The Rho family GTPases RhoA (Rho), Rac1, and Cdc42 are essential effectors of integrin-mediated cell attachment and spreading. Rho activity, which promotes formation of focal adhesions and actin stress fibers, is inhibited upon initial cell attachment to allow sampling of the new adhesive environment. The Abl-related gene (Arg) tyrosine kinase mediates adhesion-dependent inhibition of Rho through phosphorylation and activation of the Rho inhibitor p190RhoGAP-A (p190). p190 phosphorylation promotes its binding to p120RasGAP (p120). Here, we elucidate the mechanism by which p120 binding regulates p190 activation after adhesion. We show that p190 requires its p120-binding domain to undergo Arg-dependent activation in vivo. However, p120 binding does not activate p190RhoGAP activity in vitro. Instead, activation of p190 requires recruitment to the cell periphery. Integrin-mediated adhesion promotes relocalization of p190 and p120 to the cell periphery in wild-type fibroblasts, but not in arg(-/-) fibroblasts. A dominant-negative p120 fragment blocks p190:p120 complex formation, prevents activation of p190 by adhesion, and disrupts the adhesion-dependent recruitment of p190 to the cell periphery. Our results demonstrate that integrin signaling through Arg activates p190 by promoting its association with p120, resulting in recruitment of p190 to the cell periphery where it inhibits Rho.

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Abl family kinases, which include the mammalian Abl and Arg (Abl-related gene) kinases, regulate neuronal morphogenesis in developing metazoa (for review, see [1]). Activation of Abl kinase activity directs changes in actin-dependent processes such as membrane ruffling, filopodial protrusion, and cell motility. However, the mechanisms by which increased Abl or Arg kinase activity promote cytoskeletal rearrangements are unclear. We provide evidence that the Rho inhibitor p190RhoGAP (GTPase-activating protein) is an Arg substrate in the postnatal mouse brain. We show that p190RhoGAP has reduced phosphotyrosine content in postnatal arg(-/-) mouse brain extracts relative to wild-type extracts. In addition, the adhesion-dependent stimulation of p190RhoGAP phosphorylation observed in wild-type cells is not observed in arg(-/-) fibroblasts and neurons. Arg can phosphorylate p190RhoGAP in vitro and in vivo on tyrosine (Y) 1105. We find that Arg can stimulate p190RhoGAP to inhibit Rho and that Arg-mediated phosphorylation is required for this stimulation. Phosphorylation by Arg also promotes p190RhoGAP's association with p120RasGAP and stimulates p190RhoGAP's ability to induce neuritogenesis in neuroblastoma cells. Our results demonstrate that p190RhoGAP is an Arg substrate in the developing brain and suggest that Arg mediates the adhesion-dependent regulation of neuronal morphogenesis in the postnatal brain by phosphorylating p190RhoGAP.

The major cellular inhibitors of the small GTPases of the Ras superfamily are the GTPase-activating proteins (GAPs), which stimulate the intrinsic GTP hydrolyzing activity of GTPases, thereby inactivating them. The catalytic activity of several GAPs is reportedly inhibited or stimulated by various phospholipids and fatty acids in vitro, indicating a likely physiological role for lipids in regulating small GTPases. We find that the p190 RhoGAP, a potent GAP for the Rho and Rac GTPases, is similarly sensitive to phospholipids. Interestingly, however, several of the tested phospholipids were found to effectively inhibit the RhoGAP activity of p190 but stimulate its RacGAP activity. Thus, phospholipids have the ability to "switch" the GTPase substrate preference of a GAP, thereby providing a novel regulatory mechanism for the small GTPases.GTPase-activating proteins (GAPs) 1 for the small GTPases of the Ras superfamily are potent stimulators of intrinsic GTP hydrolyzing activity and are the major down-modulators of GTPase function. In vitro studies indicate that individual GAPs can regulate multiple members of the GTPase subfamilies, raising a question as to whether context-dependent regulation of the GAPs provides additional specificity in vivo (1). The in vivo regulation of GAPs is poorly understood, but it appears that protein-protein interactions, phosphorylation, and membrane translocation may all play a role (1-4).Phospholipids can also regulate GAP catalytic function in vitro. For example, the RasGAP activity of p120 RasGAP and NF1 is inhibited by various acidic phospholipids and fatty acids (5,6). Catalytic function of the RacGAP, n-chimaerin, is inhibited by some phospholipids and stimulated by others (7). Several GAPs for the Arf GTPases depend on phosphoinositides for GAP activity (8 -10).Here, we report that phospholipids strongly influence the GAP activity of the p190 RhoGAPs (p190A and p190B), which regulate both Rho and Rac GTPases (11,12). Interestingly, some of the phospholipids are potent inhibitors of p190 RhoGAP activity but are stimulators of its RacGAP activity. This finding indicates that phospholipids have the potential to "switch" the GTPase substrate preference for a GAP, thereby providing a novel regulatory mechanism for determining signaling specificity in vivo. Preparation of Recombinant Proteins-Hexa-histidine-tagged fulllength p190A and p190B proteins and the GAP domain-containing fragment of p190A (residues 1135-1513) were expressed in Sf9 insect cells and affinity purified on a nickel-Sepharose column. Prenylated baculovirus-produced Rac1 and RhoA (RhoA virus was provided by Dr. Matt Hart) were isolated from the membrane fraction of Sf9 cells and purified as described previously (13). Both GTPases were determined to be essentially pure as assessed by SDS-PAGE and Coomassie staining (not shown). Nonprenylated Rac1 and RhoA and the isolated GAP domain (residues 198 -439) and full-length p50RhoGAP were produced as glutathione S-transferase fusions in Escherichia coli. EXPERIMENTAL PRO...