Molecular studies on the effects of phosphorylation by p21-activated protein kinase Pak2 on protein kinase activation and protein interactions

The p21-activated protein kinase Pak2 is activated by binding of Cdc42(GTP) and autophosphorylation at eight sites. Ser-141, Ser-165, and Thr-402 are implicated in activation of Pak2. Conserved phosphorylation of Thr-402 in the activation loop is critical for the activity of Pak2. To examine the roles of Ser-141 and Ser-165 in Cdc42-dependent activation of Pak2, the sites were substituted with alanine or aspartate. Autophosphorylation and substrate phosphorylation by S141A are significantly reduced, as compared to wild-type Pak2 when activated by Cdc42. S141D, S165A, and S165D are similar to WT Pak2 in autophosphorylation and phosphorylation of S3. GST pull-down assays and coimmunoprecipitation show that WT, S141A, and S141D are stably associated with active Cdc42, but not with inactive Cdc42. S141A binds to Cdc42 at a 6-fold higher level than S141D. Thus, autophosphorylation of Ser-141 is required for the full extent of autophosphorylation and substrate phosphorylation by Cdc42-activated Pak2, and Ser-141 could have a regulatory role in subcellular localization and activation of Pak2 through modulating the interaction of Pak2 with Cdc42. Pak2, and Ser-141 could have a regulatory role in subcellular localization and activation of Pak2 through modulating the interaction of Pak2 with Cdc42.;The c-Abl tyrosine kinase is phosphorylated by Pak2 at Ser-619, which is located next to a PxxP motif (PTPPKRSS619S) that binds to the SH3 domain of Abl interactor proteins Abil/2. The effect of phosphorylation of Ser-619 on the association of c-Abl with Abi was examined using GST pull-down assays and coimmunoprecipitation. The phosphorylated Abl 593–730 fragment by Pak2 dramatically reduces Abi2 binding, as compared to the nonphosphorylated Abl. Coimmunoprecipitation following cotransfection of WT c-Abl and the mutants 3A (AAA620) and 3D (DDD620) with HA-Abi2 in 293T cells, shows that the amount of Abi2 bound to 3D is decreased by ∼80% as compared to WT and 3A. The tyrosine kinase activity of 3D with the substrate Crk is significantly enhanced in comparison to WT and 3A. The interaction of 3D with Crk is 2-fold higher than WT and 3A in the binding assays with GST-Crk. The data suggest a molecular mechanism whereby Pak2 phosphorylation of c-Ab1 on Ser-619 inhibits the interaction of c-Abl with Abi2 and enhances the association with Crk, leading to an increase of phosphorylation of Crk by c-Abl.