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.

丝裂原激活蛋白激酶 (MAPK) 是一个保守家族 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事件的事件有关 癌症。 这一前提得到了以下发现的加强：癌基因如 因为 raf 和 ras 激活这些途径。 虽然了解 脊椎动物 MAPK 激活才刚刚开始出现，我们了解更多 关于酵母中的类似途径。 S. 中独立但结构相关的 MAPK 激活途径。 酿酒酵母控制三种不同的生理反应。 最好的 了解这些是模拟细胞的信息素诱导途径 分化成交配能力状态。 诱导的信息素 信号通过 G 蛋白与细胞内成分耦合， 涉及五种蛋白激酶：STE20、STE11、STE7 和一对冗余蛋白激酶 MAPK 同源物 FUS3 和 KSS1。 我的目标是： [1] 使用以下方法重建 STE11-STE7-FUS3 磷酸化级联 纯化的成分。 [2] 定义信息素诱导刺激 STE7 的分子基础 和STE11。 磷酸化位点物理作图后 分析对信号转导和酶活性的影响。 因为 STE11 的 N 端负调控结构域具有抑制作用 作用，将使用分离的重组多肽进行激酶测定 测试假底物抑制模型。 最后，我们将使用一个 剂量抑制方法来识别参与的新成分 促进信号转导。 [3] 研究导致信息素脱敏的机制 感应信号。 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 脱敏反应。 基因筛选将用于鉴定新颖的 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。 我们将检查酶- 使用 STE7 和不同的磷酸化测定进行底物选择性 酵母 MAP 激酶家族成员。 我们将产生 STE7 突变 体外并使用遗传选择来识别促进的变化 与不适当的 MAPK 激活途径相互作用。