PHOSPHORYLATION OF MYOSIN LIGHT CHAIN 2 IN CARDIAC FUNCTION

心肌功能中肌球蛋白轻链 2 的磷酸化

• 批准号: 6233242
• 负责人: Nathan Maduka Jideama
• 金额: $ 6.95万
• 依托单位: CLARK ATLANTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-28 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6233242
• 关键词: SDS polyacrylamide gel electrophoresis; autoradiography; calcium transporting ATPase; cow; enzyme activity; heart cell; heart function; high performance liquid chromatography; immunoprecipitation; in situ hybridization; ion exchange chromatography; isozymes; laboratory rat; muscle contraction; myofibrils; myosin light chain kinase; phosphorylation; protein kinase C; protein structure function; site directed mutagenesis; thin layer chromatography; western blottings

The contraction of cardiac muscle is modulated by Ca2+/calmodulin- dependent myosin light chain kinase (MLCK), Ca2+ dependent protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) through the phosphorylation of myofibrillar proteins such as troponin T (TnT), troponin I (TnI) and myosin light chain 2 (MLC2). The overall goal of this proposal is to determine the concerted role(s) of myosin light chain kinase and protein kinase C isozymes in phosphorylating cardiac MLC2 and the function of the phosphorylation in cardiac physiology. Phosphorylation, activity assay, and contractile movement of myocytes will be used to determine the role(s) of MLCK and PKC isozymes in cardiac function. The effect of phosphorylation on Ca2+ stimulated MgATPase activity will be determined. Site-specific phosphorylation patterns of cardiac MLC2 by MLCK and PKC isozymes will also be determined through two-dimensional phosphopeptide mapping. Functional significance of phosphorylation of cardiac MLC2 by MLCK and PKC isozymes will be determined using cardiac recombinant MLC2 mutants in which the serine residues at the phosphorylation sites will be replaced by alanine residues. Contractility of the cardiomyocytes will be measured with and without MLCK and PKC inhibitors to determine the biochemical and physiological relevance of MLCK and PKC phosphorylation of the cardiac MLC2. It is anticipated that the data collected from this study will provide new information on the regulation of cardiac physiology by MLCK and PKC isozymes. Specific roles of MLCK and PKC isozymes in regulation cardiac function will be determined. The role(s) MLCK and PKC isozymes play in site-specific phosphorylation and in producing biochemical and physiological changes in the myofibrils will also be determined.

心肌的收缩由 Ca2+/钙调蛋白依赖性肌球蛋白轻链激酶 (MLCK)、Ca2+ 依赖性蛋白激酶 C (PKC) 和 cAMP 依赖性蛋白激酶 A (PKA) 通过肌原纤维蛋白（如肌钙蛋白 T）的磷酸化来调节。 TnT)、肌钙蛋白 I (TnI) 和肌球蛋白轻链 2 (MLC2)。该提案的总体目标是确定肌球蛋白轻链激酶和蛋白激酶 C 同工酶在磷酸化心脏 MLC2 中的协同作用以及磷酸化在心脏生理学中的功能。肌细胞的磷酸化、活性测定和收缩运动将用于确定 MLCK 和 PKC 同工酶在心脏功能中的作用。将确定磷酸化对 Ca2+ 刺激的 MgATP 酶活性的影响。 MLCK 和 PKC 同工酶对心脏 MLC2 的位点特异性磷酸化模式也将通过二维磷酸肽图谱确定。 MLCK和PKC同工酶对心脏MLC2的磷酸化的功能意义将使用心脏重组MLC2突变体来确定，其中磷酸化位点处的丝氨酸残基将被丙氨酸残基取代。将在有或没有 MLCK 和 PKC 抑制剂的情况下测量心肌细胞的收缩性，以确定心脏 MLC2 的 MLCK 和 PKC 磷酸化的生化和生理相关性。预计本研究收集的数据将为 MLCK 和 PKC 同工酶调节心脏生理学提供新信息。 MLCK 和 PKC 同工酶在调节心脏功能中的具体作用将被确定。 MLCK 和 PKC 同工酶在位点特异性磷酸化以及在肌原纤维中产生生化和生理变化中所起的作用也将被确定。