Suzuki T, Wang J H
J Biol Chem. 1987 Mar 15;262(8):3880-5.
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.
一个纯的牛磷酸受磷蛋白样品被环磷酸腺苷依赖性蛋白激酶最大程度地磷酸化，达到约1摩尔磷酸盐/摩尔蛋白（分子量25,000），而从牛心脏肌浆网（SR）囊泡中纯化的、已被该蛋白激酶预磷酸化的磷酸受磷蛋白，被发现含有4.6摩尔磷酸盐/摩尔磷酸受磷蛋白。磷酸受磷蛋白磷酸化的减少发生在免疫亲和层析步骤的蛋白纯化过程中。通过向磷酸化反应中加入亲和柱流出组分，可以恢复蛋白磷酸化。流出组分的磷酸化刺激活性对煮沸和胰蛋白酶处理有抗性，并且可被有机溶剂提取，这表明内源性因子是脂质。发现各种磷脂能够刺激环磷酸腺苷依赖性蛋白激酶对磷酸受磷蛋白的磷酸化，但只有磷脂酰肌醇能够将蛋白磷酸化刺激到与SR膜结合的磷酸受磷蛋白磷酸化所达到的水平，即约5摩尔磷酸盐/摩尔。在磷脂酰肌醇存在下磷酸化的磷酸受磷蛋白，与从磷酸化的SR囊泡中分离出的磷酸受磷蛋白相比，显示出相似的磷酸化位点和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳迁移率变化。本研究结果表明，SR中的磷酸受磷蛋白嵌入富含磷脂酰肌醇的微环境中，并且这种特定环境可能对磷酸受磷蛋白调节钙泵很重要。
