Ras-GAP controls Rho-mediated cytoskeletal reorganization through its SH3 domain.
Proteins of the Ras superfamily, Ras, Rac, Rho, and Cdc42, control the remodelling of the cortical actin cytoskeleton following growth factor stimulation. A major regulator of Ras, Ras-GAP, contains several structural motifs, including an SH3 domain and two SH2 domains, and there is evidence that they harbor a signalling function. We have previously described a monoclonal antibody to the SH3 domain of Ras-GAP which blocks Ras signalling in Xenopus oocytes. We now show that microinjection of this antibody into Swiss 3T3 cells prevents the formation of actin stress fibers stimulated by growth factors or activated Ras, but not membrane ruffling. This inhibition is bypassed by coinjection of activated Rho, suggesting that the Ras-GAP SH3 domain is necessary for endogenous Rho activation. In agreement, the antibody blocks lysophosphatidic acid-induced neurite retraction in differentiated PC12 cells. Furthermore, we demonstrate that microinjection of full-length Ras-GAP triggers 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 Ras-GAP connects the Ras and Rho pathways and, therefore, regulates the actin cytoskeleton through a mechanism which probably does not involve p190 Rho-GAP.
Rhône-Poulenc Rorer Central Research, Gene Medicine Department, Centre de Recherche de Vitry Alfortville, 94403 Vitry sur Seine, France. veronique.leblanc@exonhit.com
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D016475:3T3 Cells; D000199:Actins / Q000502:physiology / Q000648:ultrastructure; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D003599:Cytoskeleton / Q000502:physiology* / Q000648:ultrastructure*; D005260:Female; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D051379:Mice; D009414:Nerve Growth Factors / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology / Q000648:ultrastructure; D016716:PC12 Cells; D011506:Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013314:Stress, Mechanical; D014981:Xenopus; D020703:ras GTPase-Activating Proteins; D018631:ras Proteins / Q000378:metabolism*; D018909:src Homology Domains
D000199:Actins; D000911:Antibodies, Monoclonal; D020690:GTPase-Activating Proteins; D009414:Nerve Growth Factors; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D020558:GTP Phosphohydrolases; D018631:ras Proteins