Phosphorylation and Regulation of G-protein-activated Phospholipase C-β3 by cGMP-dependent Protein Kinases

Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, β-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanisms of these drugs are thought to lower agonist-induced intracellular [Ca ²⁺] by increasing intracellular cAMP and cGMP, activating their respective protein kinases. However, the physiological targets of cyclic nucleotide-dependent protein kinases are not clear. The molecular basis for the regulation of intracellular Ca²⁺ by signaling pathways coupled to cyclic nucleotides is not well defined. G-protein-activated phospholipase C (PLC-β) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphates to generate diacylglycerol and inositol 1,4,5-triphosphate, leading to the activation of protein kinase C and the mobilization of intracellular Ca ²⁺. In this study, we shown that G-protein-activated PLC enzymes are the potential targets of cGMP-dependent protein kinases (PKG). PKG can directly phosphorylate PLC-β2 and PLC-β3 in vitro with purified proteins and in vivo with metabolic labeling. Phosphorylation of PLC-β leads to the inhibition of G-protein-activated PLC-β3 activity by 50-70% in COS-7 cell transfection assays. By using phosphopeptide mapping and site-directed mutagenesis, we further identified two key phosphorylation sites for the regulation of PLC-β3 by PKG (Ser²⁶ and Ser¹¹⁰⁵). Mutation at these two sites (S26A and S1105A) of PLC-β3 completely blocked the phosphorylation of PLC-β3 protein catalyzed by PKG. Furthermore, mutation of these serine residues removed the inhibitory effect of PKG on the activation of the mutant PLC-β3 proteins by G-protein subunits. Our results suggest a molecular mechanism for the regulation of G-protein-mediated intracellular [Ca²⁺] by the NO-cGMP-dependent signaling pathway.