings suggest that posttranslational lipid modifications of Ha-Ras are required for both its localization and biological activity. Furthermore, lipid-modified Ras proteins have been shown to more efficiently activate adenylate cyclase in the yeast system (288, 376). Posttranslational modifications of Ki-Ras are also required for MAP kinase activation in a cell-free system (309). In addition, posttranslational modifications of Ha-Ras are required for activation of, but not for association with, Raf protein kinase (392, 547).

The intracellular signals that couple growth factors to MAP kinase may determine the different effects of growth factors; for example, transient activation of MAP kinase by EGF stimulates proliferation of PC12 cells, whereas sustained activation of MAP kinase by nerve growth factor (NGF) induces differentiation of PC12 cells. Activation of MAP kinase by NGF involves two distinct pathways: the initial activation of MAP kinase requires Ras proteins, but its activation is sustained by Rap1 (806) (see below). Rap1 is activated by C3G, a GEP for Rap1, and forms a stable complex with B-Raf (806). Activation of B-Raf by Rap1 represents a common mechanism to induce sustained activation of the MAP kinase cascade.

In addition to Ras proteins, a protein named 14-3-3 seems to also interact with Raf-1 and activate it (176, 197). 14-3-3 is a specific phosphoserine-binding protein (500). Raf-1 itself contains two phosphorylation sites that may interact with 14-3-3. 14-3-3 may have two different roles: first, 14-3-3 may be required for maintaining Raf-1 in an inactive conformation, as Raf-1 that is unable to stably interact with 14-3-3 is activated (467). In response to signaling events and Ras protein activation, 14-3-3 may subsequently play a second role in facilitating activation of Raf-1 and stabilizing activated Raf-1.

The observation that Raf-1 becomes hyperphosphorylated in response to many signaling events (492) has long suggested that phosphorylation plays a role in regulating Raf-1 activity. Mechanisms by which phosphorylation could regulate Raf-1 function include direct alteration of the intrinsic activity of Raf-1 and alteration of critical protein interactions, such as with 14-3-3. The rapid and transient nature of Raf-1 activation further complicates the issue, making it difficult to distinguish between activating and inactivating modifications. Nevertheless, by the use of overexpression systems and mutational analysis, the phosphorylation of tyrosine residues 340 and 341 has been shown to enhance the catalytic activity of Raf-1 (441). The tyrosine kinases implicated in phosphorylating Raf-1, and thereby enhancing its activity, include members of the Src kinase family (441, 562, 576).

A novel protein kinase that functions downstream of Ras proteins, kinase suppressor of Ras (KSR), has recently been identified by genetic screening as a suppressor of phenotypes caused by an activated Ras protein in both Drosophila and C. elegans (366, 694, 717). Epistasis analysis in Drosophila suggests that KSR functions downstream of Drosophila Ras-1 but upstream or in parallel to Raf protein (717). Characterization of a mouse KSR homolog suggests that KSR facilitates signal transmission between Raf proteins, MEK, and MAP kinase (718). In addition, upon Ras protein activation, KSR translocates to the plasma membrane, where it forms a stable complex with Raf proteins (718). Moreover, KSR, via its kinase domain, forms a stable complex with MEK in the cytosol of quiescent cells (144). Therefore, in response to an activated Ras protein, KSR might shuttle MEK from the cytosol to activated Raf proteins at the membrane.

