REGULATION OF CDC25P DURING RAS PATHWAY ACTIVATION

这项提案的广泛、长期目标是确定 酵母Ras鸟嘌呤核苷酸交换因子， CDC25p，将葡萄糖刺激的信号转导到酵母RAS中 路径。具体目标是：1.划定功能和 激活cdc25p；2.分析囊泡转运的作用 在调节酵母RAS介导的葡萄糖反应中。房协 信号通路将环境条件与细胞周期调控结合在一起 细胞增殖。RAS途径组件在结构上和 在功能上从酵母到人类都是保守的。放松对这一途径的管制 导致不受控制的增殖，在哺乳动物中，这可能导致 癌症。由于进化守恒，有利于 在遗传易驯化的酵母中研究这一途径。功能和 将通过绘制CDC25P的调节结构域来研究CDC25P的激活 利用诱变技术获得了CDC25P。此域将用于识别潜在的 在CDC25P上游发挥作用的RAS途径的组成部分通过 亲和层析和酵母双杂交系统。潜力 上游成分也将使用基因筛查进行识别。 使用这些技术确定的候选蛋白质将进一步 用遗传和生化技术来描述的。的作用 糖信号转导到RAS途径中的囊泡运输 将通过跟踪cdc25p的亚细胞定位进行分析 含有分泌途径突变的酵母菌及其作图 通过诱变，在CDC25P中存在亚细胞定位结构域。

The broad, long term objective of this proposal is to determine the mechanism through which the yeast Ras guanine nucleotide exchange factor, Cdc25p, transduces the signal from glucose stimulation into the yeast Ras pathway. The specific aims are: 1. to delineate the functional and activation of Cdc25p; and 2. to analyze the role orf vesicular transport in regulation of the yeast Ras-mediated glucose response. The Ras signaling pathway couples environmental conditions with the regulation of cellular proliferation. Ras pathway components are structurally and functionally conserved from yeast to man. Deregulation of this pathway results in uncontrolled proliferation, which in mammals, can lead to cancer. Because of the evolutionary conservation, it is advantageous to study this pathway in the genetically tractable yeast. The function and activation of Cdc25p will be studied by mapping the regulatory domains of Cdc25p using mutagenesis. This domain will be used to identify potential components of the Ras pathway functioning upstream of Cdc25p through affinity chromatography and the yeast two-hybrid system. Potential upstream components will also be identified using a genetic screen. Candidate proteins identified using these techniques will be further characterized using genetic and biochemical techniques. The role of vesicular transport in transducing the glucose signal into the Ras pathway will be analyzed by tracking the subcellular localization of Cdc25p using yeast strains containing mutations in the secretory pathway and by mapping the subcellular localization domains present in Cdc25p by mutagenesis.