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

摘要

pPhosphorylation of activation loop threonine (Thrsup505/sup) and regulatory domain tyrosine (Tyrsup311/sup) residues are key regulators of PKC (protein kinase C) δ function in platelets. In the present study, we show that Gsubq/sub and Gsub12/13/sub pathways regulate the Thrsup505/sup and Tyrsup311/sup phosphorylation on PKCδ in an interdependent manner. DiC8 (1,2-dioctanoylglycerol), a synthetic analogue of DAG (diacylglycerol), caused Thrsup505/sup, but not Tyrsup311/sup, phosphorylation on PKCδ, whereas selective activation of Gsub12/13/sub pathways by the YFLLRNP peptide failed to cause phosphorylation of either residue. However, simultaneous activation by DiC8 and YFLLRNP resulted in Thrsup505/sup and Tyrsup311/sup phosphorylation on PKCδ. In addition, we found that the activation of SFKs (Src family tyrosine kinases) is essential for Gsub12/13/sub-mediated Tyrsup311/sup phosphorylation of PKCδ. These results were confirmed using Gsubq/sub-deficient mouse platelets. Finally, we investigated whether Thrsup505/sup phosphorylation is required for Tyrsup311/sup phosphorylation. A T505A PKCδ mutant failed to be phosphorylated at Tyrsup311/sup, even upon stimulation of both Gsubq/sub and Gsub12/13/sub pathways. We conclude that (i) PKCδ binding to DAG, downstream of Gsubq/sub pathways, and its translocation results in Thrsup505/sup phosphorylation, (ii) Gsub12/13/sub pathways activate SFKs required for the phosphorylation of Tyrsup311/sup on Thrsup505/sup-phosphorylated PKCδ, and (iii) Thrsup505/sup phosphorylation is a prerequisite for Tyrsup311/sup phosphorylation on PKCδ./p