Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells.
The role of cGMP-dependent protein kinase in the regulation of intracellular Ca2+ levels in vascular smooth muscle cells was examined by studying the effects of cGMP on the phosphorylation of the Ca(2+)-ATPase regulatory protein phospholamban. Cultured rat aortic smooth muscle cells incubated with atrial natriuretic peptide II or sodium nitroprusside responded with increased phosphorylation of the 6000-Da subunit of phospholamban. The identity of phospholamban was confirmed using immunoprecipitation methods. Phosphorylation was associated with an increase in the activation of membrane-associated ATPase by Ca2+. These results indicated that at least one site of action of cGMP in smooth muscle cells is the sarcoplasmic reticulum, where phosphorylation of proteins regulating Ca2+ fluxes occurs. Studies using confocal laser scanning microscopy to define the cellular distribution of cGMP-dependent protein kinase suggested that the enzyme was localized to the same cellular region(s) as was phospholamban. Phosphorylation of proteins by cGMP in broken cell fractions from rabbit aorta was also performed. Phospholamban and other proteins were phosphorylated in the presence of cGMP but not cAMP, suggesting that only cGMP-dependent protein kinase was associated with smooth muscle membrane fractions containing phospholamban. These results suggest that one mechanism of action of cGMP in the reduction of intracellular Ca2+ is the activation of sarcoplasmic reticulum Ca(2+)-ATPase via phosphorylation of phospholamban. The data also support the concept that compartmentalization of protein kinases with substrates in the intact cell is an important factor involved in protein phosphorylation.
Cornwell
T L
TL
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688.
Pryzwansky
K B
KB
Wyatt
T A
TA
Lincoln
T M
TM
D000818:Animals; D001011:Aorta / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation; D008565:Membrane Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology* / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D008565:Membrane Proteins; D009124:Muscle Proteins; D011494:Protein Kinases; D000099211:Phospholamban