Abnormal regulation of the Ras and Rho GTPases is associated with oncogenesis. Among the key regulators of these GTPases are the GTPase activating proteins (GAPs). The GAPs act, biochemically, as potent stimulators of the weak intrinsic GTP hydrolyzing activity of small GTPases, thereby inactivating them. However, the biological function of these numerous and ubiquitous regulatory proteins is poorly understood. To determine the precise signaling mechanism and functional role for GAPs, mouse knockouts corresponding to each of two closely related RhoGAPs, known as the p190 RhoGAPs, have been generated. Each of the knockout mice exhibits a perinatal lethal phenotype, and embryo- derived cells and tissues will be used to examine the signaling role and biological requirements for these proteins. The function of the p190 RhoGAPs will be examined at the molecular, cellular, and organismal levels. For p190 RhoGAP, the hypothesis will be tested that this protein mediates an adhesion signaling pathway via the Src and PKC kinases that directs Rho-mediated actin reorganization during neural development. For p190-B RhoGAP, the hypothesis will be tested that this protein modulates Rho activity in a pathway downstream of insulin that directs CREB-dependent cell size regulation and differentiation in nervous and non-nervous tissues. The nature of an observed partial functional redundancy between these two proteins in embryonic development will be examined by inter-crossing the two knockout lines. In addition, a closely related Drosophila homologue of the p190 RhoGAPs has been identified, and loss-of- function mutations of this gene will be generated to examine the role of the protein in a genetically tractable model system. The proposed studies are expected to substantially advance the current understanding of p190 RhoGAP function, and the broad objective of the work is to gain insight into the general biological and signaling functions of the GAPs. This is an important step toward establishing an accurate picture of the regulatory mechanisms for small GTPases in vivo, and the nature of their dysregulation in human cancers.
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