CHARACTERIZATION OF A RAC1-ASSOCIATED AND RHOGDI-ASSOCIATED LIPID KINASE SIGNALING COMPLEX

Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxi dase activation. The mechanisms by,which these G proteins mediate thei r effects are unclear, although a number of downstream targets have be en identified. The interaction of most of these target proteins with R ho GTPases is GTP dependent and requires the effector domain, The acti vation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have Set been identifi ed. We previously showed that Rac interacts with a type I phosphatidyl -inositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP-and GDP-bound Rad. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrate d that the C terminus of Rac is necessary and sufficient for binding t o both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copu rify by liquid chromatography, suggesting that they bind as a complex to Rac, RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interac tion between Rac and the lipid kinases was enhanced by specific phosph olipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implic ated in several Rac-regulated processes, and they bind to the Rac C te rminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathw ays.