MECHANISMS OF RAF KINASE ACTIVATION AND SUBSTRATE PHOSPHORYLATION

Raf-1蛋白Ser/Thr激酶作为一种关键的穿梭酶， 连接刺激生长因子受体和蛋白激酶C， 细胞膜中早期生长反应基因的激活 原子核 过去一年，我们在以下方面取得了相当大的进展： 鉴定Raf-1的上游激活因子和下游效应因子 信号通路 我们已经证明， 激活的Ras与 Raf-1 Ras与Raf-1的这种特异性相互作用是否足够 用于激活Raf-1激酶活性或需要额外的辅因子 仍有待阐明。 迄今为止，在体外激活Raf-1的尝试 与Ras-GTP的合作都没有成功 为了理解 辅助因子在Raf-1激活机制中的可能作用，我们有 用细胞亚组分扩展了这项研究。 拉斯的一个后果- Raf结合是Raf/丝裂原活化蛋白激酶的活化 (MAP激酶）途径。 活化的Raf磷酸化ser 217和ser 221 MAP激酶激酶（MEK）是唯一已知的 Raf-1 去磷酸化实验与磷酸化 突变体分析表明， 足以达到最大激活。 活化的MEK磷酸化MAP 激酶，反过来，催化几个磷酸化 胞质蛋白，包括核糖体蛋白S6激酶和MAP 激酶活化蛋白激酶1和2。 此外，活化的MEK还 催化多种致癌基因类转录的磷酸化 导致基因转录变化的因素。 为了扩大 本研究的其他家庭成员的Raf（A-Raf和B-Raf），我们有 在Spodopera中表达了这些蛋白质的野生型和突变体版本 frugiperda（Sf 9）细胞，实验正在进行中。

Raf-1 protein ser/thr kinase functions as a critical shuttle enzyme that connects stimulation of growth factor receptors and protein kinase C at the membrane with activation of early growth response genes in the nucleus. During the past year we have made considerable progress in identifying the upstream activators and downstream effectors of the Raf-1 signalling pathway. We have demonstrated that the effector domain of the activated Ras interacts with the amino-terminal regulatory domain of Raf-1. Whether this specific interaction of Ras with Raf-1 is sufficient for activation of Raf-1 kinase activity or requires additional co-factors remains to be elucidated. Thus far, attempts to activate Raf-1 in vitro with Ras-GTP have been unsuccessful. In an effort to understand the possible role of the co-factor in the Raf-1 activation mechanism, we have extended this study with cellular subfractions. One consequence of Ras- Raf binding is the activation of Raf/mitogen-activated protein kinase (MAP kinase) pathway. The activated Raf phosphorylates ser217 and ser221 of MAP kinase kinase (MEK), the only known physiological substrate of Raf-1. Dephosphorylation experiments together with phosphorylation mutant analysis demonstrated that phosphorylation of either residue is sufficient for maximal activation. Activated MEK phosphorylates the MAP kinase which, in turn, catalyzes the phosphorylation of several cytoplasmic proteins, including ribosomal protein S6 kinase and MAP kinase-activated protein kinase 1 and 2. In addition, activated MEK also catalyzes the phosphorylation of multiple oncogene class transcription factors leading to changes in gene transcription. In an effort to extend this study to the other family members of Raf (A-Raf and B-Raf), we have expressed wild-type and mutant versions of these proteins in Spodopera frugiperda (Sf9) cells and experiments are in progress.