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Regulation of the Wnt signal by receptor-mediated endocytosis

Research Project

Project/Area Number 15390094
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field General medical chemistry
Research InstitutionHIROSHIMA UNIVERSITY

Principal Investigator

KIKUCHI Akira  Hiroshima University, Graduate School of Biomedical Sciences, Professor, 大学院・医歯薬学総合研究科, 教授 (10204827)

Co-Investigator(Kenkyū-buntansha) KISHIDA Shosei  Hiroshima University, Graduate School of Biomedical Sciences, Associate Professor, 大学院・医歯薬学総合研究科, 助教授 (50274064)
YAMAMOTO Hideki  Hiroshima University, Graduate School of Biomedical Sciences, Research Associate, 大学院・医歯薬学総合研究科, 助手 (20372691)
Project Period (FY) 2003 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 2004: ¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 2003: ¥10,300,000 (Direct Cost: ¥10,300,000)
KeywordsWnt / endocytosis / Rho-kinase / conditioned medium / lipidation / gycosylation / β-catenin / Frizzled / LRP / ダイナミン / 受容体 / Rhoキナーゼ / PC12細胞
Research Abstract

The following observations concerning Wnt signal and endocytosis were obtained in this research.
(1)Purification of Wnt proteins
Approximately 40〜60 μg of Wnt-3a and Wnt-5a were purified from 1 L of conditioned medium using column chromatographises. Purified Wnt-3a accumulated β-catenin and activated Rho-kinase in various cell lines. Purified Wnt-5a suppressed Wnt-3a-dependent Tcf transcriptional activation thorough the activation of NLK. Further, we also succeeded to establish cell lines producing Wnt-11.
(2)Post-translational modification of Wnt proteins
Wnt proteins has been suggested to be modified post-translationaly ; lipidation and gycosylation. However, the analyses using purified proteins have been little done. Mass spectrometory demonstarated that Wnt-5a is modified with palmitic scid. When Wnt-3a and Wnt-5a were treated with glycosidase-F, the mobility shift on the SDS-gel occurred, indicating that they are glycosylated. Palmitoylation of Wnt-5a was required for it actions. Glycosylation is necessary for the secretion but not for the actions.
(3)Receptor mediated endocytosis of Wnt
We established an assay to observe endocytosis of Frizzled 5 and LRP6 in a Wnt-response manner. Both receptors were detected on the plasma membranes without Wnt-stimulation. Wnt-3a induced the inetrnalization of Frizzled 5 and LRP6 in 30-60 min and formed a small vesicle colocalized with EEA1, an early endocytotic marker. After 2〜3 h after stimulation, Frizzled 5 and LRP6 appeared on the plasma membranes again. A dominant negative form of Dynamin, which inhibited receptor internalization, suppressed Wnt-3a-dependent accumulation of β-catenin. The siRNA for caveolin or clathrin also suppressed Wnt-3a-dependent accumulation of β-catenin. These results indicate the receptor endocytosis of Wnt are essential for the activation of Wnt signal.

Report

(3 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • Research Products

    (39 results)

All 2005 2004 2003 Other

All Journal Article (27 results) Publications (12 results)

  • [Journal Article] SUMO-1 modification of PIASy, an E3 ligase, is necessary for PIASy-dependent activation of Tcf-4.2005

    • Author(s)
      Ihara, M.
    • Journal Title

      Mol.Cell.Biol. 25

      Pages: 3506-3518

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Ubiquitin-interacting motifs of Epsin are involved in the regulation of insulin-dependent endocytosis.2005

    • Author(s)
      Sugiyama, S.
    • Journal Title

      J.Biochem.(Tokyo) 137

      Pages: 355-364

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] GSK-3β regulates phosphorylation of CRMP-2 and neuronal polarity.2005

    • Author(s)
      Yoshimura, T.
    • Journal Title

      Cell 120

      Pages: 137-149

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] GSK-3βregulates phosphorylation of CRMP-2 and neuronal polarity.2005

