Dunkley P R, Baker C M, Robinson P J
J Neurochem. 1986 Jun;46(6):1692-703. doi: 10.1111/j.1471-4159.1986.tb08486.x.
Depolarization of synaptosomes is known to cause a calcium-dependent increase in the phosphorylation of a number of proteins. It was the aim of this study to determine which protein kinases are activated on depolarization by analyzing the incorporation of 32Pi into synaptosomal phosphoproteins and phosphopeptides. The following well-characterized phosphoproteins were chosen for study: phosphoprotein "87K," synapsin Ia and Ib, phosphoproteins IIIa and IIIb, the catalytic subunits of calmodulin kinase II, and the B-50 protein. Each was initially identified as a phosphoprotein in lysed synaptosomes after incubation with [gamma-32P]ATP. Mobility on two-dimensional polyacrylamide gels and phosphorylation by specific protein kinases were the primary criteria used for identification. A technique was developed that allowed simultaneous analysis of the phosphopeptides derived from all of these proteins. Phosphopeptides were characterized in lysed synaptosomes after activating cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases in the presence of [gamma-32P]ATP. Phosphoproteins labelled in intact synaptosomes after incubation with 32Pi were then compared with those seen after ATP-labelling of lysed synaptosomes. As expected from previous work, phosphoprotein "87K," and synapsin Ia and Ib were labelled, but for the first time, phosphoproteins IIIa, IIIb, and the B-50 protein were identified as being labelled in intact synaptosomes; the calmodulin kinase II subunits were hardly phosphorylated. From a comparison of the phosphopeptide profiles it was found that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases are all active in intact synaptosomes and their activity is dependent on extrasynaptosomal calcium. The activation of cyclic AMP-stimulated protein kinases in intact synaptosomes was confirmed by the addition of dibutyryl cyclic AMP and theophylline which specifically increased the labelling of phosphopeptides in synapsin Ia and Ib and in phosphoproteins IIIa and IIIb. On depolarization of intact synaptosomes, a number of phosphopeptides showed increased labelling and the pattern suggested that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases were all activated. No new peptides were phosphorylated, suggesting that depolarization simply increased the activity of already active protein kinases and that there was no depolarization-specific increase in protein phosphorylation.
已知突触体的去极化会导致多种蛋白质的磷酸化在钙依赖的情况下增加。本研究的目的是通过分析32Pi掺入突触体磷蛋白和磷酸肽的情况，来确定去极化时哪些蛋白激酶被激活。选择了以下几种特性明确的磷蛋白进行研究：磷蛋白“87K”、突触结合蛋白Ia和Ib、磷蛋白IIIa和IIIb、钙调蛋白激酶II的催化亚基以及B-50蛋白。在与[γ-32P]ATP孵育后，每种蛋白最初都在裂解的突触体中被鉴定为磷蛋白。二维聚丙烯酰胺凝胶上的迁移率和特定蛋白激酶的磷酸化是用于鉴定的主要标准。开发了一种技术，可以同时分析源自所有这些蛋白的磷酸肽。在[γ-32P]ATP存在的情况下，激活环磷酸腺苷（cAMP）、钙调蛋白和磷脂刺激的蛋白激酶后，在裂解的突触体中对磷酸肽进行了表征。然后将用32Pi孵育后完整突触体中标记的磷蛋白与ATP标记裂解突触体后观察到的磷蛋白进行比较。正如先前工作所预期的那样，磷蛋白“87K”以及突触结合蛋白Ia和Ib被标记，但首次鉴定出磷蛋白IIIa、IIIb和B-50蛋白在完整突触体中被标记；钙调蛋白激酶II亚基几乎没有被磷酸化。通过比较磷酸肽图谱发现，cAMP、钙调蛋白和磷脂刺激的蛋白激酶在完整突触体中均有活性，且它们的活性依赖于突触体外的钙。通过添加二丁酰环磷酸腺苷和茶碱，证实了完整突触体中环磷酸腺苷刺激的蛋白激酶的激活，这特异性地增加了突触结合蛋白Ia和Ib以及磷蛋白IIIa和IIIb中磷酸肽的标记。在完整突触体去极化时，一些磷酸肽显示标记增加，模式表明cAMP、钙调蛋白和磷脂刺激的蛋白激酶均被激活。没有新的肽被磷酸化，这表明去极化只是简单地增加了已激活蛋白激酶的活性，并且蛋白磷酸化没有去极化特异性的增加。
