KINASES AND CONTROL OF CELL-TYPE SPECIALIZATION

丝裂原活化蛋白激酶(MAPKs)是一个保守的家族。 蛋白激酶。这些酶介导细胞内的磷酸化。 将受体激活与细胞增殖控制联系起来的事件 和差异化。定义以下各项的体系结构和法规 信号通路与理解导致各种不同 癌症。这一前提得到了加强，因为发现癌基因 因为英国皇家空军和皇家空军激活了这些通路。虽然理解了 脊椎动物MAPK的激活才刚刚开始，我们知道更多 关于酵母中类似的途径。 不同的MAPK激活通路在S. 酿酒酵母控制着三种不同的生理反应。最好的 理解这些的是模拟细胞的信息素诱导的途径 分化为有交配能力的状态。信息素诱导 信号通过G蛋白偶联到细胞内的成分， 涉及5个蛋白激酶，STE20、STE11、STE7和一个冗余对 MAPK同源物、FUS3和KSS1。我的目标是： [1]重组STE11-STE7-FUS3磷酸化级联 提纯的成分。 [2]确定信息素刺激STE7的分子基础 和STE11。在对磷酸化位点进行物理映射后， 分析了对信号转导和酶活性的影响。因为 STE11的N末端负调控结构域具有抑制性 角色，将使用分离的重组多肽进行激酶分析 测试一种假底物抑制模型。最后，我们将使用 一种新成分的剂量抑制识别方法 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 一种预测的蛋白质酪氨酸磷酸酶在 去化反应。基因筛查将被用于识别小说 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蛋白。我们将检测酶- STE7与Different磷酸化反应的底物选择性 酵母菌MAP-激酶家族成员。我们将产生STE7突变 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。

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.