With the use of a screen for eye development defects in Drosophila, the connector enhancer of KSR (CNK) protein has been identified as an enhancer of a KSR dominant negative mutant phenotype (719). Mutation of CNK suppresses the phenotype of activated Ras proteins or Sevenless but not Raf proteins, suggesting that it acts upstream of Raf proteins. CNK has several protein interaction domains. These domains include a sterile alpha motif (SAM) domain, a PSD-95/Dlg-A/ZO-1 (PDZ) domain, two proline-rich (potential SH3-binding) domains, and a PH domain; such domains are found in many proteins involved in signaling and suggest further interactions of CNK with other proteins and small molecules. In two-hybrid assays in S. cerevisiae, a COOH-terminal portion of CNK that contains the PH domain interacts with the Raf kinase domain (719). Thus the SAM, PDZ, and novel domains might be available for other interactions, although it is not known whether CNK also binds other proteins in the Ras-Raf signaling pathway. CNK has a molecular structure similar to that of recently identified rat neuronal proteins, named MAGUINs, that interact with the PDZ domains of PSD-95/SAP90 and S-SCAM (802). MAGUINs interact with c-Raf-1 but do not affect its enzymatic activity (803). PSD-95/SAP90 and S-SCAM are neuronal membrane-associated guanylate kinases, and these proteins function as synaptic scaffolding proteins (264). In fact, PSD-95/SAP90 further interacts with synGAP, which regulates the activity of Ras proteins (346). Therefore, MAGUINs may also bind Raf proteins and link it to PSD-95/SAP90 and S-SCAM in synaptic junctions.

A variety of candidate Ras protein effectors have been reported in addition to Raf proteins. These include Ral GDS (279, 342, 676), RIN1 (254), and phosphatidylinositol (PI) 3-kinase (608). AF6/Canoe is also suggested to be a binding partner of Ras proteins (373, 746), but this result has been called into question (434). It has recently been shown that Rap1 shows a much higher affinity to AF6 than Ras proteins do (404). p120 Ras GAP may participate in Ras protein-mediated gene expression, although it is still unclear whether p120 Ras GAP is a regulator, an effector, or both for Ras proteins. In contrast, activation of PI 3-kinase by Ras proteins may promote cell survival (334, 608). However, it has not been established whether these effector molecules other than Raf proteins really play a role in the downstream pathway of Ras proteins.

The plasma membrane localization of Ras proteins is crucial for their functions. The mechanism by which Ras proteins get to the plasma membrane has not fully been understood. It has recently been shown that Ras proteins do not directly travel to the plasma membrane from the cytosol, but interact with intracellular membranes (25, 114). Ras proteins first associate with the endoplasmic reticulum and then with the Golgi apparatus. The initial association of Ras proteins with the endoplasmic reticulum requires only the COOH-terminal Cys-A-A-X structure and farnesylation. N-Ras and Ha-Ras seem to be transported by exocytic vesicles following association with the endoplasmic reticulum and the Golgi apparatus. Ki-Ras takes a faster route that may not involve the Golgi apparatus. The hypervariable domains of the three Ras proteins are necessary and sufficient to account for their differential localizations. Inhibition of vesicle transport with brefeldin A (BFA), an inhibitor of Arf protein GEP (see below), blocks the transit of N-Ras to the plasma membrane, demonstrating the importance of vesicle transport for N-Ras function. Carboxylmethylation and A-A-X proteolysis are also necessary for proper association with the plasma membrane (255).

Mutated versions of the three human Ras genes have been detected in ~30% of all human cancers, implying an important role for aberrant Ras protein function in carcinogenesis. For example, Ras gene mutations are highly prevalent in pancreatic (90%) (9), lung (30%) (607), and colorectal (50%) (59, 756) carcinomas. Because Ras proteins regulate diverse extracellular signaling pathways for cell growth, differentiation, and apoptosis, the deregulated function of other cellular components can cause aberrant Ras protein function in the absence of mutations in the Ras genes themselves. Overexpression of ErbB2 or EGF receptor tyrosine kinase is common in breast cancers, and their transforming actions are dependent on signaling through the loss of negative Ras protein regulators (155). Similarly, the loss of function of negative Ras protein regulators, such as neurofibromin defective in type 1 neurofibromatosis-associated tumors, can cause aberrant upregulation of Ras protein function (42). Therefore, the importance of aberrant Ras protein function in human cancers may be greater than expected and may extend to tumors that do not harbor mutated Ras alleles.

