Phosphorylation of c-Abl by protein kinase Pak2 regulates differential binding of ABI2 and CRK.

The tyrosine kinase c-Abl is implicated in a variety of cellular processes that are tightly regulated by c-Abl kinase activity and/or by interactions between c-Abl and other signaling molecules. The interaction of c-Abl with the Abl interactor protein Abi2 is shown to be negatively regulated by phosphorylation of serines 637 and 638. These serines are adjacent to the PxxP motif (PTPPKRS637S638SFR) that binds the SH3 domain of Abi. Phosphorylation of the Abl 593-730 fragment by Pak2 dramatically reduces Abi2 binding ( approximately 90%). Mutation of serines 637-639 to alanine (3A) or aspartate (3D) results in an increased tyrosine kinase activity of c-Abl 3D, and a slight reduction of the activity of the 3A mutant, as compared to wild-type (WT) c-Abl. The interaction between Abi2 and c-Abl 3D is inhibited by 80%, as compared to WT c-Abl or c-Abl 3A. This is accompanied by a 2-fold increase in binding of Crk to c-Abl 3D. The data indicate a molecular mechanism whereby phosphorylation of c-Abl by Pak2 inhibits the interaction between the SH3 domain of Abi2 and the PxxP motif of c-Abl. This phosphorylation enhances the association of c-Abl with the substrate Crk and increases c-Abl-mediated phosphorylation of Crk, thus altering the association of Crk with other signaling molecules.

Regulation of the interaction of Pak2 with Cdc42 via autophosphorylation of serine 141.

Pak2, a member of the p21-activated protein kinase (Pak) family, is activated in response to a variety of stresses and is directly involved in the induction of cytostasis. At the molecular level Pak2 binds Cdc42(GTP), translocating Pak2 to the endoplasmic reticulum where it is autophosphorylated and activated. Pak2 is autophosphorylated at eight sites; Ser-141 and Ser-165 in the regulatory domain and Thr-402 in the activation loop are identified as key sites in activation of the protein kinase. The function of phosphorylation of Ser-141 and Ser-165 on the activation was analyzed with wild-type (WT) and mutants of Pak2. With S141A, the level of autophosphorylation was reduced to 65% as compared with that of WT and S141D with a concomitant 45% reduction in substrate phosphorylation, indicating that phosphorylation at Ser-141 is required for optimal activity. Autophosphorylation inhibited the interaction between WT Pak2 and Cdc42(GTP). In 293T cells, WT Pak2, S141A, and S141D formed a stable complex with the constitutively active mutant Cdc42 L61, but not with the dominant negative Cdc42 N17. As shown in glutathione S-transferase pull-down assays, S141A bound to Cdc42(GTP) at a 6-fold higher level than that of S141D. In contrast, the S165A and S165D mutants had no effect on autophosphorylation, binding to Cdc42, or activation of Pak2. In summary, autophosphorylation of Ser-141 was required for activation of Pak2 and down-regulated the interaction of Pak2 with Cdc42. A model is proposed suggesting that binding of Cdc42 localizes Pak2 to the endoplasmic reticulum, where autophosphorylation alters association of the two proteins.