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部刺激，这些外部刺激调节增殖、分化、存活和对应激的反应。 MAPK级联可以在调节细胞存活的癌蛋白和抗癌蛋白的下游和上游发挥作用。 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级联的信号转导的新兴主题是，它们经常使用支架/衔接蛋白在细胞内形成更高级的分子组装体，其中空间组织起着关键作用。 空间完整性在受体介导的信号转导途径的初始活化步骤期间是至关重要的，此时细胞质组分如支架、激酶或其他酶必须物理连接到感测刺激的膜受体和G蛋白。该提案的广泛目标是了解MAPK级联激活的分子机制，重点是导致在细胞皮层组装活性信号复合物的步骤。 对S.酿酒酵母是研究保守的受体/G蛋白偶联MAP激酶级联激活机制的极好模型系统。Ste 5支架是该途径的关键特异性决定因素。 Ste 5将MAPKKKK Ste 11、MAPKK Ste 7和MAPK Fus 3束缚成复合物，并在刺激存在下被募集到G蛋白的G β亚基。 该募集事件对于由Ste 20激活Ste 11是必不可少的，Ste 20是一种PAK样激酶，其在细胞皮质结合Cdc 42 GT3和G β。 Ste 5向G β的募集涉及一种新的定位途径，其中Ste 5首先穿梭于细胞核。 我们已经确定了一些监管机构的本地化途径。 核穿梭和招聘的监管机构是保守的细胞骨架的组成部分，这表明核穿梭和招聘之间的联系可能是普遍相关的。 该提议的具体目的是1）定义调节其定位的Ste 5的顺式元件，2）分析Ste 5的核输入的调节剂，3）分析Ste 5的核输出的调节剂，4）定义允许核穿梭、膜募集和激酶缔合的活性Ste 5寡聚体的指令，5）定义确保配对MAPK、Fus 3、只有交配信息素才能激活

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.