Nemenoff R A, Price D J, Mendelsohn M J, Carter E A, Avruch J
Howard Hughes Medical Institute Laboratories, Harvard Medical School, Massachusetts General Hospital, Boston.
J Biol Chem. 1988 Dec 25;263(36):19455-60.
Protein kinase activity toward the 40 S ribosomal protein S6 is activated 6-fold in regenerating rat liver following 70% hepatectomy. The kinase is maximally activated within 2 h after surgery, remains active up to 36 h after surgery, and declines rapidly thereafter. The post-hepatectomy S6 kinase activity exhibits structural and functional similarity to an insulin-stimulated S6 kinase in H4 hepatoma cells. Both S6 kinase activities are cAMP- and Ca2+-independent, and have a requirement for [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The regenerating liver and the insulin-stimulated H4 hepatoma S6 kinase elute at similar positions when sequentially fractionated by anion-exchange and cation-exchange chromatography. Both enzymes migrate at Mr 70,000 on fast protein liquid chromatography Superose 12 gel filtration. In H4 hepatoma cells, activation of S6 kinase activity is reversed by removal of insulin, and the cells can then be restimulated. Freshly isolated hepatocytes from normal animals show low levels of S6 kinase activity which can be stimulated by epidermal growth factor and insulin. Hepatocytes prepared from regenerating liver remnant have constitutively high levels of S6 kinase activity, which is unresponsive to insulin plus epidermal growth factor and which remains elevated at least 2 h in the absence of exogenously added growth factors. These findings demonstrate S6 protein kinase activation in vivo, in the setting of regulated cell growth; as in cultured cells, activation of S6 kinase probably represents an early step in the pleiotypic response elicited by activation of growth factor receptors.
在70%肝切除术后的再生大鼠肝脏中，针对40S核糖体蛋白S6的蛋白激酶活性被激活了6倍。该激酶在手术后2小时内被最大程度激活，在手术后36小时内保持活性，此后迅速下降。肝切除术后的S6激酶活性与H4肝癌细胞中胰岛素刺激的S6激酶在结构和功能上具有相似性。两种S6激酶活性均不依赖于cAMP和Ca2+，且需要[乙二胺四乙酸]。当通过阴离子交换和阳离子交换色谱依次分级分离时，再生肝脏和胰岛素刺激的H4肝癌S6激酶在相似位置洗脱。两种酶在快速蛋白质液相色谱Superose 12凝胶过滤中以70,000的相对分子质量迁移。在H4肝癌细胞中，去除胰岛素可逆转S6激酶活性的激活，然后细胞可被再次刺激。从正常动物新鲜分离的肝细胞显示出低水平的S6激酶活性，可被表皮生长因子和胰岛素刺激。从再生肝脏残余物制备的肝细胞具有组成性高水平的S6激酶活性，对胰岛素加表皮生长因子无反应，并且在没有外源添加生长因子的情况下至少2小时保持升高。这些发现证明了在体内调节细胞生长的情况下S6蛋白激酶的激活；与培养细胞一样，S6激酶的激活可能代表生长因子受体激活引发的多效性反应的早期步骤。
