TRANSLOCATION, ACTIVATION AND ASSOCIATION OF PROTEIN-TYROSINE KINASE (P72(SYK)) WITH PHOSPHATIDYLINOSITOL 3-KINASE ARE EARLY EVENTS DURING PLATELET ACTIVATION

We have previously reported that a non-receptor-type protein-tyrosine kinase p72(syk), exists in both membrane and cytosolic fractions in porcine platelets and is activated after thrombin stimulation. To facilitate the understanding of the function of p72(syk), we have investigated the topological features, kinase activities and the interaction with another signal-transducing molecule, namely phosphatidylinositol 3-kinase, during platelet activation. Membrane and cytosolic fractions were separated from thrombin-treated porcine platelets, and the amount of p72(syk) was quantified by the immunoblot technique or the kinase activity of each fraction was determined by an immunoprecipitation kinase assay. After stimulation by thrombin, cytosolic p72(syk) rapidly translocated to the membrane fraction within 10 s and there was also a significant increase in the amount of p72(syk) in the cytoskeletal fraction. The autophosphorylation activity of membrane-associated p72(syk) significantly increased approximately tenfold and reached a maximum at 10 s; the activity subsequently decreased to almost the basal level within 120 s. For similar time courses, association of p72(syk) with phosphatidylinositol 3-kinase and tyrosine phosphorylation of p72(syk) were observed. These results suggest that translocation, activation, and association of p72(syk) with transducing molecules such as phosphatidylinositol 3-kinase, events which occur during platelet activation, may participate in early signal-transduction events.