The Rap subfamily consists of Rap1A, Rap1B, and Rap2. Rap1 proteins have been independently isolated by three laboratories by different methods: they have been isolated as homologs of Ras proteins by hybridization (573), they have been purified as small G proteins (smg p21) by column chromatography (337, 534), and they have been identified as K-Rev1 in a screen for cDNAs that revert the morphology of Ki-Ras-transformed cells (353). Interestingly, Rap1 proteins have an effector domain virtually identical to that of Ras proteins, suggesting that both proteins theoretically interact with similar effectors and show similar or antagonistic effects. The antagonistic function of K-Rev1 on Ras-transforming activity was the first studied (353). Rap1A binds to the two Ras-binding regions of Raf-1 (RBD and CRD), and this binding of Rap1A to CRD is competitive with Ras proteins (296, 297). Rap1 does not induce Raf-1 activation in intact cells but inhibits the Ha-Ras-induced Raf-1 activation in intact cells when Rap1 is overexpressed (124). However, most extracellular signals that induce Raf-1 activation, such as PDGF and EGF, activate rather than inhibit Rap1 (828). Furthermore, a phorbol ester induces Rap1 activation in Rat1 cells, but does not inhibit the PDGF- and EGF-induced activation of MAP kinase (828). These results suggest that the suppression of Ras protein function by Rap1 is simply due to the artificially competitive inhibition of the Ras protein binding to RBD or CRD.

In contrast to the role of Rap1 antagonistic to that of Ras proteins, evidence is accumulating that Rap1 functions independently of Ras protein signaling, utilizing effectors similar or identical to those of Ras proteins, like Raf proteins. Rap1, as well as Ki-Ras, induces DNA synthesis in Swiss 3T3 cells (11, 807). Rap1, as well as Ki-Ras, binds and activates B-Raf in vitro (541). In intact PC12 cells in response to cAMP and NGF, Rap1 is activated and induces B-Raf activation, causing sustained activation of the MAP kinase cascade that is necessary for neuronal differentiation (761, 806). Most recently, CD31, an important integrin adhesion amplifier, has been shown to selectively activate Rap1, but not Ha-Ras, R-Ras, or Rap2 (587). An activated mutant of Rap1 stimulates T lymphocyte adhesion to intercellular adhesion molecule and vascular cell adhesion molecule, as does C3G. Thus Rap1 regulates ligand-induced cell adhesion, and it may play a more general role in coordinating adhesion-dependent signals. In contrast to Rap1, little is known about Rap2 (573).

Several distinct second messenger pathways, including those for calcium (194), diacylglycerol (462), phospholipase C- (462), and cAMP (10), and perhaps others (497a), are able to induce Rap1 activation. Clearly, Rap1 activation is a common event, which suggests a function that is central in signal transduction processes. C3G is a Rap1-specific GEP containing a proline-rich domain that interacts with the SH3 domain of members of the Crk adaptor proteins, Crk I, Crk II, and Crk L (239, 355). In general, this association is constitutive, but tyrosine phosphorylation of Crk may disrupt the interaction (546). The SH2 domain of Crk binds directly to various activated receptor tyrosine kinases and phosphotyrosine-containing adaptor proteins (355). This association of Crk-C3G with these complexes may enhance GEP activity of C3G (301), suggesting that complex formation and dissociation of C3G regulate Rap1 activation by tyrosine kinases. However, in a human Jurkat T cell leukemia line, T-cell receptor-dependent induction of a Cbl-Crk L-C3G signaling complex does not activate Rap1 (586). Therefore, more work will be required to clarify how C3G complex formation is coupled to Rap1 regulation. Recently, two novel GEPs specific for Rap1, named Epac/cAMP-GEFI and nRap GEP/PDZ-GEF1/Hs-RA-GEF, have been identified (147, 148, 335, 402, 540). Epac/cAMP-GEFI has cAMP-binding and Ras GEP domains; thus this GEP activity is dependent on cAMP (148, 335). nRap GEP has been isolated as a binding partner of S-SCAM, that interacts with N-methyl-D-aspartate (NMDA) receptors and neuroligin through PSD-95/Dlg-A/ZO-1 (PDZ) domains at synaptic junctions (540). In contrast to Epac/cAMP-GEFI, nRap GEP/PDZ-GEF1/Hs-RA-GEF has one PDZ, one Ras association, and one Ras GEP domains as well as one COOH-terminal consensus motif for binding to PDZ domains. However, nRap GEP/PDZ-GEF1/Hs-RA-GEF has an incomplete cAMP-binding domain and its GEP activity is independent of cAMP. SPA-1, a Rap1 GAP, has been shown to interfere with Rap1 activation by membrane-targeted C3G (729). Overexpression of SPA-1 in HeLa cells suppresses Rap1 activation upon plating on dishes coated with fibronectin and results in the reduced adhesion. In addition, overexpression of SPA-1 in promyelocytic 32D cells also inhibited both activation of Rap1 and induction of cell adhesion by granulocyte colony-stimulating factor, suggesting that Rap1 is required for the cell adhesion induced by both extracellular matrix and soluble ligands (729).

