Atypical mechanism of regulation of the Wrch-1 Rho family small GTPase

Rho family GTPases are GDP/GTP-regulated molecular switches that regulate signaling pathways controlling diverse cellular processes. Wrch-1 was identified as a Wnt-1 regulated Cdc42 homolog, upregulated by Wnt1 signaling in Wnt1-transformed mouse mammary cells, and was able to promote formation of filopodia and activate the PAK serine/threonine kinase. Wrch-1 shares significant sequence and functional similarity with the Cdc42 small GTPase. However, Wrch-1 possesses a unique N-terminal 46 amino acid sequence extension that contains putative Src homology 3 (SH3) domain-interacting motifs. We determined the contribution of the N terminus to Wrch-1 regulation and activity. We observed that Wrch-1 possesses properties that distinguish it from Cdc42 and other Rho family GTPases. Unlike Cdc42, Wrch-1 possesses an extremely rapid, intrinsic guanine nucleotide exchange activity. Although the N terminus did not influence GTPase or GDP/GTP cycling activity in vitro, N-terminal truncation of Wrch-1 enhanced its ability to interact with and activate PAK and to cause growth transformation. The N terminus associated with the Grb2 SH3 domain-containing adaptor protein, and this association increased the levels of active Wrch-1 in cells. We propose that Grb2 overcomes N-terminal negative regulation to promote Wrch-1 effector interaction. Thus, Wrch-1 exhibits an atypical model of regulation not seen in other Rho family GTPases.     

Multiple sequence elements facilitate Chp Rho GTPase subcellular location, membrane association, and transforming activity

Cdc42 homologous protein (Chp) is a member of the Rho family of small GTPases and shares significant sequence and functional similarity with Cdc42. However, unlike classical Rho GTPases, we recently found that Chp depends on palmitoylation, rather than prenylation, for association with cellular membranes. Because palmitoylation alone is typically not sufficient to promote membrane association, we evaluated the possibility that other carboxy-terminal residues facilitate Chp subcellular association with membranes. We found that Chp membrane association and transforming activity was dependent on the integrity of a stretch of basic amino acids in the carboxy terminus of Chp and that the basic amino acids were not simply part of a palmitoyl acyltransferase recognition motif. We also determined that the 11 carboxy-terminal residues alone were sufficient to promote Chp plasma and endomembrane association. Interestingly, stimulation with tumor necrosis factor-alpha activated only endomembrane-associated Chp. Finally, we found that Chp membrane association was not disrupted by Rho guanine nucleotide dissociation inhibitory proteins, which are negative regulators of Cdc42 membrane association and biological activity. In summary, the unique carboxy-terminal sequence elements that promote Chp subcellular location and function expand the complexity of mechanisms by which the cellular functions of Rho GTPases are regulated.     

Critical and distinct roles of amino- and carboxyl-terminal sequences in regulation of the biological activity of the Chp atypical Rho GTPase

Chp (Cdc42 homologous protein) shares significant sequence and functional identity with the human Cdc42 small GTPase, and like Cdc42, promotes formation of filopodia and activates the p21-activated kinase serine/threonine kinase. However, unlike Cdc42, Chp contains unique amino- and carboxyl-terminal extensions. Here we determined whether Chp, like Cdc42, can promote growth transformation and evaluated the role of the amino- and carboxyl-terminal sequences in Chp function. Surprisingly, we found that a GTPase-deficient mutant of Chp exhibited low transforming activity but that deletion of the amino terminus of Chp greatly enhanced its transforming activity. Thus, the amino terminus may serve as a negative regulator of Chp function. The carboxyl terminus of Cdc42 contains a CAAX (where C is cysteine, A is aliphatic amino acid, X is terminal amino acid) tetrapeptide sequence that signals for the posttranslational modification critical for Cdc42 membrane association and biological function. Although Chp lacks aCAAXmotif, we found that Chp showed carboxyl terminus-dependent localization to the plasma membrane and to endosomes. Furthermore, an intact carboxyl terminus was required for Chp transforming activity. However, treatment with inhibitors of protein palmitoylation, but not prenylation, caused Chp to mislocalize to the cytoplasm. Thus, Chp depends on palmitoylation, rather than isoprenylation, for membrane association and function. In summary, Chp is implicated in cell transformation, and the unique amino and carboxyl termini of Chp represent atypical mechanisms of regulation of Rho GTPase function.     

