ROLE OF RAS IN RAF COUPLING TO TRANSMEMBRANE RECEPTOR TYROSINE KINASES

多种细胞因子对细胞的刺激和转化 生长因子(GF)、GF受体和酪氨酸的致癌版本 激酶癌基因导致Raf-1蛋白的磷酸化，从而导致 迁移率的变化和激酶活性的增加。我们已经证明了 最近，另一个Raf家族成员B-RAF的激酶活性是 PC12嗜铬细胞瘤细胞的配体依赖性调控 神经生长因子诱导分化时。RAS系列GTP 结合蛋白被认为与信号通路有关，导致 增殖分化。RAS似乎在上游发挥作用 Raf-1，从与Raf-1的激酶激活突变体的工作判断 克服细胞RAS功能的障碍和Raf-1的抑制性突变， 以及Raf-1反义构建物，它们阻止生长诱导和 RAS致癌版本的转化。这些综合数据表明 RAS可能通过GF受体调控Raf的磷酸化激活。 我们使用了RAS的显性负突变体M17RAS来分析 Raf的诱导磷酸化和活化是否需要RAS- NIH/3T3细胞中的Raf-1和B-Raf-Kinase在PC12中的表达 嗜铬细胞瘤细胞。我们的结果表明：(1)M17Ras阻断了激酶 血清、TPA、TPA刺激NIH/3T3细胞后Raf-1的活化 PDFG，以及EGF。(2)类似地，Raf-1和B-Raf在 表达NGF、EGF、PDGF和TPA的PC12细胞的刺激被阻断 M17 RAS(3)Raf-1和B-Raf的胰酶磷酸肽图 表明所有的GF敏感点都被突变体RAS抑制 表达，提示RAS控制Raf激酶的活性 它调节Raf-1和B-Raf的自磷酸化。(4)玻璃纤维 受体自身磷酸化和PLC-Gamma1磷酸化 细胞不受M17 RAS的影响，表明RAS的一块 功能通常不会干扰来自GF的信令 感受器。此外，RAS和RAF之间的协同作用是 在使用两种原癌基因共转染的实验中得到证实 转化NIH3T3细胞并进行转化和转录检测 激活。

Stimulation of cells with a variety of cytokines as well as transformation with oncogenic versions of growth factors (GF), GF receptors, and tyrosine kinase oncogenes results in phosphorylation of Raf-1 protein, resulting in a mobility shift and increased kinase activity. We have demonstrated recently that the kinase activity of another Raf family member, B-raf, is regulated in a ligand-dependent fashion in PC12 pheochromocytoma cells when induced to differentiate by nerve growth factor. Ras family GTP binding proteins have been implicated in signaling pathways that result in proliferation and differentiation. Ras appears to function upstream of Raf-1, as judged from work with kinase activating mutants of Raf-1 that overcome a block to cellular Ras function and inhibitory mutants of Raf-1, as well as Raf-1 antisense constructs, which block growth induction and transformation by oncogenic versions of Ras. These combined data suggest that Ras may control phosphorylation-activation of Raf by GF receptors. We have used a dominant negative mutant of Ras, M17 Ras, to analyze whether Ras is required for induced phosphorylation and activation of Raf- 1 kinase in NIH/3T3 cells and Raf-1 as well as B-Raf-kinase in PC12 pheochromocytoma cells. Our results show that (1) M17Ras blocks kinase activation of Raf-1 in NIH/3T3 cells after stimulation with serum, TPA, PDFG, as well as EGF. (2) Similarly, activation of Raf-1 and B-Raf after NGF, EGF, PDGF, and TPA stimulation is blocked in PC12 cells expressing M17 Ras. (3) Tryptic phosphopeptide mapping of Raf-1 and B-Raf demonstrates that all GF-sensitive spots were suppressed by mutant Ras expression, suggesting that Ras controls the activity of Raf kinase kinases or that it regulates Raf-1 and B-Raf autophosphorylation. (4) GF receptor autophosphorylation and PLC-gamma1 phosphorylation in the same cells were unaffected by M17 Ras, demonstrating that a block of Ras function does not generally interfere with the signaling from GF receptors. Furthermore, a synergistic interaction between Ras and Raf was demonstrated in experiments using cotransfection of both proto-oncogenes into NIH3T3 cells and assay for transformation and transcription activation.