Terada K, Kaziro Y, Satoh T
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
J Biol Chem. 1995 Nov 17;270(46):27880-6. doi: 10.1074/jbc.270.46.27880.
It has been demonstrated that Ras is involved in interleukin 3 (IL-3)-stimulated signal transduction in various hematopoietic cultured cells (Satoh, T., Nakafuku, M., Miyajima, A., and Kaziro, Y. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3314-3318; Duronio, V., Welham, M. J., Abraham, S., Dryden, P., and Schrader, J. W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1587-1591). However, it has not been fully understood which of IL-3-promoted cellular responses, i.e. proliferation, survival, and differentiation, requires Ras function. We employed a system of inducible expression of the dominant-negative (S17N) or dominant-active (G12V) mutant of Ras in BaF3 mouse pro-B cell line to analyze the role of Ras in IL-3-stimulated signal transduction. Induction of the dominant-negative Ras(S17N) effectively inhibited the IL-3-induced activation of c-Raf-1 and mitogen-activated protein kinase (MAPK). Furthermore, the activation of fos gene promoter following IL-3 stimulation was almost completely abolished when Ras(S17N) was induced. Under these conditions, Ras(S17N) exhibited no inhibitory effect on IL-3-dependent proliferation assessed by the increase of cell numbers and a mitochondrial enzyme activity. The results indicate that Ras-dependent pathways, including the Raf/MAPK/Fos pathway, are dispensable for IL-3-induced growth stimulation. When BaF3 cells were treated with a tyrosine kinase inhibitor, herbimycin A, IL-3-dependent proliferation of the cells was impaired, suggesting that tyrosine kinase-mediated pathways are critical for growth promotion. On the other hand, apoptotic cell death caused by deprivation of IL-3 was prevented by the induction of the activated mutant Ras(G12V), although the rate of cell number increase was markedly reduced. Thus, it is likely that Ras-independent pathways play important roles to facilitate the proliferation although they may not be essential for IL-3-stimulated antiapoptotic signal transduction.
已有研究表明，Ras参与了多种造血培养细胞中白细胞介素3（IL-3）刺激的信号转导过程（佐藤，T.，中福，M.，宫岛，A.，和卡齐罗，Y.（1991年）《美国国家科学院院刊》88卷，3314 - 3318页；杜罗尼奥，V.，韦勒姆，M. J.，亚伯拉罕，S.，德莱登，P.，和施拉德，J. W.（1992年）《美国国家科学院院刊》89卷，1587 - 1591页）。然而，对于IL-3促进的细胞反应，即增殖、存活和分化，其中哪一种需要Ras功能，尚未完全明确。我们采用了一种在BaF3小鼠前B细胞系中可诱导表达Ras的显性负性（S17N）或显性活性（G12V）突变体的系统，来分析Ras在IL-3刺激的信号转导中的作用。显性负性Ras（S17N）的诱导有效抑制了IL-3诱导的c-Raf-1和丝裂原活化蛋白激酶（MAPK）的激活。此外，当诱导Ras（S17N）时，IL-3刺激后fos基因启动子的激活几乎完全被消除。在这些条件下，通过细胞数量增加和线粒体酶活性评估，Ras（S17N）对IL-3依赖性增殖没有抑制作用。结果表明，包括Raf/MAPK/Fos途径在内的Ras依赖性途径对于IL-3诱导的生长刺激是可有可无的。当用酪氨酸激酶抑制剂除草霉素A处理BaF3细胞时，细胞的IL-3依赖性增殖受到损害，这表明酪氨酸激酶介导的途径对于生长促进至关重要。另一方面，尽管细胞数量增加的速率明显降低，但通过诱导活化突变体Ras（G12V）可防止因IL-3剥夺导致的凋亡细胞死亡。因此，虽然Ras非依赖性途径对于IL-3刺激的抗凋亡信号转导可能不是必需的，但它们可能在促进增殖方面发挥重要作用。
