RAS/CDC42 DEPENDENT SIGNAL TRANSDUCTION IN FISSION YEAST

本研究的目的是研究Ras和Cdc 42依赖的信号转导途径， 在裂殖酵母，粟酒裂殖酵母中的转导。 裂变 酵母Ras蛋白同源物Ras 1是两种不同细胞 这些功能与高等生物中已知的Ras功能非常相似： (1)交配信息素诱导的MAP激酶级联反应的调节和（2） 控制依赖于细胞间隙的细胞形态。 Ras 1链接到 MAP激酶级联通过与MAP激酶激酶的直接相互作用 激酶，Byr 2。 哺乳动物Cdc 42 GTP结合蛋白的同源物 Ras 1的下游调节细胞形态，这两种蛋白质是 多蛋白信号转导复合物的一部分。 在我们的初步研究中，我们已经证明了Shk 1，一个同源的 哺乳动物p65 PAK和酿酒酵母Ste 20蛋白激酶， 是裂殖酵母中Cdc 42的下游靶标。 我们的研究结果表明 Shk 1将Ras 1/Cdc 42信号复合物与一个 未表征的形态控制途径，并参与 Ras 1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 Ras 1/Cdc 42/Shk 1模块在进化中是保守的。 我们将使用 遗传上易处理的分裂酵母作为一个强大的模型系统， 表征这些新的信号转导途径调节 发现了信号模块 我们的主要工作将集中在 表征Shk 1蛋白的生物学功能和调节 激酶。 我们将使用酵母基因筛选， Shk 1的调节子和下游靶点。 从这些分离出来的克隆 筛选将通过检查其对Ras和/或 Cdc 42依赖的信号通路使用成熟的酵母，两栖动物 （非洲爪蟾（Xenopus laevis））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物的性质和生物学作用 序列的 我们还将结合基因和生化技术 表征Shk 1功能特性的方法， 参与其调控的机制。 我们建议的研究应 增加我们对Ras和Cdc 42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癌基因之一。 人类致癌基因。 Cdc 42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 我们对Ras 1/Cdc 42/shk 1依赖的裂变信号转导进行了研究， 酵母对获得相关的信号通路的理解， 更高的生物。

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.