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カルシウム依存性ホスホリパーゼA_2(PLA_2)と相互作用する細胞因子の研究

Research Project

Project/Area Number 10212208
Research Category

Grant-in-Aid for Scientific Research on Priority Areas (B)

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

Principal Investigator

工藤 一郎  昭和大学, 薬学部, 教授 (30134612)

Co-Investigator(Kenkyū-buntansha) 有田 斉  塩野義製薬(株), 創薬第二研究所, 所長
村上 誠  昭和大学, 薬学部, 助教授 (60276607)
Project Period (FY) 1998 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥35,600,000 (Direct Cost: ¥35,600,000)
Fiscal Year 2001: ¥9,000,000 (Direct Cost: ¥9,000,000)
Fiscal Year 2000: ¥9,000,000 (Direct Cost: ¥9,000,000)
Fiscal Year 1999: ¥8,700,000 (Direct Cost: ¥8,700,000)
Fiscal Year 1998: ¥8,900,000 (Direct Cost: ¥8,900,000)
KeywordsホスホリパーゼA2 / アラキドン酸 / シクロオキシゲナーゼ / プロスタグランジン / リン脂質 / ビメンチン / ヘパラン硫酸プロテオグリカン / リン酸化 / ホスホリパーゼA_2 / ピメンチン
Research Abstract

哺乳動物細胞には、通常mMレベルのCa^<2+>を活性発現に必要とする10種の分泌性PLA_2(sPLA_2)と、μMレベルのCa^<2+>で活性化する細胞質PLA_2α(cPLA_2α)が存在する。我々は、これらCa^<2+>依存性PLA_2のうち、特定の分子種のみが効率良くプロスタグランジン(PG)産生系と連関することを明らかにしてきた。本研究班では、その効率を規定するPLA_2相互作用因子の探索を行い、以下の結果を得た。
1.cPLA_2αと相互作用する因子の検索
cPLA_2αは細胞活性化に伴う細胞質Ca^<2+>の一過的上昇に伴って核周辺膜に移行し、これにcPLA_2α分子内のC2ドメインが重要である。我々はFar-Western法を用いて、cPLA_2α結合蛋白を探索した。その結果、cPLA_2αは分子内のC2ドメインを介して、中間径フィラメント構成蛋白であるビメンチンのHead domainにCa^<2+>依存的に結合することを見出した。更に、ビメンチンのHead domainを細胞に過剰発現すると、cPLA_2α依存的なアラキドン酸遊離とPGE_2産生はdominant-negative的に抑制された。このことから、ビメンチンが核膜周縁のcPLA_2αアダプターとして機能していることが強く示唆された。
2.sPLA_2と相互作用する因子の検索
II型に属する一群sPLA_2(IIA,IID,IIE,V型)は塩基性の酵素であり、高いヘパリン親和性を有する。我々はこれら一群の酵素が細胞表層のGPIアンカー型ヘパラン硫酸プロテオグリカンであるグリピカンに結合し、炎症刺激に伴ってカベオリンに富む小胞に集積した後、核周辺膜に局在性を変化させることが明らかとなった。核周辺膜にはアラキドン酸代謝系下流酵素であるCOXやLOXが存在しており、これらsPLA_2による核周辺膜からのアラキドン酸遊離は、酵素間での効率的な基質の受け渡しにおいて合目的的である。一方、X型sPLA_2はヘパリン非結合性の分子種であり、細胞外から容易に形質膜外層のホスファチジルコリン(PC)に作用してアラキドン酸を遊離する。IIF型sPLA_2はII型様酵素の中で唯一酸性蛋白であり、他のII型様酵素とは異なりヘパリンに対する結合性を示さず、形質膜外層の膜修飾ドメインに作用してアラキドン酸遊離を惹起する。この時、IIF型酵素に特有のC末端延長配列が形質膜への結合を促進する。III型sPLA_2はハチ毒由来sPLA2と相同性の高いsPLA_2ドメインと、N末ドメイン、C末ドメインからなり、他のsPLA_2と比べて分子量が著しく大きい。sPLA_2ドメインは本酵素の触媒機能の細小単位であり、形質膜外層のPCに作用してアラキドン酸を遊離する。また、N末及びC末ドメインは本酵素を細胞接着斑の先端部に集積させる機能を持つ。

Report

(4 results)
  • 2001 Annual Research Report
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • Research Products

    (24 results)

All Other

All Publications (24 results)

  • [Publications] Murakami, M.et al.: "Distinct arachidonate-releasing functions of mammalian secreted phospholipase A_2s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms"J.Biol.Chem.. 276. 10083-10096 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Murakami, M.et al.: "Different functional aspects of the group II subfamily (Types IIA and V) and type X secretory phospholipase A_2s in regulating arachidonic acid release and prostaglandin generation.Implications of cyclooxygenase-2 induction and phospholipid scramblase-mediated cellular membrane perturbation"J.Biol.Chem.. 274. 31435-31444 (1999)

