Protein kinase-A inhibits phospholipase-C activity and alters protein phosphorylation in rat myometrial plasma membranes.
Our previous studies implicated the involvement of protein kinase-A in the inhibitory effects of isoproterenol and relaxin on oxytocin-stimulated phosphoinositide turnover in rat myometrium. To understand the possible mechanisms involved, the properties and regulation of phospholipase-C (PLC) in purified myometrial plasma membranes from estrogen-primed rats were studied. The PLC activity measured with exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate was Ca2+ dependent. The nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate stimulated PLC activity with a ED50 of 1.6 microM and shifted the calcium dependence curve to the left. Guanosine 5'-(3-O-thio)triphosphate-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis was inhibited by activation of endogenous and exogenous cAMP-dependent protein kinase (PKA). The effects of endogenous and exogenous PKA were significantly reversed by IP20, a potent synthetic peptide inhibitor of PKA. In the presence of [gamma-32Pi]ATP and exogenous PKA, 32Pi was incorporated in an IP20-sensitive manner into major bands at approximately 17,000, 20,000-24,000, 33,000, 38,000, 40,000-44,000, and other higher mol wt. These data indicate that one or more GTP-binding proteins mediate activation of membrane-bound PLC in rat myometrium. Phosphorylation of one or more membrane-associated proteins by PKA may regulate myometrial PLC activity and play a role in the inhibitory effects of isoproterenol and relaxin.
Wen
Y
Y
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77030.
Anwer
K
K
Singh
S P
SP
Sanborn
B M
BM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D001345:Autoradiography; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009215:Myometrium / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium
D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D011494:Protein Kinases; D011493:Protein Kinase C