The Ral subfamily consists of RalA and RalB (100, 101). Ral GDS, a Ral GEP, has been found to be a Ras protein effector (279, 342, 676). Moreover, insulin and EGF induce activation of Ral proteins, and this activation is inhibited by a dominant negative mutant of Ras proteins, suggesting that RalA is downstream of the Ras protein signaling pathway (783). In NIH 3T3 cells, both a dominant active mutant of Ha-Ras and Ral GDS synergize with Raf-1 in the induction of cell transformation and the activation of c-fos promoter (548, 741), and a dominant negative mutant of RalA inhibits the Ha-Ras- and Raf-1-induced transformation (741). These observations suggest that the Ral GDS-Ral protein pathway contributes to cell transformation and gene expression. However, a dominant active mutant of RalA alone cannot efficiently induce the oncogenic transformation or the c-fos induction compared with a dominant active mutant of Ha-Ras and Ral GDS (548, 741), suggesting that the transformation and the gene expression induced by Ral GDS may require other factors in addition to Ral proteins.

Three effectors for Ral proteins are known: RalBP1, phospholipase D, and filamin. RalBP1 contains a Rho GAP homology domain that exhibits the GAP activity for Rac/Cdc42 proteins, but not for Rho proteins (179). Although Rac/Cdc42 proteins contribute to the Ha-Ras-induced oncogenic transformation (582) (see below), it is unclear whether the association of Ral proteins with RalBP1 regulates the activity of these Rho/Rac/Cdc42 proteins. RalBP1 has been found to interact with POB1 and Reps1 (302, 791), which have proline-rich sequences responsible for interaction with Grb2 and Crk, and an Eps15 homology domain. Ral proteins are involved in endocytosis of the growth factor receptors probably through RalBP1, POB1, Eps15, and Epsin (511). Another Ral protein effector, phospholipase D, is also implicated in vesicle trafficking (179, 316). The activity of phospholipase D is induced by Src and Ras proteins. A dominant negative mutant of RalA inhibits both v-Src- and v-Ras-induced phospholipase D activity (316). The third effector protein of Ral is filamin (537). Either a dominant negative mutant of RalA or the RalA-binding domain of filamin blocks Cdc42-induced filopodium formation. A dominant active mutant of RalA elicits actin-rich filopodia, but it does not generate filopodia in filamin-deficient cells. Thus the Ral signaling appears to regulate vesicle trafficking, cytoskeletal organization, gene expression, and cell transformation. The GAP proteins for RalA were characterized and partially purified (48, 170); however, the molecular cloning of these proteins has not yet been achieved.

