Characterization of Ca2+/calmodulin-dependent protein kinase I as a myosin II regulatory light chain kinase in vitro and in vivo

Futoshi Suizu, Yasuaki Fukuta, Kozue Ueda, Takahiro Iwasaki, Hiroshi Tokumitsu, Hiroshi Hosoya

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Ca2+/calmodulin (CaM)-dependent protein kinase I (CaM-KI), which is a member of the multifunctional CaM-K family, is thought to be involved in various Ca2+-signalling pathways. In this report, we demonstrate that CaM-KI activated by an upstream kinase (CaM-K kinase), but not unactivated CaM-KI, phosphorylates myosin II regulatory light chain (MRLC) efficiently (Kcat, 1.7 s-1) and stoichiometrically (≈0.8 mol of phosphate/mol) in a Ca2+/CaM-dependent manner in vitro. One-dimensional phosphopeptide mapping and mutational analysis of MRLC revealed that the activated CaM-KI monophosphorylates only Ser-19 in MRLC. Transient expression of the Ca2-/CaM-independent form of CaM-KI (CaM-KI1-293) in HeLa cells induced Ser-19 phosphorylation of myosin, II accompanied by reorganization of actin filaments in the peripheral region of the cells. CaM-KI-induced reorganization of actin filaments was suppressed by co-expression of non-phosphorylatable MRLC mutants (S19A and T18AS19A). Furthermore, a kinase-negative form of CaM-KI (CaM-KI1-293.E49E) significantly reduced reorganization of actin filaments, indicating a dominant negative effect. This is the first demonstration that the activation of the CaM-KI cascade induces myosin II phosphorylation, resulting in regulation of actin filament organization in mammalian cells.

Original languageEnglish
Pages (from-to)335-345
Number of pages11
JournalBiochemical Journal
Volume367
Issue number2
DOIs
Publication statusPublished - Oct 15 2002
Externally publishedYes

Keywords

  • Actin filament
  • Phosphorylation
  • Stress fibre

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Characterization of Ca<sup>2+</sup>/calmodulin-dependent protein kinase I as a myosin II regulatory light chain kinase in vitro and in vivo'. Together they form a unique fingerprint.

  • Cite this