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Levels of active Rac1 at epithelial junctions are partially modulated via interaction with Ajuba, an actin binding and scaffolding protein. Here we demonstrate that Ajuba interacts with the Cdc42 GTPase activating protein CdGAP, a GAP for Rac1 and Cdc42, at cell-cell contacts. CdGAP recruitment to junctions does not require Ajuba; rather Ajuba seems to control CdGAP residence at sites of cell-cell adhesion. CdGAP expression potently perturbs junctions and Ajuba binding inhibits CdGAP activity. Ajuba interacts with Rac1 and CdGAP via distinct domains and can potentially bring them in close proximity at junctions to facilitate activity regulation. Functionally, CdGAP-Ajuba interaction maintains junctional integrity in homeostasis and diseases: (i) gain-of-function CdGAP mutants found in Adams-Oliver Syndrome patients strongly destabilize cell-cell contacts and (ii) CdGAP mRNA levels are inversely correlated with E-cadherin protein expression in different cancers. We present conceptual insights on how Ajuba can integrate CdGAP binding and inactivation with the spatio-temporal regulation of Rac1 activity at junctions. Ajuba provides a novel mechanism due to its ability to bind to CdGAP and Rac1 via distinct domains and influence the activation status of both proteins. This functional interplay may contribute towards conserving the epithelial tissue architecture at steady-state and in different pathologies.

The loss of E-cadherin causes dysfunction of the cell-cell junction machinery, which is an initial step in epithelial-to-mesenchymal transition (EMT), facilitating cancer cell invasion and the formation of metastases. A set of transcriptional repressors of E-cadherin (CDH1) gene expression, including Snail1, Snail2 and Zeb2 mediate E-cadherin downregulation in breast cancer. However, the molecular mechanisms underlying the control of E-cadherin expression in breast cancer progression remain largely unknown. Here, by using global gene expression approaches, we uncover a novel function for Cdc42 GTPase-activating protein (CdGAP) in the regulation of expression of genes involved in EMT. We found that CdGAP used its proline-rich domain to form a functional complex with Zeb2 to mediate the repression of E-cadherin expression in ErbB2-transformed breast cancer cells. Conversely, knockdown of CdGAP expression led to a decrease of the transcriptional repressors Snail1 and Zeb2, and this correlated with an increase in E-cadherin levels, restoration of cell-cell junctions, and epithelial-like morphological changes. In vivo, loss of CdGAP in ErbB2-transformed breast cancer cells impaired tumor growth and suppressed metastasis to lungs. Finally, CdGAP was highly expressed in basal-type breast cancer cells, and its strong expression correlated with poor prognosis in breast cancer patients. Together, these data support a previously unknown nuclear function for CdGAP where it cooperates in a GAP-independent manner with transcriptional repressors to function as a critical modulator of breast cancer through repression of E-cadherin transcription. Targeting Zeb2-CdGAP interactions may represent novel therapeutic opportunities for breast cancer treatment.

Mutations in the CdGAP/ARHGAP31 gene, which encodes a GTPase-activating protein for Rac1 and Cdc42, have been reported causative in the Adams-Oliver developmental syndrome often associated with vascular defects. However, despite its abundant expression in endothelial cells, CdGAP function in the vasculature remains unknown. Here, we show that vascular development is impaired in CdGAP-deficient mouse embryos at E15.5. This is associated with superficial vessel defects and subcutaneous edema, resulting in 44% embryonic/perinatal lethality. VEGF-driven angiogenesis is defective in CdGAP(-/-) mice, showing reduced capillary sprouting from aortic ring explants. Similarly, VEGF-dependent endothelial cell migration and capillary formation are inhibited upon CdGAP knockdown. Mechanistically, CdGAP associates with VEGF receptor-2 and controls VEGF-dependent signaling. Consequently, CdGAP depletion results in impaired VEGF-mediated Rac1 activation and reduced phosphorylation of critical intracellular mediators including Gab1, Akt, PLCγ and SHP2. These findings are the first to demonstrate the importance of CdGAP in embryonic vascular development and VEGF-induced signaling, and highlight CdGAP as a potential therapeutic target to treat pathological angiogenesis and vascular dysfunction.

