ACTIVATION OF PHOSPHOINOSITIDE 3-KINASE ACTIVITY BY CDC42HS BINDING TO P85

The Ras-like GTPase Cdc42 is essential for cell polarity and bud site assembly in Saccharomyces cerevisiae by regulating cell cycle dependen t reorganization of cortical cytoskeletal elements. However, its role in mammalian cells is unknown. To identify potential effecters of Cdc4 2Hs, we incubated lysates from NIH 3T3 fibroblasts or PC12 cells with immobilized glutathione S-transferase (GST)-Cdc42Hs fusion proteins bo und to different guanine nucleotides and observed a specific associati on between the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase ( PI 3-kinase) and GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-bou nd GST-Cdc42Hs. Recombinant p85 formed a complex with GTP gamma S-boun d CST-Cdc42Hs and with a GTPase defective GTP-bound GST-Cdc42Hs-Q61L m utant, but not with a GTP gamma S-bound, effector domain GST-Cdc42HsT3 5A mutant. Both the Rho-GAP homology domain of p85 and the Cdc42Hs-GAP competitively inhibited the binding of recombinant p85 to Cdc42Hs. In addition, PI 3-kinase activity immunoprecipitated from cell lysates w ith anti-p85 antibody was stimulated 2-4-fold by GST-Cdc42-GTP gamma S . Similar interactions were observed between p85 and GST-Rac1- GTP gam ma S but not between p85 and GST-RhoA-GTP gamma S. These findings sugg est that PI 3-kinase, through the Rho GAP homology domain of p85, can couple to the effector domain of Cdc42Hs and that p85 may be a target for the GTP bound forms of Cdc42Hs and Rac1.