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活性酸素産生機構における脂質シグナルのダイナミックイメージング解析

Research Project

Project/Area Number 16017261
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionKobe University

Principal Investigator

齋藤 尚亮  神戸大学, バイオシグナル研究センター, 教授 (60178499)

Co-Investigator(Kenkyū-buntansha) 白井 康仁  神戸大学, バイオシグナル研究センター, 助教授 (60263399)
Project Period (FY) 2004 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥9,200,000 (Direct Cost: ¥9,200,000)
Fiscal Year 2005: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 2004: ¥4,600,000 (Direct Cost: ¥4,600,000)
Keywordsファゴサイトーシス / プロテインキナーゼC / GFP / イメージング / 活性酸素 / Rac / マクロファージ / 活性酵素
Research Abstract

貪食細胞での微生物の殺菌は、Noxの細胞膜成分(cytochromeb558)と4つ細胞質成分(p47phox,p67phox,p40phox,Rac)により構成されるNADPHoxidase(Nox)に由来する活性酸素種により行われる。本研究は、この厳密に制御された活性酸素産生の分子機構を、機能蛋白質特にPKCおよびRacに焦点を当て、リアルタイムで可視化することにより解明することを目的とした。【今年度の成果】(1)Rac分子種の貪食IgGビーズへの集積度に違いがあることを見出した(Rac1>Rac3>Rac2)。この違いは、C末側の塩基性配列(PB)の違いに依存することを、イメージング解析により示した。また、protein-lipid overlay assayにより、この領域に脂質結合特異性があることを示した。さらに、Rac分子種のPBを入れ替えたキメラ蛋白を用いることにより、各分子種のPBの活性酸素産生に果たす重要性を明らかにした。また、Rac2の活性型変異体(Rac(Q61L))の集積を観察すると、小胞体などの細胞内器官の膜に局在し、貪食時にそれらの小器官膜が線状に貪胞膜へ癒合するのが観察された。(2)GFP融合PKCを貪食細胞に発現させIgGビーズを貧食させると、7種のPKC分子種の中beta I-PKCとepsilon-PKCのみが食胞に集積する。その結果、貧食時のepsilon-PKCの集積は、そのC1BドメインにPLC-gamma1経由で形成されるdiacylglycerol(DAG)が結合することにより起こることを明らかにした。【考察】(1)Rac1は貪食時にPBの高い正電荷とその脂質結合能を利用し食胞膜に集積する。Rac2はPBの正電荷を利用した弱い集積機序の他に、まず細胞内器官膜にターゲットし、この膜が貪胞膜に融合することを利用した、PBの正電荷に依存しない集積機序を持つことがわかった。これらのことから、3種すべてのRac分子種が活性酸素産生に関与する能力を有するが、マクロファージではRac1が、白血球ではRac2が優位に関与することが推察された。(2)同じC1BドメインをもつPKC分子種の中でも、特にepsilon-PKCが、貪食時食胞に強く集積することは、C1Bの脂質結合性の違いがPKCの分子種特異的シグナリングに重要な役割を果たしていることが推察された。

Report

(2 results)
  • 2005 Annual Research Report
  • 2004 Annual Research Report
  • Research Products

    (23 results)

All 2006 2005 2004 Other

All Journal Article (23 results)

  • [Journal Article] Targeting of PKC-ε during Fc γR-Dependent Phagocytosis Requires the εC1B domain and Phospholipase C-γ12006

    • Author(s)
      K L.Cheeseman.他
    • Journal Title

      Mol.Biol.Cell 17

      Pages: 799-813

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Protein kinase C-α mediates TNF release process in RBL 2H3 mast cells-Running title : αPKC mediates TNF release in RBL-2H3 cells-2005

    • Author(s)
      Abdel-Raheem, I.T.他
    • Journal Title

      Br.J.Pharmacol. 145

      Pages: 405-414

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Effects of different anti-tau antibodies on tau fibrillogenesis : RTA-1 and RTA-2 counteract tau aggregation.2005

    • Author(s)
      Taniguchi, T.他
    • Journal Title

      FEBS Lett. 579

      Pages: 1399-1404

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Importance of chroman ring and tyrosine phosphorylation in the subtype-specific translocation and activation of diacyiglycerol kinase alpha by d-alpha-tocophenol.2005

    • Author(s)
      Fukunaga-Takenaka, R.他
    • Journal Title

      Genes to Cells. 10

      Pages: 311-319

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Phosphorylation of PKC activation loop plays an important role in receptor-mediated translocation of PKC.2005

