ACTIVITY REGULATION OF CYTOSOLIC RAF-1 PROTEIN KINASE BY PHOSPHORYLATION

多种跨膜受体酪氨酸激酶的配体调节 包括血小板衍生生长因子在内的RAF-1活性，集落刺激 因子-1、胰岛素、成纤维细胞生长因子和表皮生长因子 (EGF)。我们选择了表皮生长因子受体(EGFR)系统来检测 Raf-1的磷酸化调控位点。HER-14细胞的体外孵育 EGF导致凝胶迁移率的变化，这是由 丝氨酸残基上的RAF蛋白的磷酸盐掺入增加。 Raf-1激酶的比活性在以下条件下被刺激约6倍 这些条件。激活的RAF-I在物理上与EGFR关联 EGF以配体依赖的方式进行处理。多克隆Raf-I抗血清 共沉淀EGFR，单抗EGFR共沉淀约 Raf-1人群的1%。然而，Raf-1免疫沉淀来自32P EGF后标记细胞未见磷酸盐掺入T-酪氨酸 相反，它显示出磷酸丝氨酸含量增加了两倍。 HER-14细胞蛋白激酶C(PKC)表达下调 12-0-十四酮佛波醇-13-乙酸酯不阻断EGF诱导的移位 在Raf-1的电泳迁移率中，提示PKC不是 对EGF处理的细胞中RAF-I的激活是必不可少的。 因此，这个系统表明，必须有一个非PKC中介的 丝氨酸磷酸化，激活Raf-I激酶活性。 可能与EGF触发的Raf-1相关的候选激酶 激活包括MAP2和S6Kinase。我们目前正在进行 表皮生长因子中Raf-I优势丝氨酸磷酸化位点的确定 在刺激下，PKC下调了她的14个细胞。

A variety of ligands for transmembrane receptor tyrosine kinases regulate raf-1 activity including platelet-derived growth factor, colony stimulating factor-1, insulin, fibroblast growth factor and epidermal growth factor (EGF). We have chosen the EGF receptor (EGFR) system for examination of the regulatory phosphorylation site of Raf-1. Incubation of HER 14 cells with EGF leads to a shift in the electrophoretic mobility, which is caused by an increased phosphate incorporation into the raf protein on serine residues. The specific activity of the Raf-1 kinase is stimulated about sixfold under these conditions. Activated Raf-I physically associates with the EGFR upon EGF treatment in a ligand-dependent manner. Polyclonal Raf-I antisera coprecipitate the EGFR, and monoclonal EGFR antibodies coprecipitate about 1% of the Raf-1 population. However, Raf-1 immunoprecipitates from 32P labeled cells show no phosphate incorporation into T-tyrosine after EGF treatment and instead show a twofold elevated phosphoserine content. Downregulation of protein kinase C (PKC) in HER 14 cells with 12-0-tetradeconyl-phorbol-13-acetate does not block the EGF induced shift in the electrophoretic mobility of Raf-1, suggesting that PKC is not essential for raf-I activation in EGF-treated cells. Therefore, this system demonstrates that there must be a non-PKC mediated serine phosphorylation, which activates the Raf-I kinase activity. Candidate kinases which may be responsible for EGF triggered Raf-1 activation include MAP2 and S6 Kinase. We are currently in the process of determining the predominant serine phosphorylation site of Raf-I in EGF stimulated, PKC downregulated HER 14 cells.