Regulation of hematopoietic-specific G-protein Gα₁₅ and Gα₁₆ by protein kinase C

Heterotrimeric G proteins mediate cell growth and differentiation by coupling cell surface receptors to intracellular effector enzymes. The G-protein α subunit, Gα₁₆ and its murine homologue Gα₁₅, are expressed specifically in hematopoietic cells and their expression is highly regulated during differentiation of normal and leukemic cells. In this study, we examined the phosphorylation of Gα₁₅/Gα₁₆ and its role in receptor and effector coupling. We observed a PMA-stimulated intact cell phosphorylation of Gα₁₅ in COS7 cells transfected with Gα₁₅ and protein kinase Cα (PKCα), and phosphorylation of endogenous Gα₁₆ in HL60 cells. We also showed that peptides derived from the two G-proteins were phosphorylated in vitro using purified brain PKC. Furthermore, we identified the putative phosphorylation site and showed that mutation or deletion of this PKC phosphorylation site inhibited phospholipase C (PLC) activation. The behavior of double mutants with the constitutively active G-protein mutation (QL-mutant) and mutation in the putative phosphorylation site suggests that the phosphorylation site of Gα_15/16 is essential for receptor-coupled activation of PLC, but not for direct interaction of the G-protein with PLC-β.