Lee S A, Holz R W
J Biol Chem. 1986 Dec 25;261(36):17089-98.
The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.
研究了佛波酯、二辛酰甘油（DiC8）和微摩尔浓度的Ca2+对洋地黄皂苷处理的嗜铬细胞中蛋白质磷酸化和儿茶酚胺分泌的影响。[γ-32P]ATP用作通透细胞中磷酸化的底物。12-O-十四酰佛波醇-13-乙酸酯（TPA）增强了洋地黄皂苷通透细胞中Ca2+依赖性儿茶酚胺的分泌。这种增强需要MgATP。只有那些在体外激活蛋白激酶C的佛波酯才能增强儿茶酚胺分泌和蛋白质磷酸化。DiC8在体外激活蛋白激酶C并在原位模拟佛波酯的作用，也增强了儿茶酚胺分泌和蛋白质磷酸化。完整细胞预先用TPA或DiC8孵育对于随后洋地黄皂苷处理的嗜铬细胞中儿茶酚胺分泌和蛋白质磷酸化的最大效应是必需的。TPA诱导的蛋白质磷酸化增强几乎完全不依赖Ca2+，而DiC8诱导的蛋白质磷酸化增强主要依赖Ca2+。单独的微摩尔浓度Ca2+也增强了大量蛋白质的磷酸化。在没有外源蛋白激酶C激活剂的情况下，大多数响应TPA或与Ca2+联合被DiC8增强磷酸化的蛋白质也被微摩尔浓度的Ca2+磷酸化。在完整细胞中，1,1-二甲基-4-苯基哌嗪鎓（DMPP）诱导至少17种蛋白质的Ca2+依赖性磷酸化，这些蛋白质通过二维凝胶电泳检测到。与1,1-二甲基-4-苯基哌嗪鎓孵育时磷酸化的所有蛋白质在洋地黄皂苷处理的细胞中与微摩尔浓度Ca2+孵育时也被磷酸化。这些结果表明，TPA或DiC8增强的Ca2+依赖性儿茶酚胺分泌与蛋白质磷酸化增强有关，这可能由蛋白激酶C介导，并且蛋白激酶C的激活调节洋地黄皂苷处理的嗜铬细胞中儿茶酚胺的分泌。
