Mechanism and Specificity of MAP Kinases

MAP 激酶的机制和特异性

• 批准号: 6636337
• 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/6636337
• 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.

说明(申请人摘要)：丝裂原活化蛋白激酶(MAPK) 是信号转导中极其重要的酶。一场战争的后果 这些酶的正常控制的崩溃可能会导致许多毁灭性的 癌症等疾病。在人类中发现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 每个子科。为了实现这一目标，一种可靠的化学-生物学方法是 采用，在那里酶学和分子生物学相结合来建立 酶的基本性质。第一个焦点是定义动力学 细胞外信号调节蛋白激酶ERK2的作用机制 被发现，并检验ADP释放是限速的假设。 精密的预稳态急冷流和停流荧光 技术和平衡约束研究将被用于量化和 确定单独的酶促步骤。第二个焦点建立在动能的基础上 对蛋白质相互作用调节ERK2的假说进行建模和检验 活动。结构分析-将确定ERK2-底物相互作用和 底物诱导的髓鞘碱性蛋白构象转变及其机制 截短蛋白底物c-Myc(1-100)和Ets-1(1-138)，使用 痕量标记实验。一种强大的定点突变组合 而预稳态动力学将关键地探索其机理 ERK2介导的蛋白质-蛋白质相互作用的含义，重点是 最近发现在许多ERK2底物中发现了模块结合结构域。这个 Third Focus将使用肽噬菌体展示技术绘制表位图 ERK2-蛋白质相互作用和检验蛋白质-蛋白质相互作用假说 ERK2介导的相互作用是由对小模块的识别驱动的 结合序列已经进化为形成瞬时稳定的复合体。这个 这一时期的具体目标是：L)明确 ERK2；2)进行结构-功能研究以检测ERK2底物 蛋白质-蛋白质相互作用；以及3)鉴定紧密结合的模块化多肽 用噬菌体展示序列并与野生型进行功能比较 模数序列。这将是第一次全面的机理研究。 ERK2和新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 人类参与的一种主要酶的调节崩溃 癌症和疾病。