    • Author(s)
      Seki, T.他
    • Journal Title

      Genes to Cells 10

      Pages: 225-239

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Two types of chorine acetyltransferase of rat differ in intracellular translocation.2005

    • Author(s)
      Matsuo, A.他
    • Journal Title

      Neurochem.Int 46

      Pages: 423-433

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Involvement of γPKC in estrogen-induced neuroprotection on focal brain ischemia through G protein-coupled estrogen receptor.2005

    • Author(s)
      Hayashi, S.他
    • Journal Title

      J Neurochem. 93

      Pages: 883-891

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Transgenic mice expressing mutant (N279K) human tau show mutation dependent cognitive deficits without neurofibrillary tangle formation.2005

    • Author(s)
      Taniguchi, T.他
    • Journal Title

      FEBS Lett. 579

      Pages: 5704-5712

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Mutant protein kinase C gamma found in spinocerebellar ataxia type 14 is susceptible to aggregate and cause cell death.2005

    • Author(s)
      Seki, T.他
    • Journal Title

      J.Biol.Chem 280

      Pages: 29096-29106

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Immunocytochemical localization of a neuron-specific diacylglycerol kinase β and γ in the developing rat brain.2005

    • Author(s)
      Adachi, N.他
    • Journal Title

      Mol.Brain Res. 139

      Pages: 288-299

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Isoform-specific Membrane Targeting Mechanism of Rac During FcγR-mediated Phagocytosis.-Positive charge-dependent and independent targeting mechanism of Rac to the phagosome-2005

    • Author(s)
      Ueyama, T.他
    • Journal Title

      J.Immunol. 175

      Pages: 2381-2390

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Effects of different anti-tau antibodies on tau fibrillogenesis : RTA-1 and RTA-2 counteract tau aggregation.2005

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

      FEBS Left. 579

      Pages: 1399-1404

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Activation and translocation of PKCδ is necessary for VEGF-induced ERK activation through KDR in HEK293T cells.2004

    • Author(s)
      Kuriyama, M
    • Journal Title

      Biochem.Biophys.Res.Commun. 325

      Pages: 843-851

    • Related Report
      2004 Annual Research Report
  • [Journal Article] In vivo transgene expression using an adenoviral tetracycline-regulated system with neuron-specific enolase promoter.2004

    • Author(s)
      Bhattacharjee, A.K.
    • Journal Title

      Biochem.Biophys.Res.Commun. 317

      Pages: 1144-1148

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Superoxide (O_2) production at phagosomal cup/phagosome through βI PKC during FcγR-mediated phagocytosis in microglia.2004

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

      J Immunol. 173

      Pages: 4582-4589

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Propagation of γPKC translocation along the dendrites of Purkinje cell in γPKC-GFP transgenic mice.2004

    • Author(s)
      Sakai, N.
    • Journal Title

      Genes to Cells 9

      Pages: 945-957

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Down-regulation of Melanogenesis by Phospholipase D2 through Ubiquitin Proteasome-mediated Degradation of Tyrosinase.2004

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

      J.Biol.Chem. 279

      Pages: 27774-27780

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Nuclear transportation of diacylglycerol kinase γ and its possible function in the nucleus

    • Author(s)
      Matsubara, T.他
    • Journal Title

      J.Biol.Chem (印刷中)

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Protein kinase C-a mediates TNF release process in RBL 2H3 mast cells-Running title : αPKC mediates TNF release in RBL-2H3 cells-

    • Author(s)
      Abdel-Raheem, I.T.
    • Journal Title

      Br.J.Pharmacol. (未定)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Importance of chroman ring and tyrosine phosphorylation in the subtype-specific translocation and activation of diacylglycerol kinase alpha by d-alpha-tocophenol.

    • Author(s)
      Fukunaga-Takenaka, R
    • Journal Title

      Genes to Cells (未定)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Phosphorylation of PKC activation loop plays an important role in receptor-mediated translocation of PKC.

    • Author(s)
      Seki, T
    • Journal Title

      Genes to Cells (未定)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Two types of choline acetyltransferase of rat differ in intracellular translocation.

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

      Neurochem.Int (未定)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Involvement of γPKC in estrogen-induced neuroprotection on focal brain ischemia through G protein-coupled estrogen receptor.

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

      J Neurochem. (未定)

    • Related Report
      2004 Annual Research Report

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Published: 2004-04-01   Modified: 2018-03-28  

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