Cdc42 GTPase-activating protein (CdGAP) interacts with the SH3D domain of Intersectin through a novel basic-rich motif

The small GTPases Rac1 and Cdc42 are key regulators of the cytoskeleton. We have previously identified the endocytic protein Intersectin as a binding partner and regulator of Cdc42 GTPase-activating protein (CdGAP) with activity towards Rac1 and Cdc42. This interaction is mediated through the SH3D domain of Intersectin and the central domain of CdGAP, which does not contain any typical proline-rich domain or known SH3-binding motif. Here, we have characterized the Intersectin-SH3D/CdGAP interaction. We show that Intersectin-SH3D interacts directly with a small region of CdGAP highly enriched in basic residues and comprising a novel conserved xKx(K/R)K motif.     

Extracellular signal-regulated kinase 1 interacts with and phosphorylates CdGAP at an important regulatory site

Rho GTPases regulate multiple cellular processes affecting both cell proliferation and cytoskeletal dynamics. Their cycling between inactive GDP- and active GTP-bound states is tightly regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We have previously identified CdGAP (for Cdc42 GTPase-activating protein) as a specific GAP for Rac1 and Cdc42. CdGAP consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP is a member of the impressively large number of mammalian RhoGAP proteins that is well conserved among both vertebrates and invertebrates. In mice, we find two predominant isoforms of CdGAP differentially expressed in specific tissues. We report here that CdGAP is highly phosphorylated in vivo on serine and threonine residues. We find that CdGAP is phosphorylated downstream of the MEK-extracellular signal-regulated kinase (ERK) pathway in response to serum or platelet-derived growth factor stimulation. Furthermore, CdGAP interacts with and is phosphorylated by ERK-1 and RSK-1 in vitro. A putative DEF (docking for ERK FXFP) domain located in the proline-rich region of CdGAP is required for efficient binding and phosphorylation by ERK1/2. We identify Thr776 as an in vivo target site of ERK1/2 and as an important regulatory site of CdGAP activity. Together, these data suggest that CdGAP is a novel substrate of ERK1/2 and mediates cross talk between the Ras/mitogen-activated protein kinase pathway and regulation of Rac1 activity.     

A stretch of polybasic residues mediates Cdc42 GTPase-activating protein (CdGAP) binding to phosphatidylinositol 3,4,5-trisphosphate and regulates its GAP activity

The Rho family of small GTPases are membrane-associated molecular switches involved in the control of a wide range of cellular activities, including cell migration, adhesion, and proliferation. Cdc42 GTPase-activating protein (CdGAP) is a phosphoprotein showing GAP activity toward Rac1 and Cdc42. CdGAP activity is regulated in an adhesion-dependent manner and more recently, we have identified CdGAP as a novel molecular target in signaling and an essential component in the synergistic interaction between TGFβ and Neu/ErbB-2 signaling pathways in breast cancer cells. In this study, we identified a small polybasic region (PBR) preceding the RhoGAP domain that mediates specific binding to negatively charged phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In vitro reconstitution of membrane vesicles loaded with prenylated Rac1 demonstrates that the PBR is required for full activation of CdGAP in the presence of PI(3,4,5)P3. In fibroblast cells, the expression of CdGAP protein mutants lacking an intact PBR shows a significant reduced ability of the protein mutants to induce cell rounding or to mediate negative effects on cell spreading. Furthermore, an intact PBR is required for CdGAP to inactivate Rac1 signaling into cells, whereas it is not essential in an in vitro context. Altogether, these studies reveal that specific interaction between negatively charged phospholipid PI(3,4,5)P3 and the stretch of polybasic residues preceding the RhoGAP domain regulates CdGAP activity in vivo and is required for its cellular functions.     

