Mechanism and Specificity of MAP Kinases

MAP 激酶的机制和特异性

• 批准号: 6727675
• 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/6727675
• 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）：丝裂原激活蛋白激酶（MAPK） 是信号转导中极其重要的酶。的后果是 这些酶的正常控制的崩溃可能会导致许多毁灭性的后果 癌症等疾病。人类中已鉴定出三个主要亚科 长期目标是确定抑制特定成员的方法 每个亚科。为了实现这一目标，可靠的化学生物学方法是 结合酶学和分子生物学建立 酶的基本性质。第一个重点是定义动力学 细胞外信号调节激酶 ERK2（第一个 MAPK）的机制 被发现，并检验 ADP 释放是限速的假设。 复杂的预稳态猝灭流和停流荧光 技术和平衡结合研究将用于量化和 确定单个酶促步骤。第二个焦点建立在动力学上 模型并检验蛋白质-蛋白质相互作用调节 ERK2 的假设 活动。结构分析 - 将确定 ERK2-底物相互作用和 髓磷脂碱性蛋白和底物诱导的构象转变 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。定点诱变的强大组合 和前稳态动力学将批判性地探讨其机制 ERK2 介导的蛋白质-蛋白质相互作用的影响，重点关注 最近在许多 ERK2 底物中发现了模块化结合域。这 第三个重点将使用肽噬菌体展示技术来绘制表位图 ERK2-蛋白质相互作用并检验蛋白质-蛋白质的假设 ERK2介导的相互作用是由小模块的识别驱动的 已进化形成瞬时稳定复合物的结合序列。这 这一时期的具体目标是： 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 通过肽噬菌体展示序列并将其功能与野生型进行对比 模块化序列。这将是第一个全面的机制研究 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了解 人类参与的主要酶之一的调节崩溃 癌症和疾病。