Cell Cycle Arrest and Signal Transduction in Yeast

所有真核细胞都使用多个丝裂原活化蛋白激酶(MAPK)级联反应来响应许多外界刺激，这些刺激调控着细胞的增殖、分化、存活和对应激的反应。MAPK级联信号通路可以作用于调节细胞存活的癌蛋白和抗癌蛋白的下游和上游。因此，了解MAPK级联激活和维持通路特异性的基本机制具有普遍的相关性和重要性。通过MAPK级联途径进行信号转导的一个新的主题是，它们经常利用支架/接头蛋白在细胞内形成更高阶的分子组装，其中空间组织起着关键作用。在受体介导的信号转导通路的初始激活步骤中，空间完整性是至关重要的，此时细胞质成分，如支架、激酶或其他酶必须物理地连接到膜受体和感知刺激的G蛋白。这项建议的主要目标是了解MAPK级联激活的分子机制，重点是导致在细胞皮质组装活性信号复合体的步骤。酿酒酵母的交配途径是研究保守的受体/G蛋白偶联的MAP蛋白激活级联反应机制的一个很好的模型系统。Ste5支架是该途径的关键特异性决定因素。Ste5将MAPKKKK Ste11、MAPKK Ste7和MAPK Fus3连接成一个复合体，并在刺激存在的情况下被招募到G蛋白的Gbeta亚基。这种招募活动对于Ste20激活Ste11是必不可少的，Ste20是一种类似PAK的激酶，在细胞皮质结合CDC42 GTPase和Gbeta。Ste5向Gbeta的募集涉及一种新的定位途径，即Ste5首先穿梭于细胞核中。我们已经确定了这一本地化途径的一些调节因子。核穿梭和招募的几个调节因子是细胞骨架的保守组成部分，这表明核穿梭和招募之间的联系可能总体上是相关的。本提案的具体目的是1)确定调节其定位的Ste5顺式元件，2)分析Ste5核进口的调控因子，3)分析Ste5核出口的调控因子，4)定义允许核穿梭、膜募集和激酶结合的活性Ste5寡聚体的指令，5)定义特异性决定因素，确保交配MAPK Fus3只被交配信息素激活。

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. MAPK cascades can function both downstream and upstream of oncoproteins and anti-oncoproteins that regulate cell survival. Thus, understanding basic mechanisms involved in MAPK cascade activation and the maintenance of pathway specificity is of general relevance and importance. The emerging theme of signal transduction through MAPK cascades is that they often use scaffold/adapter proteins to form higher order molecular assemblies within cells, with spacial organization playing a critical role. Spatial integrity is critical during the initial activation step of receptor-mediated signal transduction pathways, when cytoplasmic components such as scaffolds, kinases or other enzymes must be physically linked to membrane receptors and G proteins that sense the stimuli. The broad goals of this proposal are to understand the molecular mechanism of activation of a MAPK cascade, with an emphasis on the steps that lead to the assembly of an active signaling complex at the cell cortex. The mating pathway of S. cerevisiae is an excellent model system to study the mechanism of activation of a conserved receptor/G protein-coupled MAP kinase cascade. The Ste5 scaffold is a key specificity determinant for this pathway. Ste5 tethers the MAPKKKK Ste11, MAPKK Ste7 and MAPK Fus3 into a complex and is recruited to the Gbeta subunit of the G protein in the presence of stimulus. This recruitment event is essential for activation of Ste11 by Ste20, a PAK-like kinase that binds both Cdc42 GTPase and Gbeta at the cell cortex. The recruitment of Ste5 to Gbeta involves a novel localization pathway in which Ste5 first shuttles through the nucleus. We have identified a number of regulators of this localization pathway. Several of the regulators of nuclear shuttling and recruitment are conserved components of the cytoskeleton, suggesting that the link between nuclear shuttling and recruitment may be generally relevant. The Specific Aims of this proposal are to 1) Define the cis-elements of Ste5 that regulate its localization, 2) Analyze regulators of nuclear import of Ste5, 3) Analyze regulators of nuclear export of Ste5, 4) Define the dictates of an active Ste5 oligomer that permit nuclear shuttling, membrane recruitment and kinase association, 5) Define specificity determinants that ensure that the mating MAPK, Fus3, is only activated by mating pheromone.