Cohn J A
Department of Medicine, Duke University, Durham, North Carolina 27710.
Am J Physiol. 1990 Feb;258(2 Pt 1):C227-33. doi: 10.1152/ajpcell.1990.258.2.C227.
T84 cell monolayers were used to study the cholinergic regulation of protein phosphorylation in epithelial cells. When T84 cell monolayers are labeled with 32Pi and stimulated with carbachol, six proteins exhibit altered phosphorylation. The most prominent response is a fivefold increase in labeling of p83, an acidic protein of Mr 83,000. Increasing labeling of p83 parallels stimulated secretion with respect to the onset of agonist action, agonist potency, and antagonism by atropine. However, the p83 and secretory responses differ in that the p83 response is more sustained. When T84 cell fractions are incubated with [gamma-32P]ATP, Ca2(+)-phospholipid stimulates p83 labeling. Phosphorylation of p83 also occurs when a T84 cell extract is incubated with purified protein kinase C and when intact cells are exposed to phorbol myristate acetate. p83 does not become phosphorylated in cell fractions incubated with adenosine 3',5'-cyclic monophosphate (cAMP) or in monolayers stimulated with agonists acting via cAMP. Thus carbachol stimulates the phosphorylation of an endogenous substrate for protein kinase C in T84 cells. The duration of this phosphorylation response suggests that protein kinase C may mediate a sustained response to carbachol, possibly acting to limit the duration of stimulated secretion.
采用T84细胞单层来研究上皮细胞中蛋白质磷酸化的胆碱能调节。当T84细胞单层用³²Pi标记并用卡巴胆碱刺激时，有六种蛋白质的磷酸化发生改变。最显著的反应是p83（一种分子量为83,000的酸性蛋白质）的标记增加了五倍。就激动剂作用的起始、激动剂效力和阿托品拮抗作用而言，p83标记的增加与刺激分泌平行。然而，p83反应和分泌反应的不同之处在于，p83反应更持久。当T84细胞组分与[γ-³²P]ATP一起孵育时，Ca²⁺-磷脂刺激p83标记。当T84细胞提取物与纯化的蛋白激酶C一起孵育以及完整细胞暴露于佛波酯肉豆蔻酸酯时，p83也会发生磷酸化。在用3',5'-环磷酸腺苷（cAMP）孵育的细胞组分中或在用通过cAMP起作用的激动剂刺激的单层细胞中，p83不会发生磷酸化。因此，卡巴胆碱刺激T84细胞中蛋白激酶C的内源性底物的磷酸化。这种磷酸化反应的持续时间表明，蛋白激酶C可能介导对卡巴胆碱的持续反应，可能起到限制刺激分泌持续时间的作用。
