Cell Cycle Arrest and Signal Transduction in Yeast

描述（由申请人提供）：所有真核细胞都使用多个有丝分裂原活化蛋白激酶（MAPK）级联来响应许多外部刺激，这些刺激调节所有真核细胞的增殖、分化、存活和对应激的反应。MAPK级联作用于调节细胞存活的癌蛋白和抗癌蛋白的上游和下游。了解MAPK级联激活和通路特异性的基本机制具有普遍的相关性和重要性。本研究的目标是了解酿酒酵母交配MAPK级联的激活机制，强调导致细胞皮层受体连接的G蛋白上活性Ste5支架激酶信号复合物组装的步骤，随后的形态学事件和信号的保真度。典型的MAPK支架Ste5在激活相关的MAPK级联中起着多个核心作用，并且是动态的，Ste5的核穿梭对于招募和MAPK激活很重要。我们发现Ste5的寡聚化与高活性、核输出增强、与Ste11和招募相关。Ste5通过与鸟嘌呤交换因子Cdc24的相互作用而基本独立于G蛋白募集。在信息素信号传导过程中，Ste5定位到皮层需要Rho1、Bni1、Myo2和肌动蛋白电缆。Ste5通过不同的机制激活Fus3和Kss1。G蛋白和Ste5基本激活Fus3和Kss1，但MAPK磷酸酶对Fus3的选择性抑制和Kss1对磷酸酶的免疫促进了途径特异性的增殖而不是交配。本研究的具体目的是：1)确定Ste5是如何通过Cdc24稳定募集的，以及稳定募集的作用；2)确定Rho1-Bni1-Myo2如何定位Ste5； 3)确定Ste5的活性和非活性形式以及Fus3和Kss1的差异控制；4)确定MAPK磷酸酶选择性基础抑制Fus3的途径特异性机制和Kss1对磷酸酶的免疫。

DESCRIPTION (provided by applicant): All eukaryotic cells use multiple mitogen-activated protein kinase (MAPK) cascades to respond to many external stimuli that regulate proliferation, differentiation, survival and response to stress in all eukaryotes. MAPK cascades act downstream and upstream of oncoproteins and anti-oncoproteins that regulate cell survival. Understanding basic mechanisms involved in MAPK cascade activation and pathway specificity is of general relevance and importance. The goals of this proposal are to understand the mechanism of activation of the model S. cerevisiae mating MAPK cascade, emphasizing steps that lead to the assembly of an active Ste5 scaffold-kinase signaling complex at a receptor-linked G protein at the cell cortex, subsequent morphological events and fidelity of signaling. The prototypical MAPK scaffold Ste5 plays multiple central roles in activating an associated MAPK cascade and is dynamic, with nuclear shuttling of Ste5 being important for recruitment and MAPK activation. We found that oligomerization of Ste5 correlates with high activity, enhanced nuclear export, association with Ste11 and recruitment. Ste5 was basally recruited independent of G protein through interactions with the guanine exchange factor Cdc24. Localization of Ste5 to the cortex during pheromone signaling required Rho1, Bni1, Myo2 and actin cables. Ste5 was found to activate Fus3 and Kss1 by distinct mechanisms. The G protein and Ste5 were found to basally activate both Fus3 and Kss1, but selective repression of Fus3 by MAPK phosphatase and immunity of Kss1 to phosphatases promoted pathway specificity for proliferation rather than mating. The Specific Aims of this proposal are to 1) Determine how Ste5 is stably recruited through Cdc24 and the role of stable recruitment, 2) Determine how Rho1-Bni1-Myo2 localize Ste5, 3) Define active and inactive forms of Ste5 and the differential control of Fus3 and Kss1, 4) Determine the mechanism of pathway specificity of selective basal repression of Fus3 by MAPK phosphatase and Kss1 immunity to phosphatases.