    • Author(s)
      Yoshimura, T.
    • Journal Title

      Cell 120

      Pages: 137-149

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] GSK-3 β regulates phosphorylation of CRMP-2 and neuronal polarity."2005

    • Author(s)
      Yoshimura, T.
    • Journal Title

      Cell 120

      Pages: 137-149

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Wnt-3a and Dvl induce neurite retraction by activating Rho-associated kinase.2004

    • Author(s)
      Kishida, S.
    • Journal Title

      Mol.Cell.Biol. 24

      Pages: 4487-501

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] Early embryonic death in mice lacking the β-catenin-binding protein Duplin.2004

    • Author(s)
      Nishiyama, M.
    • Journal Title

      Mol.Cell.Biol. 24

      Pages: 8386-8394

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] Glycogen synthase kinase-3β is involved in the phosphorylation and suppression of androgen receptor activity.2004

    • Author(s)
      Salas, T.R.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 19191-19200

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] The receptor tyrosine kinase Ror2 associates with and is activate by casein kinase Iε.2004

    • Author(s)
      Kani, S.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 50102-50109

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Galectin-3 is a novel binding partner of β-catenin.2004

    • Author(s)
      Shimura, T.
    • Journal Title

      Cancer Res. 64

      Pages: 6363-6367

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] SUMO-1 modification of the synergy control motif of Ad4BP/SF-1 regulates synergistic transcription between Ad4BP/SF-1 and Sox9.2004

    • Author(s)
      Komatsu, T.
    • Journal Title

      Mol.End. 18

      Pages: 2451-2462

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] The receptor tyrosine kinase Ror2 associates with and is activate by casein kinase I ε.2004

    • Author(s)
      Kani, S.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 50102-50109

    • Related Report
      2004 Annual Research Report
  • [Journal Article] XSENP1, a novel sumo-specific protease in Xenopus, inhibits normal head formation by down-regulation of Wnt/β-catenin signaling.2004

    • Author(s)
      Yukita, A.
    • Journal Title

      Genes Cells 9

      Pages: 732-726

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Sall1, a causative gene for Townes-Brocks syndrome, enhances the canonical Wnt signaling by localizing to heterochromatin.2004

    • Author(s)
      Sato, A.
    • Journal Title

      Biochem.Biophys.Res.Commun. 319

      Pages: 103-113

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Immunohistochemical analysis and mutational analyses of β-catenin, Axin family and APC genes in hepatocellular carcinomas.2004

    • Author(s)
      Ishizaki, Y.
    • Journal Title

      Int.J.Oncol. 24

      Pages: 1077-1083

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Sumoylation is involved in β-catenin-dependent activation of Tcf-4.2003

    • Author(s)
      Yamamoto H.
    • Journal Title

      EMBO J. 22

      Pages: 2047-2059

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Yeast glycogen synthase kinase-3 activates Msn2-dependent transcription of stress responsive genes.2003

    • Author(s)
      Hirata Y.
    • Journal Title

      Mol.Biol.Cell 14

      Pages: 302-312

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Casein kinase Iε enhances the binding of Dvl-1 to Frat-1 and is essential for Wnt-3a-induced accumulatin of β-catenin.2003

    • Author(s)
      Hino S.
    • Journal Title

      J.Biol.Chem. 278

      Pages: 14066-14073

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Identification and characterization of a novel Dvl-binding protein that suppresses Wnt signaling pathway.2003

    • Author(s)
      Oshita, A.
    • Journal Title

      Genes Cells 8

      Pages: 1005-1017

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Tumor formation by genetic mutations in the components of the Wnt signaling pathway.2003

    • Author(s)
      Kikuchi, A.
    • Journal Title

      Cancer Sci. 94

      Pages: 225-229

    • NAID

      50000783892

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Impaired degradation of inhibitory subunit of NF-kB (IkB) and β-catenin as a result of targeted disruption of the β-TrCP1 gene.2003

