Functional interaction of protein kinase Cα with the tyrosine kinases Syk and Src in human platelets

There is a high degree of cross-talk between tyrosine phosphorylation and the serine/threonine phosphorylation signaling pathways. Here we show a physical and functional interaction between the classical protein kinase C isoform (cPKC), PKCalpha, and two major nonreceptor tyrosine kinases in platelets, Syk and Src. In the presence of the cPKC-selective inhibitor Go6976, platelet 5-hydroxytryptamine release was abolished in response to co-activation of glycoproteins VI and Ib-IX-V by the snake venom alboaggregin A, whereas platelet aggregation was substantially inhibited. Of the two platelet cPKCs, PKCalpha but not PKCbeta was activated, occurring in an Syk- and phospholipase C-dependent manner. Syk and PKCa associate in a stimulation-dependent manner, requiring Syk but not PKC activity. PKCa and Syk also co-translocate from the cytosol to the plasma membrane upon platelet activation, in a manner dependent upon the activities of both kinases. Although PKCa is phosphorylated on tyrosine downstream of Syk, we provide evidence against phosphorylation of Syk by PKC a, consistent with a lack of effect of PKC a inhibition on Syk activity. PKCa also associates with Src; although in contrast to interaction with Syk, PKCa activity is required for the association of these kinases but not the stimulation-induced translocation of Src to the cell membrane. Finally, the activity of Src is negatively regulated by PKC, as shown by potentiation of Src activity in the presence of the PKC inhibitors GF109203X or Go6976. Therefore, there is a complex interplay between PKCa, Syk, and Src involving physical interaction, phosphorylation, translocation within the cell, and functional activity regulation.