Differential regulation of threonine and tyrosine phosphorylations on protein kinase Cδ by G-protein-mediated pathways in platelets

Phosphorylation of activation loop threonine (Thr 505 ) and regulatory domain tyrosine (Tyr 311 ) residues are key regulators of PKC (protein kinase C) δ function in platelets. In the present study, we show that G q and G 12/13 pathways regulate the Thr 505 and Tyr 311 phosphorylation on PKCδ in an interdependent manner. DiC8 (1,2-dioctanoylglycerol), a synthetic analogue of DAG (diacylglycerol), caused Thr 505 , but not Tyr 311 , phosphorylation on PKCδ, whereas selective activation of G 12/13 pathways by the YFLLRNP peptide failed to cause phosphorylation of either residue. However, simultaneous activation by DiC8 and YFLLRNP resulted in Thr 505 and Tyr 311 phosphorylation on PKCδ. In addition, we found that the activation of SFKs (Src family tyrosine kinases) is essential for G 12/13 -mediated Tyr 311 phosphorylation of PKCδ. These results were confirmed using G q -deficient mouse platelets. Finally, we investigated whether Thr 505 phosphorylation is required for Tyr 311 phosphorylation. A T505A PKCδ mutant failed to be phosphorylated at Tyr 311 , even upon stimulation of both G q and G 12/13 pathways. We conclude that (i) PKCδ binding to DAG, downstream of G q pathways, and its translocation results in Thr 505 phosphorylation, (ii) G 12/13 pathways activate SFKs required for the phosphorylation of Tyr 311 on Thr 505 -phosphorylated PKCδ, and (iii) Thr 505 phosphorylation is a prerequisite for Tyr 311 phosphorylation on PKCδ.