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 raf-1活性，包括血小板衍生生长因子、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EGF受体（EGFR）系统进行检查， Raf-1的调节磷酸化位点。HER 14细胞与 EGF导致电泳迁移率的变化，这是由一种 丝氨酸残基上的磷酸盐掺入到RAF蛋白中增加。 Raf-1激酶的比活性在100 μ mol/L的条件下被刺激约6倍。 了以下条件激活的Raf-I与EGFR物理结合， EGF的作用机制多克隆Raf-I抗血清 共沉淀EGFR，单克隆EGFR抗体共沉淀约 占Raf-1人口的1%然而，Raf-1与32 P 标记的细胞在EGF后没有显示磷酸盐掺入T-酪氨酸 处理，而是显示磷酸丝氨酸含量增加两倍。 蛋白激酶C在HER 14细胞中的表达下调 12-0-十四烷基-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，表明PKC不是 EGF处理的细胞中raf-I活化所必需的。 因此，该系统表明，必须存在非PKC介导的 丝氨酸磷酸化，其激活Raf-I激酶活性。 可能负责EGF触发Raf-1的候选激酶 活化包括MAP 2和S6激酶。我们目前正在 确定EGF中Raf-I的主要丝氨酸磷酸化位点 PKC抑制HER 14细胞增殖。

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.