Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities
Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities
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DOI：
10.1073/pnas.1600633113
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发表时间：
2016-07-05
影响因子：
11.1
通讯作者：
Stull, James T.
中科院分区：
文献类型：
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作者：
Chang, Audrey N.;Mahajan, Pravin;Stull, James T.
众所周知的肌肉特异性平滑肌肌球蛋白轻链激酶（MLCK）（smMLCK）和骨骼肌球蛋白轻链激酶（skMLCK）是由催化核心的自动调节区段C末端调节的专用蛋白激酶，其在不存在Ca 2 +/钙调蛋白（CaM）的情况下阻断肌球蛋白调节轻链（RLC）结合和磷酸化。尽管已知最近发现的心脏MLCK（cMLCK）是体内正常RLC磷酸化和生理心脏性能所必需的，但关于cMLCK生化性质的信息是有限的。我们发现，第四个未表征的MLCK，MLCK 4，也表达在心肌与其他MLCK的高催化结构域序列相似性，但缺乏自抑制段。其晶体结构显示催化结构域处于其活性构象，具有短的C-末端“假调节螺旋”，由于缺少接头区域而不能抑制催化。MLCK 4仅具有Ca 2 +/CaM非依赖性活性，对于不同的RLC具有相当的V-max和K-m值。相比之下，cMLCK的V-max值比其他三种MLCK家族成员的V-max值低几个数量级，而其K-m（RLC和ATP）和K-CaM值相似。与在不存在Ca 2 +/CaM的情况下缺乏活性的smMLCK和skMLCK相反，cMLCK具有由Ca 2 +/CaM刺激的组成型活性。用skMLCK和cMLCK的嵌合体分析自身调节片段对cMLCK活性的潜在贡献。cMLCK的组成性低活性似乎是其催化核心结构所固有的，而不是自身抑制片段。因此，心肌中的RLC磷酸化可能由两种具有不同生化调节特性的不同蛋白激酶调节。
The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca2+/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca2+/CaM-independent activity with comparable V-max and K-m values for different RLCs. In contrast, the V-max value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its K-m (RLC and ATP) and K-CaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca2+/CaM, cMLCK has constitutive activity that is stimulated by Ca2+/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties.