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Analysis of calmodulin-kinase cascade by using functional proteomics.

Research Project

Project/Area Number 17570115
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Functional biochemistry
Research InstitutionKagawa University

Principal Investigator

TOKUMITSU Hiroshi  Kagawa University, Faculty of Medicine, Assistant Professor, 医学部, 助教授 (20237077)

Co-Investigator(Kenkyū-buntansha) KOBAYASHI Ryoji  Kagawa University, Faculty of Medicine, Professor, 医学部, 教授 (00020917)
HATANO Naoya  Rare Sugar Research Center, Assistant Professor, 希少糖研究センター, 客員助教授 (10332280)
Project Period (FY) 2005 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
KeywordsCaM-kinase cascade / Intracellular calcium / CaM-KK / CaM-KI / Signal transduction / Protein phosphorylation / Numb / Numbl
Research Abstract

To search for the substrates of Ca^<2+>/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr^<177>) GST-fused CaM-KI catalytic domain (residues 1-293, Lys^<49>Glu) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr^<177>-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser^<264> in Numb and Ser^<304> in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phosphoNumb/Numbl antibody, we observed the phosphorylation of … More Numb family proteins in various rat tissue extracts and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser^<264> in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl blocked dephosphorylation of Ser^<304>. Numb is thought to participate in clathrin-dependent endocytosis by directly interacting with the clathrin-associated adaptor complex AP-2, although the underlying mechanisms are unknown. Pull-down experiments showed that the phosphorylation of Numb impaired its binding to the AP-2 complex and simultaneously recruited 14-3-3 proteins in vitro. Based on experiments using Numb mutants, both the initial phosphorylation of Ser^<264> and the subsequent phosphorylation of Ser^<283> are sufficient to abolish the binding of Numb to AP-2 and to promote the interaction with 14-3-3 protein. These findings suggest a novel mechanism for the regulation of Numb-mediated endocytosis, namely through direct phosphorylation. Less

Report

(3 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • Research Products

    (9 results)

All 2007 2006 2005

All Journal Article (9 results)

  • [Journal Article] Knockdown of nuclear Ca^<2+>/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish.2007

    • Author(s)
      Takaki Nimura et al.
    • Journal Title

      Archives of Biochemistry and Biophysics 457

      Pages: 205-216

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] Spatiotemporal expression of four isoforms of Ca^<2+>/calmodulin-dependent protein kinase I in brain and its possible roles in hippocampal dendritic growth.2007

    • Author(s)
      Akifumi Kamata et al.
    • Journal Title

      Neuroscience Research 57

      Pages: 86-97

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] Spatiotemporal expression of four isoforms of Ca^<2+>/calmodulin-dependent protein kinase I in brain and its possible roles in hippocampal dendritic growth.2007

    • Author(s)
      A.Kamata, H.Sakagami, H.Tokumitsu, Y.Owada, K.Fukunaga, H.Kondo
    • Journal Title

      Neuroscience Research 57(1)

      Pages: 86-97

    • NAID

      10021188190

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Knockdown of nuclear Ca^<2+>/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish.2007

    • Author(s)
      T.Nimura, N.Sueyoshi, A.Ishida, Y.Yoshimura, M.Ito, H.Tokumitsu, Y.Shigeri, N.Nozaki, I.Kameshita
    • Journal Title

      Archives of Biochemistry and Biophysics 457(2)

      Pages: 205-216

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2.2006

    • Author(s)
      Hiroshi Tokumitsu et al.
    • Journal Title

      FEBS Letters 580

      Pages: 5797-5801

    • NAID

      120007015428

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2.2006

    • Author(s)
      H.Tokumitsu, N.Hatano, S.Yokokura, Y.Sueyoshi, N.Nozaki, R.Kobayashi
    • Journal Title

      FEBS Letters 580(24)

      Pages: 5797-5801

    • NAID

      120007015428

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Phosphorylation of Numb family proteins. : Possible involvement of Ca^<2+>/calmodulin-dependent protein kinases.2005

    • Author(s)
      Hiroshi Tokumitsu et al.
    • Journal Title

      Journal of Biological Chemistry 280

      Pages: 35108-35118

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Phosphorylation of Numb family proteins. : Possible involvement of Ca^<2+>/calmodulin-dependent protein kinases.2005

    • Author(s)
      H.Tokumitsu, N.Hatano, H.Inuzuka, Y.Sueyoshi, S.Yokokura, T.Ichimura, N.Nozaki, R.Kobayashi
    • Journal Title

      Journal of Biological Chemistry 280(42)

      Pages: 35108-35118

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Phosphorylation of Numb family proteins :Possible involvement of Ca^<2+>/calmodulin-dependent protein kinases.2005

    • Author(s)
      Hiroshi Tokumitsu et al.
    • Journal Title

      Journal of Biological Chemistry 280

      Pages: 35108-35118

    • Related Report
      2005 Annual Research Report

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Published: 2005-04-01   Modified: 2016-04-21  

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