GC-GAP,a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2

Gab1 and Gab2 are scaffolding proteins acting downstream of cell surface receptors and interact with a variety of cytoplasmic signaling proteins such as Grb2, Shp-2, phosphatidylinositol 3-kinase, Shc, and Crk. To identify new binding partners for GAB proteins and better understand their functions, we performed a yeast two-hybrid screening with hGab2-(120-587) as bait. This work led to identification of a novel GTPase-activating protein (GAP) for Rho family GTPases. The GAP domain shows high similarity to the recently cloned CdGAP and displays activity toward RhoA, Rac1, and Cdc42 in vitro. The protein was named GC-GAP for its ability to interact with GAB proteins and its activity toward Rac and Cdc42. GC-GAP is predominantly expressed in the brain with low levels detected in other tissues. Antibodies directed against GC-GAP recognized a protein of approximately 200 kDa. Expression of GC-GAP in 293T cells led to a reduction in active Rac1 and Cdc42 levels but not RhoA. Suppression of GC-GAP expression by siRNA inhibited proliferation of C6 astroglioma cells. In addition, GC-GAP contains several classic proline-rich motifs, and it interacts with the first SH3 domain of Crk and full-length Nck in vitro. We propose that Gab1 and Gab2 in cooperation with other adapter molecules might regulate the cellular localization of GC-GAP under specific stimuli, acting to regulate precisely Rac and Cdc42 activities. Given that GC-GAP is specifically expressed in the nervous system and that it is localized to the dendritic processes of cultured neurons, GC-GAP may play a role in dendritic morphogenesis and also possibly in neural/glial cell proliferation.     

Rich, a rho GTPase-activating protein domain-containing protein involved in signaling by Cdc42 and Rac1.

A previously unidentified Rho GTPase-activating protein (GAP) domain-containing protein was found in a yeast two-hybrid screen for cDNAs encoding proteins binding to the Src homology 3 domain of Cdc42-interacting protein 4 (CIP4). The protein was named RICH-1 (RhoGAP interacting with CIP4 homologues), and, in addition to the RhoGAP domain, it contained an N-terminal domain with endophilin homology and a C-terminal proline-rich domain. Transient transfections of RICH-1 indicated that it bound to CIP4 in vivo, as shown by co-immunoprecipitation experiments, as well as co-localization assays. In vitro assays demonstrated that the RhoGAP domain of RICH-1 catalyzed GTP hydrolysis on Cdc42 and Rac1, but not on RhoA. Ectopic expression of the RhoGAP domain as well as the full-length protein interfered with platelet-derived growth factor BB-induced membrane ruffling, but not with serum-induced stress fiber formation, further emphasizing the notion that, in vivo, RICH-1 is a GAP for Cdc42 and Rac1.     

The human orthologue of CdGAP is a phosphoprotein and a GTPase-activating protein for Cdc42 and Rac1 but not RhoA

BACKGROUND INFORMATION: Rho GTPases regulate a wide range of cellular functions affecting both cell proliferation and cytoskeletal dynamics. They cycle between inactive GDP- and active GTP-bound states. This cycle is tightly regulated by GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase-activating proteins). Mouse CdGAP (mCdc42 GTPase-activating protein) has been previously identified and characterized as a specific GAP for Rac1 and Cdc42, but not for RhoA. It consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP-related genes are present in both vertebrates and invertebrates. We have recently reported that two predominant isoforms of CdGAP (250 and 90 kDa) exist in specific mouse tissues. RESULTS: In the present study, we have identified and characterized human CdGAP (KIAA1204) which shares 76% sequence identity to the long isoform of mCdGAP (mCdGAP-l). Similar to mCdGAP, it is active in vitro and in vivo on both Cdc42 and Rac1, but not RhoA, and is phosphorylated in vivo on serine and threonine residues. In contrast with mCdGAP-l, human CdGAP interacts with ERK1/2 (extracellular-signal-regulated kinase 1/2) through a region that does not involve a DEF (docking site for ERK Phe-Xaa-Phe-Pro) domain. Also, the tissue distribution of CdGAP proteins appears to be different between human and mouse species. Interestingly, we found that CdGAP proteins cause membrane blebbing in COS-7 cells. CONCLUSIONS: Our results suggest that CdGAP properties are well conserved between human and mouse species, and that CdGAP may play an unexpected role in apoptosis.     