R-Ras has been shown to be involved in multiple biological functions: the ability to transform NIH 3T3 cells, the promotion of cell adhesion, and the regulation of apoptotic response in hematopoietic cells (128, 621, 695, 816). Unlike other Ras family members, R-Ras does not activate Raf proteins or MAP kinases in cells, whereas it stimulates PKB/Akt effectively through PI 3-kinase (444). TC21 has a highly oncogenic potential and is found mutated in some human tumors and overexpressed in breast cancer (37, 94, 300). As to the activation of Raf proteins by TC21, it is controversial (242, 611). Recently, new members of the Ras family, Rit and Rin, have been identified by an expression cloning screen (385). Rit is ubiquitously expressed, whereas Rin is expressed only in neural tissue. A unique feature of their structures is that they lack a known recognition signal for COOH-terminal prenylation. Nonetheless, both proteins localize on the plasma membrane, probably through a COOH-terminal cluster of basic amino acids. Rin binds calmodulin through a COOH-terminal motif, suggesting that Rin may be involved in calcium-mediated signaling in neurons (385). Rad is another member of Ras-like proteins that has originally been isolated as a gene overexpressed in the skeletal muscle of humans with type II diabetes (594). Kir/Gem has also been cloned as a gene that is overexpressed in cells transformed by abl tyrosine kinase (123) or cloned from mitogen-induced human peripheral blood T cells (429). Kir/Gem and Rad constitute a new family of Ras-related proteins. The distinct structural features of this family include the G3 GTP-binding motif, extensive NH₂- and COOH-terminal extensions beyond the Ras-related domain, and a motif that determines membrane association (429). Rheb, another Ras protein-related molecule, has been isolated by differential cloning techniques to identify genes that are rapidly induced in brain neurons by synaptic activity (790). Expression of Rheb is rapidly and transiently induced in hippocampal granule cells by seizures and by NMDA-dependent synaptic activity (790). The amino acid sequence of Rheb is most closely homologous to yeast Ras1 and human Rap2. In the developing brain, Rheb mRNA is expressed at relatively high levels. Its close homology with Ras proteins and its rapid inducibility by receptor-dependent synaptic activity suggest that Rheb may play an important role in long-term activity-dependent neuronal responses (790). More recently, Ras protein-like proteins, named B-Ras1 and B-Ras2, have been identified (181). These proteins interact with IB and IB, which are inhibitors for the nuclear transcription factor kappa B, NF-B, and decrease the rate of degradation of IBs. In cells, B-Ras proteins are associated only with NF-B:IB complexes and therefore may provide an explanation for the slower rate of degradation of IB compared with IB (181).

	IV. RHO/RAC/CDC42 PROTEINS AS REGULATORS OF BOTH CYTOSKELETAL REORGANIZATION AND GENE EXPRESSION

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The mammalian Rho family consists of at least 14 distinct members as shown in Table 1 and Figure 1. The function of the Rho family was first demonstrated in yeast (5, 45, 320). Phenotypes of the mutants that carry mutations in these genes indicated that Rho/Cdc42 proteins are involved in the budding process, presumably through reorganization of the actin cytoskeleton (320, 798). In mammals, the function of Rac proteins was the first to be clarified. GTP-Rac1, in addition to two other cytosolic proteins, p47phox and p67phox, were shown to be required for the activation of NADPH oxidase of phagocytic cells (1, 2, 18, 358, 479, 649). Then, the function of mammalian Rho proteins was elucidated by use of an exoenzyme of Clostridium botulinum, named C3, that specifically ADP-ribosylates Rho proteins (6, 343, 512). C3 ADP-ribosylates an amino acid (Asn-41) in the effector region of RhoA and inhibits its function by preventing interaction with downstream effectors (651). By the use of C3, Rho proteins were first suggested to be involved in cytoskeletal control (97, 564). Rho proteins were subsequently shown to regulate formation of stress fibers and focal adhesions in fibroblasts by use of its dominant active mutant and Rho GDI (475, 600, 601) and to regulate Ca²⁺ sensitivity of smooth muscle contraction (276). In contrast, Rac and Cdc42 proteins regulate formation of lamellipodia and filopodia, respectively (368, 522, 602). It has now been established that at least Rho/Rac/Cdc42 proteins regulate primarily cytoskeletal reorganization in response to extracellular signals in mammalian cells. Evidence has also accumulated that they may play additional roles in gene expression (126, 272, 473, 567, 692, 776). Furthermore, involvement of Rho/Rac/Cdc42 proteins in diverse cellular events, such as cell growth (341, 420, 552, 581-583, 794), membrane trafficking (4, 69, 86, 365, 380), development (227), and axon guidance (412) and extension (277, 314, 369), have been reported. In these cellular events, it is not known whether Rho/Rac/Cdc42 proteins directly regulate them or indirectly regulate them through cytoskeletal reorganization and gene expression. Many upstream regulators and downstream effectors have been identified for Rho/Rac/Cdc42 proteins, and although their modes of activation and action have gradually been elucidated, our understanding remains incomplete. Posttranslational modifications of Rho proteins are also crucial for their various functions including cell shape change, cell motility, cytoplasmic division of Xenopus embryo, and regulation of 1,3--glucan synthase of S. cerevisiae (305, 352, 475, 705).

