The mammalian ras genes encode 21 kilodalton GTPases that mediate a variety of signal transduction pathways involved in cellular proliferation and differentiation. These genes have been found to undergo specific point mutations at a high frequency in a variety of human tumor types, which has led to the suggestion that they can function as dominant oncogenes. The rho genes, which encode GTPase that are about 35% identical to the ras GTPases, have been found to play a role in cytoskeletal organization and cell morphology. Although the rho genes do not appear to have a similar oncogenic activity, a connection between the ras and rho-mediated pathways has recently emerged. Experimental evidence suggests that these two GTPases might function on the same signalling cascade, although the mechanism by which the pathways are integrated is unknown. The ras GTPase activating protein (rasGAP), which binds to the ras """"""""effector domain"""""""" and stimulates the intrinsic GTPase activity of ras by several hundred fold, has emerged as the best candidate thus far for an effector target of activated ras protein. In growth factor-stimulated and tyrosine kinase-transformed cells most of the rasGAP enters into a tight complex with the cellular protein p190. We have recently clone cDNAs encoding p190, and have found in subsequent studies that this protein has a GAP activity of its own that is specific for the rho family GTPases. Thus, the complex of rasGAP and p190 in growth-simulated cells establishes a direct physical connection between the ras and rho-mediated signalling pathways. It ma;y be that coupling of these pathways through this complex serves to coordinate progression through the cell cycle with cytoskeletal reorganization. In addition to having rhoGAP activity, we have found that p190 is likely to function as a GTPase, and additionally that p190 has a partial nuclear localization, suggesting that it might play a role in transducing signals across the nuclear membrane. We propose to extend our initial observations about p190 to clarify its role in cell signalling. We will conduct an in vitro analysis of p190 using a mutational approach to address the biochemical and structural properties of the protein. To complement these studies, we will conduct an in vivo functional analysis of p190 and the rasGAP;p190 complex aimed at defining the role of these proteins in ras and rho-mediated signal transduction.
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