Department of Molecular Pathogenesis, Medical Research Institute, School of Biomedical Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan.
J Biol Chem. 2010 Oct 29;285(44):33680-90. doi: 10.1074/jbc.M110.122390. Epub 2010 Aug 27.
Phosphorylation of myosin regulatory light chain (MLC) plays a regulatory role in muscle contraction, and the level of MLC phosphorylation is balanced by MLC kinase and MLC phosphatase (MLCP). MLCP consists of a catalytic subunit, a large subunit (MYPT1 or MYPT2), and a small subunit. MLCP activity is regulated by phosphorylation of MYPTs, whereas the role of small subunit in the regulation remains unknown. We previously characterized a human heart-specific small subunit (hHS-M(21)) that increased the sensitivity to Ca(2+) in muscle contraction. In this study, we investigated the role of hHS-M(21) in the regulation of MLCP phosphorylation. Two isoforms of hHS-M(21), hHS-M(21)A and hHS-M(21)B, preferentially bound the C-terminal one-third region of MYPT1 and MYPT2, respectively. Amino acid substitutions at a phosphorylation site of MYPT1, Ser-852, impaired the binding of MYPT1 and hHS-M(21). The hHS-M(21) increased the phosphorylation level of MYPT1 at Thr-696, which was attenuated by Rho-associated kinase (ROCK) inhibitors and small interfering RNAs for ROCK. In addition, hHS-M(21) bound ROCK and enhanced the ROCK activity. These findings suggest that hHS-M(21) is a heart-specific effector of ROCK and plays a regulatory role in the MYPT1 phosphorylation at Thr-696 by ROCK.
肌球蛋白调节轻链（MLC）的磷酸化在肌肉收缩中起调节作用，MLC 的磷酸化水平由 MLC 激酶和 MLC 磷酸酶（MLCP）平衡。MLCP 由一个催化亚基、一个大亚基（MYPT1 或 MYPT2）和一个小亚基组成。MLCP 的活性受 MYPT 的磷酸化调节，而小亚基在调节中的作用尚不清楚。我们之前鉴定了一种人心肌特异性小亚基（hHS-M(21)），它增加了肌肉收缩对 Ca(2+)的敏感性。在这项研究中，我们研究了 hHS-M(21)在调节 MLCP 磷酸化中的作用。hHS-M(21)有两种同工型，hHS-M(21)A 和 hHS-M(21)B，分别优先结合 MYPT1 和 MYPT2 的 C 端三分之一区域。MYPT1 上一个磷酸化位点 Ser-852 的氨基酸取代，损害了 MYPT1 与 hHS-M(21)的结合。hHS-M(21)增加了 MYPT1 在 Thr-696 处的磷酸化水平，ROCK 抑制剂和针对 ROCK 的小干扰 RNA 可减弱该作用。此外，hHS-M(21)与 ROCK 结合并增强了 ROCK 的活性。这些发现表明 hHS-M(21)是 ROCK 的一种心脏特异性效应物，在 ROCK 介导的 MYPT1 在 Thr-696 处的磷酸化中起调节作用。
