Phosphorylation and regulation of G-protein-activated phospholipase C-beta3 by cGMP-dependent protein kinases

Among the drugs that are known to relax the vascular smooth muscle and regu late other cellular functions, beta -adrenergic agonists and nitric oxide-c ontaining compounds are some of the most effective ones. The mechanisms of these drugs are thought to lower agonist-induced intracellular [Ca2+] by in creasing intracellular cAMP and cGMP, activating their respective protein k inases, However, the physiological targets of cyclic nucleotide-dependent p rotein kinases are not clear, The molecular basis for the regulation of int racellular Ca2+ by signaling pathways coupled to cyclic nucleotides is not well defined. G-protein-activated phospholipase C (PLC-beta) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphrosphates to generate diacylgly cerol and inositol 1,4,5-triphosphate, leading to the activation of protein kinase C and the mobilization of intracellular Ca2+ In this study, we show n that G-protein-activated PLC enzymes are the potential, targets of cGMP-d ependent protein kinases (PKG), PKG can directly phosphorylate PLC-beta2 an d PLC-beta3 in vitro with purified proteins and in vivo with metabolic labe ling. Phosphorylation of PLC-beta leads to the inhibition of G-protein-acti vated PLC-beta3 activity by 50-70% in COS-7 cell transfection assays. By us ing phosphopeptide mapping and site-directed mutagenesis, we further identi fied two key phosphorylation sites for the regulation of PLC-beta3 by PKG ( Ser(26) and Ser(1105)). Mutation at these two sites (S26A and S1105A) of PL C-beta3 completely blocked the phosphorylation of PLC-beta3 protein catalyz ed by PKG. Furthermore, mutation of these serine residues removed the inhib itory effect of PKG on the activation of the mutant PLC-beta3 proteins by G -protein subunits, Our results suggest a molecular mechanism for the regula tion of G-protein-mediated intracellular [Ca2+] by the NO-cGMP-dependent si gnaling pathway.