Investigation on the functional significance of myosin light chain kinase and myosin light chain phosphorylation

肌球蛋白轻链激酶和肌球蛋白轻链磷酸化的功能意义研究

• 批准号: 15590721
• 负责人: YAMASHITA Hiroshi
• 金额: $ 1.92万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590721/
• 关键词: Cardiac myosin; Myosin light chain kinase; Myosin light chain phosphorylation; Laser trap; In vitro motility assay; Actin-activated ATPase activity; Telokin; Subfragment-2; ミオシン軽鎖アイソフォーム; カーボンファイバー; ミオシン連鎖リン酸化; 細胞内カルシウム濃度; 単離心筋細胞

Myosin light chain kinase (MLCK) is a multifunctional protein with kinase domain in the middle, actin-binding domain in the amino-terminus, and myosin binding domain in the carboxyl-terminus of the molecule. In smooth muscles, MLCK phosphorylates the regulatory light chain (RLC) to activate myosin ATPase activity and start muscle shortening. In cardiac muscles, however, MLCK also phosphorylates RLC and has a modulatory effect on muscle contraction, i.e. phorphorylation of RLC increases calcium sensitivity of isometric force production. Recently, Kohama et al. reported that MLCK could enhance molecular function by directly binding to smooth muscle myosin molecules without phosphorylating RLC. Although similar MLCK isoform is expressed and localized on the myosin filaments in cardiac muscles, the functional role of MLCK is not well elucidated. Therefore, we investigated the effect of MLCK on the molecular function of cardiac myosin in vitro.An amino-terminal MLCK fragment (MF) containing … More myosin-binding region but lacking the kinase domain was expressed in E.coli using bovine stomach MLCK cDNA and purified. Myosin was purified from rat cardiac muscles. Actin-activated ATPase activity and actin filament velocity in the in vitro motility assay were measured in the presence and absence of 5mmol/L of MF. The filament velocity was 22% higher in the presence of MF compared to the control condition (5.6±0.5 v.s. 4.6±0.4 mm/sec, p<0.01). The ATPase activity was not different between these conditions. On the urea polyacrylamide gels, the phosphorylation level was not affected by the addition of MF, either.The MLCK fragment studied in the present study is expressed in smooth muscle cells as an independent protein, telokin. In smooth muscles, telokin is proposed to bind to the S-1-S-2 junction of myosin molecule and induce a conformational change. Taken together, the present results suggest that MLCK may modulate cardiac function not only by phosphorylating RLC to increase calcium sensitivity but also by directly binding to the myosin molecule to change conformation. Less

肌球蛋白轻链激酶（MLCK）是一种多功能蛋白质，其中间具有激酶结构域，氨基端具有肌动蛋白结合结构域，羧基端具有肌球蛋白结合结构域。在平滑肌中，MLCK磷酸化调节轻链（RLC）以激活肌球蛋白ATP酶活性并开始肌肉缩短。然而，在心肌中，MLCK也使RLC磷酸化，并对肌肉收缩具有调节作用，即RLC的磷酸化增加等长力产生的钙敏感性。最近，Kohama等报道MLCK可以通过直接结合平滑肌肌球蛋白分子而不磷酸化RLC来增强分子功能。虽然类似的MLCK亚型在心肌中的肌球蛋白丝上表达和定位，但MLCK的功能作用尚未得到很好的阐明。因此，我们在体外研究了MLCK对心肌肌球蛋白分子功能的影响。 ...更多信息 使用牛胃MLCK cDNA在大肠杆菌中表达肌球蛋白结合区但缺乏激酶结构域，并纯化。从大鼠心肌中纯化肌球蛋白。在体外运动实验中，在有和无5 mmol/L MF的情况下，测定肌动蛋白激活的ATP酶活性和肌动蛋白丝速度。与对照条件相比，MF存在下的细丝速度高22%（5.6±0.5 vs. 4.6±0.4 mm/sec，p<0.01）。ATP酶活性在这些条件下没有差异。在尿素聚丙烯酰胺凝胶上，磷酸化水平不受MF的加入的影响，在本研究中研究的MLCK片段在平滑肌细胞中作为一个独立的蛋白质，telokin表达。在平滑肌中，telokin被认为与肌球蛋白分子的S-1-S-2连接结合并引起构象变化。两者合计，目前的结果表明，MLCK可能不仅通过磷酸化RLC，以增加钙敏感性，但也通过直接结合到肌球蛋白分子，改变构象来调节心脏功能。少

项目成果

Unloaded shortening increases peak of Ca2+ transients but accelerates their decay in ra single cardiac myocytes.

未加载的缩短会增加 Ca2 瞬变的峰值，但会加速它们在单个心肌细胞中的衰减。

• 发表时间: 2003
• 期刊: American Journal of Physiology - Heart & Circulatory Physiology 285
• 作者: Osanai T;et al.;Sato T.;保田 壮一郎
• 通讯作者: 保田 壮一郎

山下 尋史: "Myosin light chain isoforms modify force-generating ability of cardioac myosin by changing the kinetics of actin-myosin interaction"Cardiovascular Research. 60. 580-588 (2003)

Hirofumi Yamashita：“肌球蛋白轻链异构体通过改变肌动蛋白-肌球蛋白相互作用的动力学来改变心肌肌球蛋白的力生成能力”心血管研究60。580-588（2003）。

Unloaded shortening increases peak of Ca2+ transients but accelerates their decay in rat single cardiac myocytes

• DOI: 10.1152/ajpheart.00012.2003
• 发表时间: 2003-08-01
• 期刊: AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
• 影响因子: 4.8
• 作者: Yasuda, S;Sugiura, S;Sugi, H
• 通讯作者: Sugi, H

Single cell mechanics of rat cardiomyocytes under isometric, unloaded, and physiologically loaded conditions

• DOI: 10.1152/ajpheart.00948.2003
• 发表时间: 2004-07-01
• 期刊: AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
• 影响因子: 4.8
• 作者: Nishimura, S;Yasuda, S;Sugiura, S
• 通讯作者: Sugiura, S

西村 智: "Single cell mechanics of rat cardiomyocytes under isometric, unloaded and physiologically loaded conditions"American Journal of Physiology. (in press).

Satoshi Nishimura：“等长、无负载和生理负载条件下大鼠心肌细胞的单细胞力学”美国生理学杂志（正在出版）。