Cdc42 GTPase Activity Research Articles

A new immunomodulatory protein from Ganoderma microsporum inhibits epidermal growth factor mediated migration and invasion in A549 lung cancer cells

Dysregulation of human epidermal growth factor receptor pathways by over-expression or constitutive activation can promote lung tumor processing including angiogenesis and metastasis and is associated with poor prognosis in non-small cell lung cancers. Ganoderma also known as Lingzhi has been one of the most popular chemoprevention mushrooms in East Asia for centuries. Among many bioactive components identified from Ganoderma, an immunomodulatory protein is the major ingredient for the treatment of lung cancer. Recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. However, knowledge on the pharmacological and molecular mechanisms in suppressing EGF-mediated tumor invasion and metastasis is poorly understood. The goal of study is investigate in suppressing tumor invasion and metastasis activity of GMI. GMI exhibited an inhibitory effect on EGF-induced migration and invasion. GMI treatment with EGF presented the most potent anti-migration and anti-invasion properties on Boyden chamber assay. GMI inhibited EGF-induced phosphorylation and activation of EGFR and AKT pathway kinases in a dose-dependent manner. Additionally, the EGF-induced activation of Cdc42 GTPase was inhibited by GMI, while GMI had little effect on the EGF-induced activation of Rac1 GTPase. GMI also inhibited the EGF-induced microfilament depolymerization. These findings are the first to reveal the novel functions of GMI in tumor anti-metastasis and the molecular basis for its anticancer action.

Crossveinless‐c, the Drosophila homolog of tumor suppressor DLC1, regulates directional elongation of dendritic branches via down‐regulating Rho1 activity

Diverse neuronal subtypes develop distinctive morphologies of dendritic arbors that receive synaptic or sensory inputs. Dendritic arbors of many subtypes take on a polarized shape, and one underlying mechanism is unidirectionally biased elongation of dendritic branches. As reported herein, we found that Drosophila Crossveinless-c (Cv-c) was a key regulator for such directional growth. In the cv-c mutant, two subclass of multidendritic sensory neurons examined formed dorsally directed branches; however, dendritic branches had difficulty in growing along the anterior-posterior (A-P) body axis. Cv-c belongs to the family of Rho GTPase-activating proteins (RhoGAPs) and is the homolog of human tumor suppressor DLC1. The RhoGAP activity of Cv-c was required cell-autonomously for the A-P-oriented growth, and Cv-c elevated the GTPase activity of Rho1 and Cdc42 in a cell-free assay. Our analysis of genetic interactions suggested that Rho1 was the target of Cv-c in vivo. All of our results suggest that Cv-c contributes to sprouting and subsequent growth of the A-P-oriented branches through negative regulation of Rho1. We discuss a role of Cv-c in dendritic growth in response to environmental cues.

P210Bcr−Abl desensitizes Cdc42 GTPase signaling for SDF-1α-directed migration in chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) is a lethal hematological disorder caused by the p210(Bcr-Abl) oncogene. Previous studies have suggested that p210(Bcr-Abl) transformation contributes to homing and retention defects, typical of immature myeloid cells in CML, by attenuating chemotactic response to stromal-derived factor-1alpha (SDF-1alpha). As Rho family GTPases are key regulators of the cytoskeleton and have been previously found to interact with p210(Bcr-Abl), this study aimed to determine whether p210(Bcr-Abl) signaling affects SDF-1alpha chemotaxis through Rho GTPase signaling. We found that SDF-1alpha stimulated Cdc42 GTPase activation in myeloid progenitor 32D, but not in p210(Bcr-Abl)-transformed (32Dp210) cells. In fact, the basal level of active Cdc42 was elevated in 32Dp210 cells and mononuclear cells isolated from bone marrow of CML patients. Inhibition of p210(Bcr-Abl) kinase activity decreased basal Cdc42 activity and restored SDF-1alpha-induced Cdc42 and migration responses. Transduction of active Tat-Cdc42V12 abolished this reconstituted chemotactic response. As Cdc42 is particularly important in cytoskeletal remodeling and directional sensing, these results suggest that sustained activation of Cdc42 GTPase through p210(Bcr-Abl) tyrosine kinase signaling in CML cells contributes to defects in SDF-1alpha-chemotactic response due to desensitization of the actin polarization signal required for directional migration.

