Cobb M H, Rosen O M
J Biol Chem. 1983 Oct 25;258(20):12472-81.
Particulate preparations from insulin-treated 3T3-L1 cells retain the enhanced ability to incorporate 32P from [gamma-32P]ATP into ribosomal protein S6 (Smith, C. J., Rubin, C. S., and Rosen, O. M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2641-2645). A cyclic AMP-independent protein kinase that phosphorylates S6 and casein and that may be involved in the increase in S6 phosphorylation produced by insulin has been isolated based upon the observation that there is 1.5-3.0-fold higher activity in particulate preparations derived from insulin-treated cells than there is in comparable preparations from control cells. The enzyme activity was purified 2071-fold by KCl extraction, phosphocellulose chromatography, and gel filtration. The S6 phosphorylating activity was also characterized by its behavior on casein-Sepharose and DEAE-cellulose chromatography and its sedimentation in glycerol gradients. None of these procedures resolved the S6 and casein kinase activities. Some of the properties of this kinase, including a molecular weight of about 35,000, inhibition by F- or phosphate, chromatography on DEAE-cellulose and phosphocellulose, and insensitivity to inhibition by GTP, are similar to those of a previously described enzyme, casein kinase I (Dahmus, M. E. (1981) J. Biol. Chem. 256, 3319-3325; Hathaway, G. M., and Traugh, J. A. (1979) J. Biol. Chem. 254, 762-768).
来自胰岛素处理过的3T3-L1细胞的微粒制剂仍保持着将[γ-32P]ATP中的32P掺入核糖体蛋白S6的增强能力（史密斯，C.J.，鲁宾，C.S.，和罗森，O.M.（1980年）《美国国家科学院院刊》77，2641 - 2645）。基于在源自胰岛素处理细胞的微粒制剂中的活性比在来自对照细胞的类似制剂中高1.5 - 3.0倍这一观察结果，已分离出一种不依赖环磷酸腺苷的蛋白激酶，该激酶使S6和酪蛋白磷酸化，并且可能参与胰岛素产生的S6磷酸化增加过程。通过氯化钾提取、磷酸纤维素层析和凝胶过滤，该酶活性被纯化了2071倍。S6磷酸化活性还通过其在酪蛋白 - 琼脂糖和二乙氨基乙基纤维素层析上的行为以及在甘油梯度中的沉降来表征。这些方法均未分离出S6和酪蛋白激酶活性。这种激酶的一些特性，包括分子量约为35000、受氟或磷酸盐抑制、在二乙氨基乙基纤维素和磷酸纤维素上的层析行为以及对鸟苷三磷酸抑制不敏感，与先前描述的一种酶——酪蛋白激酶I（达姆斯，M.E.（1981年）《生物化学杂志》256，3319 - 3325；哈撒韦，G.M.，和特劳，J.A.（1979年）《生物化学杂志》254，762 - 768）相似。
