Thrombin-stimulated phosphatidylinositol 3-kinase activity in platelets isassociated with activation of PYK2 tyrosine kinase: Activation of both enzymes is aggregation independent

In this study, we investigated the activation of a new member of the focal adhesion kinase family of tyrosine kinases, the proline-rich tyrosine kinas e, or PYK2, in platelets. We show that PYK2 is tyrosine phosphorylated and its activity is increased during early stages of platelet aggregation. This activation coincided with increased association of phosphatidylinositol (P I) 3-kinase and PYK2, as determined by both anti-PI 3-kinase and anti-PYK2 immunoprecipitates. However, under basal conditions, some association of PY K2 and PI 3-kinase was consistently observed, even though little or no tyro sine phosphorylated PYK2 could be detected. In addition, both increased PI 3-kinase activity and increased PYK2 activity could be detected in immunopr ecipitates following thrombin stimulation. All of these events were unaffec ted by blocking platelet aggregation with arginine-glycine-aspartate-serine (RGDS) peptide, which interferes with binding of the platelet integrin alp ha(IIb)beta(3) to fibrinogen. Neither was the activation of the PYK2 kinase activity affected by blocking PI 3-kinase activity. These results support a model in which PYK2 is associated with PI 3-kinase in unstimulated platel ets and following activation of platelets, there is an increase in tyrosine phosphorylation of PYK2, increased PYK2 activity, and increased associatio n of PYK2 with PI 3-kinase, which may contribute to the increase in PI 3-ki nase activity. All of these were found to be early events independent of su bsequent platelet aggregation. J. Cell. Physiol. 183:314-320, 2000. (C) 200 0 Wiley-Liss. Inc.