Identification and characterization of a novel Rho GTPase activating protein implicated in receptor-mediated endocytosis

Cbl-interacting protein of 85 kDa (CIN85) is a recently identified adaptor protein involved in the endocytic process of several receptor tyrosine kinases. Here we have identified a novel RhoGAP, CIN85 associated multi-domain containing Rho1 (CAMGAP1) as a binding protein for CIN85. CAMGAP1 is composed of an Src homology 3 (SH3) domain, multiple WW domains, a proline-rich region, a PH domain and a RhoGAP domain, and has the domain architecture similar to ARHGAP9 and ARHGAP12. CAMGAP1 mRNA is widely distributed in murine tissues. Biochemical assays showed its GAP activity toward Rac1 and Cdc42. Protein binding and expression studies indicated that the second SH3 domain of CIN85 binds to a proline-rich region of CAMGAP1. Overexpression of a truncated form of CAMGAP1 interferes with the internalization of transferrin receptors, suggesting that CAMGAP1 may play a role in clathrin-mediated endocytosis.     

The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases.

We previously described IQGAP1 as a human protein related to a putative Ras GTPase-activating protein (RasGAP) from the fission yeast Schizosaccharomyces pombe. Here we report the identification of a liver-specific human protein that is 62% identical to IQGAP1. Like IQGAP1, the novel IQGAP2 protein harbors an N-terminal calponin homology motif which functions as an F-actin binding domain in members of the spectrin, filamin, and fimbrin families. Both IQGAPs also harbor several copies of a novel 50- to 55-amino-acid repeat, a single WW domain, and four IQ motifs and have 25% sequence identity with almost the entire S. pombe sar1 RasGAP homolog. As predicted by the presence of IQ motifs, IQGAP2 binds calmodulin. However, neither full-length nor truncated IQGAP2 stimulated the GTPase activity of Ras or its close relatives. Instead, IQGAP2 binds Cdc42 and Racl but not RhoA. This interaction involves the C-terminal half of IQGAP2 and appears to be independent of the nucleotide binding status of the GTPases. Although IQGAP2 shows no GAP activity towards Cdc42 and Rac1, the protein did inhibit both the intrinsic and RhoGAP-stimulated GTP hydrolysis rates of Cdc42 and Rac1, suggesting an alternative mechanism via which IQGAPs might modulate signaling by these GTPases. Since IQGAPs harbor a potential actin binding domain, they could play roles in the Cdc42 and Rac1 controlled generation of specific actin structures.     

ArhGAP15, a novel human RacGAP protein with GTPase binding property

We have previously described a partial cDNA sequence encoding a RhoGAP protein, GAP25 that is homologous to the recently reported ArhGAP9 and ArhGAP12. We now describe a related new member ArhGAP15 that shares a number of domain similarities, including a pleckstrin homology (PH) domain, a RhoGAP domain and a novel motif N-terminal to the GAP domain. This novel motif was found to be responsible for nucleotide-independent Rac1 binding. Using swop mutants of Rac/Cdc42, we have established that the binding is through the C-terminal half of Rac1. The GAP domain of ArhGAP15 showed specificity towards Rac1 in vitro. The PH domain is required for ArhGAP15 to localize to cell periphery and over-expression of the full-length ArhGAP15, but not the mutant with a partial deletion of the PH domain, resulted in an increase in actin stress fibers and cell contraction. These morphological effects can be attenuated by the co-expression of dominant negative Rac1(N17). HeLa cells expressing ArhGAP15 were also resistant to phorbol myristatate acetate treatment, suggesting that ArhGAP15 is a potential regulator of Rac1.     

Characterization of a brain-specific Rho GTPase-activating protein,p200RhoGAP

The Rho GTPase-activating proteins (RhoGAPs) are a family of multifunctional molecules that transduce diverse intracellular signals by regulating Rho GTPase activities. A novel RhoGAP family member, p200RhoGAP, is cloned in human and mouse. The murine p200RhoGAP shares 86% sequence identity with the human homolog. In addition to a conserved RhoGAP domain at the N terminus, multiple proline-rich motifs are found in the C-terminal region of the molecules. Northern blot analysis revealed a brain-specific expression pattern of p200RhoGAP. The RhoGAP domain of p200RhoGAP stimulated the GTPase activities of Rac1 and RhoA in vitro and in vivo, and the conserved catalytic arginine residue (Arg-58) contributed to the GAP activity. Expression of the RhoGAP domain of p200RhoGAP in Swiss 3T3 fibroblasts inhibited actin stress fiber formation stimulated by lysophosphatidic acid and platelet-derived growth factor-induced membrane ruffling but not Bradykinin-induced filopodia formation. Endogenous p200RhoGAP was localized to cortical actin in naive N1E-115 neuroblastoma cells and to the edges of extended neurites of differentiated N1E-115 cells. Transient expression of the RhoGAP domain and the full-length molecule, but not the catalytic arginine mutants, readily induced a differentiation phenotype in N1E-115 cells. Finally, p200RhoGAP was capable of binding to the Src homology 3 domains of Src, Crk, and phospholipase Cgamma in vitro and became tyrosine-phosphorylated upon association with activated Src in cells. These results suggest that p200RhoGAP is involved in the regulation of neurite outgrowth by exerting its RhoGAP activity and that its cellular activity may be regulated through interaction with Src-like tyrosine kinases.     