C-terminal di-arginine motif of Cdc42 protein is essential for binding to phosphatidylinositol 4,5-bisphosphate-containing membranes and inducing cellular transformation

Rho GTPases regulate a diverse range of processes that are dependent on their proper cellular localization. The membrane localization of these GTPases is due in large part to their carboxyl-terminal geranylgeranyl moiety. In addition, most of the Rho family members contain a cluster of positively charged residues (i.e. a "polybasic domain"), directly preceding their geranylgeranyl moiety, and it has been suggested that this domain serves to fine-tune their localization among different cellular membrane sites. Here, we have taken a closer look at the role of the polybasic domain of Cdc42 in its ability to bind to membranes and induce the transformation of fibroblasts. A FRET assay for the binding of Cdc42 to liposomes of defined composition showed that Cdc42 associates more strongly with liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)) when compared either with uncharged control membranes or with liposomes containing a charge-equivalent amount of phosphatidylserine. The carboxyl-terminal di-arginine motif (Arg-186 and Arg-187) was shown to play an essential role in the binding of Cdc42 to PIP(2)-containing membranes. We further showed that substitutions for the di-arginine motif, when introduced within a constitutively active ("fast cycling") Cdc42(F28L) background, had little effect on the ability of the activated Cdc42 mutant to induce microspikes/filopodia in NIH 3T3 cells, whereas they eliminated its ability to transform fibroblasts. Taken together, these findings suggest that the di-arginine motif within the carboxyl terminus of Cdc42 is necessary for this GTPase to bind at membrane sites containing PIP(2), where it can initiate signaling activities that are essential for the oncogenic transformation of cells.     

Swf1p, a member of the DHHC-CRD family of palmitoyltransferases, regulates the actin cytoskeleton and polarized secretion independently of its DHHC motif

Rho and Rab family GTPases play a key role in cytoskeletal organization and vesicular trafficking, but the exact mechanisms by which these GTPases regulate polarized cell growth are incompletely understood. A previous screen for genes that interact with CDC42, which encodes a Rho GTPase, found SWF1/PSL10. Here, we show Swf1p, a member of the DHHC-CRD family of palmitoyltransferases, localizes to actin cables and cortical actin patches in Saccharomyces cerevisiae. Deletion of SWF1 results in misorganization of the actin cytoskeleton and decreased stability of actin filaments in vivo. Cdc42p localization depends upon Swf1p primarily after bud emergence. Importantly, we revealed that the actin regulating activity of Swf1p is independent of its DHHC motif. A swf1 mutant, in which alanine substituted for the cysteine required for the palmitoylation activity of DHHC-CRD proteins, displayed wild-type actin organization and Cdc42p localization. Bgl2p-marked exocytosis was found wild type in this mutant, although invertase secretion was impaired. These data indicate Swf1p has at least two distinct functions, one of which regulates actin organization and Bgl2p-marked secretion. This report is the first to link the function of a DHHC-CRD protein to Cdc42p and the regulation of the actin cytoskeleton.     

Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the β subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43Δ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species.     

ARHGAP30 is a Wrch-1-interacting protein involved in actin dynamics and cell adhesion

The atypical Rho GTPase Wrch-1 has been proposed roles in cell migration, focal adhesion dissolution, stress fibre break down and tight junction heterogeneity. A screen for Wrch-1 binding-partners identified the novel RhoGAP protein, ARHGAP30, as a Wrch-1 interactor. ARHGAP30 is related to the Cdc42- and Rac1-specific RhoGAP CdGAP, which was likewise found to bind Wrch-1. In contrast to CdGAP, ARHGAP30 serves as a Rac1- and RhoA-specific RhoGAP. Ectopic expression of ARHGAP30 results in membrane blebbing and dissolution of stress-fibres and focal adhesions. Our data suggest roles for ARHGAP30 and CdGAP in regulation of cell adhesion downstream of Wrch-1.     

