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

肌球蛋白轻链激酶（Myosin light chain kinase， MLCK）是一种多功能蛋白，其激酶结构域位于分子的中间，肌动蛋白结合结构域位于分子的氨基端，肌动蛋白结合结构域位于分子的羧基端。在平滑肌中，MLCK磷酸化调控轻链（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也磷酸化RLC并对肌肉收缩具有调节作用，即RLC的磷酸化增加了等长力产生的钙敏感性。最近，Kohama等人报道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。虽然在心肌的肌球蛋白丝上也有类似的MLCK异构体表达和定位，但MLCK的功能作用尚不清楚。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。利用牛胃MLCK cDNA在大肠杆菌中表达了一个含有更多肌球蛋白结合区但缺乏激酶结构域的氨基末端MLCK片段（MF）。肌球蛋白是从大鼠心肌中纯化的。在5mmol/L MF存在和不存在的情况下，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存在下的纤维速度提高了22%（5.6±0.5 v.s. 4.6±0.4 mm/sec, p<0.01）。三磷酸腺苷酶活性在这些条件下没有差异。在尿素聚丙烯酰胺凝胶上，MF的加入也不影响其磷酸化水平。本研究中研究的MLCK片段在平滑肌细胞中作为一种独立的蛋白——端粒蛋白表达。在平滑肌中，端粒蛋白被认为与肌球蛋白分子的S-1-S-2连接并诱导构象改变。综上所述，目前的研究结果表明，MLCK不仅可以通过磷酸化RLC来增加钙敏感性，还可以通过直接结合肌球蛋白分子来改变构象来调节心功能。少

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