Lipid products of phosphoinositide 3-kinase interact with rad GTPase and stimulate GDP dissociation

A number of reports suggest that under different conditions leading to cytoskeleton reorganization the GTPase Rad and possibly RhoA are downstream targets of phosphoinositide 3-kinase (PI S-kinase). In order to gain more insight into this particular signaling pathway, we have addressed the question of a possible direct interaction of PI 3-kinase products with the Rho family GTPases RhoA, Rad, and Cdc42. Using recombinant proteins, we found that Rad and, to a lesser extent, RhoA but not Cdc42 were capable to selectively bind to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) in a mixture of crude brain phosphoinositides. Nucleotide-depleted Rad was the most efficient, but the GDP- and GTP-bound forms retained significant PtdIns(3,4,5)P-3 binding activity. This protein-lipid association involved electrostatic as well as hydrophobic interactions, since both phosphate groups located at specific positions of the inositol ring and fatty-acyl chains were absolutely required. Based on the sequence of Rac1, two potential binding sites were identified, one at the C terminus and one in the extra alpha-helical domain. Deletion of these two domains resulted in a complete loss of binding to PI 3-kinase products. Finally, PtdIns(3,4,5)P-3 strongly stimulated GDP dissociation from Rad. in a dose-dependent manner. In agreement, data obtained in intact cells suggest that PtdIns(3,4,5)P-3 might target Rac1 to peculiar membrane domains, allowing formation of specific clusters containing not only small GTPases but other partners bearing pleckstrin homology domains such as specific exchange factors required for Rad and RhoA activation.