CELL CYCLE ARREST AND SIGNAL TRANSDUCTION IN YEAST

描述（改编自申请人的摘要）：在萌芽中交配 酵母酿酒酿酒酵母为负增长提供了一个很好的范例 高等真核生物的控制和分化。 对肽的反应 信息素，分裂的单倍体细胞在G1期停止分裂，并表达 细胞间接触和融合所需的特殊基因。 交配是 由一个保守的MAP激酶级联调节，该级联由两个MAP 激酶、MEK、MEKK和PAK型激酶。 该级联反应由 异源三聚体和Rho型G蛋白，SH 3结构域蛋白，和一种新的 一种叫做Ste 5的支架蛋白，它在空间上组织蛋白质 激酶。 本建议旨在研究信号的两个基本问题 与生长和分化有关的所有细胞的 真核生物-（1）MAP激酶级联被激活的机制， (2)MAP激酶级联如何执行特定功能。 授出 由不同的和相互关联的项目组成，所有这些项目都是 继续开展上一个赠款期内完成的工作。 PI建议：1. 研究不同的可能功能， 通过分离和分析Ste 5对蛋白激酶的调节作用 Ste 5突变体在特定功能和与蛋白质相互作用方面的缺陷 激酶和G蛋白。 突变蛋白质将被生物化学表征 对于结合通路组分、激活激酶和引导 底物磷酸化 2. 细胞生物学和遗传学方法将用于研究 Ste 5定位的细胞周期和信息素依赖性调节的作用 以确定它是否对隔离MAPK级联酶重要， 不同的细胞区室来进行特定的反应。 3. 将分析MEKK Ste 11的激活机制， 与Ste 11和Ste 5二聚化以及调控蛋白之间的关联有关。 Ste 11、Ste 5和Ste 50的结构域。 4. 将研究Ste 5在介导途径特异性中的作用 通过分离和分析突变形式的Ste 5和Ste 11， 其他MAP激酶级联。

DESCRIPTION (Adapted from the applicant's abstract): Mating in the budding yeast S. cerevisiae provides an excellent paradigm for negative growth control and differentiation in higher eukaryotes. In response to peptide pheromones, dividing haploid cells stop dividing in G1 phase and express specialized genes required for cell-cell contact and fusion. Mating is regulated by a conserved MAP kinase cascade that consists of two MAP kinases, a MEK, a MEKK, and a PAK-type kinase. The cascade is regulated by heterotrimeric and Rho-type G proteins, an SH3 domain protein, and a novel scaffolding protein called Ste5 that spatially organizes the protein kinases. This proposal aims to study two fundamental problems of signal transduction that pertain to growth and differentiation in all eukaryotes--(1) the mechanism by which a MAP kinase cascade is activated and (2) how a MAP kinase cascade performs specialized functions. The grant consists of distinct and interrelated projects, all of which are a continuation of work done during the previous granting period. The PI proposes to: 1. Investigate the different possible functions of Ste5 with respect to protein kinase regulation by isolating and analyzing Ste5 mutants defective in specific functions and interactions with protein kinases and G proteins. Mutant proteins will be characterized biochemically for the ability to bind to pathway components, activate kinases, and direct substrate phosphorylation. 2. Cell biological and genetic approaches will be used to investigate the role of cell cycle- and pheromone-dependent regulation of Ste5 localization to determine whether it is important for sequestering MAPK cascade enzymes to different cellular compartments for specific responses. 3. Mechanisms of activation of the MEKK Ste11 will be analyzed with respect to both Ste11 and Ste5 dimerization and association between the regulatory domain of Ste11 and Ste5 and Ste50. 4. The role of Ste5 in mediating pathway specificity will be investigated by isolating and analyzing mutant forms of Ste5 and Ste11 that activate other MAP kinase cascades.