GTP-binding protein-like domain of AGAP1 is protein binding site that allosterically regulates ArfGAP protein catalytic activity

AGAPs are a subtype of Arf GTPase-activating proteins (GAPs) with 11 members in humans. In addition to the Arf GAP domain, the proteins contain a G-protein-like domain (GLD) with homology to Ras superfamily proteins and a PH domain. AGAPs bind to clathrin adaptors, function in post Golgi membrane traffic, and have been implicated in glioblastoma. The regulation of AGAPs is largely unexplored. Other enzymes containing GTP binding domains are regulated by nucleotide binding. However, nucleotide binding to AGAPs has not been detected. Here, we found that neither nucleotides nor deleting the GLD of AGAP1 affected catalysis, which led us to hypothesize that the GLD is a protein binding site that regulates GAP activity. Two-hybrid screens identified RhoA, Rac1, and Cdc42 as potential binding partners. Coimmunoprecipitation confirmed that AGAP1 and AGAP2 can bind to RhoA. Binding was mediated by the C terminus of RhoA and was independent of nucleotide. RhoA and the C-terminal peptide from RhoA increased GAP activity specifically for the substrate Arf1. In contrast, a C-terminal peptide from Cdc42 neither bound nor activated AGAP1. Based on these results, we propose that AGAPs are allosterically regulated through protein binding to the GLD domain.     

Characterization of a novel GTPase-activating protein associated with focal adhesions and the actin cytoskeleton

In the present study we characterize a novel RhoGAP protein (RC-GAP72) that interacts with actin stress fibers, focal adhesions, and cell-cell adherens junctions via its 185-amino acid C-terminal region. Overexpression of RC-GAP72 in fibroblasts induces cell rounding with partial or complete disruption of actin stress fibers and formation of membrane ruffles, lamellipodia, and filopodia. RC-GAP72 mutant truncated downstream of the GTPase-activating protein (GAP) domain retains the ability to stimulate membrane protrusions but fails to affect stress fiber integrity or induce cell retraction. A mutant protein consisting of the C terminus of RC-GAP72 and lacking the GAP domain does not exert any visible effect on cellular morphology. Inactivation of the GAP domain by a point mutation does not abolish the effect of RC-GAP72 on actin stress fibers but moderates its capability to induce membrane protrusions. Our data imply that the cytoskeletal localization of RC-GAP72 and its interaction with GTPases are essential for its effect on the integrity of actin stress fibers, whereas the induction of lamellipodia and filopodia depends on the activity of the GAP domain irrespective of binding to the actin cytoskeleton. We propose that RC-GAP72 affects cellular morphology by targeting activated Cdc42 and Rac1 GTPases to specific subcellular sites, triggering local morphological changes. The overall physiological functions of RC-GAP72 are presently unknown, yet our data suggest that RC-GAP72 plays a role in regulating cell morphology and cytoskeletal organization.     

CdGAP associates with actopaxin to regulate integrin-dependent changes in cell morphology and motility