Rho family GTPases act as molecular switches regulating actin cytoskeleton dynamics. Attenuation of their signaling capacity is provided by GTPase-activating proteins (GAPs), including p190A, that promote the intrinsic GTPase activity of Rho proteins. In the current study we have performed a small-scale ENU mutagenesis screen and identified a novel loss of function allele of the p190A gene Arhgap35, which introduces a Leu1396 to Gln substitution in the GAP domain. This results in decreased GAP activity for the prototypical Rho-family members, RhoA and Rac1, likely due to disrupted ordering of the Rho binding surface. Consequently, Arhgap35-deficient animals exhibit hypoplastic and glomerulocystic kidneys. Investigation into the cystic phenotype shows that p190A is required for appropriate primary cilium formation in renal nephrons. P190A specifically localizes to the base of the cilia to permit axoneme elongation, which requires a functional GAP domain. Pharmacological manipulations further reveal that inhibition of either Rho kinase (ROCK) or F-actin polymerization is able to rescue the ciliogenesis defects observed upon loss of p190A activity. We propose a model in which p190A acts as a modulator of Rho GTPases in a localized area around the cilia to permit the dynamic actin rearrangement required for cilia elongation. Together, our results establish an unexpected link between Rho GTPase regulation, ciliogenesis and glomerulocystic kidney disease.

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.

Regulation of cell proliferation and motility is essential for normal development. The Rho family of GTPases plays a critical role in the control of cell polarity and migration by effecting the cytoskeleton, membrane trafficking, and cell adhesion. We investigated a recognized developmental disorder, Adams-Oliver syndrome (AOS), characterized by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). Through a genome-wide linkage analysis, we detected a locus for autosomal-dominant ACC-TTLD on 3q generating a maximum LOD score of 4.93 at marker rs1464311. Candidate-gene- and exome-based sequencing led to the identification of independent premature truncating mutations in the terminal exon of the Rho GTPase-activating protein 31 gene, ARHGAP31, which encodes a Cdc42/Rac1 regulatory protein. Mutant transcripts are stable and increase ARHGAP31 activity in vitro through a gain-of-function mechanism. Constitutively active ARHGAP31 mutations result in a loss of available active Cdc42 and consequently disrupt actin cytoskeletal structures. Arhgap31 expression in the mouse is substantially restricted to the terminal limb buds and craniofacial processes during early development; these locations closely mirror the sites of impaired organogenesis that characterize this syndrome. These data identify the requirement for regulated Cdc42 and/or Rac1 signaling processes during early human development., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

RhoA, Rac1 and Cdc42, the best-characterized members of the Rho family of small GTPases, are critical regulators of many cellular activities. Cdc42 GTPase-activating protein (CdGAP) is a serine- and proline-rich RhoGAP protein showing GAP activity against both Cdc42 and Rac1 but not RhoA. CdGAP is phosphorylated downstream of the MEK-ERK (extracellular signal-regulated kinase) pathway in response to serum and is required for normal cell spreading and polarized lamellipodia formation. In this study, we found that CdGAP protein and mRNA levels are highly increased in mammary tumor explants expressing an activated Neu/ErbB-2 (Neu-NT) receptor. In response to transforming growth factor-β (TGFβ) stimulation, Neu-NT-expressing mammary tumor explants demonstrate a clear induction in cell motility and invasion. We show that downregulation of CdGAP expression by small interfering RNA abrogates the ability of TGFβ to induce cell motility and invasion of Neu-NT-expressing mammary tumor explants. However, it has no effect on TGFβ-mediated cell adhesion on type 1 collagen and fibronectin. Interestingly, protein expression of E-Cadherin is highly increased in Neu-NT-expressing mammary tumor explants depleted of CdGAP. In addition, complete loss of E-Cadherin expression is not observed in CdGAP-depleted cells during TGFβ-mediated epithelial to mesenchymal transition. Downregulation of the CdGAP expression also decreases cell proliferation of Neu-NT-expressing mammary tumor explants independently of TGFβ. Rescue analysis using re-expression of various CdGAP deletion-mutant proteins revealed that the proline-rich domain (PRD) but not the GAP domain of CdGAP is essential to mediate TGFβ-induced cell motility and invasion. Finally, we found that TGFβ induces the expression and phosphorylation of CdGAP in mammary epithelial NMuMG cells. Taken together, these studies identify CdGAP as a novel molecular target in TGFβ signaling and implicate CdGAP as an essential component in the synergistic interaction between TGFβ and Neu/ErbB-2 signaling pathways in breast cancer cells.