Morelloflavone, a Biflavonoid, Inhibits Tumor Angiogenesis by Targeting Rho GTPases and Extracellular Signal-Regulated Kinase Signaling Pathways

Morelloflavone, a biflavonoid extracted from Garcinia dulcis, has shown antioxidative, antiviral, and anti-inflammatory properties. However, the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date. In this study, we postulated that morelloflavone might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We showed that morelloflavone could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, invasion, and capillary-like tube formation of primary cultured human umbilical vascular endothelial cells in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the mouse aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. Furthermore, morelloflavone inhibited tumor growth and tumor angiogenesis of prostate cancer cells (PC-3) in xenograft mouse tumor model in vivo, suggesting that morelloflavone inhibited tumorigenesis by targeting angiogenesis. To understand the underlying mechanism of morelloflavone on the inhibitory effect of tumor growth and angiogenesis, we showed that morelloflavone could inhibit the activation of both RhoA and Rac1 GTPases but have little effect on the activation of Cdc42 GTPase. Additionally, morelloflavone inhibited the phosphorylation and activation of Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway kinases without affecting VEGF receptor 2 activity. Together, our results indicate that morelloflavone exerts antiangiogenic action by targeting the activation of Rho-GTPases and ERK signaling pathways. These findings are the first to reveal the novel functions of morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action.

Actin Pedestal Formation by Enteropathogenic Escherichia coli Is Regulated by IQGAP1, Calcium, and Calmodulin

During infection, enteropathogenic Escherichia coli (EPEC) injects effector proteins into the host cell to manipulate the actin cytoskeleton and promote formation of actin pedestals. IQGAP1 is a multidomain protein that participates in numerous cellular functions, including Rac1/Cdc42 and Ca(2+)/calmodulin signaling and actin polymerization. Here we report that IQGAP1, Ca(2+), and calmodulin modulate actin pedestal formation by EPEC. Infection with EPEC promotes both the interaction of IQGAP1 with calmodulin and the localization of IQGAP1 and calmodulin to actin pedestals while reducing the interaction of IQGAP1 with Rac1 and Cdc42. IQGAP1-null fibroblasts display a reduced polymerization of actin in response to EPEC. In addition, antagonism of calmodulin or chelation of intracellular Ca(2+) reduces EPEC-dependent actin polymerization. Furthermore, IQGAP1 specifically interacts with Tir in vitro and in cells. Together these data identify IQGAP1, Ca(2+), and calmodulin as a novel signaling complex regulating actin pedestal formation by EPEC.

A dual role for IQGAP1 in regulating exocytosis

Polarized secretion is a tightly regulated event generated by conserved, asymmetrically localized multiprotein complexes, and the mechanism(s) underlying its temporal and spatial regulation are only beginning to emerge. Although yeast Iqg1p has been identified as a positional marker linking polarity and exocytosis cues, studies on its mammalian counterpart, IQGAP1, have focused on its role in organizing cytoskeletal architecture, for which the underlying mechanism is unclear. Here, we report that IQGAP1 associates and co-localizes with the exocyst-septin complex, and influences the localization of the exocyst and the organization of septin. We further show that activation of CDC42 GTPase abolishes this association and inhibits secretion in pancreatic beta-cells. Whereas the N-terminus of IQGAP1 binds the exocyst-septin complex, enhances secretion and abrogates the inhibition caused by CDC42 or the depletion of IQGAP1, the C-terminus, which binds CDC42, inhibits secretion. Pulse-chase experiments indicate that IQGAP1 influences protein-synthesis rates, thus regulating exocytosis. We propose and discuss a model in which IQGAP1 serves as a conformational switch to regulate exocytosis.

Rho GDP dissociation inhibitor-mediated disruption of Rho GTPase activity impairs lens fiber cell migration, elongation and survival

To explore the role of the Rho GTPases in lens morphogenesis, we overexpressed bovine Rho GDP dissociation inhibitor (RhoGDIα), which serves as a negative regulator of Rho, Rac and Cdc42 GTPase activity, in a lens-specific manner in transgenic mice. This was achieved using a chimeric promoter of δ-crystallin enhancer and αA-crystallin, which is active at embryonic day 12. Several individual transgenic (Tg) lines were obtained, and exhibited ocular specific phenotype comprised of microphthalmic eyes with lens opacity. The overexpression of bovine RhoGDIα disrupted membrane translocation of Rho, Rac and Cdc42 GTPases in Tg lenses. Transgenic lenses also revealed abnormalities in the migration pattern, elongation and organization of lens fibers. These changes appeared to be associated with impaired organization of the actin cytoskeleton and cell–cell adhesions. At E14.5, the size of the RhoGDIα Tg lenses was larger compared to wild type (WT) and the central lens epithelium and differentiating fibers exhibited an abnormal increase of bromo-deoxy-uridine incorporation. Postnatal Tg eyes, however, were much smaller in size compared to WT eyes, revealing increased apoptosis in the disrupted lens fibers. Taken together, these data demonstrate a critical role for Rho GTPase-dependent signaling pathways in processes underlying morphogenesis, fiber cell migration, elongation and survival in the developing lens.

