Translocation, Activation and Association of Protein-tyrosine Kinase (p72syk) with Phosphatidylinositol 3-Kinase are Early Events During Platelet Activation

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Abstract
We have previously reported that a non‐receptor‐type protein‐tyrosine kinase p72syk, exists in both membrane and cytosolic fractions in porcine platelets and is activated after thrombin stimulation. To facilitate the understanding of the function of p72syk, 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 p72syk 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 p72syk rapidly translocated to the membrane fraction within 10 s and there was also a significant increase in the amount of p72syk in the cytoskeletal fraction. The autophosphorylation activity of membrane‐associated p72syk 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 p72syk with phosphatidylinositol 3‐kinase and tyrosine phosphorylation of p72syk were observed. These results suggest that translocation, activation, and association of p72syk with transducing molecules such as phosphatidylinositol 3‐kinase, events which occur during platelet activation, may participate in early signal‐transduction events.