Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain

The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain i n tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by prot ein-protein and/or protein-lipid interactions. Here we show that Dbl PH dom ain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinosit ol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the P H domain significantly inhibits the Cdc42 interactive activity of the DH do main suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transf orming activity. Immunofluorescence analysis of NIH3T3 transfectants reveal ed that whereas the PH domain localizes to actin stress fibers and plasma m embrane, the PH mutants are no longer detectable on the plasma membrane. Th ese results suggest that modulation of PIPs in both the GEF catalytic activ ity and the targeting to plasma membrane determines the outcome of the biol ogic activity of Dbl.