REGULATION OF MAPK MODULE ASSEMBLY AND ACTIVATION

MAPK(丝裂原活化蛋白激酶)级联从酵母到哺乳动物都是保守的。在酿酒酵母交配途径中，MAPK模块由与Ste5支架蛋白形成复合体的顺序作用的蛋白激酶Ste11、Ste7和Fus3/Kss1组成。这种组装的必要性尚不清楚，但它可能有助于信号转导和/或信号隔离，以防止MAPK通路之间的串扰。了解这些协会是如何受到监管的，以及议会发生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，通过该模型，MAPK模块在生产和非生产状态之间循环。我们的假设预测，一种磷酸酶可能调节这一循环。我们将使用共刺激方案和磷酸酶活性分析来测试这一模型。(目的2)此外，由于Fus3和Ste5的细胞内靶向是信息素诱导的，Ste7也可能表现出不同的定位模式。此外，由于Ste7在激活环和饲喂部位的调节结构域中被磷酸化，Ste7的磷酸化状态可能会影响其定位。我们将使用GFP标记的Ste7变体来分析Ste7在信息素诱导下的定位。

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.