Multiple Rho proteins regulate the subcellular targeting of PAK5

We investigated the regulatory mechanisms controlling the subcellular localization of p21-activated kinase 5 (PAK5) and found that the Cdc42/Rac interactive binding (CRIB) domain within PAK5 is critical for proper targeting within the cell. We also observed that PAK5 interacts with RhoD and RhoH in addition to Cdc42, and that interaction with RhoD targets PAK5 to subcellular locations that are distinct from those stimulated by Cdc42. Through deletion analysis we observed that the mitochondrial localization of PAK5 is controlled by multiple domains, providing evidence that the kinase activity of PAK5 is critical to its ability to cycle on and off mitochondria, and demonstrate that expression of kinase-inactive PAK5 elicits dramatic effects on mitochondrial morphology. These data indicate that PAK5 is directed to distinct subcellular locations by different Rho family small G proteins as well as by intrinsic targeting sequences.     

Characterization of TCL, a new GTPase of the rho family related to TC10 andCcdc42

GTPases of the Rho family control a wide variety of cellular processes such as cell morphology, motility, proliferation, differentiation, and apoptosis. We report here the characterization of a new Rho member, which shares 85% and 78% amino acid similarity to TC10 and Cdc42, respectively. This GTPase, termed as TC10-like (TCL) is encoded by an unexpectedly large locus, made of five exons spanning over 85 kilobases on human chromosome 14. TCL mRNA is 2.5 kilobases long and is mainly expressed in heart. In vitro, TCL shows rapid GDP/GTP exchange and displays higher GTP dissociation and hydolysis rates than TC10. Using the yeast two-hybrid system and GST pull-down assays, we show that GTP-bound but not GDP-bound TCL protein directly interacts with Cdc42/Rac interacting binding domains, such as those found in PAK and WASP. Despite its overall similarity to TC10 and Cdc42, the constitutively active TCL mutant displays distinct morphogenic activity in REF-52 fibroblasts, producing large and dynamic F-actin-rich ruffles on the dorsal cell membrane. Interestingly, TCL morphogenic activity is blocked by dominant negative Rac1 and Cdc42 mutants, suggesting a cross-talk between these three Rho GTPases.     

Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification

Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1- and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt.     

The TRE17 oncogene encodes a component of a novel effector pathway for Rho GTPases Cdc42 and Rac1 and stimulates actin remodeling

The Rho family GTPases Cdc42 and Rac1 play fundamental roles in transformation and actin remodeling. Here, we demonstrate that the TRE17 oncogene encodes a component of a novel effector pathway for these GTPases. TRE17 coprecipitated specifically with the active forms of Cdc42 and Rac1 in vivo. Furthermore, the subcellular localization of TRE17 was dramatically regulated by these GTPases and mitogens. Under serum-starved conditions, TRE17 localized predominantly to filamentous structures within the cell. Epidermal growth factor (EGF) induced relocalization of TRE17 to the plasma membrane in a Cdc42-/Rac1-dependent manner. Coexpression of activated alleles of Cdc42 or Rac1 also caused complete redistribution of TRE17 to the plasma membrane, where it partially colocalized with the GTPases in filopodia and ruffles, respectively. Membrane recruitment of TRE17 by EGF or the GTPases was dependent on actin polymerization. Finally, we found that a C-terminal truncation mutant of TRE17 induced the accumulation of cortical actin, mimicking the effects of activated Cdc42. Together, these results identify TRE17 as part of a novel effector complex for Cdc42 and Rac1, potentially contributing to their effects on actin remodeling. The present study provides insights into the regulation and cellular function of this previously uncharacterized oncogene.     