BACKGROUND: Integrin signaling, stimulated by cell adhesion to the extracellular matrix, plays a critical role in coordinating changes in cell morphology and migration. The requisite remodeling of the cytoskeleton is controlled by the Rho family of small GTPases, which are, in turn, regulated via activation by guanine-nucleotide exchange factors (GEFs) and inactivation by GTPase-activating proteins (GAPs). However, the mechanisms contributing to the precise spatial and temporal regulation of these Rho GTPase modulators remain poorly understood. RESULTS: The Cdc42/Rac GAP CdGAP has previously been implicated as an inhibitor of growth-factor-induced lamellipodia formation. Herein, CdGAP is shown to localize to focal adhesions, potentially through its direct association with the amino terminus of actopaxin, a paxillin and actin binding protein. CdGAP activity is regulated in an adhesion-dependent manner and, through the overexpression of wild-type CdGAP and a GAP-deficient mutant, as well as RNA interference, is shown to be required for normal cell spreading, polarized lamellipodia formation, and cell migration. Introduction of an actopaxin mutant defective for CdGAP binding, or reduction of actopaxin by using RNAi, significantly attenuated these effects. CONCLUSIONS: We have established that CdGAP is an important regulator of integrin-induced Rho family signaling to the cytoskeleton and that its interaction with the focal-adhesion protein actopaxin is critical for the correct spatial and/or temporal regulation of CdGAP function. A complete understanding of the coordination of signaling events downstream of integrin engagement with the extracellular matrix will provide valuable insight into the regulation of cell migration during processes such as wound repair, development, and tumor cell metastasis.     

Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets

Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that – beside the GAP domain – the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation.     

Concerted regulation of cell dynamics by BNIP-2 and Cdc42GAP homology/Sec14p-like,proline-rich,and GTPase-activating protein domains of a novel Rho GTPase-activating protein,BPGAP1

RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.     

The C-terminal domain of Rac1 contains two motifs that control targeting and signaling specificity

Rho-like GTPases control a wide range of cellular functions such as integrin- and cadherin-mediated adhesion, cell motility, and gene expression. The hypervariable C-terminal domain of these GTPases has been implicated in membrane association and effector binding. We found that cell-permeable peptides, encoding the C termini of Rac1, Rac2, RhoA, and Cdc42, interfere with GTPase signaling in a specific fashion in a variety of cellular models. Pull-down assays showed that the C terminus of Rac1 does not associate to either RhoGDI or to Pak. In contrast, the C terminus of Rac1 (but not Rac2 or Cdc42) binds to phosphatidylinositol 4,5-phosphate kinase (PIP5K) via amino acids 185-187 (RKR). Moreover, Rac1 associates to the adapter protein Crk via the N-terminal Src homology 3 (SH3) domain of Crk and the proline-rich stretch in the Rac1 C terminus. These differential interactions mediate Rac1 localization, as well as Rac1 signaling, toward membrane ruffling, cell-cell adhesion, and migration. These data show that the C-terminal, hypervariable domain of Rac1 encodes two distinct binding motifs for signaling proteins and regulates intracellular targeting and differential signaling in a unique and non-redundant fashion.     

The Cool-2/alpha-Pix protein mediates a Cdc42-Rac signaling cascade

BACKGROUND: Cloned-out of library-2 (Cool-2)/PAK-interactive exchange factor (alpha-Pix) was identified through its ability to bind the Cdc42/Rac target p21-activated kinase (PAK) and has been implicated in certain forms of X-linked mental retardation as well as in growth factor- and chemoattractant-coupled signaling pathways. We recently found that the dimeric form of Cool-2 is a specific guanine nucleotide exchange factor (GEF) for Rac, whereas monomeric Cool-2 is a GEF for Cdc42 as well as Rac. However, unlike many GEFs, Cool-2 binds to activated forms of Cdc42 and Rac. Thus, we have investigated the functional consequences of these interactions. RESULTS: We show that the binding of activated Cdc42 to the Cool-2 dimer markedly enhances its ability to associate with GDP bound Rac1, resulting in a significant activation of Rac-GEF activity. While the Rac-specific GEF activity of Cool-2 is mediated through the Dbl homology (DH) domain from one monomer and the Pleckstrin homology domain from the other, activated Cdc42 interacts with the DH domain, most likely opposite the DH domain binding site for GDP bound Rac. Activated Rac also binds to Cool-2; however, it strongly inhibits the GEF activity of dimeric Cool-2. CONCLUSIONS: We provide evidence for novel mechanisms of allosteric regulation of the Rac-GEF activity of the Cool-2 dimer, involving stimulatory effects by Cdc42 and feedback inhibition by Rac. These findings demonstrate that by serving as a target for GTP bound Cdc42 and a GEF for Rac, Cool-2 mediates a GTPase cascade where the activation of Cdc42 is translated into the activation of Rac.     

Intersectin 1L guanine nucleotide exchange activity is regulated by adjacent src homology 3 domains that are also involved in endocytosis

Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L.     

Coactivation of Rac1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor

A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.