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重复，以调节对一系列细胞外刺激的不同反应。尽管这种情况存在广泛串扰的可能性，但通常观察到，给定途径的MAPK仅在适当的刺激下被激活。这种特异性似乎是保持的，因为给定级联的酶在稳定的复合体中彼此关联。这种模块化组织对信号放大、调节和整合的影响是理解控制正常增殖、发育和炎症反应的事件的核心。虽然脊椎动物途径的模块化组织正在变得明显，但酵母中类似的途径是这些级联反应的体系结构和动力学的范例。我们将利用已知的关于酵母交配压力和细胞完整性级联的知识来了解模块化组织对信号传输机制和路径间通信的影响。我们的目标是：[1]定义脚手架关联在信号中的调节和功能。MAPK激酶Ste7与支架-Ste5结合有缺陷的突变体将被分离出来，以鉴定结合区域，并了解磷酸化如何调节Ste7-Ste5结合。这些突变体还将被用作工具，以了解Ste7-Ste5关联在体内信号传递和体外磷转移反应中所起的作用。[2]确定控制信号组件开启和关闭状态转换的蛋白质(S)。将使用两个混合筛选和一种生化方法来分离调节子，这些调节子促进Ste7与交配途径模块的其他组件从非生产性相互作用过渡到生产性相互作用。[3]研究共享成分的分布是否调节通路活动。MAPK激酶Ste11的结合缺陷突变体将被分离出来，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中的信号传递。PBS2-Ste11的共同定位将在体内使用共聚焦荧光显微镜在信号传递过程中进行检查。{4}定义一个刺激激活两个MAPK通路的机制。信息素激活交配的MAPK级联，其输出然后刺激细胞完整性的MAPK级联。基因方法将被用来识别协调这两条途径活动的“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.