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.     

Integrin-mediated Signals Regulated by Members of the Rho Family of GTPases

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal–regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.     

Targeting and activation of Rac1 are mediated by the exchange factor beta-Pix

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator beta-Pix (p21-activated kinase [Pak]-interacting exchange factor). The interaction with beta-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1-beta-Pix interaction is required for Rac1 activation by beta-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the beta-Pix-binding kinase Pak1, we show that Pak1 regulates the Rac1-beta-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor beta-Pix.     

The Shank family of postsynaptic density proteins interacts with and promotes synaptic accumulation of the beta PIX guanine nucleotide exchange factor for Rac1 and Cdc42

The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and beta PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of beta PIX. Shank colocalizes with beta PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with beta PIX and beta PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of beta PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits beta PIX and PAK to spines for the regulation of postsynaptic structure.     

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.     

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.     

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.     

Analysis of small GTPase signaling pathways using p21-activated kinase mutants that selectively couple to Cdc42

p21-activated kinase 1 (Pak1) is an effector for the small GTPases Cdc42 and Rac. Because Pak1 binds to and is activated by both these GTPases, it has been difficult to precisely delineate the signaling pathways that link extracellular stimuli to Pak1 activation. To separate activation of Pak1 by Cdc42 versus activation by Rac, we devised a genetic screen in yeast that enabled us to create and identify Pak1 mutants that selectively couple to Cdc42 but not Rac1. We recovered several such Pak1 mutants and found that the residues most often affected lie within the p21 binding domain, a region previously known to mediate Pak1 binding to GTPases, but that several mutations also map outside the borders of the p21 binding domain. Pak1 mutants that associate with Cdc42 but not Rac1 were also activated by Cdc42 but not Rac1. In rat 3Y1 cells expressing oncogenic Ha-Ras, the Pak1 mutants defective in Rac1 binding are not activated, suggesting that Ras signals through a GTPase other than Cdc42 to activate Pakl. Similar results were obtained when epidermal growth factor was used to activate Pak1. However, Pak1 mutants that are unable to bind Rac are nonetheless well activated by calf serum, implying that this stimulus may induce Pak activation independent of Rac.     

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

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