Background

Myosin light chain kinase (MLCK) is a Ca²⁺-calmodulin-dependent enzyme dedicated to phosphorylate and activate myosin II to provide force for various motile processes. In smooth muscle cells and many other cells, small MLCK (S-MLCK) is a major isoform. S-MLCK is an actomyosin-binding protein firmly attached to contractile machinery in smooth muscle cells. Still, it can leave this location and contribute to other cellular processes. However, molecular mechanisms for switching the S-MLCK subcellular localization have not been described.

Methods

Site-directed mutagenesis and in vitro protein phosphorylation were used to study functional roles of discrete in-vivo phosphorylated residues within the S-MLCK actin-binding domain. In vitro co-sedimentation analysis was applied to study the interaction of recombinant S-MLCK actin-binding fragment with filamentous actin. Subcellular distribution of phosphomimicking S-MLCK mutants was studied by fluorescent microscopy and differential cell extraction.

Results

Phosphorylation of S-MLCK actin-binding domain at Ser25 and/or Thr56 by proline-directed protein kinases or phosphomimicking these posttranslational modifications alters S-MLCK binding to actin filaments both in vitro and in cells, and induces S-MLCK subcellular translocation with no effect on the enzyme catalytic properties.

Conclusions

Phosphorylation of the amino terminal actin-binding domain of S-MLCK renders differential subcellular targeting of the enzyme and may, thereby, contribute to a variety of context-dependent responses of S-MLCK to cellular and tissue stimuli.

General significance

S-MLCK physiological function can potentially be modulated via phosphorylation of its actin recognition domain, a regulation distinct from the catalytic and calmodulin regulatory domains.

中文翻译：

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小肌球蛋白轻链激酶的磷酸化依赖性亚细胞再分布

背景

肌球蛋白轻链激酶 (MLCK) 是一种 Ca ²⁺ -钙调蛋白依赖性酶，专用于磷酸化和激活肌球蛋白 II，为各种运动过程提供动力。在平滑肌细胞和许多其他细胞中，小 MLCK (S-MLCK) 是主要的异构体。S-MLCK 是一种肌动球蛋白结合蛋白，与平滑肌细胞的收缩机制紧密相连。尽管如此，它仍然可以离开这个位置并为其他细胞过程做出贡献。然而，尚未描述切换 S-MLCK 亚细胞定位的分子机制。

方法

定点诱变和体外蛋白质磷酸化用于研究 S-MLCK 肌动蛋白结合域内离散的体内磷酸化残基的功能作用。体外共沉淀分析用于研究重组S-MLCK肌动蛋白结合片段与丝状肌动蛋白的相互作用。通过荧光显微镜和差异细胞提取研究了拟磷 S-MLCK 突变体的亚细胞分布。

结果

S-MLCK 肌动蛋白结合域在 Ser25 和/或 Thr56 处被脯氨酸定向蛋白激酶磷酸化或磷酸化这些翻译后修饰改变 S-MLCK 在体外和细胞中与肌动蛋白丝的结合，并诱导 S-MLCK 亚细胞易位，无酶催化性能的影响。

结论

S-MLCK 氨基末端肌动蛋白结合结构域的磷酸化导致酶的亚细胞靶向性不同，因此可能有助于 S-MLCK 对细胞和组织刺激的各种背景依赖性反应。

一般意义

S-MLCK 生理功能可能通过其肌动蛋白识别域的磷酸化进行调节，这是一种不同于催化和钙调蛋白调节域的调节。