Ohta Y, Ohba T, Fukunaga K, Miyamoto E
Department of Pharmacology, Kumamoto University Medical School, Japan.
J Biol Chem. 1988 Aug 15;263(23):11540-7.
A 50-kDa protein was recognized in rat embryo fibroblast 3Y1 cells with an affinity-purified antibody against rat brain Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). When the cytosolic extract from quiescent 3Y1 cells was immunoprecipitated with the antibody, the 50-kDa protein in the immunoprecipitate became phosphorylated in a Ca2+- and calmodulin-dependent manner following exposure to [gamma-32P]ATP. Moreover, the reaction proceeded through an intramolecular mechanism. These results suggest that the 50-kDa protein is a subunit of CaM kinase II in rat 3Y1 cells. The addition of 10% fetal calf serum to quiescent 3Y1 cells caused a rapid increase in the phosphorylation of the 50-kDa protein, which was immunoprecipitated with the affinity-purified anti-CaM kinase II antibody. The phosphorylation of CaM kinase II was detected as early as 20 s after the addition of serum, reached the maximal level at 2 min, and decreased to the basal level within 60 min. Platelet-derived growth factor and epidermal growth factor also elicited the phosphorylation of the 50-kDa protein in quiescent 3Y1 cells, while neither insulin nor 12-O-tetradecanoylphorbol-13-acetate did. Calcium ionophores, A23187 and ionomycin, also caused the phosphorylation of the protein in 3Y1 cells. Moreover, phosphopeptide mappings of the phosphorylated 50-kDa subunit generated in response to serum, EGF, and A23187 yielded patterns similar to that generated from the immunoprecipitated 50-kDa subunit phosphorylated in vitro. Phosphoamino acid analysis of the phosphorylated subunit demonstrated that serine residue was the major amino acid labeled under any condition. These results suggest that CaM kinase II undergoes phosphorylation in response to various stimuli that can increase the free Ca2+ concentration in the cytoplasm of quiescent fibroblast cells and therefore probably mediates at least some of the biological actions of growth factors.
用针对大鼠脑钙调蛋白依赖性蛋白激酶II（CaM激酶II）的亲和纯化抗体在大鼠胚胎成纤维细胞3Y1中识别出一种50 kDa的蛋白质。当用该抗体对静止3Y1细胞的胞质提取物进行免疫沉淀时，免疫沉淀物中的50 kDa蛋白质在暴露于[γ-32P]ATP后以钙和钙调蛋白依赖性方式发生磷酸化。此外，反应通过分子内机制进行。这些结果表明，50 kDa蛋白质是大鼠3Y1细胞中CaM激酶II的一个亚基。向静止3Y1细胞中添加10%胎牛血清导致50 kDa蛋白质的磷酸化迅速增加，该蛋白质用亲和纯化的抗CaM激酶II抗体进行免疫沉淀。早在添加血清后20秒就检测到CaM激酶II的磷酸化，在2分钟时达到最大水平，并在60分钟内降至基础水平。血小板衍生生长因子和表皮生长因子也能诱导静止3Y1细胞中50 kDa蛋白质的磷酸化，而胰岛素和12-O-十四烷酰佛波醇-13-乙酸酯则不能。钙离子载体A23187和离子霉素也能导致3Y1细胞中该蛋白质的磷酸化。此外，对血清、表皮生长因子和A23187反应产生的磷酸化50 kDa亚基的磷酸肽图谱与体外磷酸化的免疫沉淀50 kDa亚基产生的图谱相似。对磷酸化亚基的磷酸氨基酸分析表明，丝氨酸残基是在任何条件下标记的主要氨基酸。这些结果表明，CaM激酶II在响应各种能增加静止成纤维细胞细胞质中游离钙浓度的刺激时会发生磷酸化，因此可能介导生长因子的至少一些生物学作用。