Get to grips: steering local actin dynamics with IQGAPs

IQGAPs are actin-binding proteins that scaffold numerous interaction partners, transmitting extracellular signals that influence mitogenic, morphological and migratory cell behaviour. However, the precise mechanisms by which IQGAP proteins influence actin dynamics and actin filament structures have been elusive. Now that IQGAP1 has emerged as a potential key regulator of actin-cytoskeletal dynamics by recruiting both the actin related protein (Arp)2/3 complex and/or formin-dependent actin polymerizing machineries, we propose that IQGAP1 might coordinate the function of mechanistically different actin nucleators for cooperative localized actin filament production in various cellular processes.

Regulation of Cdc42 GTPase Activity in the Formation of Hyphae inCandida albicans

The human fungal pathogen Candida albicans can switch between yeast, pseudohyphal, and hyphal morphologies. To investigate whether the distinctive characteristics of hyphae are due to increased activity of the Cdc42 GTPase, strains lacking negative regulators of Cdc42 were constructed. Unexpectedly, the deletion of the Cdc42 Rho guanine dissociation inhibitor RDI1 resulted in reduced rather than enhanced polarized growth. However, when cells lacking both Cdc42 GTPase-activating proteins, encoded by RGA2 and BEM3, were grown under pseudohyphal-promoting conditions the bud was highly elongated and lacked a constriction at its base, so that its shape resembled a hyphal germ tube. Moreover, a Spitzenkörper was present at the bud tip, a band of disorganized septin was present at bud base, true septin rings formed within the bud, and nuclei migrated out of the mother cell before the first mitosis. These are all characteristic features of a hyphal germ tube. Intriguingly, we observed hyphal-specific phosphorylation of Rga2, suggesting a possible mechanism for Cdc42 activation during normal hyphal development. In contrast, expression of Cdc42(G12V), which is constitutively GTP bound because it lacks GTPase activity, resulted in swollen cells with prominent and stable septin bars. These results suggest the development of hyphal-specific characteristics is promoted by Cdc42-GTP in a process that also requires the intrinsic GTPase activity of Cdc42.

Hyaluronan-CD44 Interaction with IQGAP1 Promotes Cdc42 and ERK Signaling, Leading to Actin Binding, Elk-1/Estrogen Receptor Transcriptional Activation, and Ovarian Cancer Progression

In this study, we have examined the interaction of hyaluronan (HA)-CD44 with IQGAP1 (one of the binding partners for the Rho GTPase Cdc42) in SK-OV-3.ipl human ovarian tumor cells. Immunological and biochemical analyses indicated that IQGAP1 (molecular mass of approximately 190 kDa) is expressed in SK-OV-3.ipl cells and that IQGAP1 interacts directly with Cdc42 in a GTP-dependent manner. Both IQGAP1 and Cdc42 were physically linked to CD44 in SK-OV-3.ipl cells following HA stimulation. Furthermore, the HA-CD44-induced Cdc42-IQGAP1 complex regulated cytoskeletal function via a close association with F-actin that led to ovarian tumor cell migration. In addition, the binding of HA to CD44 promoted the association of ERK2 with the IQGAP1 molecule, which stimulated both ERK2 phosphorylation and kinase activity. The activated ERK2 then increased the phosphorylation of both Elk-1 and estrogen receptor-alpha (ER alpha), resulting in Elk-1- and estrogen-responsive element-mediated transcriptional up-regulation. Down-regulation of IQGAP1 (by treating cells with IQGAP1-specific small interfering RNAs) not only blocked IQGAP1 association with CD44, Cdc42, F-actin, and ERK2 but also abrogated HA-CD44-induced cytoskeletal function, ERK2 signaling (e.g. ERK2 phosphorylation/activity, ERK2-mediated Elk-1/ER alpha phosphorylation, and Elk-1/ER alpha-specific transcriptional activation), and tumor cell migration. Taken together, these findings indicate that HA-CD44 interaction with IQGAP1 serves as a signal integrator by modulating Cdc42 cytoskeletal function, mediating Elk-1-specific transcriptional activation, and coordinating "cross-talk" between a membrane receptor (CD44) and a nuclear hormone receptor (ER alpha) signaling pathway during ovarian cancer progression.

