Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on alpha IIb beta 3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2

Proline-rich tyrosine kinase 2 (Pyk2) (also known as RAFTK, CAK beta or CAD TK) has been identified as a member of the focal adhesion kinase (FAK) fami ly of protein-tyrosine kinases and it has been suggested that the mode of P yk2 activation is distinct from that of FAK. In the present study we invest igated the mode of Pyk2 activation in human platelets. When platelets were stimulated with thrombin, Pyk2, as well as FAK, was markedly tyrosine-phosp horylated, in a manner mostly dependent on alpha IIb beta 3 integrin-mediat ed aggregation. The residual Pyk2 tyrosine phosphorylation observed in the absence of platelet aggregation was completely abolished by pretreatment wi th BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxy methyl ester]. The Pyk2 phosphorylation was inhibited by protein kinase C ( PKC) inhibitors at concentrations that inhibited platelet aggregation. In c ontrast, direct activation of PKC with the active phorbol ester PMA induced the tyrosine phosphorylation of Pyk2 and FAK but only when platelets were fully aggregated with the exogenous addition of fibrinogen (the ligand for alpha IIb beta 3 integrin). Furthermore, PMA-induced Pyk2 (and FAK) tyrosin e phosphorylation was also observed when platelets adhered to immobilized f ibrinogen. The activation of the von Willebrand factor (vWF)-glycoprotein I b pathway with botrocetin together with vWF failed to induce Pyk2 (and FAK) tyrosine phosphorylation. Most Pyk2 and FAK was present in the cytosol and membrane skeleton fractions in unstimulated platelets. When platelets were stimulated with thrombin, both Pyk2 and FAK were translocated to the cytos keleton in an aggregation-dependent manner. In immunoprecipitation studies, Pyk2, as well as FAK, seemed to associate with Shc through Grb2. With the use of glutathione S-transferase fusion proteins containing Shc-SH2, Grb2-S H2, and Grb2 N-terminal and C-terminal SH3 domains, it was implied that the proline-rich region of Pyk2 (and FAK) binds to the N-terminal SH3 domain o f Grb2 and that the phosphotyrosine residue of Shc binds to the SH2 domain of Grb2. Although Pyk2 and FAK have been reported to be differentially regu lated in many cell types, our results suggest that, in human platelets, the mode of Pyk2 activation is mostly similar to that of FAK, in terms of aIIb beta 3 integrin-dependent and PRC-dependent tyrosine phosphorylation. Furt hermore, Pyk2 as well as FAK, might have one or more important roles in pos t-aggregation tyrosine phosphorylation events, in association with the cyto skeleton and through interaction with adapter proteins including Grb2 and S hc.