Rho GTPases regulate a multitude of cellular processes from cytoskeletal reorganization to gene transcription and are negatively regulated by GTPase-activating proteins (GAPs). Cdc42 GTPase-activating protein (CdGAP) is a ubiquitously expressed GAP for Rac1 and Cdc42. In this study, we set out to identify CdGAP-binding partners and, using a yeast two-hybrid approach, glycogen synthase kinase 3alpha (GSK-3alpha) was identified as a partner for CdGAP. GSK-3 exists in two isoforms, alpha and beta, and is involved in regulating many cellular functions from insulin response to tumorigenesis. We show that GSK-3alpha and -beta interact with CdGAP in mammalian cells. We also demonstrate that GSK-3 phosphorylates CdGAP both in vitro and in vivo on Thr-776, which we have previously shown to be an ERK 1/2 phosphorylation site involved in CdGAP regulation. We report that the mRNA and protein levels of CdGAP are increased upon serum stimulation and that GSK-3 activity is necessary for the up-regulation of the protein levels of CdGAP but not for the increase in mRNA. We conclude that GSK-3 is an important regulator of CdGAP and that regulation of CdGAP protein levels by serum presents a novel mechanism for cells to control Cdc42/Rac1 GTPase signaling pathways.

The Rho GTPases are implicated in almost every fundamental cellular process. They act as molecular switches that cycle between an active GTP-bound and an inactive GDP-bound state. Their slow intrinsic GTPase activity is greatly enhanced by RhoGAPs (Rho GTPase-activating proteins), thus causing their inactivation. To date, more than 70 RhoGAPs have been identified in eukaryotes, ranging from yeast to human, and based on sequence homology of their RhoGAP domain, we have grouped them into subfamilies. In the present Review, we discuss their regulation, biological functions and implication in human diseases.

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.

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.

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.

The GTPase-activating proteins (GAPs) accelerate the hydrolysis of GTP to GDP by small GTPases. The GTPases play diverse roles in many cellular processes, including proliferation, cell motility, endocytosis, nuclear import/export, and nuclear membrane formation. Little is known about GAP-domain proteins in spermatogenesis. We isolated a novel RhoGAP domain-containing tGAP1 protein from male germ cells that exhibits unusual properties. The tGAP1 is expressed at low levels in early spermatogonia. Robust transcription initiates in midpachytene spermatocytes and continues after meiosis. The 175-kDa tGAP1 protein localizes to the cytoplasm of spermatocytes and to the cytoplasm and nucleus in spermatids. The protein contains four GAP domain-related sequences, in contrast to all other GAP proteins that harbor one such domain. No activity toward RhoA, Rac1, or Cdc42 could be detected. Results of transfection studies in various somatic cells indicated that low-level tGAP1 expression significantly slows down the cell cycle. Expression of higher levels of tGAP1 by infection of somatic cells with recombinant adenoviruses demonstrated that tGAP1 efficiently induces apoptosis, which to our knowledge is the first such demonstration for a RhoGAP protein. Based on its subcellular location in spermatids and its activity, tGAP1 may play a role in nuclear import/export.