RHO GTPase Signaling for Axon Extension: Is Prenylation Important?

Many lines of evidence indicate the importance of the Rho family guanine nucleotide triphosphatases (GTPases) in directing axon extension and guidance. The signaling networks that involve these proteins regulate actin cytoskeletal dynamics in navigating neuronal growth cones. However, the intricate patterns that regulate Rho GTPase activation and signaling are not yet fully defined. Activity and subcellular localization of the Rho GTPases are regulated by post-translational modification. The addition of a geranylgeranyl group to the carboxy (C-) terminus targets Rho GTPases to the plasma membrane and promotes their activation by facilitating interaction with guanine nucleotide exchange factors and allowing sequestering by association with guanine dissociation inhibitors. However, it is unclear how these modifications affect neurite extension or how subcellular localization alters signaling from the classical Rho GTPases (RhoA, Rac1, and Cdc42). Here, we review recent data addressing this issue and propose that Rho GTPase geranylgeranylation regulates outgrowth.     

Identification and characterization of the Cdc42-binding site of IQGAP1

IQGAP1 is a multi-domained protein that integrates signaling of the Rho family GTPase Cdc42 with regulation of the cytoskeleton. Using SPOT analysis and in vitro peptide competition assays we have identified a 24 amino acid region of IQGAP1 that is necessary for Cdc42 binding. Both in vitro and in vivo analyses reveal that deletion of this sequence abolishes binding of IQGAP1 to Cdc42. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. An IQGAP1 mutant lacking the Cdc42 binding site mislocalizes to the cell periphery. These observations specifically define a short sequence of IQGAP1 that is required for its interaction with Cdc42 and demonstrate that Cdc42 binding is necessary for the normal subcellular distribution of IQGAP1.     

The Function of Two Rho Family GTPases Is Determined by Distinct Patterns of Cell Surface Localization

