Cell Cycle Arrest and Signal Transduction in Yeast

所有真核细胞都使用多个有丝分裂原活化蛋白激酶（MAPK）级联反应来响应许多外部刺激，调节增殖、分化、生存和对应激的反应。MAPK级联可以在调节细胞存活的癌蛋白和抗癌蛋白的上游和下游发挥作用。因此，了解MAPK级联激活和通路特异性维持的基本机制具有普遍的相关性和重要性。通过MAPK级联进行信号转导的新主题是，它们经常使用支架/适配器蛋白在细胞内形成高阶分子组装，其中空间组织起着关键作用。在受体介导的信号转导途径的初始激活步骤中，空间完整性至关重要，因为细胞质成分（如支架、激酶或其他酶）必须与感知刺激的膜受体和G蛋白物理连接。本研究的主要目标是了解MAPK级联激活的分子机制，重点研究导致细胞皮层活跃信号复合物组装的步骤。酿酒酵母的交配途径是研究保守受体/G蛋白偶联MAP激酶级联激活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连接成一个复合物，并在刺激存在的情况下被招募到G蛋白的Gbeta亚基上。这一招募事件对于Ste20激活Ste11至关重要，Ste20是一种结合Cdc42 GTPase和细胞皮层Gbeta的pak样激酶。Ste5向Gbeta的募集涉及一种新的定位途径，其中Ste5首先穿梭于细胞核中。我们已经确定了这种定位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成分，这表明核穿梭和招募之间的联系可能是普遍相关的。本提案的具体目标是：1)确定Ste5中调节其定位的顺式元件，2)分析Ste5的核进口调节因子，3)分析Ste5的核出口调节因子，4)确定允许核穿梭、膜募集和激酶结合的活性Ste5寡聚物的支配，5)确定特异性决定因素，确保交配MAPK Fus3仅被交配信息素激活。

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.