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Physiological role of myosin light chain kinase in regulating smooth muscle contraction : An approach by gene targeting followed by rescue.

Research Project

Project/Area Number 06454156
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field General pharmacology
Research InstitutionGunma University

Principal Investigator

KOHAMA Kazuhiro  Gunma University, School of Medicine, Department of Pharmacology Professor, 医学部, 教授 (30101116)

Co-Investigator(Kenkyū-buntansha) ISHIKAWA Ryoki  Assistant Professor, 医学部, 助手 (20212863)
OKAGAKI Tsuyoshi  Lecturer, 医学部, 講師 (80185412)
Project Period (FY) 1994 – 1995
Project Status Completed (Fiscal Year 1995)
Budget Amount *help
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1995: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1994: ¥5,700,000 (Direct Cost: ¥5,700,000)
Keywordssmooth muscle / myosin light chain kinase / calcium ion / antisence DNA / regulation of contraction / regulatory protein / phosphorylation / カルシウムイオン / アクチン / ミオシン
Research Abstract

Smooth muscle contraction is induced by an ATE-dependent interaction between actin and myosin, on which Ca^<2+> exerts regulatory activity, The site of action of Ca^<2+> is calmodulin (CaM). The role of CaM is to activate myosin light chain kinase (MLCK), which is able to phosphorylate the 20 kDa regulatory light chain of myosin. The myosin thus phosphorylated is in an active form that is able to interact with actin ATP-dependently. But the effect of Ca^<2+> on the actual contraction of smooth muscle is much more complex. There are regulatory ways by Ca^<2+> which are not subject to phosphorylation.
To approach such a problem, we are interested in the actin binding activity of MLCK, a property that has been known for many years. We examined the effect of the actin-binding activity, and found that the activity regulates the interaction in association with CaM.Thus, MLCK regulates the interaction by its kinase activity as well as its actin-binding activity.
We transfected plasmid containing antisence DNA of MLCK into smooth muscle cells and observed a reduction in the amount of MLCK.We also produced MLCK fragments by transfecting expression plasmieds containing various length of sence DNA of MLCIK into E.Coli. We obtained a few fragments that regulate the actin-myosin interaction. These results enable the study to examine physiological role of MLCK by introducing the recombinant fragments into the cells where endogenouse levels of MLCK is lowered.

Report

(3 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • Research Products

    (26 results)

All Other

All Publications (26 results)

