Cell Cycle Arrest and Signal Transduction in Yeast

描述(申请人提供)：所有真核细胞使用多个丝裂原活化蛋白激酶(MAPK)级联反应许多外部刺激，这些刺激调节所有真核细胞的增殖、分化、生存和对应激的反应。MAPK级联作用于调节细胞存活的癌蛋白和抗癌蛋白的下游和上游。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。该建议的目标是了解模型酿酒酵母交配MAPK级联的激活机制，强调导致活性Ste5支架信号复合体在细胞皮质受体连接的G蛋白处组装的步骤、随后的形态事件和信号的保真度。典型的MAPK支架Ste5在激活相关的MAPK级联过程中扮演着多个中心角色，并且是动态的，Ste5的核穿梭对招募和MAPK激活是重要的。我们发现，Ste5的寡聚化与高活性、核输出增强、与Ste11相关和招募相关。STE5是通过与鸟嘌呤交换因子CDc24相互作用而独立于G蛋白被招募的。在信息素信号传递过程中，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可以基本激活Fus3和Kss1，但MAPK磷酸酶选择性抑制Fus3和Kss1对磷酸酶的免疫促进了增殖而不是交配的途径特异性。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的活性和失活形式以及对Fus3和Kss1的不同调控；4)确定MAPK磷酸酶选择性基础抑制Fus3的通路特异性和Kss1对磷酸酶免疫的机制。

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.