Physical and Functional Interaction between Protein Kinase C δ and Fyn Tyrosine Kinase in Human Platelets

Abstract

An increasing number of tyrosine kinases have been shown to associate with isoforms of the protein kinase C (PKC) family. Here, we show evidence for physical and functional interaction between PKCδ and the Src family kinase Fyn in human platelets activated by alboaggregin-A, a snake venom capable of activating both GPIbV-IX and GPVI adhesion receptors. This interaction involves phosphorylation of PKCδ on tyrosine and is specific in that other isoforms of PKC, PKCε and λ, which also become tyrosine-phosphorylated, do not interact with Fyn. In addition, PKCδ does not interact with other platelet-expressed tyrosine kinases Syk, Src, or Btk. Stimulation also leads to activation of both Fyn and PKCδ and to serine phosphorylation of Fyn within a PKC consensus sequence. Alboaggregin-A-dependent activation of Fyn is blocked by bisindolylmaleimide I, suggesting a role for PKC isoforms in regulating Fyn activity. Platelet activation with alboaggregin-A induces translocation of the two kinases from cytoplasm to the plasma membrane of platelets, as observed by confocal immunofluorescence microscopy. Translocation of Fyn and PKCδ are blocked by PP1 and bisindolylmaleimide I, showing a dependence upon Src and PKC kinase activities. Although PKC activity is required for translocation, it is not required for association between the two kinases, because this was not blocked by bisindolylmaleimide I. Rottlerin, which inhibited PKCδ activity, did not block translocation of either PKCδ or Fyn but potentiated platelet aggregation, 5-hydroxytryptamine secretion, and the calcium response induced by alboaggregin-A, indicating that this kinase plays a negative role in the control of these processes.