RAS/CDC42 DEPENDENT SIGNAL TRANSDUCTION IN FISSION YEAST

裂殖酵母中 RAS/CDC42 依赖性信号转导

基本信息

批准号：
6180955
负责人：
STEVAN MARCUS
金额：
$ 18.67万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-04-01 至 2002-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6180955
关键词：
G protein Schizosaccharomyces pombe Xenopus oocyte alternatives to animals in research biological signal transduction cell cycle proteins computer assisted sequence analysis fungal genetics microtubule associated protein molecular cloning oncogenes phosphorylation protein kinase site directed mutagenesis

项目摘要

The objective of this proposal is to study Ras and Cdc42-dependent signal transduction in the fission yeast, Schizosaccharomyces pombe. The fission yeast Ras protein homolog, Ras1, is required for two distinct cellular functions that closely parallel known Ras functions in higher organisms: (1) regulation of mating pheromone-induced MAP kinase cascade and (2) control of cytoskeletal-dependent cellular morphology. Ras1 is linked to the MAP kinase cascade via a direct interaction with the MAP kinase kinase kinase, Byr2. A homolog of the mammalian Cdc42 GTP-binding protein acts downstream of Ras1 in regulating cell morphology, and the two proteins are part of a multiprotein signal transduction complex. In our PRELIMINARY STUDIES, we have demonstrated that Shk1, a homolog of the mammalian p65PAK and Saccharomyces cerevisiae Ste20 protein kinases, is a downstream target of Cdc42 in fission yeast. Our results suggest that Shk1 links the Ras1/Cdc42 signaling complex to an as yet uncharacterized morphology control pathway and participates in the regulation of the Ras1-dependent MAP kinase cascade. Our results, combined with data from others, suggest that signaling modules homologous to the Ras1/Cdc42/Shk1 module are conserved in evolution. We will use the genetically tractable fission yeast as a powerful model system for characterizing the signal transduction pathways regulated by these newly discovered signaling modules. Our primary efforts will focus on characterizing the biological functions and regulation of the Shk1 protein kinase. We will use yeast genetic screens to identify potential regulators and downstream targets of Shk1. Clones isolated from these screens will be characterized by examining their effects on Ras- and/or Cdc42-dependent signaling pathways using well-established yeast, amphibian (Xenopus laevis), and mammalian in vivo and in vitro assay systems. These experiments should provide important information on the functional properties and biological roles of the products encoded by our cloned sequences. We will also use a combination of genetic and biochemical approaches to characterize the functional properties of Shk1 and mechanisms involved in its regulation. Our proposed studies should increase our fundamental understanding of Ras and Cdc42-dependent signal transduction. Mutationally activated Ras oncogenes are among the most prevalent of known human oncogenes. Cdc42-related G proteins have recently been implicated as playing essential roles in Ras-induced transformation of mammalian cells. Our long-term objective is to apply the knowledge obtained from our studies on Ras1/Cdc42/shk1-dependent signal transduction in fission yeast toward gaining an understanding of related signaling pathways in higher organisms.

该建议的目的是研究RAS和CDC42依赖性信号裂变酵母的转导，schizosacchomyces pombe。裂变两个不同的细胞需要酵母Ras蛋白同源物Ras1 紧密平行的RAS在较高生物体中起作用的功能：（1）调节配偶信息素诱导的MAP激酶级联反应和（2）控制细胞骨架依赖性细胞形态。 RAS1链接到通过与MAP激酶激酶直接相互作用的MAP激酶级联激酶，BYR2。哺乳动物CDC42 GTP结合蛋白作用的同源物 RAS1在调节细胞形态中的下游，两种蛋白质是多蛋白信号转导复合物的一部分。在我们的初步研究中，我们证明了SHK1是哺乳动物p65pak和酿酒酵母Ste20蛋白激酶，是裂变酵母中Cdc42的下游目标。我们的结果表明 SHK1将RAS1/CDC42信号复合复合综述链接到未表征的形态控制途径，并参与 RAS1依赖性MAP激酶级联反应的调节。我们的结果，结合来自其他数据的数据，建议信号模块同源到Ras1/cdc42/shk1模块在进化中是保守的。我们将使用遗传上可牵引的裂变酵母作为强大的模型系统表征由这些新近调节的信号转导途径发现的信号模块。我们的主要努力将重点放在表征SHK1蛋白的生物学功能和调节激酶。我们将使用酵母遗传筛选来识别潜力 SHK1的调节器和下游目标。与这些孤立的克隆屏幕将通过检查其对RAS和/或的影响来表征使用良好的酵母，两栖动物依赖Cdc42的信号通路（Xenopus laevis）和体内哺乳动物和体外测定系统。这些实验应提供有关功能的重要信息我们克隆编码的产品的特性和生物学作用序列。我们还将结合遗传和生化表征SHK1和SHK1的功能特性的方法其调节涉及的机制。我们提出的研究应该增加我们对RAS和CDC42依赖性信号的基本理解转导。突变激活的Ras癌基因是最普遍的已知人发育基因。最近与CDC42相关的G蛋白有关作为在RAS引起的哺乳动物转变中扮演重要角色细胞。我们的长期目标是应用从我们对RAS1/CDC42/SHK1依赖性信号转导的研究酵母要了解相关信号通路更高的生物。