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

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Abstract
Phosphorylation of activation loop threonine (Thr505) and regulatory domain tyrosine (Tyr311) residues are key regulators of PKC (protein kinase C) δ function in platelets. In the present study, we show that Gq and G12/13 pathways regulate the Thr505 and Tyr311 phosphorylation on PKCδ in an interdependent manner. DiC8 (1,2-dioctanoylglycerol), a synthetic analogue of DAG (diacylglycerol), caused Thr505, but not Tyr311, phosphorylation on PKCδ, whereas selective activation of G12/13 pathways by the YFLLRNP peptide failed to cause phosphorylation of either residue. However, simultaneous activation by DiC8 and YFLLRNP resulted in Thr505 and Tyr311 phosphorylation on PKCδ. In addition, we found that the activation of SFKs (Src family tyrosine kinases) is essential for G12/13-mediated Tyr311 phosphorylation of PKCδ. These results were confirmed using Gq-deficient mouse platelets. Finally, we investigated whether Thr505 phosphorylation is required for Tyr311 phosphorylation. A T505A PKCδ mutant failed to be phosphorylated at Tyr311, even upon stimulation of both Gq and G12/13 pathways. We conclude that (i) PKCδ binding to DAG, downstream of Gq pathways, and its translocation results in Thr505 phosphorylation, (ii) G12/13 pathways activate SFKs required for the phosphorylation of Tyr311 on Thr505-phosphorylated PKCδ, and (iii) Thr505 phosphorylation is a prerequisite for Tyr311 phosphorylation on PKCδ.

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