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

Chunzhi Xia,Zhenmin Bao,Caiping Yue,Barbara M Sanborn,Mingyao Liu

doi:10.1074/jbc.m006266200

Abstract

Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, beta-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 Ca(2+) 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-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-beta2 and PLC-beta3 in vitro with purified proteins and in vivo with metabolic labeling. Phosphorylation of PLC-beta leads to the inhibition of G-protein-activated PLC-beta3 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-beta3 by PKG (Ser(26) and Ser(1105)). Mutation at these two sites (S26A and S1105A) of PLC-beta3 completely blocked the phosphorylation of PLC-beta3 protein catalyzed by PKG. Furthermore, mutation of these serine residues removed the inhibitory effect of PKG on the activation of the mutant PLC-beta3 proteins by G-protein subunits. Our results suggest a molecular mechanism for the regulation of G-protein-mediated intracellular [Ca(2+)] by the NO-cGMP-dependent signaling pathway.

Highlights

Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, ␤-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones
Direct phosphorylation of phospholipase C (PLC)-␤ isozymes by cGMP-dependent protein kinases has not been reported, it has been known that an increase in intracellular cGMP can inhibit the hydrolysis of phosphatidylinositide and the release of intracellular Ca2ϩ
In order to understand the regulation of G-protein-mediated PLCCa2ϩ signaling by pathway coupled to intracellular cGMP, we extended our studies on the phosphorylation of PLC-␤ isozymes by PKG both in vitro and in the smooth muscle cells

Summary

Introduction

Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, ␤-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanism by which cyclic nucleotides (cAMP and cGMP) could relax the smooth muscle is believed to involve the inhibition of intracellular free Ca2ϩ concentration as a result of activating cyclic nucleotide-dependent protein kinases [9]. Our recent work [22, 39], together with others [37], has suggested that PKA could phosphorylate G-protein-activated phospholipase C isozymes, regulating the G-protein-mediated intracellular Ca2ϩ signaling.

Results

Conclusion