Cell Cycle Arrest and Signal Transduction in Yeast

所有真核细胞都使用多种丝裂原激活蛋白激酶 (MAPK) 级联反应来响应许多调节增殖、分化、生存和应激反应的外部刺激。 MAPK 级联可以在调节细胞存活的癌蛋白和抗癌蛋白的下游和上游发挥作用。 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意义和重要性。 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更高阶的分子组装体，其中空间组织发挥着关键作用。 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，此时细胞质成分（例如支架、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。该提案的主要目标是了解 MAPK 级联激活的分子机制，重点是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优秀模型系统。 Ste5 支架是该通路的关键特异性决定因素。 Ste5 将 MAPKKKK Ste11、MAPKK Ste7 和 MAPK Fus3 连接成复合物，并在存在刺激的情况下被招募到 G 蛋白的 Gbeta 亚基上。 这种募集事件对于 Ste20 激活 Ste11 至关重要，Ste20 是一种 PAK 样激酶，可在细胞皮层结合 Cdc42 GTPase 和 Gbeta。 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。 我们已经确定了该本地化途径的许多监管者。 核穿梭和募集的几个调节因子是细胞骨架的保守成分，这表明核穿梭和募集之间的联系可能普遍相关。 该提案的具体目标是 1) 定义调节其定位的 Ste5 顺式元件，2) 分析 Ste5 核输入的调节因子，3) 分析 Ste5 核输出的调节因子，4) 定义允许核穿梭、膜募集和激酶关联的活性 Ste5 寡聚物的指令，5) 定义确保交配 MAPK Fus3 仅由交配信息素激活的特异性决定因素。

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.