Phosphorylation and regulation of G-protein-activated phospholipase C-beta 3 by cGMP-dependent protein kinases.
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.
Xia
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Department of Medical Biochemistry and Genetics, Center for Cancer Biology and Nutrition, Institute of Biosciences and Technology, Texas A & M University System Health Science Center, Houston, Texas 77030, USA.
Bao
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Yue
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Sanborn
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Liu
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D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D054799:Phospholipase C beta; D010766:Phosphorylation; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*
D007527:Isoenzymes; D017869:Cyclic GMP-Dependent Protein Kinases; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D002118:Calcium