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.

丝裂原活化蛋白激酶（MAPK）是一个保守的丝裂原活化蛋白激酶家族。 蛋白激酶 这些酶介导细胞内磷酸化 将受体激活与细胞增殖控制联系起来的事件 和差异化。 定义这些的架构和监管 信号通路与理解导致各种 癌的 这一假设得到了以下发现的支持： 因为RAF和RAS激活这些途径。 虽然理解 脊椎动物的MAPK激活才刚刚开始出现，我们知道更多 酵母中类似的途径。 S. 酿酒酵母控制三种不同的生理反应。 最好的 据了解，这些是信息素诱导的途径，模拟细胞 才能分化成有交配能力的状态 信息素诱导 信号通过G蛋白偶联到细胞内成分， 涉及五种蛋白激酶，STE 20，STE 11，STE 7和一个冗余对 MAPK同源物，FUS 3和KSS 1。 我的目标是： [1]使用以下物质重构STE 11-STE 7-FUS 3磷酸化级联： 纯化的成分。 [2]确定信息素诱导刺激STE 7的分子基础 STE11。 在磷酸化位点的物理作图后， 分析对信号转导和酶活性的影响。 因为 STE 11 N-末端负调控结构域具有抑制性 作用，将使用分离的重组多肽的激酶测定 以测试假底物抑制模型。 最后，我们将使用 剂量抑制方法，以确定涉及 促进信号传导。 [3]调查导致对信息素脱敏的机制 感应信号 我们将评估是否反馈磷酸化， 一种预测的蛋白酪氨酸磷酸酶在 去硒化反应 基因筛选将用于识别新的 促进脱敏的成分。 [4]评估防止结构交叉相互作用的参数 不同信号通路中的相关激酶。 我们会检查酶- 使用STE 7和不同的磷酸化测定的底物选择性 酵母MAP激酶家族成员。 我们将产生STE 7突变， 体外和使用遗传选择来识别促进 与不适当的MAPK激活途径相互作用。