KINASES AND CONTROL OF CELL-TYPE SPECIALIZATION

激酶和细胞类型特化的控制

基本信息

批准号：
6476490
负责人：
BEVERLY ERREDE
金额：
$ 30.87万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-07-01 至 2004-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6476490
关键词：
biological signal transduction cell differentiation confocal scanning microscopy enzyme activity fungal genetics gene mutation mitogen activated protein kinase pheromone phosphorylation protein localization yeast two hybrid system yeasts

项目摘要

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.

丝裂原激活蛋白激酶 (MAPK) 级联由三种顺序作用的蛋白激酶组成。相关的级联在细胞中重复，以介导对大量细胞外刺激的不同反应。尽管这种情况可能会导致广泛的串扰，但通常观察到给定通路的 MAPK 仅由适当的刺激激活。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彼此结合。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变得越来越明显，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压力和细胞完整性级联来了解模块化组织对信号传输机制和通路间通信的影响。我们的目标是： [1] 定义支架关联在信号传导中的调节和功能。将分离出无法与支架-Ste5 结合的 MAPK 激酶 Ste7 突变体，以识别结合区域并了解磷酸化如何调节 Ste7-Ste5 关联。这些突变体还将用作了解 Ste7-Ste5 关联对体内信号传导和体外磷酸转移反应的作用的工具。 [2] 识别控制信号组件开启和关闭状态之间转换的蛋白质。将使用两种混合筛选和生化方法来分离调节因子，以促进 Ste7 与交配途径模块的其他组件从非生产性相互作用转变为生产性相互作用。 [3] 研究共享成分的分布是否调节通路活动。 MAPK 激酶激酶 Ste11 的结合缺陷突变体可区分交配模块中的 Ste5 和应激模块中的 MAPK 激酶 Pbs2，并将其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导。在信号传导过程中，将使用共焦荧光显微镜在体内检查 Pbs2-Ste11 共定位。 {4} 定义一种刺激激活两种 MAPK 通路的机制。信息素激活交配 MAPK 级联，其输出随后刺激细胞完整性 MAPK 级联。遗传学方法将用于识别协调这两条途径的活动的“第二信使”。