Reorganization of the actin cytoskeleton plays crucial roles in many cellular functions such as cell shape change, cell motility, cell adhesion, and cytokinesis. The actin cytoskeleton is composed of actin filaments and many specialized actin-binding proteins (671, 688, 826). Filamentous actin is generally organized into a number of discrete structures (Fig. 6): 1) actin stress fibers: bundles of actin filaments that traverse the cell and are linked to the extracellular matrix through focal adhesions; 2) lamellipodia: thin protrusive actin sheets that dominate the edges of cultured fibroblasts and many migrating cells; membrane ruffles observed at the leading edge of the cell result from lamellipodia that lift up off the substratum and fold backward; and 3) filopodia: fingerlike protrusions that contain a tight bundle of long actin filaments in the direction of the protrusion. They are found primarily in motile cells and neuronal growth cones. It is important, therefore, that the polymerization and depolymerization of cortical actin be tightly regulated. For the most part, this regulation of actin polymerization is orchestrated by Rho/Rac/Cdc42 proteins. Rho proteins regulate stress fiber formation (475, 600), while Rac proteins regulate ruffling and lamellipodia formation (602), and Cdc42 regulates filopodium formation (368, 522).

View larger version (29K): [in this window] [in a new window]   Fig. 6. Mode of action of Rho/Rac/Cdc42 proteins in cytoskeletal reorganization. A: mode of action of Rho proteins. B: mode of action of Rac proteins. C: mode of action of Cdc42. Rho, Rho proteins; Rac, Rac proteins.

The activation and inactivation of Rho/Rac/Cdc42 proteins are regulated by essentially the same mechanism as Ras proteins by GEPs and GAPs, respectively. However, they are further regulated by another class of regulator, GDIs (206, 275, 280, 704, 732). In the cytosol, Rho/Rac/Cdc42 proteins are complexed with the GDI and maintained in the GDP-bound inactive form. The GDP-bound form is first released from a GDI by a still unknown mechanism and is converted to the GTP-bound form by the action of a GEP. The GTP-bound form then interacts with the downstream effector(s). Thereafter, the GTP-bound form is converted to the GDP-bound form by the action of a GAP. The GDP-bound form then forms a complex with the GDI and returns to the cytosol.

Rho/Rac/Cdc42 proteins are posttranslationally modified with lipid as described above and therefore they have to be in complex with GDIs to remain soluble in the cytosol. However, it is unknown whether all the GDP-bound form of Rho/Rac/Cdc42 proteins are complexed with GDIs and remain in the cytosol. Some amount of the GDP-bound form may be associated with membranes, and it may be converted to the GTP-bound form and exert its function on the membrane. In this case, GDIs would not be essential for their cyclical activation and inactivation.

