REGULATION OF MAPK MODULE ASSEMBLY AND ACTIVATION

MAPK 模块组装和激活的调节

• 批准号: 6385239
• 负责人: ANISA SCOTT
• 金额: $ 0.03万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6385239
• 关键词: Saccharomyces cerevisiae; biological signal transduction; chimeric proteins; enzyme activity; enzyme mechanism; fluorescence microscopy; microorganism reproduction; mitogen activated protein kinase; molecular assembly /self assembly; mutant; phosphoprotein phosphatase; phosphorylation; protein localization; protein protein interaction

MAPK (mitogen activated protein kinase) cascades are conserved from yeast to mammals. In the S. cerevisiae mating pathway, the MAPK module consists of the sequentially acting kinases Ste 11, Ste7, and Fus3/Kss1 that physically associate in a complex with the Ste5 scaffolding protein. The necessity for this assembly is unknown but it may facilitate signal transduction and/or signal insulation to prevent cross-talk among the MAPK pathways. Understanding how the associations are regulated and the consequences for changes in the assembly are unknown. These questions are the foundation upon which our two specific aims have been established: (Aim 1) We have proposed a model whereby the MAPK module cycles between productive and nonproductive states. Our hypothesis predicts a phosphatase may regulate this cycle. We will employ a copurification scheme and phosphatase activity assays to test this model. (Aim 2) Also, since intracellular targeting of Fus3 and Ste5 is pheromone inducible, Ste7 may also display different patterns of localization. Furthermore, since Ste7 becomes phosphorylated in the activation loop and in the regulatory domain at feedack sites, the phosphorylation state of Ste7 may influence its localization. We will use GFP-tagged Ste7 variants to analyze localization of Ste7 upon pheromone induction.

MAPK（丝裂原活化蛋白激酶）级联从酵母到哺乳动物是保守的。在S.在酿酒酵母交配途径中，MAPK模块由顺序作用的激酶Ste 11、Ste 7和Fus 3/Kss 1组成，它们与Ste 5支架蛋白在复合物中物理缔合。这种组装的必要性是未知的，但它可能有助于信号转导和/或信号绝缘，以防止MAPK通路之间的串扰。了解关联是如何被调节的，以及组件中变化的后果是未知的。这些问题是我们建立两个具体目标的基础：（目标1）我们提出了一个模型，使MAPK模块在生产和非生产状态之间循环。我们的假设预测磷酸酶可能调节这个周期。我们将采用共纯化方案和磷酸酶活性测定来测试这个模型。(Aim 2）此外，由于Fus 3和Ste 5的细胞内靶向是信息素可诱导的，所以Ste 7也可以显示不同的定位模式。此外，由于Ste 7在激活环和反馈位点的调节结构域中被磷酸化，因此Ste 7的磷酸化状态可能影响其定位。我们将使用GFP标记的Ste 7变体来分析Ste 7在信息素诱导后的定位。