    • Author(s)
      Nakayama, K.
    • Journal Title

      Proc.Natl.Acad.Sci.USA 100

      Pages: 8752-8757

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GAP function towards Ras.2003

    • Author(s)
      Yunoue, S.
    • Journal Title

      J.Biol.Chem. 278

      Pages: 26958-26969

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Alleviating the suppression of glycogen synthase kinase-3β by Akt leads to the phosphorylation of CREB and its transactivation in intact cells nuclei.2003

    • Author(s)
      Salas, T.R.
    • Journal Title

      J.Biol.Chem. 278

      Pages: 41338-41346

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Siah-1 facilitates ubiquitination and degradation of synphilin-1, and is involved in dopamine release and biosynthesis.2003

    • Author(s)
      Nagano, Y.
    • Journal Title

      J.Biol.Chem. 278

      Pages: 51504-51514

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Alleviating the suppression of glycogen synthase kinase -3β by Akt leads to the phosphorylation of CREB and its transactivation in intact cells nuclei.2003

    • Author(s)
      Salas, T.R.
    • Journal Title

      J.Biol.Chem. 278

      Pages: 41338-41346

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] SUMO-1 modification of PIASy, an E3 ligase, is necessary for PIASy-dependent activation of Tcf-4.

    • Author(s)
      Ihara, M.
    • Journal Title

      Mol.Cell.Biol. (in press)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Ubiquitin-interacting motifs of Epsin are involved in the regulation of insulin-dependent endocytosis.

    • Author(s)
      Sugiyama, S.
    • Journal Title

      J.Biochem. (in press)

    • Related Report
      2004 Annual Research Report
  • [Publications] Yamamoto H.: "Sumoylation is involved in β-catenin-dependent activation of Tcf-4"EMBO J.. 22・9. 2047-2059 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Hirata Y.: "Yeast glycogen synthase kinase-3 activates Msn2-dependent transcription of stress responsive genes"Mol.Biol.Cell. 14・1. 302-312 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Hino S.: "Casein kinase Iε enhances the binding of Dvl-1 to Frat-1 and is essential for Wnt-3a-induced accumulatin of β-catenin"J.Biol.Chem.. 278・16. 14066-14073 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Yunoue S.: "Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GAP function towards Ras"J.Biol.Chem.. 278・29. 26958-26969 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Salas, T.R.: "Alleviating the suppression of glycogen synthase kinase-3β by Akt leads to the phosphorylation of CREB and its transactivation in intact cells nuclei"J.Biol.Chem.. 278・42. 41338-41346 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Nagano Y.: "Siah-1 facilitates ubiquitination and degradation of synphilin-1, and is involved in dopamine release and biosynthesis"J.Biol.Chem.. 278・58. 51504-51514 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Nakayama K.: "Impaired degradation of inhibitory subunit of NF-κB (IκB) and β-catenin as a result of targeted disruption of the β-TrCP1 gene"Proc.Natl.Acad.Sci.USA. 100・15. 8752-8757 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Oshita A.: "Identification and characterization of a novel Dvl-binding protein that suppresses Wnt signaling pathway"Genes Cells. 8・12. 1005-1017 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Sakashita G.: "Regulation of type 1 protein phosphatase/inhibitor-2 complex by glycogen synthase kinase-3β in intact cells"J.Biochem.. 133・2. 165-171 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kshida S.: "Wnt-3a and Dvl induces neurite retraction by activating Rho-associated kinase"Mol.Cell.Biol.. (in press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Salas, T.R.: "Glycogen synthase kinase-3β is involved in the phosphorylation and suppression of androgen receptor activity"J.Biol.Chem.. (in press). (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kikuchi A.: "Tumor formation by genetic mutations in the components of the Wnt signaling Pathway"Cancer Sci.. 94・3. 225-229 (2003)

    • Related Report
      2003 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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