Synthesis of phosphatidylinositol 3,4-bisphosphate is regulated by protein-tyrosine phosphorylation but the p85α subunit of phosphatidylinositol 3-kinase may not be a target for tyrosine kinases in thrombin-stimulated human platelets

Abstract

To elucidate the mechanism involving synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2), which is the main species of 3-phosphorylated phosphoinositides in activated blood platelets, we observed a correlation among protein-tyrosine phosphorylation, protein kinase C (PKC) activation, and PtdIns(3,4)P 2 synthesis in these anucleate cells. Thrombin (1 U/ml) elicited marked protein-tyrosine phosphorylation, PKC activation, and PtdIns(3,4)P 2 synthesis. In contrast, 1 μM 12- O-tetradecanoylphorbol 13-acetate barely induced tyrosine phosphorylation and PtdIns(3,4)P 2 synthesis although it strongly activated PKC. A variety of kinase inhibitors were tested for their ability to inhibit the thrombin effects. Both staurosporine and tyrphostin inhibited thrombin-stimulated tyrosine phosphorylation and PtdIns(3,4)P 2 synthesis. H-7, which specifically, although weakly, inhibited PKC activation, had no effect on tyrosine phosphorylation and PtdIns(3,4)P 2 production. Among the various kinase inhibitors tested, staurosporine was the most potent inhibitor of protein tyrosine phosphorylation and PtdIns(3,4)P 2 synthesis, and there was a good correlation of the inhibition between these two parameters, although it also inhibited PKC activation. To examine the involvement of PtdIns 3-kinase, which is believed to play an important role in 3-phosphorylated phosphoinositide synthesis, we studied tyrosine phosphorylation and the association with tyrosine-phosphorylated proteins of the p85α subunit of PtdIns 3-kinase in thrombin-stimulated platelets. We did not detect tyrosine-phosphorylated protein by Western blotting where p85α was located. Similarly, when platelet lysates were precipitated with anti-p85α antibodies and then blotted with anti-phosphotyrosine antibodies, tyrosine-phosphorylated p85α was undetectable. Furthermore, when the cell lysates were precipitated with anti-phosphotyrosine antibodies, no p85α was found in the immunoprecipitates. These results show that PtdIns(3,4)P 2 synthesis in stimulated platelets is mediated by tyrosine phosphorylation, as it is in proliferating cells, but the p85α subunit of PtdIns 3-kinase may not be a target for tyrosine kinases and that staurosporine, though non-specific, would be a useful tool for elucidating signal transduction involving d-3-phosphorylated phosphoinositide generation and protein-tyrosine phosphorylation in blood platelets.