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促分裂原活化蛋白激酶（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、存活和应激反应。MAPK级联在调节细胞存活的癌蛋白和抗癌蛋白的下游和上游起作用。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。这个提议的目的是了解模型S的激活机制。酿酒酵母交配MAPK级联，强调步骤，导致在细胞皮层的受体连接的G蛋白，随后的形态学事件和信号的保真度的活性Ste 5支架激酶信号传导复合物的组装。原型MAPK支架Ste 5在激活相关MAPK级联中起多个中心作用并且是动态的，其中Ste 5的核穿梭对于募集和MAPK激活是重要的。 我们发现Ste 5的寡聚化与高活性、增强的核输出、与Ste 11的关联和募集相关。Ste 5通过与鸟嘌呤交换因子Cdc 24的相互作用而独立于G蛋白被基本招募。Ste 5在信息素信号传导过程中定位到皮层需要Rho 1、Bni 1、Myo 2和肌动蛋白电缆。发现Ste 5通过不同的机制激活Fus 3和Kss 1。G蛋白和Ste 5被发现基本激活Fus 3和Kss 1，但选择性阻遏Fus 3 MAPK磷酸酶和免疫Kss 1磷酸酶促进途径特异性增殖，而不是交配。该提议的具体目的是1）确定Ste 5如何通过Cdc 24稳定募集以及稳定募集的作用，2）确定Rho 1-Bni 1-Myo 2如何定位Ste 5，3）定义Ste 5的活性和非活性形式以及Fus 3和Kss 1的差异控制，4）确定MAPK磷酸酶选择性基础抑制Fus 3的途径特异性和Kss 1对磷酸酶的免疫机制。

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.