Jefferson A B, Schulman H
Department of Pharmacology, Stanford University School of Medicine, California 94305-5332.
J Biol Chem. 1991 Jan 5;266(1):346-54.
The rat pituitary cell line GH3 contains a high molecular weight microtubule-associated protein with properties characteristic of microtubule-associated protein-2 (MAP-2). The 280-kDa protein is selectively immunoprecipitated by antibodies to authentic bovine brain MAP-2 and is phosphorylated at appropriate sites by cAMP-dependent protein kinase (cAMP kinase) and multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Although MAP-2 is a minor cellular constituent, it can be immunoprecipitated from [32P]Pi-labeled GH3 cells and shown to contain a high level of basal phosphorylation. Vasoactive intestinal peptide, forskolin, 3-isobutyl-1-methylxanthene, or cholera toxin, treatments which increase cellular cAMP levels, or dibutyryl cAMP stimulate phosphorylation of specific sites on MAP-2 without significantly increasing its high state of basal phosphorylation. Phosphopeptide mapping reveals that the sites phosphorylated by cAMP kinase in vitro are the same sites whose phosphorylation in situ increases following stimulation of GH3 with agents that activate cAMP kinase. Increasing intracellular Ca2+ levels in GH3 cells also stimulates phosphorylation of MAP-2 but at sites distinct from those phosphorylated following treatment with cAMP inducing agonists. Phosphopeptide mapping indicates that the sites phosphorylated by CaM kinase in vitro are the same sites whose phosphorylation in situ increases following Ca2(+)-mediated stimulation. We conclude that activation of cAMP- and Ca2(+)-based signaling pathways leads to phosphorylation of MAP-2 in GH3 cells and that cAMP kinase and CaM kinase mediate phosphorylation by these pathways, respectively.
大鼠垂体细胞系GH3含有一种高分子量微管相关蛋白，其具有微管相关蛋白-2（MAP-2）的特性。这种280 kDa的蛋白能被抗牛脑源MAP-2的抗体选择性免疫沉淀，且能被环磷酸腺苷依赖性蛋白激酶（cAMP激酶）和多功能钙/钙调蛋白依赖性蛋白激酶（CaM激酶）在合适位点磷酸化。尽管MAP-2是细胞中的一种次要成分，但可从[32P]Pi标记的GH3细胞中免疫沉淀出来，并显示其含有高水平的基础磷酸化。血管活性肠肽、福斯可林、3-异丁基-1-甲基黄嘌呤或霍乱毒素（这些处理可提高细胞内cAMP水平），或二丁酰环磷腺苷可刺激MAP-2上特定位点的磷酸化，但不会显著增加其基础磷酸化的高水平状态。磷酸肽图谱分析显示，cAMP激酶在体外磷酸化的位点与用激活cAMP激酶的试剂刺激GH3细胞后原位磷酸化增加的位点相同。提高GH3细胞内的Ca2+水平也会刺激MAP-2的磷酸化，但位点与用cAMP诱导激动剂处理后磷酸化的位点不同。磷酸肽图谱分析表明，CaM激酶在体外磷酸化的位点与Ca2+介导刺激后原位磷酸化增加的位点相同。我们得出结论，基于cAMP和Ca2+的信号通路激活会导致GH3细胞中MAP-2的磷酸化，且cAMP激酶和CaM激酶分别介导这些通路的磷酸化。
