Growth factor-coupled signaling promotes cell cycle progression and cytoskeletal rearrangements that are responsible for changes in cell morphology and essential for cell motility and cytokinesis. A subgroup of Ras-related GTP-binding proteins that includes the Rho, Rac, and Cdc42 proteins are likely to play a critical role, together with Ras, in mediating these cytoskeletal changes. The human Cdc42 protein (Cdc42Hs) represents an especially interesting member of this family because it may feed into this pathway through an activation of PI 3-kinase activity and as an outcome of a direct interaction with the dbI oncogene product. Dbl strongly catalyzes the exchange of GTP for GDP on Cdc42Hs and is a prototype for a family of potent oncoproteins and cell growth regulators. The overall goal of the proposed studies is to take advantage of recent developments in the identification of cellular proteins that directly influence the GTP-binding/GTPase cycle of Cdc42Hs, or serve as putative targets for Cdc42Hs, to determine the molecular mechanisms underlying the regulation of this GTP-binding protein and its signaling pathway. These studies will constitute the following specific aims. 1.) Characterization of oncogenic Dbl and related proteins that stimulate the guanine nucleotide exchange activity of Cdc42Hs. 2.) Determination of the mechanisms by which the GDP-dissociation inhibitor (GDI) regulates different biochemical activities of Cdc42Hs. 3.) Determination of the mechanisms underlying the actions of the Cdc42Hs-GTPase-activating protein (GAP) and related GAP molecules. 4.) Identification and biochemical characterization of Cdc42Hs target/effector molecules. During the course of these studies a number of important questions will be addressed. These include a.) does the transformation activity of oncogenic Dbl require the specific activation of Cdc42Hs, b.) what is the role of the potentially new signaling motif, the pleckstrin-homology domain-, in the actions of oncogenic Dbl and related proteins, c.) how does the GDI interfere with the actions of Dbl arid the Cdc42Hs-GAP, d.) how does the GDI stimulate the release of Cdc42Hs and related Rho-subtype proteins from membranes, e.) what regions of the Cdc42Hs-GAP and related GAP molecules are responsible for binding Cdc42Hs versus catalysis of its GTPase activity, f.) does Cdc42Hs regulate PI 3-kinase activity in vivo, and g.) how does a 65 kDa serine/threonine kinase that is related to the PAK and Ste2O kinases participate in Cdc42Hs signaling? These studies should provide new insights into the regulation of Cdc42 and its actions in mammalian cells, and thus represent an important step toward understanding the biochemical pathways that direct cell polarity and regulate cell shape, motility, and cytokinesis. Studies of the different protein-protein interactions that influence Cdc42Hs activity also are relevant to an understanding of the regulation of related GTP-binding proteins, such as the Rac and Rho proteins, that impact on cell cytoskeleton and morphology and thus influence a diversity of cellular processes and disease states including cancer and metastasis.