Many GEPs for Rho/Rac/Cdc42 proteins have been isolated and characterized as shown in Table 2 and numbers are still increasing. Most GEPs have been isolated as oncogenes. The GEPs thus far identified share a common motif, designated the Dbl-homology (DH) domain, for which the Dbl oncogene product is the prototype (93). Biochemical analysis has confirmed that DH domains of GEPs indeed show GEP activity on Rho/Rac/Cdc42 proteins in a cell-free assay system (259, 262, 468). In addition to the DH domain, GEPs share a PH domain, which may be involved in proper cellular localization presumably through interaction with PIP₂ (469, 824). Some members of GEPs, such as Dbl and Vav1, have been shown to exhibit exchange activity in vitro for a broad range including Rho/Rac/Cdc42 proteins, whereas others appear to be more specific. Lbc, for example, and more recently discovered oncoproteins Lfc and Lsc are specific for Rho proteins (226, 823), whereas FGD1 and frabin are specific for Cdc42 (532, 738, 822). Although Vav1 is a GEP for Rac proteins (133, 245), Vav2, a GEP closely related to Vav1, functions preferably as a GEP for Rho proteins (642, 643). Some GEPs, such as Dbl, prefer the lipid-modified form of the substrate small G proteins to the lipid-unmodified ones (788).

In addition to the PH and DH domains, many GEPs have other domains that are commonly found in signaling molecules, such as the SH2 domain for Vav or the SH3 domain for Vav and Dbs, suggesting that they may have additional functions (93, 133, 642, 778). A GEP for Rho proteins, named p115 Rho GEF, that contains the regulator of G protein (RGS) domain has recently been identified (262, 367). RGS stimulates the intrinsic GTPase activity of the -subunit of G₁₂ and G₁₃. p115 Rho GEF acts as an intermediary in the regulation of Rho proteins by G₁₂ and G₁₃ (260, 367). In addition, another Rho GEP (named PDZ-Rho GEF) that contains RGS and PDZ domains has been reported (204). These findings have provided a new model for a signaling pathway for Rho proteins from membrane receptors. Recently, SHP-2, a protein tyrosine phosphatase containing SH2 domains, has been demonstrated to suppress the activity of Vav2 and consequently to reduce the Rho's ability to form stress fibers and focal adhesions (360). SHP-2 thereby positively regulates the hepatocyte growth factor (HGF)/scatter factor (SF)-induced cell scattering. However, detailed information regarding signaling cascades coupling the extracellular stimuli to activation of GEPs for Rac/Cdc42 proteins is still limited. Some GEPs like Tiam1 (248) and Ras GRF (667) carry a second PH domain. For Tiam1 and Ras GEF, this second, NH₂-terminal PH domain mediates localization to cell membranes (74, 469). In addition to the PH domain, frabin has an actin-binding domain at its NH₂-terminal region (532). The actin-binding domain in addition to the DH and first PH domains is essential for the filopodium formation mediated by frabin through Cdc42 (532, 738). Frabin furthermore induces lamellipoidum formation through indirect activation of Rac (553). The COOH-terminal FYVE and second PH domains, which associate with an unidentified membrane structure, in addition to the DH and first PH domains are necessary for this action (553).

The first GAP protein specific for Rho proteins was biochemically purified from human spleen and bovine adrenal gland (211, 212, 488). This protein, designated p50 Rho GAP, has GAP activity toward Rho/Rac/Cdc42 proteins in vitro (381). A number of proteins that exhibit GAP activity for Rho/Rac/Cdc42 proteins have subsequently been identified in mammalian cells (Table 2). These proteins all share a related GAP domain that spans ~140 amino acids of the protein but bears no significant resemblance to Ras GAP. The substrate specificity of Rho GAPs toward Rho/Rac/Cdc42 proteins varies with each GAP protein. Although some of these proteins exhibit GAP activity for several small G proteins in cell-free assay systems, their substrate specificities in vivo appear to be more restricted. For instance, the substrate spectrum of p50 Rho GAP in vitro encompasses Rho/Rac/Cdc42 proteins; however, in vivo, it appears to be restricted to Rho proteins only (603). Although first identified as a tyrosine-phosphorylated p120 Ras GAP-associated protein in Src-transformed cells and in growth factor-treated cells (169, 657), p190 Rho GAP was later shown to possess GAP activity for Rho proteins (656). Although the biological function of p190 Rho GAP is not well understood, the interaction of p190 Rho GAP with p120 Ras GAP has been suggested to induce a conformational change in p120 Ras GAP, resulting in increased accessibility of the effector binding surface of its SH3 domain (298). A role for p190 Rho GAP in regulating Rho protein function in cells undergoing cytoskeletal rearrangements has been suggested (95, 603), but it is not known whether this effect is induced by p190 Rho GAP as a downstream effector of Rho proteins.

