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 down stream targets of phosphoinositide 3-kinase (PI S-kinase). In order to gain more insight into this particular signaling pathway, we have add ressed the question of a possible direct interaction of PI 3-kinase pr oducts with the Rho family GTPases RhoA, Rad, and Cdc42. Using recombi nant 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 phosphoin ositides. Nucleotide-depleted Rad was the most efficient, but the GDP- and GTP-bound forms retained significant PtdIns(3,4,5)P-3 binding act ivity. This protein-lipid association involved electrostatic as well a s hydrophobic interactions, since both phosphate groups located at spe cific positions of the inositol ring and fatty-acyl chains were absolu tely required. Based on the sequence of Rac1, two potential binding si tes were identified, one at the C terminus and one in the extra alpha- helical domain. Deletion of these two domains resulted in a complete l oss of binding to PI 3-kinase products. Finally, PtdIns(3,4,5)P-3 stro ngly 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 formatio n of specific clusters containing not only small GTPases but other par tners bearing pleckstrin homology domains such as specific exchange fa ctors required for Rad and RhoA activation.