CLONING AND EXPRESSION OF A HUMAN CDC42 GTPASE-ACTIVATING PROTEIN REVEALS A FUNCTIONAL SH3-BINDING DOMAIN

CDC42, a member of the Rho family of small GTP-binding proteins, regul ates cytoskeletal rearrangements required for cell division. Activatin g mutations in CDC42 that are refractory to GTPase activation confer a phenotype of large, multinucleated cells. Like other small GTP-bindin g proteins, CDC42 is activated by a guanosine exchange factor and inac tivated by a GTPase-activating protein (GAP). An unidentified 25-kDa p latelet protein has been shown to function as a specific CDC42GAP. Her e we report the cloning of a cDNA encoding this GAP from a human plate let-precursor cell line. Sequence analysis reveals the presence of thr ee consensus box regions characteristic of rhoGAPs. A glutathione S-tr ansferase fusion protein containing the three boxes derived from the n ew clone strongly stimulated the GTPase activity of CDC42 but was much less effective on other Rho proteins. This indicates that the cDNA cl one encodes a specific GAP for CDC42. Sequence analysis also reveals a potential proline-rich Src homology 3 (SH3)-binding domain preceding the first consensus box. Binding experiments show that this motif can interact with the SH3 domains of p85alpha and of c-Src. Thus, CDC42GAP may function as a link between CDC42 and other signaling pathways.