Coincident regulation of PKCδ in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling

PKC (protein kinase C)8 plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr(311) is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr(311) and Tyr(565) are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKC delta. Phosphorylation of both Tyr(311) and Tyre. is dependent on Sire kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCS with PMA also induced phosphorylation of Tyr(311) and Tyr(565), and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCS is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKC delta. Both thrombin and PMA induce recruitment of PKCS to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKC delta, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCS is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity.