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 po rcine platelets and is activated after thrombin stimulation. To facili tate the understanding of the function of p72(syk), we have investigat ed the topological features, kinase activities and the interaction wit h another signal-transducing molecule, namely phosphatidylinositol 3-k inase, during platelet activation. Membrane and cytosolic fractions we re separated from thrombin-treated porcine platelets, and the amount o f p72(syk) was quantified by the immunoblot technique or the kinase ac tivity of each fraction was determined by an immunoprecipitation kinas e assay. After stimulation by thrombin, cytosolic p72(syk) rapidly tra nslocated to the membrane fraction within 10 s and there was also a si gnificant increase in the amount of p72(syk) in the cytoskeletal fract ion. 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 w ithin 120 s. For similar time courses, association of p72(syk) with ph osphatidylinositol 3-kinase and tyrosine phosphorylation of p72(syk) w ere observed. These results suggest that translocation, activation, an d association of p72(syk) with transducing molecules such as phosphati dylinositol 3-kinase, events which occur during platelet activation, m ay participate in early signal-transduction events.