RAS-GAP CONTROLS RHO-MEDIATED CYTOSKELETAL REORGANIZATION THROUGH ITSSH3 DOMAIN

Proteins of the Ras superfamily, Ras, Rac, Rho, and Cdc42, control the remodelling of the cortical actin cytoskeleton following growth facto r stimulation. A major regulator of Pas, Ras-GAP, contains several str uctural motifs, including an SH3 domain and two SH2 domains, and there is evidence that they harbor a signalling function. We have previousl y described a monoclonal antibody to the SH3 domain of Ras-GAP which b locks Ras signalling in Xenopus oocytes, We now show that microinjecti on of this antibody into Swiss 3T3 cells prevents the formation of act in stress fibers stimulated by growth factors or activated Ras, but no t membrane ruffling. This inhibition is bypassed by coinjection of act ivated Rho, suggesting that the Ras-GAP SH3 domain is necessary for en dogenous Rho activation. In agreement, the antibody blocks lysophospha tidic acid-induced neurite retraction in differentiated PC12 cells. Fu rthermore, we demonstrate that microinjection of full-length Ras-GAP t riggers stress fiber polymerization in fibroblasts in an SH3-dependent manner, strongly suggesting an effector function besides its role as a Ras downregulator. These results support the idea that Pas-GAP conne cts the Ras and Rho pathways and, therefore, regulates the actin cytos keleton through a mechanism which probably does not involve p190 Rho-G AP.