KINASES AND CONTROL OF CELL-TYPE SPECIALIZATION

激酶和细胞类型特化的控制

• 批准号: 2022213
• 负责人: BEVERLY ERREDE
• 金额: $ 28.12万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022213
• 关键词: G protein; alternatives to animals in research; biological signal transduction; cell differentiation; cell type; enzyme activity; fungal genetics; genetic regulation; high performance liquid chromatography; mass spectrometry; mitogen activated protein kinase; mutant; pheromone; phosphorylation; protein tyrosine phosphatase; recombinant proteins

Mitogen-activated protein kinases (MAPKs) are a conserve family of protein kinases. These enzymes mediate intracellular phosphorylation events that link receptor activation to the control of cell proliferation and differentiation. Defining the architecture and regulation of these signal pathways is pertinent to understanding events that cause various cancers. This premise is reinforced by the finding that oncogenes such as raf and ras activate these pathways. While an understanding of vertebrate MAPK activation is just beginning to emerge, we know more about analogous pathways in yeast. Separate but structurally related MAPK activation pathways in S. cerevisiae control three distinct physiological responses. The best understood of these is the pheromone induced pathway that simulates cells to differentiate into a mating competent state. The pheromone induced signal is coupled though a G protein to the intracellular components that involves five protein kinases, STE20, STE11, STE7 and a redundant pair of MAPK homologs, FUS3 and KSS1. My objective are to: [1] Reconstitute the STE11-STE7-FUS3 phosphorylation cascade using purified components. [2] Define the molecular basis for pheromone induced stimulation of STE7 and STE11. After physical mapping of phosphorylation sites will be analyzed for effects on signal transduction and enzyme activity. Because the N-terminal negative regulatory domain of STE11 has an inhibitory role,kinase assays with isolated recombinant polypeptides will be used to test a pseudosubstrate inhibition model. Finally, we will use a dosage suppression approach to identity novel components involved in promoting signal transduction. [3] Investigate mechanisms causing desensitization to the pheromone induced signal. We will evaluate whether feed back phosphorylation and a predicted protein tyrosine phosphatase have specific roles in the desenitization response. A genetic screen will be used to identity novel components that promote desensitization. [4] Evaluate parameters that prevent cross interactions of structurally related kinases in different signal pathways. We will examine enzyme- substrate selectivity using phosphorylation assay with STE7 and different yeast MAP-kinase family members. We will generate STE7 mutations in vitro and use genetic selection to identity changes that promote interactions with inappropriate MAPK activation pathways.

有丝分裂原激活的蛋白激酶（MAPK）是一个保护家族 蛋白激酶。 这些酶介导细胞内磷酸化 将受体激活与控制细胞增殖的控制的事件 和分化。 定义这些结构和调节 信号途径与理解导致各种事件有关 癌症。 这种前提是通过发现这样的癌症来加强了这种前提的 随着RAF和RAS激活这些途径。 虽然对 脊椎动物MAPK激活刚刚开始出现，我们知道更多 关于酵母中的类似途径。 单独但与结构相关的MAPK激活途径。 酿酒酵母控制三个不同的生理反应。 最好的 了解这些是信息素诱导的途径，该途径模拟了细胞 分化为一个胜任状态。 信息素诱导 信号通过G蛋白耦合到细胞内成分 涉及五个蛋白激酶，STE20，Ste11，Ste7和一对 MAPK同源物，FUS3和KSS1。 我的目标是： [1]使用使用Ste11-Ste7-Fus3磷酸化级联反应使用 纯化的组件。 [2]定义了信息素诱导Ste7刺激的分子基础 和Ste11。 磷酸化位点的物理映射之后 分析了对信号转导和酶活性的影响。 因为 Ste11的N末端负调控域具有抑制 角色，将使用具有孤立重组多肽的激酶测定 测试假基底抑制模型。 最后，我们将使用 对身份的剂量抑制方法涉及的新颖成分 促进信号转导。 [3]研究导致信息素脱敏的机制 诱导信号。 我们将评估是否反馈磷酸化和 预测的蛋白酪氨酸磷酸酶在 去苯响应。 遗传屏幕将用于身份小说 促进脱敏的组件。 [4]评估可以防止结构上交叉相互作用的参数 不同信号途径中的相关激酶。 我们将检查酶 - 使用Ste7和不同的磷酸化测定法和不同的底物选择性 酵母地图激酶家庭成员。 我们将在 体外并使用遗传选择来促进身份变化 与不适当的MAPK激活途径的相互作用。