The Rho GTPases RhoA, Rac1, and Cdc42 play a major role in regulating the reorganization of the actin cytoskeleton. We recently identified CdGAP, a novel GTPase-activating protein with activity toward Rac1 and Cdc42. CdGAP consists of a N-terminal GAP domain, a central domain, and a C-terminal proline-rich domain. Here we show that through a subset of its Src homology 3 domains, the endocytic protein intersectin interacts with CdGAP. In platelet-derived growth factor-stimulated Swiss 3T3 cells, intersectin co-localizes with CdGAP and inhibits its GAP activity toward Rac1. Intersectin-Src homology 3 also inhibits CdGAP activity in GAP assays in vitro. Although the C-terminal proline-rich domain of CdGAP is required for the regulation of its GAP activity by intersectin both in vivo and in vitro, it is not necessary for CdGAP-intersectin interaction. Our data suggest that the central domain of CdGAP is required for CdGAP-intersectin interaction. Thus, we propose a model in which intersectin binding results in a change of CdGAP conformation involving the proline-rich domain that leads to the inhibition of its GAP activity. These observations provide the first demonstration of a direct regulation of RhoGAP activity through a protein-protein interaction and suggest a function for intersectin in Rac1 regulation and actin dynamics.

Microphthalmia with linear skin defects (MLS) is an X-linked dominant, male-lethal syndrome characterized by microphthalmia, aplastic skin and agenesis of the corpus callosum, and is caused by the deletion of a 500 kb critical region in Xp22.3. Our laboratory isolated a novel rho GTPase-activating protein (rhoGAP) gene named ARHGAP6 from the MLS region. ARHGAP6 contains 14 exons encoding a 974 amino acid protein with three putative SH3-binding domains. Because exons 2-14 are deleted in all MLS patients, we hypothesized that ARHGAP6 may be responsible for some of the phenotypic features of MLS. We pursued two approaches to study the function of ARHGAP6 and its role in the pathogenesis of MLS: gene targeting of the rhoGAP domain in mouse embryonic stem cells and in vitro expression studies. Surprisingly, loss of the rhoGAP function of Arhgap6 does not cause any detectable phenotypic or behavioral abnormalities in the mutant mice. Transfected mammalian cells expressing ARHGAP6 lose their actin stress fibers, retract from the growth surface and extend thin, branching processes resembling filopodia. The ARHGAP6 protein co-localizes with actin filaments through an N-terminal domain and recruits F-actin into the growing processes. Mutation of a conserved arginine residue in the rhoGAP domain prevents the loss of stress fibers but has little effect on process outgrowth. These results suggest that ARHGAP6 has two independent functions: one as a GAP with specificity for RhoA and the other as a cytoskeletal protein that promotes actin remodeling.

Cdc42 mediates several signaling pathways leading to actin reorganization, transcriptional activation, and cell cycle control. Mutational analysis of Cdc42 has revealed that actin reorganization and transcriptional activation are induced through independent signaling pathways. The Y40C effector mutant of Cdc42 no longer interacts with many of its known target proteins, such as p65(PAK) and WASP, yet this mutant can still induce filopodia formation. To identify Cdc42 targets involved in actin rearrangements, we have screened a yeast two-hybrid cDNA library using the Y40C mutant of Cdc42 as a bait. We report here the identification of a novel serine- and proline-rich GTPase-activating protein, CdGAP, which is active in vitro on both Cdc42 and Rac. Microinjection of CdGAP into serum-starved fibroblasts inhibits both platelet-derived growth factor-induced lamellipodia and bradykinin-induced filopodia mediated by Rac and Cdc42, respectively. CdGAP does not show in vitro activity toward Rho, and it has no effect on lysophosphatidic acid-induced stress fiber formation when microinjected into fibroblasts. The carboxyl terminus of CdGAP reveals potential protein kinase C phosphorylation sites and five SH3 binding motifs. Thus, CdGAP is a novel GAP that is likely to participate in Cdc42- and Rac-induced signaling pathways leading to actin reorganization.