Integrin Signaling through Arg Activates p190RhoGAP by Promoting Its Binding to p120RasGAP and Recruitment to the Membrane

Abstract

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.

The Abl-related Gene Tyrosine Kinase Acts through p190RhoGAP to Inhibit Actomyosin Contractility and Regulate Focal Adhesion Dynamics upon Adhesion to Fibronectin

“…We hypothesized that the more prominent FAs and stress fibers observed in argϪ/Ϫ cells ( Figure 2B), might result from hyperactive Rho signaling. We have previously shown that Arg is required for inhibition of Rho after integrin-mediated adhesion to FN (Bradley et al, 2006). Re-expression of Arg restores adhesiondependent inhibition of Rho to argϪ/Ϫ cells (Bradley et al, 2006).…”

“…We have previously shown that Arg is required for inhibition of Rho after integrin-mediated adhesion to FN (Bradley et al, 2006). Re-expression of Arg restores adhesiondependent inhibition of Rho to argϪ/Ϫ cells (Bradley et al, 2006). Re-expression of an Arg mutant lacking the C-terminal F-actin-and microtubule-binding domains (Arg⌬C-YFP) in argϪ/Ϫ cells restores adhesion-dependent Rho inhibition at 10 and 20 min; however, active Rho levels are slightly higher than wt cells at 30 min ( Figure 4A).…”

“…Arg phosphorylates and activates the Rho inhibitor p190RhoGAP after integrin-mediated adhesion (Hernandez et al, 2004b;Bradley et al, 2006). We monitored FA and stress fiber structure in p190rhogapaϪ/Ϫ (p190Ϫ/Ϫ) and littermate control p190rhogapa ϩ/ϩ (p190 ϩ/ϩ ) fibroblasts expressing YFP or Arg-YFP (Figure 4, C-F).…”

“…Integrin receptor engagement induces Arg-dependent phosphorylation of p190RhoGAP (Hernandez et al, 2004b), which triggers p190RhoGAP localization to the cell membrane where it inhibits Rho activity (Bradley et al, 2006). These findings led us to examine a possible role for integrin signaling through Arg, p190RhoGAP, and Rho in regulating cell adhesion and contractility during cell migration.…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…It is presently not clear how MAPK regulates Rho and ROCK activities downstream of FAK, although logical targets would include the GEFs and GAPs that set GTP loading of Rho. Integrin engagement activates p190RhoGAP, a negative regulator of Rho [105], through the tyrosine kinases Src [106][107][108][109][110], FAK [111] and Arg [112,113]. p190RhoGAP localizes to structures relatively poor in stress fibers [108] and arc-like structures in EGF stimulated cells with concomitant reduction in actin stress fibers [114], suggesting a functional link between the RhoGAP and actin dynamics during cell spreading and growth factor signaling [114,115].…”