  • [Publications] Sato,M.: "Myosin light chain kinase from vascular smooth muscle inhibits the ATP-dependent interaction between actin and myosin by binding to actin." J.Biochem.118. 1-3 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Hayakawa,K.: "Reversible effects of Okadaic acid and Microcyotin-LR on the ATP-dependent interaction between actin and myosin." J.Biochem.117. 509-514 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Higashi-Fujime,S.: "The fastest actin-based motor protein from the green alga,Cnara,and its distinct mode of interaction with actin." FEBS Lett.375. 151-154 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Ishikawa,R.: "Purification of an ATP-dependent actin-binding protein from a lower eukaryote,physarum polycepharum." Biochem.Biophys.Res.Commun.212. 347-352 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kohama,K.: "Smooth muscle contraction. New regulatory modes." Japan Sci.Soc.Press & S.Karger, 159 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Maruyama,K.: "Calcium as cell Signal" Igaku-Shoin, 293 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] K.Hayakawa, T.Okagaki, S.Higashi-Fujime and K.Kohama: "Bundling of actin-bilaments by myosin light chain kinase from smooth muscle." Biochem.Biophys.Res.Commun.199. 786-791 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Y.Sasaki, F.Tsunomori, T.Yamashita, K.Horie, H.Usuki, R.Ishikawa and K.Kohama: "Local enviromental change from the G-to F-form of the actin molule detcted on anisotropy decay measurement." J.Biochem.116. 236-238 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] L.-H.Ye, K.Hayakawa, Y.Lin, T.Okagaki, K.Fujita and K.KOhama: "The regulatory role of myosin light chain kinase as an actin-binding protein." J.Biochem.116. 1377-1382 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Y.Lin, R.Ishikawa, T.Okagaki, L.-H.Ye and K.Kohama: "Stimulation of the ATP-dependent interaction between actin and myosin-binding fragment of smooth muscle caldesmon." Cell Motil.Cytoskeleton. 29. 250-258 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] R.Ishikawa, K.Hayashi, T.Shirao, Y.Xue, T.Takagi, Y.Sasaki and K.Kohama: "Drebrin, a development-associated brain protein from rat embryo cases the dissociation of tropomyosin from actin filaments." J.Biol.Chem.269. 29928-29933 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] M.Sato, L.-H.Ye, K.Kohama: "Myosin light chain kinase from vascular smooth muscle inhibits the ATP-dependent interaction between actin and myosin by binding to actin." J.Biochem.118. 1-3 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] K.Hayakawa and K.Kohama: "Reversible effects of Okadaic acid and Microcyotin-LR on the ATP-dependent interaction between actin and myosin." J.Biochem.117. 509-514 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] S.Higashi-Fujime, R.Ishikawa, H.Iwasawa, O.Kagami E.Kurimoto, K.Kohama and T.Hozumi: "The fastest actin-based motor protein from the green alga, Chara, and its distinct mode of interaction with actin." FEBS Lett.375. 151-154 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Sato, M.: "Myosin light chain kinase from vascular smooth muscle inhibits the ATP-dependent interaction between actin and myosin by binding to actin" J. Biochem.118. 1-3 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Hayakawa, K.: "Reversible effects of Okadaic acid and Microcyotin-LR on the ATP-dependent interaction between actin and myosin." J. Biochem.117. 509-514 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Higashi-Fujime, S.: "The fastest actin-based motor protein from the green alga, Chara, and its distinct mode of interaction with actin." FEBS Lett.375. 151-154 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Ishikawa, R.: "Purification of an ATP-dependent actin-binding protein from a lower eukaryote, Physarum polycepharum." Biochem. Biophys. Res. Commun.212. 347-352 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kohama, K.: "Smooth muscle contraction. New regulatory modes." Japan Sci. Soc. Press & S. Karger, 159 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Maruyama, K.: "Calcium as cell signal" Igaku-Shoin, 293 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] K.Hayakawa,T.Okagaki,S.Higashi-Fujime anx K.Kohama: "Bundling of actin-filaments by myosin light chain kinase from smooth muscle." Biochem.Biophys.Res.Commun.199. 786-791 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Y.Sasaki,F.Tsunomori,T.Yamashita,K.Horie,H.Usuki,R.Ishikawa and K.Kohama: "Local enviromental change from the G-to F-form of the actin molule detcted on anisotropy decay measurement." J.Biochem.116. 236-238 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] L.-H.Ye,K.Hayakawa,Y.Lin,T.Okagaki,K.Fujita and K.Kohama: "The regulatory role of myosin light chain kinase as an actin-binding protein." J.Biochem.116. 1377-1382 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Y.Lin,R.Ishikawa,T.Okagaki,L.-H.Ye and K.Kohama: "Stimulation of the ATP-dependent interaction between actin and myosin-binding fragment of smooth muscle caldesmon." Cell Motil.Cytoskeleton. 29. 250-258 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] R.Ishikawa,K.Hayashi,T.Shirao,Y.Xue,T.Takagi,Y.Sasaki and K.Kohama: "Drebrin,a development-associated brain protein from rat embryo causes the dissociation of tropomyosin from actin filaments." J.Biol.Chem.269. 29928-29933 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Kohama,K & Saida,K: "Smooth Muscle Contraction" Japan Sci.Soc.Press,Tokyo/Drrger,Basel, 159 (1995)

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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