The Ras and Rho GTPases mediate signaling pathways associated with normal cellular growth control as well as oncogenesis. Although these two groups of GTPases interact with different effector targets, it is now clear that their coordinated activity is responsible for their biological functions in cell proliferation and morphology. The mechanism by which Ras and Rho pathways are coupled is presently unknown, however, a mitogen-promoted cellular complex that forms between a Ras-specific and a Rho-specific GTPase activating protein (GAP) establishes a direct physical connection between Ras and Rho signals. The role of this complex, the RasGAPpl9O complex, in Ras- and Rho-mediated cellular signaling is being investigated. In previous studies from this laboratory, several biochemical properties of p190 have been established, including its association with RasGAP, its activity as a RhoGAP, and its GTPase activity. In addition, using a knockout strategy in mice, a specific biological role for pl9O has been identified in the developing nervous system, where the protein is required for axonal pathfinding at the midline. Moreover, these studies have identified p190 as the single major tyrosine-phosphorylated protein in the brain, suggesting that it performs a general signaling function in the nervous system. In the newly proposed studies, three specific questions will be addressed: l. Where does p190 fit into signaling pathways initiated by extracellular mitogens? 2. How do the identified biochemical properties of p190 and its protein interactions account for its putative function as a coordinator of Ras- and Rho-mediated signaling pathways? 3. What is the role of the RasGAP-pl90 complex in a defined biological system, namely, axon guidance? To address the signaling role of pl9O, the kinase pathways that lead to pl9O phosphorylation will be identified, and the cellular consequences of disrupting the RasGAP-p190 complex will be examined. Protein interactions with pl90 will be investigated by the characterization of several candidate pl9O-interactors identified in a yeast two-hybrid screen. The apparent role of p190 in axonal pathfinding suggests that pl9O might mediate the downstream signals of extracellular axon guidance cues. This will be tested directly in two documented signaling systems associated with axon guidance. The pl90 knockout mice will be used to confirm the biological relevance of such observations. These studies should help to establish a connection between the role of pl9O in Ras- and Rho-mediated signal transduction and a specific biological function that is likely to require the coordinated activities of both Ras and Rho GTPases.
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