REGULATION OF CDC25P DURING RAS PATHWAY ACTIVATION

本提案的广泛、长期目标是确定 酵母Ras鸟嘌呤核苷酸交换因子， Cdc 25 p，将葡萄糖刺激的信号转导到酵母Ras中 通路具体目标是：1.描述功能和 Cdc 25 p的活化;和2.为了分析囊泡运输的作用， 调节酵母Ras介导的葡萄糖反应。的Ras 信号通路将环境条件与 细胞增殖Ras途径组分在结构上和 从酵母到人的功能上保守。 导致不受控制的增殖，在哺乳动物中， 癌由于进化保守性， 在遗传上易处理的酵母中研究这一途径。的功能和 Cdc 25 p的激活将通过绘制Cdc 25 p的调控结构域来研究。 cdc 25 p的突变。此域将用于识别潜在的 Ras途径的组分在Cdc 25 p的上游起作用， 亲和层析和酵母双杂交系统。潜在 还将使用遗传筛选来鉴定上游成分。 使用这些技术鉴定的候选蛋白质将被进一步研究。 用遗传学和生物化学技术表征。的作用 将葡萄糖信号转导到Ras途径中的囊泡转运 将通过跟踪Cdc 25 p的亚细胞定位进行分析， 在分泌途径中含有突变的酵母菌株， 亚细胞定位结构域存在于Cdc 25 p通过诱变。

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.