MECHANISM OF A-RAF KINASE REGULATION

RAF家族蛋白激酶在有丝分裂中的第二信使作用 信号转导事件。到目前为止，大部分工作已经完成 C-Raf-1同工酶。我们现在已经进行了生化表征 内源性A-Raf激酶。与c-Raf-1一样，A-Raf的蛋白质 原癌基因是一种丝氨酸/苏氨酸激酶，在 血小板衍生生长因子等对细胞的刺激作用 有丝分裂原。与这些有丝分裂原孵育会导致 电泳率，这是由增加的磷酸盐引起的 整合到A-Raf蛋白中。 为了研究依赖RAS的A-Raf激酶的调节，我们使用了 PC12嗜铬细胞瘤细胞的稳定转染体 显性负性RAS突变体(M17)和地塞米松的表达 在NIH/3T3细胞中诱导突变。RAS突变体片段的表达 受刺激细胞中A-Raf的磷酸化和激酶活性 C-Raf-1的磷酸化和激活途径被阻断。 在瞬时转染法中，显示N-末端的一半， A-Raf激酶的调节部分，阻断RAS的诱导 C-Raf-1依赖的报告基因表达与 C-Raf-1激酶本身的调节部分。这表明了对 这些同工酶通过一种共同的机制。

Raf family protein kinases function as second messengers in mitogenic signal transduction events. So far, most of the work has been done with the c-Raf-1 isozyme. We have now performed biochemical characterization of the endogenous A-Raf kinase. Like c-Raf-1, the protein of the A-Raf proto-oncogene is a serine/ threonine kinase that is activated after stimulation of cells with platelet derived growth factor and other mitogens. Incubation with these mitogens leads to a shift in electrophoretic mobility, which is caused by an increased phosphate incorporation into the A-Raf protein. To investigate the Ras-dependent regulation of the A-Raf kinase, we used stable transfectants of PC12 pheochromocytoma cells with constitutive expression of a dominant negative Ras mutant (M17) and a dexamethasone inducible mutant in NIH/3T3 cells. The expression of the Ras mutant blocks A-Raf phosphorylation and kinase activity of stimulated cells in the same way as c-Raf-1 phosphorylation and activation is blocked. In transient transfection assays, it is shown that the N-terminal half, the regulatory portion of the A-Raf kinase, is blocking Ras induction of c-Raf-1-dependent reporter gene expression to the same extent as the regulatory part of the c-Raf-1 kinase itself. This indicates regulation of these isozymes by a common mechanism.