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.

有丝分裂原活化蛋白激酶（MAPK）级联反应由三个顺序作用的蛋白激酶组成。在细胞中重申相关的级联反应，以介导对各种细胞外刺激的不同反应。尽管这种情况有可能进行广泛的交叉谈话，但通常观察到，给定途径的MAPK仅被适当的刺激激活。由于给定级联的酶在稳定的复合物中相互关联，因此似乎维持了这种特异性。该模块化组织对信号扩增，调节和整合的影响对于了解控制正常增殖，发育和炎症反应的事件至关重要。尽管脊椎动物途径的模块化组织变得显而易见，但酵母中的类似途径是这些级联体系结构和动态的范式。我们将利用有关酵母交配应力和细胞完整性级联反应的了解，以了解模块化组织对信号传输和踏板间通信的机制的影响。我们的目的是：[1]定义脚手架关联在信号传导中的调节和功能。与脚手架STE5结合有缺陷的MAPK激酶Ste7的突变体将分离为身份结合区域，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关联在体内和体外磷酸转移反应中的作用的工具。 [2]确定信号组件的开关状态之间控制过渡的蛋白质。将使用两个杂种筛选和生化方法来隔离调节剂，以促进从非生产性的STE7与Mating Pathway模块的其他组件的过渡到生产性相互作用。 [3]研究共享组件的分布是否调节途径活动。 MAPK激酶激酶Ste11的结合有缺陷的突变体，该突变体在交配模块中与Ste5结合的结合和应力模块中的MAPK激酶PBS2的结合将被隔离，并用于学习其与另一个模块限制在另一个模块中的相互作用。使用共聚焦荧光显微镜在信号传导过程中，将在体内检查PBS2-STE11共定位。 {4}定义一个刺激激活两个MAPK途径的机制。信息素激活交配的MAPK级联反应，然后刺激细胞融合MAPK级联反应。遗传方法将用于识别“第二使者”协调这两种途径的活动。