    • Related Report
      2001 Annual Research Report
  • [Publications] Murakami, M.et al.: "Functional association of type IIA secretory phospholipase A_2 with the glycosylphosphatidylinositolanchored heparan sulfate proteoglycan in the cyclooxygenase-2-mediated delayed prostanoid-biosynthetic pathway"J.Biol.Chem.. 274. 29927-29936 (1999)

    • Related Report
      2001 Annual Research Report
  • [Publications] Murakami, M.et al.: "Functional coupling between various phospholipase A_2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways"J.Biol.Chem.. 274. 3103-3115 (1999)

    • Related Report
      2001 Annual Research Report
  • [Publications] Murakami, M.et al.: "The functions of five distinct mammalian phospholipase A_2s in regulating arachidonic acid release.Type Iia and type V secretory phospholipase A_2s are functionally redundant and act in concert with cytosolic phospholipase A_<2->"J.Biol.Chem.. 273. 14411-14423 (1998)

    • Related Report
      2001 Annual Research Report
  • [Publications] Atsumi, G.et al.: "Fas-induced arachidonic acid release is mediated by Ca^<2+>-independent phospholipase A_2 but not cytosolic phospholipase A_2, which undergoes proteolytic inactivation"J.Biol.Chem.. 273. 13870-13877 (1998)

    • Related Report
      2001 Annual Research Report
  • [Publications] T.Tanioka: "Molecular identification of cytosolic prostaglandin E_2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E_2 biosynthesis"J.Biol.Chem.. 275. 32775-32782 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] M.Murakami: "Regulation of prostaglandin E_2 biosynthesis by inducible membrane-associated prostaglandin E_2 synthase that acts in concert with cyclooxygenase-2"J.Biol.Chem.. 275. 32783-32792 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] N.Ueno: "Functional crosstalk between phospholipase D_2 and signaling phospholipase A_2/cyclooxygenase-2-mediated prostaglandin biosynthetic pathways."FEBS Lett.. 475. 242-246 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] G.Atsumi: "Distinct roles of two intracellular phospholipase A_2s in fatty acid release in the cell death pathway."J.Biol.Chem.. 275. 18248-18258 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] A.Enomoto: "Redundant and segregated functions of granule-associated heparin-binding group II subfamily of secretory phospholipases A_2 in the regulation of degranulation and prostaglandin D_2 synthesis in mast cells"J.Immunol.. 165. 4007-4014 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] H.Kuwata: "Studies on a mechanism by which cytosolic phospholipase A_2 regulates the expression and function of type IIA secretory phospholipase A_2"J.Immunol.. 165. 4024-4031 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nakatani, Y. et al.: "Identification of a cellular protein that functionally interacts with the C2 domain of cytosolic phospholipase A_2α"J. Biol. Chem.. 275. 1161-1168 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Murakami, M. et al.: "Different functional aspects of the group II subfamily (type IIA and V) and type X sPLA_2s in regulating arachidonic acid release and PG generation"J. Biol. Chem.. 274. 31435-31444 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Murakami, M. et al.: "Functional association of type IIA sPLA_2 with the GPI-Anchored heparan sulfate proteoglycan in the COX-2-mediated delayed PG biosynthetic pathway"J. Biol. Chem.. 274. 29927-29936 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Murakami, M. et al.: "Functional coupling between various phospholipase A_2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways"J. Biol. Chem.. 274. 3103-3115 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kambe, T. et al.: "Polyunsaturated fatty acids potentiate IL-1-stimulated arachidonic acid release by cells overexpressing type IIA sPLA_2"FEBS Lett.. 453. 81-84 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Sawada, H. et al.: "Regulation of type V phospholipase A_2 expression and function by proinflammatory stimuli"Eur. J. Biochem.. 263. 826-835 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Murakami,M.et al.: "Functional coupling between various phospholipase A_2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways." J.Biol.Chem.274. 3103-3115 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] Tada,K.et al.: "Induction of COX-2 by sPLA_2s in NGF-stimulated rat serosal mast cells is facilitated by interaction with fibroblasts and mediated by a mechamism independent of their enzymatic functions." J.Immunol.161. 5008-5015 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Murakami,M.et al.: "The functions of five distinct mammalian PLA_2s in regulating arachidonic acid release:type IIA and type V sPLA_2s are functionally redundant and act in concert with cPLA_2." J.Biol.Chem.273. 14411-14423 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Atsumi,G.et al.: "Fas-induced arachidonic acid release is mediated by Ca^<2+>-independent phospholipase A_2 but not cytosolic phospholipase A_2,which undergoes proteolytic inactivation." J.Biol.Chem.273. 13870-13877 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kuwata,H.et al.: "cPLA_2 is required for cytokine-induced expression of type IIA sPLA_2 that mediates optimal COX-2-dependent delayed PGE_2 generation in rat 3Y1 fibroblasts." J.Biol.Chem.273. 1733-1740 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Naraba,H.et al.: "Segregated coupling of phospholipases A_2, cyclooxygenases, and terminal prostanoid synthases in different phases of prostanoid biosynthesis in rat peritoneal macrophages." J.Immunol.160. 2974-2982 (1998)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2018-03-28  

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