Le Peuch C J, Le Peuch D A, Demaille J G
Ann N Y Acad Sci. 1982;402:549-57. doi: 10.1111/j.1749-6632.1982.tb25774.x.
We recently reported that phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump, is phosphorylated by both cAMP-dependent protein kinase and a membrane-bound, Ca2+/calmodulin-dependent phospholamban kinase. Phospholamban kinase and glycogen phosphorylase b kinase share the same substrate specificity. They differ however in that phospholamban kinase exhibits an absolute requirement for exogenous calmodulin. In line with the latter observation, phospholamban kinase is shown in this report to be inhibited by fluphenazine. Lower concentrations of the drug induced an activation of the kinase, presumably by hydrophobic interaction with either membrane phospholipids or integral proteins. Also, phospholamban kinase was found to be totally insensitive to antibodies elicited against phosphorylase kinase. Since antipsychotic drugs fail to inhibit the delta-subunit-dependent activity of phosphorylase kinase, the above findings confirm that the two kinases are distinct molecular entities. After detergent solubilization of the sarcoplasmic reticulum, the phospholamban-ATPase complex remains a substrate for phospholamban kinase activity, which retains the ability to catalyze the phosphorylation of exogenous phosphorylase b. However, the Ca2+ dependence is entirely lost upon solubilization and no kinase activity is retained on calmodulin-Sepharose in the presence of Ca2+ ions. Phospholamban and phosphorylase kinase activities copurify with the pump-phospholamban complex upon fractionation of the solubilized proteins by density gradient ultracentrifugation, suggesting a tight interaction between the ATPase, its activator, and the phospholamban kinase. A tentative schematic representation of this supramolecular assembly is based upon the results described in this and preceding papers.
我们最近报道，心肌肌浆网钙泵的激活剂受磷蛋白被环磷酸腺苷依赖性蛋白激酶和一种膜结合的钙调蛋白依赖性磷蛋白激酶磷酸化。磷蛋白激酶和糖原磷酸化酶b激酶具有相同的底物特异性。然而，它们的不同之处在于，磷蛋白激酶对外源性钙调蛋白有绝对需求。与后一观察结果一致，本报告显示氟奋乃静可抑制磷蛋白激酶。较低浓度的该药物可诱导激酶激活，推测是通过与膜磷脂或整合蛋白的疏水相互作用实现的。此外，发现磷蛋白激酶对针对磷酸化酶激酶产生的抗体完全不敏感。由于抗精神病药物无法抑制磷酸化酶激酶的δ亚基依赖性活性，上述发现证实这两种激酶是不同的分子实体。在用去污剂溶解肌浆网后，磷蛋白 - ATP酶复合物仍然是磷蛋白激酶活性的底物，该激酶保留了催化外源性磷酸化酶b磷酸化的能力。然而，溶解后完全失去了对钙离子的依赖性，并且在钙离子存在的情况下，钙调蛋白 - 琼脂糖上没有保留激酶活性。通过密度梯度超速离心对溶解的蛋白质进行分级分离时，磷蛋白和磷酸化酶激酶活性与泵 - 磷蛋白复合物一起纯化，这表明ATP酶、其激活剂和磷蛋白激酶之间存在紧密相互作用。基于本论文及之前论文中描述的结果，对这种超分子组装进行了初步的示意性表示。