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.

CD28 engagement promotes actin polymerization through the activation of the small Rho GTPase Cdc42 in human T cells.

Engagement of the costimulatory molecule CD28 is an important step in the optimal activation of T cells. Nevertheless, the specific role of CD28 in the formation of the immunological synapse and cytoskeletal changes that occur upon TCR/CD3 complex engagement is still poorly understood. Using Ab-coated surfaces, we show that CD28 engagement in the absence of any other signal induced the formation of cytoplasmic elongations enriched in filamentous actin (F-actin), in this work called filopodia or microspikes. Such structures were specific for engagement of CD28 on mAb-coated surfaces because they could not be observed in surfaces coated with either poly(L-lysine) or anti-CD3 mAb. The signaling pathway coupling CD28 to cytoskeletal rearrangements required Src-related kinase activity and promoted Vav phosphorylation and Cdc42 activation independently of the zeta-chain-associated kinase (ZAP-70). CD28-induced filopodia required Cdc42 GTPase activity, but not the related Rho GTPase Rac1. Moreover, Cdc42 colocalized to areas of increased F-actin. Our results support a specific role for the activation of the small Rho GTPase Cdc42 in the actin reorganization mediated by CD28 in human T cells.

IQGAP1 Is a Component of Cdc42 Signaling to the Cytoskeleton

The Ras-GAP related protein IQGAP1 binds several proteins, including actin, calmodulin, E-cadherin and the Rho family GTPase Cdc42. To gain insight into its in vivo function, IQGAP1 was overexpressed in mammalian cells. Transfection of IQGAP1 significantly increased the levels of active, GTP-bound Cdc42, resulting in the formation of peripheral actin microspikes. By contrast, transfection of an IQGAP1 mutant lacking part of the GAP-related domain (IQGAP1deltaGRD) substantially decreased the amount of GTP-bound Cdc42 in cell lysates. Consistent with these findings, IQGAP1DeltaGRD blocked Cdc42 function in cells that stably overexpress constitutively active Cdc42 and abrogated the effect of bradykinin on Cdc42. In cells transfected with IQGAP1deltaGRD, bradykinin was unable to activate Cdc42, translocate Cdc42 to the membrane fraction, or induce filopodia production. IQGAP1deltaGRD transfection altered cellular morphology, producing small, round cells that closely resemble Cdc42-/- cells. Some insight into the mechanism was provided by in vitro analysis, which revealed that IQGAP1deltaGRD increased the intrinsic GTPase activity of Cdc42, thereby increasing the amount of inactive, GDP-bound Cdc42. These data imply that IQGAP1 has a crucial role in transducing Cdc42 signaling to the cytoskeleton.

Regulation of CDC42 GTPase by proline-rich tyrosine kinase 2 interacting with PSGAP, a novel pleckstrin homology and Src homology 3 domain containing rhoGAP protein.

Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain-dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

Tyrosine Phosphorylation of the Bcl-2-associated Protein BNIP-2 by Fibroblast Growth Factor Receptor-1 Prevents Its Binding to Cdc42GAP and Cdc42

Fibroblast growth factor (FGF) receptor tyrosine kinases are involved in the regulation of cell growth, development, and differentiation in a variety of tissues. To isolate potential signaling molecules in the FGF signaling pathway, we have initiated a yeast two-hybrid screening using the cytosolic domain of FGF receptor-1 (Flg). Here we report the identification of BNIP-2, a previously cloned Bcl-2- and adenovirus E1B-associated protein, as a putative substrate of the receptor. When cotransfected in 293T cells, BNIP-2 was tyrosine-phosphorylated via Flg, but their interaction was transient and could only be seen by "capture" experiments with catalytically inert kinase mutants. When responsive cells were challenged with basic FGF, endogenous tyrosine-phosphorylated BNIP-2 could be precipitated with a BNIP-2 antibody. In addition, the recombinant BNIP-2 expressed in bacteria could be phosphorylated by active Flg in vitro. BNIP-2 shares a region of homology with the noncatalytic domain of Cdc42GAP, a GTPase-activating protein for the small GTP-binding molecule, Cdc42. We show here that BNIP-2 and Cdc42GAP could directly bind to each other and they also compete for the binding to the same target, Cdc42. Unexpectedly, BNIP-2, either produced as a bacterial recombinant protein or expressed in 293T cells, could stimulate the intrinsic GTPase activity of Cdc42. In all cases, tyrosine phosphorylation of BNIP-2 severely impaired its association with Cdc42GAP and its induced GTPase-activating protein-like activity toward Cdc42. These findings should allow us to further characterize the integration of signaling between receptor tyrosine kinases, GTP-binding molecules, and apoptotic pathways.

