Phosphorylation-dependent subcellular redistribution of small myosin light chain kinase

Asker Y. Khapchaev*, D. Martin Watterson, Vladimir P. Shirinsky

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article number119104
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Issue number11
StatePublished - Oct 2021


  • Actin-binding
  • Myosin light chain kinase
  • Phosphorylation
  • Protein-protein interaction
  • Subcellular localization

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology


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