肌球蛋白轻链激酶(MLCK)是一种Ca2-钙调蛋白依赖性酶,致力于磷酸化和激活肌球蛋白II以为各种运动过程提供力。在平滑肌细胞和许多其他细胞中,小MLCK(S-MLCK)是一种主要的亚型。S-MLCK是一种肌动球蛋白结合蛋白,牢固地附着在平滑肌细胞的收缩机制上。尽管如此,它可以离开这个位置,并有助于其他细胞过程。然而,尚未描述切换S-MLCK亚细胞定位的分子机制。
定点诱变和体外蛋白质磷酸化用于研究S-MLCK肌动蛋白结合结构域内的离散体内磷酸化残基的功能作用。体外共沉降分析用于研究重组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磷酸化S-MLCK突变体的亚细胞分布。
S-MLCK肌动蛋白结合结构域在Ser25和/或Thr56上通过脯氨酸指导的蛋白激酶或这些翻译后修饰的磷酸化改变了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,对酶的催化特性没有影响。
S-MLCK的氨基末端肌动蛋白结合域的磷酸化导致酶的差异亚细胞靶向,因此,有助于S-MLCK对细胞和组织刺激的各种环境依赖性反应。
S-MLCK的生理功能可能通过其肌动蛋白识别域的磷酸化来调节,不同于催化和钙调蛋白调节域的调节。
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.
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.
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.
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.
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.