IQGAP1 Integrates Ca2+/Calmodulin and Cdc42 Signaling

Calmodulin regulates diverse Ca2+-dependent cellular processes, including cell cycle progression and cytoskeletal rearrangement. A recently identified calmodulin-binding protein, IQGAP1, interacts with both actin and Cdc42. In this study, evidence is presented that, in the absence of Ca2+, IQGAP1 bound to Cdc42, which maintained Cdc42 in the active GTP-bound state. Addition of Ca2+ both directly abrogated the effect of IQGAP1 on the intrinsic GTPase activity of Cdc42 and, in the presence of calmodulin, dissociated Cdc42 from IQGAP1. In addition, in vitro binding assays revealed that calmodulin associated with both the calponin homology domain and the IQ motifs of IQGAP1. Moreover, F-actin competed with Ca2+/calmodulin for binding to the calponin homology domain, but not the IQ motifs, of IQGAP1. Analysis of cell lysates revealed that calmodulin bound to IQGAP1 in a ternary complex with Cdc42. Increasing the Ca2+ concentration enhanced the interaction between calmodulin and IQGAP1, with a concomitant decrease in the association of IQGAP1 with Cdc42. Our data suggest that IQGAP1 functions as a scaffolding protein, providing a molecular link between Ca2+/calmodulin and Cdc42 signaling.

MgcRacGAP, A New Human GTPase-activating Protein for Rac and Cdc42 Similar to Drosophila rotundRacGAP Gene Product, Is Expressed in Male Germ Cells

In a search for new partners of the activated form of Rac GTPase, we have isolated through a two-hybrid cloning procedure a human cDNA encoding a new GTPase-activating protein (GAP) for Rho family GTPases. A specific mRNA of 3.2 kilobases was detected in low abundance in many cell types and found highly expressed in testis. A protein of the predicted size 58 kDa, which we call MgcRacGAP, was detected in human testis as well as in germ cell tumor extracts by immunoblotting with antibodies specific to recombinant protein. In vitro, the GAP domain of MgcRacGAP strongly stimulates Rac1 and Cdc42 GTPase activity but is almost inactive on RhoA. N-terminal to its GAP domain, MgcRacGAP contains a cysteine-rich zinc finger-like motif characteristic of the Chimaerin family of RhoGAPs. The closest homolog of MgcRacGAP is RotundRacGAP, a product of the Drosophila rotund locus. In situ hybridization experiments in human testis demonstrate a specific expression of mgcRacGAP mRNA in spermatocytes similar to that of rotundRacGAP in Drosophila testis. Therefore, protein sequence similarity and analogous developmental and tissue specificities of gene expression support the hypothesis that RotundRacGAP and MgcRacGAP have equivalent functions in insect and mammalian germ cells. Since rotundRacGAP deletion leads to male sterility in the fruit fly, the mgcRacGAP gene may prove likewise to play a key role in mammalian male fertility.

Cloning and expression of a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain.

CDC42, a member of the Rho family of small GTP-binding proteins, regulates cytoskeletal rearrangements required for cell division. Activating mutations in CDC42 that are refractory to GTPase activation confer a phenotype of large, multinucleated cells. Like other small GTP-binding proteins, CDC42 is activated by a guanosine exchange factor and inactivated by a GTPase-activating protein (GAP). An unidentified 25-kDa platelet protein has been shown to function as a specific CDC42GAP. Here we report the cloning of a cDNA encoding this GAP from a human platelet-precursor cell line. Sequence analysis reveals the presence of three consensus box regions characteristic of rhoGAPs. A glutathione S-transferase fusion protein containing the three boxes derived from the new clone strongly stimulated the GTPase activity of CDC42 but was much less effective on other Rho proteins. This indicates that the cDNA clone encodes a specific GAP for CDC42. Sequence analysis also reveals a potential proline-rich Src homology 3 (SH3)-binding domain preceding the first consensus box. Binding experiments show that this motif can interact with the SH3 domains of p85 alpha and of c-Src. Thus, CDC42GAP may function as a link between CDC42 and other signaling pathways.