Mechanism and Specificity of MAP Kinases

MAP 激酶的机制和特异性

• 批准号: 6520074
• 负责人: Kevin N Dalby
• 金额: $ 20.19万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520074
• 关键词: biological signal transduction; chemical kinetics; enzyme mechanism; epitope mapping; fluorescent dye /probe; mitogen activated protein kinase; myelin basic proteins; protein protein interaction; protein sequence; protein structure function; site directed mutagenesis; stop flow technique

DESCRIPTION (Applicant's abstract): Mitogen-activated protein kinases (MAPKs) are extremely important enzymes in signal transduction. The consequence of a breakdown in the normal control of these enzymes can lead to many devastating diseases such as cancer. Three major subfamilies have been identified in humans and long-term goals are to identify methods of inhibiting specific members of each subfamily. To achieve this goal a solid chemical-biology approach is taken, where enzymology and molecular biology is combined to establish fundamental properties of the enzymes. The first focus is to define the kinetic mechanism of the extracellular signal-regulated kinase, ERK2, the first MAPK to be discovered, and to test the hypothesis that ADP release is rate-limiting. Sophisticated pre-steady state quench-flow and stopped-flow fluorescence techniques, and equilibrium binding studies, will be used to quantify and identify individual enzymatic steps. The second focus builds on the kinetic model and tests the hypothesis that protein-protein interactions modulate ERK2 activity. A structural analysis-will identify ERK2-substrate interactions and substrate-induced conformational transitions with myelin basic protein and the truncated protein substrates c-Myc(1-100) and Ets-1 (1-138), using trace-labeling experiments. A powerful combination of site-directed mutagenesis and pre-steady state kinetics will critically probe the mechanistic implications of protein-protein interactions mediated by ERK2, focusing on a recently discovered modular binding domain found in many ERK2 substrates. The third focus will use peptide phage display technology to epitope-map ERK2-protein interactions and to test the hypothesis that protein-protein interactions mediated by ERK2 are driven by the recognition of small modular binding sequences that have evolved to form transiently stable complexes. The specific aims during this period are: l) to define the kinetic mechanism of ERK2; 2) to perform structure-function studies to examine ERK2 substrate protein-protein interactions; and 3) to identify tight binding, modular peptide sequences by peptide phage display and contrast their function with wild type modular sequences. This will be the first comprehensive mechanistic study of ERK2 and the discovery of novel peptide inhibitors will set the scene for future structural and cell biology approaches aimed at understanding the breakdown in regulation of one of the primary enzymes in involved in human cancer and disease.

描述（申请人的摘要）：有丝分裂原激活的蛋白激酶（MAPK） 是信号转导中极为重要的酶。一个 这些酶正常控制的崩溃可能导致许多毁灭性的 癌症等疾病。人类已经确定了三个主要的亚家族 长期目标是确定抑制特定成员的方法 每个亚家族。为了实现这一目标，一种可靠的化学生物学方法是 采取的酶学和分子生物学合并以建立 酶的基本特性。第一个重点是定义动力学 细胞外信号调节激酶ERK2的机制，第一个MAPK 被发现，并检验ADP释放是限制速率的假设。 精致的稳态淬灭流和停止流量荧光 技术和平衡结合研究将用于量化和 确定单个酶促步骤。第二个焦点以动力学为基础 模型并检验了蛋白质 - 蛋白质相互作用调节ERK2的假设 活动。结构分析将确定ERK2-SUBSTRATE相互作用和 与髓磷脂碱性蛋白的底物诱导的构象过渡和 截短的蛋白质底物C-Myc（1-100）和ETS-1（1-138），使用 痕量标记实验。站点定向诱变的强大组合 稳态的状态动力学将严重探究机械 ERK2介导的蛋白质 - 蛋白质相互作用的含义，重点是 最近在许多ERK2底物中发现的模块化结合结构域。这 第三重点将使用肽噬菌体显示技术来表位图 ERK2-蛋白质相互作用并检验蛋白质蛋白的假设 ERK2介导的相互作用是由小模块的识别驱动的 结合序列已演变为形成瞬时稳定复合物。这 在此期间的具体目的是：l）定义动力学机制 erk2; 2）进行结构功能研究以检查ERK2底物 蛋白质蛋白质相互作用； 3）识别紧密结合的模块化肽 肽噬菌体序列显示并与野生类型对比 模块化序列。这将是首次对 ERK2和新型肽抑制剂的发现将为 未来的结构和细胞生物学方法旨在理解 对人类涉及的主要酶之一的调节分解 癌症和疾病。