Recent studies have shown that a cycle of inactivation and activation of Rho/Rac/Cdc42 proteins is necessary for dynamic cell functions such as growth factor-induced cell scattering. Expression of dominant active mutants of Rho/Rac/Cdc42 proteins inhibits HGF/SF-induced cell scattering (303, 331, 599), whereas C3 or Rho GDI blocks HGF/SF-induced cell scattering (706). The mode of action of Rho proteins in cell scattering remains to be clarified, but the Rho protein-regulated assembly and disassembly of stress fibers and focal adhesions have been suggested to be, at least in part, involved in this process (303, 331, 599, 706). It is not known how inactivation by GAPs is induced. In one case, integrin-induced formation of stress fibers inhibits Rho protein activation as part of a feedback inhibition system (593).

Rho GDI was originally isolated as a cytosolic protein that preferentially associated with GDP-RhoA and GDP-RhoB and thereby inhibited the dissociation of GDP (206, 732). Rho GDI requires the posttranslational lipid modifications of RhoA for its activity (286). Rho GDI prefers GDP-RhoA and GDP-RhoB to the corresponding GTP-bound forms and forms a ternary complex with the GDP-bound form (628, 732). Rho GDI is also capable of inhibiting GTP hydrolysis by Rho proteins (116, 261, 628), blocking both intrinsic and GAP-catalyzed GTPase activity. Rho GDI dissociates the GDP-bound form of prenylated RhoB from the membrane (308). Based on these properties of Rho GDI, it has been proposed that Rho GDI is involved not only in the regulation of the activation of Rho proteins but also in their translocation between the cytosol and the membrane (630, 702, 704). The GDP-bound forms of Rho proteins are complexed with Rho GDI and remain in the cytosol. When the GDP-bound form is released from Rho GDI, it is converted to the GTP-bound form by the action of Rho GEPs. The GTP-bound form then activates its specific downstream effector(s) until the GTP-bound form is converted to the GDP-bound form by Rho GAPs. Once the GDP-bound form is produced on the membrane, it is captured by Rho GDI and the complex returns to the cytosol.

Rho GDI has also been shown to be active not only on Rho proteins but also on Rac/Cdc42 proteins (18, 275, 391). In addition to Rho GDI, at least two other isoforms, named D4/Ly-GDI and Rho GDI-3, have been identified (390, 635, 809). Now, the originally identified Rho GDI is referred to as Rho GDI or Rho GDI1; D4/Ly-GDI is named Rho GDI or Rho GDI2; and Rho GDI3 is named Rho GDI or Rho GDI3. Recent NMR studies have shown that Rho GDI has a pocket that masks the lipid moieties of Rho proteins (238), consistent with biochemical analyses (286). More recently, the X-ray crystallographic structure of the Cdc42/Rho GDI complex has revealed two major sites of interaction between Rho GDI and Cdc42 (280). The NH₂-terminal regulatory region of Rho GDI binds to the switch I and II domains of Cdc42, leading to inhibition of both GDP dissociation and GTP hydrolysis. In addition, the geranylgeranyl moiety of Cdc42 inserts into a hydrophobic pocket within the immunoglobulin-like domain of the GDI molecule, keeping GDP-Cdc42 in the cytosol and permitting the dissociation of GDP-Cdc42 from membranes.

Although the mechanism by which Rho proteins are released from Rho GDI has largely been unknown, it has been shown that ERM, which consists of ezrin, radaxin, and moesin, has the capacity to displace the GDP-bound form of Rho proteins from Rho GDI (700). ERM has two functional domains; the NH₂-terminal plasma membrane-binding and COOH-terminal F