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：
2900850
负责人：
STEVAN MARCUS
金额：
$ 17.97万
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 2001-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2900850
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.

该提案的目的是研究 Ras 和 Cdc42 依赖性信号裂殖酵母裂殖酵母中的转导。裂变酵母 Ras 蛋白同源物 Ras1 是两种不同细胞所必需的与高等生物中已知的 Ras 功能非常相似的功能： (1) 交配信息素诱导的 MAP 激酶级联的调节和 (2) 细胞骨架依赖性细胞形态的控制。 Ras1 链接到 MAP 激酶级联通过与 MAP 激酶激酶直接相互作用激酶，Byr2。哺乳动物 Cdc42 GTP 结合蛋白的同源物发挥作用 Ras1 下游调节细胞形态，这两种蛋白是多蛋白信号转导复合物的一部分。在我们的初步研究中，我们已经证明了 Shk1 的同系物哺乳动物 p65PAK 和酿酒酵母 Ste20 蛋白激酶，是裂殖酵母中 Cdc42 的下游靶标。我们的结果表明 Shk1 将 Ras1/Cdc42 信号复合体与迄今为止的未表征的形态控制途径并参与 Ras1 依赖性 MAP 激酶级联的调节。我们的结果，结合其他人的数据，表明信号模块同源 Ras1/Cdc42/Shk1 模块在进化中是保守的。我们将使用遗传上易处理的裂殖酵母作为强大的模型系统表征这些新调节的信号转导途径发现了信号模块。我们的主要努力将集中在表征 Shk1 蛋白的生物学功能和调节激酶。我们将使用酵母基因筛选来识别潜在的 Shk1 的监管机构和下游目标。从这些中分离出克隆屏幕将通过检查其对 Ras 和/或的影响来表征使用成熟酵母、两栖动物的 Cdc42 依赖性信号通路（非洲爪蟾）和哺乳动物体内和体外测定系统。这些实验应该提供有关功能的重要信息我们的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和功能特性的方法其调控机制。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 癌基因是已知最普遍的癌基因之一人类致癌基因。 Cdc42相关G蛋白最近被牵连在 Ras 诱导的哺乳动物转化中发挥重要作用细胞。我们的长期目标是应用从以下领域获得的知识我们对裂变中 Ras1/Cdc42/shk1 依赖性信号转导的研究酵母以获得对相关信号通路的理解高等生物。