Rho family GTPases are critical regulators in determining and maintaining cell polarity. In Saccharomyces cerevisiae, Rho3 and Cdc42 play important but distinct roles in regulating polarized exocytosis and overall polarity. Cdc42 is highly polarized during bud emergence and is specifically required for exocytosis at this stage. In contrast, Rho3 appears to play an important role during the isotropic growth of larger buds. Using a novel monoclonal antibody against Rho3, we find that Rho3 localizes to the cell surface in a dispersed pattern which is clearly distinct from that of Cdc42. Using chimeric forms of these GTPases, we demonstrate that a small region at the N terminus is necessary and sufficient to confer Rho3 localization and function onto Cdc42. Analysis of this domain reveals two essential elements responsible for distinguishing function. First, palmitoylation of a cysteine residue by the Akr1 palmitoyltransferase is required both for the switch of function and the switch of localization properties of this domain. Second, two basic residues distal to the palmitoylation site are required for regulating binding affinity with the Exo70 and Sec3 effectors. This demonstrates the importance of localization and effector binding in determining how these GTPases evolved specific functions at distinct stages of polarized growth.Cell polarity is a highly conserved feature of eukaryotic cells and is important for a number of events in animal cell biology, including embryonic development, cell migration, and epithelial function (21). The budding yeast Saccharomyces cerevisiae provides a simple model system in which to understand cell polarity, as much of the machinery that is responsible for polarity between yeast and animal cells is highly conserved. Rho/Cdc42 family GTPases are examples of this conservation and have been shown to be critical determinants of polarity in both yeast and animal cells. Rho GTPases are thought to exert their effects on cell polarization through regulation of a number of cellular processes, including the cytoskeleton and polarized delivery of new membrane to sites of active growth.Previous studies have demonstrated that Rho3 and Cdc42 have direct roles in regulating exocytosis which are independent of their role in regulating the polarity of the actin cytoskeleton (1, 2). Studies from a number of laboratories have shown that a multisubunit vesicle tethering complex known as the exocyst is likely to be a critical effector for Rho/Cdc42 signaling during polarized exocytosis (1, 2, 11, 22). A number of models have been suggested to describe the action of Rho GTPases in regulating exocytic function (28, 30). Analysis of specific loss-of-function alleles of RHO3 and CDC42 demonstrated that defects in secretion could be distinguished not only from actin polarity but from the polarization of the exocytic machinery as well. This led to the suggestion that Rho GTPases act by local activation rather than recruitment of the exocytic machinery (25).Genetic analysis suggests that the pathway by which Cdc42 regulates secretion is closely linked to that of Rho3. Secretion-defective alleles in each of these GTPases are suppressed by a common set of genes, and the mutants exhibit synthetic lethality when combined in the same cell (1). Recent work has provided direct evidence that the Exo70 subunit of the exocyst both genetically and physically interacts with both Rho3 and Cdc42 (29).Although Rho3 and Cdc42 share a effector and have overlapping functions, there are different characteristics in how these two proteins regulate exocytosis in yeast. Analysis of the Rho3 and Cdc42 secretory mutants by electron microscopy and secretory assays revealed that cdc42-6 mutants showed defects only in cells with small or emerging buds; in contrast, rho3-V51 mutants exhibited secretory defects throughout bud growth (1, 2). These phenotypes suggested that the exocytic function of Rho3 and Cdc42 is required at overlapping but distinct stages of bud growth.Most small GTPases require multiple elements to promote their association with the membrane on which they engage their downstream targets (26). Modification of the C-terminal CAAX motif by prenylation is common to both Rho3 and Cdc42, with Rho3 predicted to be farnesylated and Cdc42 shown to be geranylgeranylated (14, 17, 19). However, as with other small GTPases, C-terminal prenylation by itself is not sufficient for stable membrane association (10, 18). As with many other small GTPases, a second site of interaction is thought to be required for both Rho3 and Cdc42 GTPases. Sequence alignment of Rho3 and Cdc42 revealed that Rho3 has a long N-terminal extension, which contains a site (a cysteine at position 5) for palmitoylation (24). In contrast, Cdc42 is not palmitoylated but instead contains a polybasic domain adjacent to the CAAX motif at its C terminus, which is thought to act as a membrane targeting signal via the electrostatic interactions with phospholipids at the plasma membrane (6, 12).In this study we examine how the function and localization of two Rho GTPases are specified at distinct stages of polarized growth in yeast. Using a novel monoclonal antibody, we find that the pattern of cell surface localization observed for the Rho3 GTPase is clearly distinct from that of Cdc42. Using chimeric forms of these GTPases, we find that the N terminus plays a particularly important role in this specification. The functional effect imparted by the N terminus appears to have two key elements. One element involves palmitoylation of a cysteine in the N terminus of Rho3 that is critical in generating the dispersed pattern of localization observed for Rho3. A second element regulates the affinity of the GTPases for a common effector, the exocyst complex. Taken together, this work provides a model for how these GTPases have evolved distinct functions by adopting sequence elements that affect both the pattern of localization and the ability to engage the downstream effector in a way that allows each GTPase to function at different stages of polarized growth.     

RhoD localization and function is dependent on its GTP/GDP-bound state and unique N-terminal motif

The atypical Rho GTPase RhoD has previously been shown to have a major impact on the organization and function of the actin filament system. However, when first discovered, RhoD was found to regulate endosome trafficking and dynamics and we therefore sought to investigate this regulation in more detail. We found that exogenously expressed RhoD in human fibroblasts localized to vesicles and the plasma membrane and that the active GTP-bound conformation was required for the plasma membrane localization but not for vesicle localization. In contrast to the GTPase deficient atypical Rho GTPases, which have a stalled GTPase activity, RhoD has an elevated intrinsic GDP/GTP exchange activity, rendering the protein constitutively active. Importantly, RhoD can still hydrolyze GTP and we found that an intact GTPase activity was required for efficient fusion of RhoD-positive vesicles. RhoD has a unique N-terminal extension of 14 amino acid residues, which is not present in the classical Rho GTPases RhoA, Cdc42 and Rac1. Deletion of this N-terminal motif often lead to clustering of RhoD positive vesicles, which were found accumulated at the peripheral membrane border. In addition, the number of vesicles per cell was increased manifold, suggesting that the N-terminal motif has an important regulatory role in vesicle dynamics.     

