KINASES AND CONTROL OF CELL-TYPE SPECIALIZATION

丝裂原活化蛋白激酶（MAPK）级联由三个顺序作用的蛋白激酶组成。相关的级联反应在细胞内反复进行，以介导对一系列细胞外刺激的不同反应。尽管这种情况可能导致广泛的串扰，但通常观察到，特定通路的mapk仅被适当的刺激激活。这种特异性似乎得以维持，因为给定级联的酶在稳定的复合物中相互关联。这种模块化组织对信号放大、调节和整合的影响对于理解控制正常增殖、发育和炎症反应的事件至关重要。虽然脊椎动物途径的模块化组织越来越明显，但酵母中的类似途径是这些级联的结构和动力学的范例。我们将利用已知的酵母交配应激和细胞完整性级联来了解模块化组织对信号传递机制和通路间通信的影响。我们的目标是：定义信号传导中支架联合的调控和功能。将分离MAPK激酶Ste7与支架- ste5结合有缺陷的突变体，以鉴定结合区域，并了解磷酸化如何调节Ste7- ste5结合。这些突变体也将被用作工具，以了解Ste7-Ste5结合在体内信号传导和体外磷转移反应中的作用。识别控制信号组件开与关状态转换的蛋白。将使用两种混合筛选和生化方法分离调节因子，这些调节因子促进Ste7与交配途径模块的其他组分从非生产性相互作用过渡到生产性相互作用。研究共享成分的分布是否调节通路活动。将分离MAPK激酶Ste11的结合缺陷突变体，这些突变体区分了在交配模块中与Ste5的结合和在胁迫模块中与MAPK激酶Pbs2的结合，并用于了解其与一个模块的相互作用是否限制了另一个模块的信号传导。Pbs2-Ste11共定位将在体内使用共聚焦荧光显微镜进行检测。{4}定义一个刺激激活两个MAPK通路的机制。信息素激活配对MAPK级联，其输出刺激细胞完整性MAPK级联。遗传方法将用于识别协调这两种途径活动的“第二信使”。

Mitogen activated protein kinase (MAPK) cascades are comprised of three sequentially acting protein kinases. Related cascades are reiterated in the cell to mediate distinct responses to a host of extracellular stimuli. Despite the potential this situation presents for extensive cross talk, it is generally observed that the MAPKs of a given pathway are activated only by the appropriate stimuli. This specificity appears to be maintained because the enzymes of a given cascade are associated with each other in stable complexes. The ramifications that this modular organization has on signal amplification, regulation and integration is central to understanding events that control normal proliferation, development and inflammatory responses. While a modular organization of vertebrate pathways is becoming apparent, analogous pathways in yeast are the paradigms for the architecture and dynamics of these cascades. We will exploit what is known about the yeast mating stress and cell- integrity cascades to learn what consequences a modular organization has on the mechanics of signal transmission and on inter-pathway communication. Our aims are to: [1] Define the regulation and function of scaffold associations in signaling. Mutants of the MAPK kinase Ste7 that are defective for binding to the scaffold-Ste5 will be isolated to identity the binding region and learn how phosphorylation regulates the Ste7-Ste5 association. The mutants also will be used as tools to learn what role the Ste7-Ste5 association has on signaling in vivo and on phosphotransfer reactions in vitro. [2] IdentifY protein(s) controlling transitions between on and off states of signaling assemblies. Two hybrid screens and a biochemical approach will be used to isolate regulators that facilitate a transition from a non-productive to productive interaction of Ste7 with other components of the mating pathway module. [3] Investigate whether distribution of shared components regulates pathway activities. Binding defective mutants of the MAPK kinase kinase Ste11 that discriminate between binding to Ste5 in the mating module and the MAPK kinase Pbs2 in the stress module will be isolated and used to learn if its interaction with one module limits signaling in the other. Pbs2-Ste11 co-localization will be examined in vivo during signalling using confocal fluorescence microscopy. {4} Define the mechanism by which one stimulus activates two MAPK pathways. Pheromone activates the mating MAPK cascade whose output then stimulates the cell-integrity MAPK cascade. Genetic methods will be used to identity the "second messengers" coordinating the of activities of these two pathways.