CELL CYCLE ARREST AND SIGNAL TRANSDUCTION IN YEAST

描述(摘自申请者的摘要)：萌芽中的交配 酿酒酵母为负生长提供了一个极好的范例 高等真核生物的调控和分化。对多肽的反应 信息素，分裂单倍体细胞在G1期停止分裂并表达 细胞间接触和融合所需的特殊基因。交配就是 由由两个MAP组成的保守的MAP激酶级联调控 一种蛋白激酶、一种MEK、一种MEKK和一种PAK类型的激酶。级联受以下因素调节 异三聚体和Rho型G蛋白--一个SH3结构域蛋白和一个新的 一种称为Ste5的支架蛋白，它在空间上组织该蛋白 激活剂。这项建议旨在研究信号的两个基本问题 所有与生长和分化有关的转导 真核生物--(1)MAP激酶级联被激活的机制和 (2)MAP激酶级联如何发挥特殊功能。助学金 由不同且相互关联的项目组成，所有这些项目都是 继续在前一批准期内完成的工作。 PI建议：1.调查不同可能的功能 通过分离和分析STE5对蛋白激酶的调节 Ste5突变体在特定功能和与蛋白质的相互作用方面存在缺陷 激酶和G蛋白。突变的蛋白质将被生化鉴定 能够与途径组件结合，激活激酶，并直接 底物磷酸化。 2.将使用细胞生物学和遗传学方法来研究 细胞周期和信息素依赖的STE5定位调控作用 确定它对隔离MAPK级联酶是否重要 不同的细胞隔间进行特定的反应。 3.对Mekk Ste11的激活机制进行了分析 对Ste11和Ste5的二聚化和调控之间的关联 Ste11、Ste5和Ste50的域。 4.将研究Ste5在介导通路特异性中的作用 通过分离和分析激活Ste5和Ste11的突变形式 其他MAPK级联反应。

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.