Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP.

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

The CEACAM1-L glycoprotein associates with the actin cytoskeleton and localizes to cell-cell contact through activation of Rho-like GTPases.

Associations between plasma membrane-linked proteins and the actin cytoskeleton play a crucial role in defining cell shape and determination, ensuring cell motility and facilitating cell-cell or cell-substratum adhesion. Here, we present evidence that CEACAM1-L, a cell adhesion molecule of the carcinoembryonic antigen family, is associated with the actin cytoskeleton. We have delineated the regions involved in actin cytoskeleton association to the distal end of the CEACAM1-L long cytoplasmic domain. We have demonstrated that CEACAM1-S, an isoform of CEACAM1 with a truncated cytoplasmic domain, does not interact with the actin cytoskeleton. In addition, a major difference in subcellular localization of the two CEACAM1 isoforms was observed. Furthermore, we have established that the localization of CEACAM1-L at cell-cell boundaries is regulated by the Rho family of GTPases. The retention of the protein at the sites of intercellular contacts critically depends on homophilic CEACAM1-CEACAM1 interactions and association with the actin cytoskeleton. Our results provide new evidence on how the Rho family of GTPases can control cell adhesion: by directing an adhesion molecule to its proper cellular destination. In addition, these results provide an insight into the mechanisms of why CEACAM1-L, but not CEACAM1-S, functions as a tumor cell growth inhibitor.

Activation of the small GTPase Rac is sufficient to disrupt cadherin-dependent cell-cell adhesion in normal human keratinocytes.

To achieve strong adhesion to their neighbors and sustain stress and tension, epithelial cells develop many different specialized adhesive structures. Breakdown of these structures occurs during tumor progression, with the development of a fibroblastic morphology characteristic of metastatic cells. During Ras transformation, Rac-signaling pathways participate in the disruption of cadherin-dependent adhesion. We show that sustained Rac activation per se is sufficient to disassemble cadherin-mediated contacts in keratinocytes, in a concentration- and time-dependent manner. Cadherin receptors are removed from junctions before integrin receptors, suggesting that pathways activated by Rac can specifically interfere with cadherin function. We mapped an important region for disruption of junctions to the putative second effector domain of the Rac protein. Interestingly, although this region overlaps the domain necessary to induce lamellipodia, we demonstrate that the disassembly of cadherin complexes is a new Rac activity, distinct from Rac-dependent lamellipodia formation. Because Rac activity is also necessary for migration, Rac is a good candidate to coordinately regulate cell-cell and cell-substratum adhesion during tumorigenesis.

Activation of cdc42, rac, PAK, and rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation.

Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, processes that play crucial roles in tumor development, invasion, and metastasis. Little is known about the Met-dependent proximal signals that regulate these events. We show that HGF stimulation of epithelial cells leads to activation of the Rho GTPases, Cdc42 and Rac, concomitant with the formation of filopodia and lamellipodia. Notably, HGF-dependent activation of Rac but not Cdc42 is dependent on phosphatidylinositol 3-kinase. Moreover, HGF-induced lamellipodia formation and cell spreading require phosphatidylinositol 3-kinase and are inhibited by dominant negative Cdc42 or Rac. HGF induces activation of the Cdc42/Rac-regulated p21-activated kinase (PAK) and c-Jun N-terminal kinase, and translocation of Rac, PAK, and Rho-dependent Rho-kinase to membrane ruffles. Use of dominant negative and activated mutants reveals an essential role for PAK but not Rho-kinase in HGF-induced epithelial cell spreading, whereas Rho-kinase activity is required for the formation of focal adhesions and stress fibers in response to HGF. We conclude that PAK and Rho-kinase play opposing roles in epithelial-mesenchymal transition induced by HGF, and provide new insight regarding the role of Cdc42 in these events.

The scaffold protein Ajuba suppresses CdGAP activity in epithelia to maintain stable cell-cell contacts.

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 Cdc42/Rac1 regulator CdGAP is a novel E-cadherin transcriptional co-repressor with Zeb2 in breast cancer.

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.

CdGAP is required for transforming growth factor ß- and Neu/ErbB-2-induced breast cancer cell motility and invasion.

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-ß (TGFß) 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 TGFß to induce cell motility and invasion of Neu-NT-expressing mammary tumor explants. However, it has no effect on TGFß-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 TGFß-mediated epithelial to mesenchymal transition. Downregulation of the CdGAP expression also decreases cell proliferation of Neu-NT-expressing mammary tumor explants independently of TGFß. 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 TGFß-induced cell motility and invasion. Finally, we found that TGFß induces the expression and phosphorylation of CdGAP in mammary epithelial NMuMG cells. Taken together, these studies identify CdGAP as a novel molecular target in TGFß signaling and implicate CdGAP as an essential component in the synergistic interaction between TGFß and Neu/ErbB-2 signaling pathways in breast cancer cells.

CdGAP, a novel proline-rich GTPase-activating protein for Cdc42 and Rac.

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.

Functional analysis of ARHGAP6, a novel GTPase-activating protein for RhoA.

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.