Recently, a new superfamily of single chain GTP-binding proteins (M-r 20- 30 kDa), for which the ras proteins are prototypes, has emerged. For the most part, little is known regarding the physiological functions of the different members of this family. However, the well-documented transforming properties of the ras proteins, together with the finding that the rap1A GTP-binding protein appears to counteract ras-induced transformation, suggests that members of this protein family may play key roles in cellular growth pathways. We have been studying one of the members of this family (originally designated as Gp, or G25K) as a possible participant in the signaling pathway of the epidermal growth factor receptor/tyrosine kinase. The molecular cloning of this GTP- binding protein led to the discovery that it represents the structural and functional homolog of the Saccharomuces cerevisiae cell-division- cycle protein, CDC42, and thus we now designate the protein as CDC42Hs. The primary goals of the studies proposed here are to identify and to characterize other proteins that are directly involved in the regulation of different activities of the CDC42Hs protein. Four areas of study will be pursued; these different areas comprise the specific aims of the proposal. The first involves an examination of the role of serine/threonine phosphorylation in the regulation of CDC42Hs function. We will examine the ability of different serine/threonine kinases (e.g. protein kinase A, protein kinase C) to phosphorylate this GTP-binding protein and to alter its ability to bind guanine nucleotides or associate with phospholipid bilayers in vitro. This data will be correlated with in vivo experiments using human platelets and various fibroblast cell lines. The second line of investigation involves the purification and characterization of a specific GTPase-activating protein (GAP) for CDC42Hs that we have recently identified. The abilities of different mutated forms of CDC42Hs to interact with the CDC42Hs-GAP, and the effects of phosphorylation (of either the CDC42Hs or the GAP) on these interactions, will be examined. The third regulatory protein which we recently have purified from brain cytosol. This protein stabilizes the GDP-bound state of CDC42Hs and therefore will be designated from here on as CDC42Hs-GDI.
The fourth aim will involve the identification of other cellular proteins that regulate CDC42Hs function; of particular interest will be the identification of a protein responsible for catalyzing the exchange of bound GDP for GTP on CDC42Hs. One line of approach will be to utilize recombinant forms of the CDC42Hs protein to develop affinity resins to facilitate the isolation of an exchange factor and/or to identify other CDC42Hs-regulatory proteins. The high degree of conservation of structure and function of the yeast and human forms of CDC42 suggests that these proteins play a key role in cell biology. The identification and characterization of those proteins involved in directly regulating the function of CDC42Hs represents an essential step toward the delineation of the signaling pathway for this ras-related GTP- binding protein in mammalian cells.
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