Smith M M, Merlie J P, Lawrence J C
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1989 Aug 5;264(22):12813-9.
Mouse BC3H1 myocytes were incubated with 32Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the 32P found in the receptor was bound to the delta subunit. Two phosphorylation sites in this subunit were resolved by reverse phase high performance liquid chromatography after exhaustive proteolysis of the protein with trypsin. Sites 1 and 2 were phosphorylated to approximately the same level in control cells. The divalent cation ionophore, A23187, increased 32P in site 1 by 40%, but did not affect the 32P content of site 2. In contrast, isoproterenol increased 32P in site 2 by more than 60%, while increasing 32P in site 1 by only 20%. When dephosphorylated receptor was incubated with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase, the delta subunit was phosphorylated to a maximal level of 1.6 phosphates/subunit. Approximately half of the phosphate went into site 2, with the remainder going into a site not phosphorylated in cells. The alpha subunit was phosphorylated more slowly, but phosphorylation of both alpha and delta subunits was blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. Phosphorylation of the receptor was also observed with preparations of phosphorylase kinase. In this case phosphorylation occurred in the beta subunit and site 1 of the delta subunit, neither of which were phosphorylated by cAMP-dependent protein kinase. The rate of receptor phosphorylation by phosphorylase kinase was slow relative to that catalyzed by cAMP-dependent protein kinase. Therefore, it can not yet be concluded that phosphorylase kinase phosphorylates the beta subunit and the delta subunit site 1 in cells. However, the results strongly support the hypothesis that phosphorylation by cAMP-dependent protein kinase accounts for phosphorylation of the alpha subunit and the delta subunit site 2 in response to elevations in cAMP.
在对乙酰胆碱受体进行溶解、免疫沉淀并进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳之前，将小鼠BC3H1肌细胞与32Pi一起孵育。在受体中发现的32P中，超过90%与δ亚基结合。在用胰蛋白酶对该蛋白进行彻底蛋白水解后，通过反相高效液相色谱法解析了该亚基中的两个磷酸化位点。在对照细胞中，位点1和位点2的磷酸化水平大致相同。二价阳离子载体A23187使位点1中的32P增加了40%，但不影响位点2中的32P含量。相比之下，异丙肾上腺素使位点2中的32P增加了60%以上，而使位点1中的32P仅增加了20%。当将去磷酸化的受体与[γ-32P]ATP和cAMP依赖性蛋白激酶的催化亚基一起孵育时，δ亚基被磷酸化至最大水平，即1.6个磷酸/亚基。大约一半的磷酸进入位点2，其余的进入细胞中未被磷酸化的位点。α亚基的磷酸化较慢，但α和δ亚基的磷酸化均被cAMP依赖性蛋白激酶的热稳定蛋白抑制剂阻断。用磷酸化酶激酶制剂也观察到了受体的磷酸化。在这种情况下，磷酸化发生在β亚基和δ亚基的位点1，而这两个位点均未被cAMP依赖性蛋白激酶磷酸化。相对于cAMP依赖性蛋白激酶催化的速率，磷酸化酶激酶对受体的磷酸化速率较慢。因此，尚不能得出磷酸化酶激酶在细胞中使β亚基和δ亚基位点1磷酸化的结论。然而，这些结果有力地支持了这样一种假说，即cAMP依赖性蛋白激酶的磷酸化导致α亚基和δ亚基位点2在cAMP升高时发生磷酸化。
