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

Fingerprint

Calcium-Calmodulin-Dependent Protein Kinase Type 1
Myosin Type II
Myosin-Light-Chain Kinase
Calmodulin
Phosphotransferases
Myosin Light Chains
Actin Cytoskeleton
Actins
Phosphorylation
In Vitro Techniques
Phosphopeptides

Keywords

  • Actin filament
  • Phosphorylation
  • Stress fibre

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of Ca2+/calmodulin-dependent protein kinase I as a myosin II regulatory light chain kinase in vitro and in vivo. / Suizu, Futoshi; Fukuta, Yasuaki; Ueda, Kozue; Iwasaki, Takahiro; Tokumitsu, Hiroshi; Hosoya, Hiroshi.

In: Biochemical Journal, Vol. 367, No. 2, 15.10.2002, p. 335-345.

Research output: Contribution to journalArticle

Suizu, Futoshi ; Fukuta, Yasuaki ; Ueda, Kozue ; Iwasaki, Takahiro ; Tokumitsu, Hiroshi ; Hosoya, Hiroshi. / Characterization of Ca2+/calmodulin-dependent protein kinase I as a myosin II regulatory light chain kinase in vitro and in vivo. In: Biochemical Journal. 2002 ; Vol. 367, No. 2. pp. 335-345.
@article{3218dd7a4ffc4be49d800d4219c3d2c7,
title = "Characterization of Ca2+/calmodulin-dependent protein kinase I as a myosin II regulatory light chain kinase in vitro and in vivo",
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.",
keywords = "Actin filament, Phosphorylation, Stress fibre",
author = "Futoshi Suizu and Yasuaki Fukuta and Kozue Ueda and Takahiro Iwasaki and Hiroshi Tokumitsu and Hiroshi Hosoya",
year = "2002",
month = "10",
day = "15",
doi = "10.1042/BJ20020536",
language = "English",
volume = "367",
pages = "335--345",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "2",

}

TY - JOUR

T1 - Characterization of Ca2+/calmodulin-dependent protein kinase I as a myosin II regulatory light chain kinase in vitro and in vivo

AU - Suizu, Futoshi

AU - Fukuta, Yasuaki

AU - Ueda, Kozue

AU - Iwasaki, Takahiro

AU - Tokumitsu, Hiroshi

AU - Hosoya, Hiroshi

PY - 2002/10/15

Y1 - 2002/10/15

N2 - 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.

AB - 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.

KW - Actin filament

KW - Phosphorylation

KW - Stress fibre

UR - http://www.scopus.com/inward/record.url?scp=0037107342&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037107342&partnerID=8YFLogxK

U2 - 10.1042/BJ20020536

DO - 10.1042/BJ20020536

M3 - Article

C2 - 12081505

AN - SCOPUS:0037107342

VL - 367

SP - 335

EP - 345

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 2

ER -