The phosphorylation of purified phospholamban by cyclic AMP-dependent protein kinase is stimulated by phosphatidylinositol.
A pure bovine phospholamban sample was phosphorylated by cyclic AMP-dependent protein kinase maximally to about 1 mol of phosphate/mol of protein (Mr 25,000), whereas phospholamban purified from bovine cardiac SR (sarcoplasmic reticulum) vesicle prephosphorylated by the protein kinase was found to contain 4.6 mol of phosphate/mol of phospholamban. The decrease in phospholamban phosphorylation occurred during the protein purification at the immunoaffinity chromatography step. The protein phosphorylation could be restored by the addition of the affinity column flow-through fraction to the phosphorylation reaction. The phosphorylation-stimulating activity of the flow-through fraction was resistant to boiling and trypsin treatment and extractable by organic solvent, suggesting that the endogenous factor(s) is lipid. Various phospholipids were found capable of stimulating the phosphorylation of phospholamban by cyclic AMP-dependent protein kinase, but only phosphatidylinositol could stimulate the protein phosphorylation to a level achieved by the phosphorylation of SR membrane-bound phospholamban, about 5 mol of phosphate/mol. Phospholamban phosphorylated in the presence of phosphatidylinositol showed similar sites of phosphorylation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shifts as the phospholamban isolated from phosphorylated SR vesicles. Results of the present study suggest that phospholamban in SR is embedded in a phosphatidylinositol-rich microenvironment, and that this specific environment may be important for the regulation of Ca2+ pump by phospholamban.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D010716:Phosphatidylinositols / Q000494:pharmacology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D002135:Calcium-Binding Proteins; D010716:Phosphatidylinositols; D011494:Protein Kinases; D000099211:Phospholamban