RAS/CDC42 DEPENDENT SIGNAL TRANSDUCTION IN FISSION YEAST

这项建议目的是研究RAS和CdC42依赖的信号 裂殖酵母中的转导。核裂变 酵母Ras蛋白同源物，Ras1，是两个不同的细胞所必需的 与高等生物体中已知的RAS功能密切相关的功能： (1)交配信息素诱导的MAP激酶级联的调节；(2) 依赖细胞骨架的细胞形态的控制。RAS1链接到 MAPK通过与MAPK的直接相互作用而级联 Kinase，Byr2。哺乳动物CDC42 GTP结合蛋白ACTS的同源物 在Ras1下游调节细胞形态，这两个蛋白是 多蛋白信号转导复合体的一部分。 在我们的初步研究中，我们已经证明了Shk1，一个与 哺乳动物p65PAK和酿酒酵母Ste20蛋白激酶， 是裂解酵母中cdc42的下游靶标。我们的结果表明 Shk1将Ras1/CDC42信号复合体连接到AN 未特化的形态调控途径，并参与了 调节依赖于Ras1的MAPK级联反应。我们的结果， 结合其他人的数据，表明信号模块同源 到Ras1/Cdc42/Shk1模块，在进化上是保守的。我们将使用 遗传易驯化的分裂酵母作为一种强大的模式系统 这些新基因调控的信号转导通路的特征 已发现信令模块。我们的主要努力将集中在 Shk1蛋白的生物学功能及其调控研究 激活剂。我们将使用酵母基因筛查来确定潜在的 Shk1的监管机构和下游目标。从这些克隆中分离出来的 屏幕的特征将是检查它们对RAS和/或 使用成熟的酵母和两栖动物的依赖于CDC42的信号通路 (非洲爪哇)，以及哺乳动物体内和体外检测系统。这些 实验应该提供关于功能的重要信息 克隆的编码产物的性质和生物学作用 序列。我们还将使用遗传和生化的组合 Shk1和Shk1功能性质的表征方法 参与其调控的机制。我们建议的研究应该 增强我们对RAS和Cdc42依赖信号的基本理解 转导。 突变激活的RAS癌基因是已知的最普遍的 人类致癌基因。最近发现与CDC42相关的G蛋白 AS在RAS诱导哺乳动物转化中的重要作用 细胞。我们的长期目标是将从 Ras1/Cdc42/shk1依赖的裂变信号转导研究 酵母菌对了解相关信号通路的作用 高等有机体。

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.