A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCzeta signaling and cell transformation

BACKGROUND: Rac and Cdc42 are members of the Rho family of small GTPases. They modulate cell growth and polarity, and contribute to oncogenic transformation by Ras. The molecular mechanisms underlying these functions remain elusive, however. RESULTS: We have identified a novel effector of Rac and Cdc42, hPar-6, which is the human homolog of a cell-polarity determinant in Caenorhabditis elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKCzeta, and forms a stable ternary complex with Rac1 or Cdc42 and PKCzeta. This association results in stimulation of PKCzeta kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKCzeta-dependent pathway distinct from the pathway mediated by Raf. CONCLUSIONS: These findings indicate that Rac/Cdc42 can regulate cell growth through Par-6 and PKCzeta, and suggest that deregulation of cell-polarity signaling can lead to cell transformation.     

Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation

Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo. In this study, we evaluated the role of phosphoinositide binding to the PH domain in regulating the DH domain function of Dbs in vitro and in vivo. We determined that mutation of basic amino acids located within the beta1-beta2 and beta3-beta4 loops of the PH domain resulted in impaired phospholipid binding in vitro, yet full guanine nucleotide exchange activity in vitro was retained for RhoA and Cdc42. Surprisingly, these mutants were compromised in their ability to activate Rho GTPases in vivo and to cause transformation of NIH 3T3 cells. However, Dbs subcellular localization was impaired by these PH domain mutations, supporting a role for phospholipid interactions in facilitating membrane association. Despite the importance of phospholipid binding for Dbs function in vivo, we found that Dbs signaling and transforming activity was not stimulated by phosphatidylinositol 3-kinase activation. We suggest that the PH domain of Dbs facilitates two distinct roles in the regulation of DH domain function, one critical for GTPase association and activation in vitro and one critical for phosphoinositide binding and GTPase interaction in vivo, that together promote Dbs association with membranes.     

Role of the guanine nucleotide exchange factor Ost in negative regulation of receptor endocytosis by the small GTPase Rac1

The Rho family of GTPases has been implicated in the regulation of intracellular vesicle trafficking. Here, we investigated the mechanism underlying the negative regulation of clathrin-mediated endocytosis of cell surface receptors mediated by the Rho family protein Rac1. Contrary to previous reports, only the activated mutant of Rac1, but not other Rho family members including RhoA and Cdc42, suppressed internalization of the transferrin receptor. On the other hand, down-regulation of Rac1 expression by RNA interference resulted in enhanced receptor internalization, suggesting that endogenous Rac1 in fact functions as a negative regulator. We identified a guanine nucleotide exchange factor splice variant designated Ost-III, which contains a unique C-terminal region including an Src homology 3 domain, as a regulator of Rac1 involved in the inhibition of receptor endocytosis. In contrast, other splice variants Ost-I and Ost-II exerted virtually no effect on receptor endocytosis. We also examined subcellular localization of synaptojanin 2, a putative Rac1 effector implicated in negative regulation of receptor endocytosis. Each Ost splice variant induced distinct subcellular localization of synaptojanin 2, depending on Rac1 activation. Furthermore, we isolated gamma-aminobutyric acid type A receptor-associated protein (GABARAP) as a protein that binds to the C-terminal region of Ost-III. When ectopically expressed, GABARAP was co-localized with Ost-III and potently suppressed the Ost-III-dependent Rac1 activation and the inhibition of receptor endocytosis. Lipid modification of GABARAP was necessary for the suppression of Ost-III. These results are discussed in terms of subcellular region-specific regulation of the Rac1-dependent signaling pathway that negatively regulates clathrin-mediated endocytosis.