The principal goal of this application is to define the role of phosphatidylinositol kinases in signaling by the small GTP-binding protein Rac. Rac is involved in regulating the actin cytoskeleton in membrane ruffling, motility, cell adherence and maintenance of cell shape. The applicant presents evidence that Rac interacts with and may regulate a PI-4 5K and PI-3 kinase (PI 3K) and findings suggesting that Rac's regulation of the cytoskeleton may be mediated by production of specific phospholipids, since phosphatidylinositol-4,5-bisphosphate (PIP2) binds to and regulates function of actin regulatory proteins. Experiments are proposed to determine the role of PI-4 5K in vivo and to determine effects of Rac on this enzyme's activity and localization in cells. The Rac-associated PI-4 5K will be purified and a cDNA clone obtained. The regions of Rac and the kinase necessary for interacting with one another will be identified, and mutants constructed with specific loss of ability to participate in this interaction, for use as dominant negative reagents in studying Rac/PIP2/cytoskeleton signaling. Interactions among Rac, PI-4 5K and other regulatory proteins (RhoGDI, exchange proteins, etc.) will be studied with pure recombinant proteins to determine the mechanism of activation of PIP2 synthesis. In addition, experiments are described to determine whether a Rac/PI 3K complex is necessary for cell adherence and whether PI 3K acts downstream of Rac, using a PI 3K dominant negative construct.
