MYOSIN PHOSPHORYLATION BY MYOSIN LIGHT CHAIN KINASE

肌球蛋白轻链激酶磷酸化肌球蛋白

• 批准号: 6571145
• 负责人: Zenon Grabarek
• 金额: $ 27.63万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6571145
• 关键词: actins; active sites; affinity chromatography; calmodulin; catalyst; chemical binding; conformation; crosslink; cysteine; electron microscopy; enzyme activity; enzyme induction /repression; fluorescent dye /probe; immunoelectron microscopy; laboratory mouse; laboratory rabbit; muscle contraction; myosin light chain kinase; myosins; nuclear magnetic resonance spectroscopy; nucleotide analog; nucleotides; phosphorylation; protein localization; protein structure function; smooth muscle

This project is aimed at understanding the molecular mechanisms by which calmodulin (CaM) modulates the enzymatic activity of the myosin light chain kinase (MLCK) and leads to the calcium dependent phosphorylation/activation of smooth muscle myosin. Specifically, this project is focused on the following problems: (1) The conformational transitions in the catalytic/regulatory domain of rabbit smooth muscle MLCK induced by its interaction with CaM and with the myosin regulatory light chain (RLC). The hypothesis of the intrasteric inhibitory mechanism will be tested be measuring the distance between resonance energy transfer chromophores placed at key positions in the native MLCK and in specifically designed mutants. Distances between landmark sites in the binary and ternary complexes of MLCK, CaM and RLC will be measured. The effects of nucleotides and nucleotide analogues on such complexes will be evaluated. (2) The functional role of MLCK segments outside the catalytic core. Sites interacting with actin and myosin heavy chain will be identified. The structural and functional properties of the N-terminal repetitive segment specific for the mammalian smooth muscle MLCK will be characterized. (3) In situ localization of MLCK in the smooth muscle cell. Distribution of MLCK in relation to other protein components under various contractile conditions will be determined by immunoelectron microscopy using antibodies specific to various segments of MLCK.

该项目旨在了解分子机制 钙调蛋白（CAM）调节肌球蛋白光的酶活性 链激酶（MLCK），导致钙依赖性 平滑肌肌球蛋白的磷酸化/激活。具体来说，这是 项目的重点是以下问题：（1）构象 兔平滑肌的催化/调节结构域的过渡 MLCK与CAM的相互作用以及与肌球蛋白调节的相互作用引起的 轻链（RLC）。固定抑制机制的假设 将测试测量共振能量传递之间的距离 在本机MLCK和在 专门设计的突变体。地标地点之间的距离 将测量MLCK，CAM和RLC的二元和三元复合物。这 核苷酸和核苷酸类似物对此类复合物的影响将是 评估。 （2）MLCK段的功能作用 核。与肌动蛋白和肌球蛋白重链相互作用的站点将是 确定。 N末端的结构和功能特性 针对哺乳动物平滑肌MLCK的重复段将是 特征。 （3）MLCK在平滑肌细胞中的原位定位。 MLCK与其他蛋白质成分之间的分布 收缩条件将由免疫电子显微镜确定 使用针对MLCK各个段的抗体。