Hoshi M, Nishida E, Sakai H
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
J Biol Chem. 1988 Apr 15;263(11):5396-401.
Treatment of quiescent human embryonic lung fibroblastic cells (TIG-3) with 10 nM epidermal growth factor (EGF) resulted in 4-6-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein 2 (MAP2) on serine and threonine residues in vitro. The half-maximal activation of the kinase activity occurred within 5 min after EGF treatment, and the maximal level was attained at 15 min. Casein and histone were very poor substrates for this EGF-stimulated MAP2 kinase activity. The activation of the kinase activity persisted after brief dialysis. Interestingly, the EGF-stimulated MAP2 kinase activity was sensitive to micromolar concentrations of free Ca2+; it was inhibited 50% by 0.5 microM Ca2+ and almost totally inhibited by 2 microM Ca2+. The activated MAP2 kinase activity was recovered in flow-through fractions on phosphocellulose column chromatography, while kinase activities that phosphorylate 40 S ribosomal protein S6 (S6 kinase activities) were mostly retained on the column and eluted at 0.5 M NaCl. Platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, phorbol esters (12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate), and fresh fetal calf serum also induced activation of the MAP2 kinase in the quiescent TIG-3 cells. The activated MAP2 kinase activity in cells stimulated by platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, 12-O-tetradecanoylphorbol 13-acetate, phorbol 12,13-dibutyrate, or fetal calf serum was almost completely inhibited by 2 microM Ca2+, like the EGF-stimulated kinase. In addition, MAP2 phosphorylated by the kinase activated by different stimuli gave very similar phosphopeptide mapping patterns. These results suggest that several growth factors, phorbol esters, and serum activate a common, Ca2+-inhibitable protein kinase which is distinct from S6 kinase in quiescent human fibroblasts.
用10 nM表皮生长因子（EGF）处理静止的人胚肺成纤维细胞（TIG-3），导致细胞提取物中的一种蛋白激酶活性激活4至6倍，该激酶在体外可使微管相关蛋白2（MAP2）的丝氨酸和苏氨酸残基磷酸化。激酶活性的半最大激活在EGF处理后5分钟内发生，最大水平在15分钟时达到。酪蛋白和组蛋白是这种EGF刺激的MAP2激酶活性非常差的底物。短暂透析后激酶活性的激活持续存在。有趣的是，EGF刺激的MAP2激酶活性对微摩尔浓度的游离Ca2+敏感；0.5 microM Ca2+可抑制50%，2 microM Ca2+几乎完全抑制。在磷酸纤维素柱色谱的流通部分中回收了激活的MAP2激酶活性，而使40 S核糖体蛋白S6磷酸化的激酶活性（S6激酶活性）大多保留在柱上并在0.5 M NaCl时洗脱。血小板衍生生长因子、成纤维细胞生长因子、胰岛素样生长因子-I、胰岛素、佛波酯（12-O-十四酰佛波醇13-乙酸酯和佛波醇12,13-二丁酸酯）以及新鲜胎牛血清也诱导静止的TIG-3细胞中MAP2激酶的激活。与EGF刺激的激酶一样，血小板衍生生长因子、成纤维细胞生长因子、胰岛素样生长因子-I、胰岛素、12-O-十四酰佛波醇13-乙酸酯、佛波醇12,13-二丁酸酯或胎牛血清刺激的细胞中激活的MAP2激酶活性几乎完全被2 microM Ca2+抑制。此外，由不同刺激激活的激酶磷酸化的MAP2给出非常相似的磷酸肽图谱模式。这些结果表明，几种生长因子、佛波酯和血清激活一种共同的、Ca2+可抑制的蛋白激酶，该激酶在静止的人成纤维细胞中与S6激酶不同。
