Thrombin-stimulated phosphatidylinositol 3-kinase activity in platelets is associated 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 kinase, 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 (PI) 3-kinase and PYK2, as determined by both anti-PI 3-kinase and anti-PYK2 immunoprecipitates. However, under basal conditions, some association of PYK2 and PI 3-kinase was consistently observed, even though little or no tyrosine phosphorylated PYK2 could be detected. In addition, both increased PI 3-kinase activity and increased PYK2 activity could be detected in immunoprecipitates following thrombin stimulation. All of these events were unaffected by blocking platelet aggregation with arginine-glycine-aspartate-serine (RGDS) peptide, which interferes with binding of the platelet integrin alpha(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 platelets and following activation of platelets, there is an increase in tyrosine phosphorylation of PYK2, increased PYK2 activity, and increased association of PYK2 with PI 3-kinase, which may contribute to the increase in PI 3-kinase activity. All of these were found to be early events independent of subsequent platelet aggregation.
Sayed
M R
MR
Department of Medicine, University of British Columbia and Vancouver Hospital and Health Sciences Centre, Jack Bell Research Centre, Vancouver, British Columbia, Canada.
Sheid
M P
MP
Stevens
C M
CM
Duronio
V
V
D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D004789:Enzyme Activation; D051418:Focal Adhesion Kinase 2; D006801:Humans; D066298:In Vitro Techniques; D009842:Oligopeptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000097:blood*; D019000:Phosphotyrosine / Q000097:blood; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000097:blood*; D013917:Thrombin / Q000494:pharmacology / Q000502:physiology*
D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D019000:Phosphotyrosine; C046617:arginyl-glycyl-aspartyl-serine; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D013917:Thrombin