細胞質から核への蛋白質輸送の分子機構:その多様性とエネルギー要求性

• Project/Area Number: 11237202
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Osaka University
• Principal Investigator: 米田 悦啓 大阪大学, 生命機能研究科, 教授 (80191667)
• Co-Investigator(Kenkyū-buntansha): 原口 徳子 独立行政法人通信総合研究所, 関西先端研究センター・生物情報研究室, 主任研究員
• Project Period (FY): 1999 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥192,500,000 (Direct Cost: ¥192,500,000); Fiscal Year 2003: ¥37,100,000 (Direct Cost: ¥37,100,000); Fiscal Year 2002: ¥37,100,000 (Direct Cost: ¥37,100,000); Fiscal Year 2001: ¥37,000,000 (Direct Cost: ¥37,000,000); Fiscal Year 2000: ¥40,400,000 (Direct Cost: ¥40,400,000); Fiscal Year 1999: ¥40,900,000 (Direct Cost: ¥40,900,000)
• Keywords: 核蛋白質輸送 / importin β / p27 / 14-3-3蛋白質 / importin α / ストレス応答 / 核局在化シグナル / 結晶構造 / importin / exportin / Ran / 核膜孔複合体 / セミインタクト細胞 / transportin / 分子イメージング / CaM kinase IV / green fluorescent protein (GFP) / β-catenin / 核膜孔 / レプトマイシンB / 転写因子 / Importin / CaMキナーゼIV / ノックアウトマウス / 細胞内情報伝達 / 遺伝子破壊

Research Abstract

核蛋白質輸送の多様性に着目した研究として、転写因子SREBP-2(sterol regulatory element binding protein-2)を指標として研究を進め、これまでに、ホモ二量体を形成したSREBP-2が、importin αを介することなくimportin βに直接結合することによって核へ輸送されることを明らかにしてきたが、importin βがimportin αとSREBP-2という2種類の機能も構造も全く異なる分子を別々に結合する様式に関する分子基盤は不明であった。本年度、importin βとSREBP-2二量体の複合体の結晶構造解析に成功し,importin βが持つ19個のHEATモチーフの中で、他よりも長いヘリックス構造を取る2つのHEATモチーフ部分がSREBP-2分子をつかむような形で両分子が結合していることを明らかにした。また、細胞周期と核蛋白質輸送の関連に着目した研究を進め、細胞周期の調節因子の1つであるp27に関して、その核局在化シグナルに含まれるスレオニン残基がリン酸化を受けると14-3-3蛋白質が結合して核局在化シグナルが輸送因子importin αによって認識されなくなることを発見した。このことから、p27の核局在化シグナルが機能しなくなることにより、正常な細胞周期進行が妨げられて細胞の癌化が誘導される可能性が示唆された。さらに、輸送因子の1つであるimportin αが、熱ショックなどの細胞ストレスに応じて核内に集積する現象を発見し、ストレス条件下では、通常の核蛋白質輸送経路が抑制され、その代わりに、ストレス応答性の輸送経路が活発に機能する状態になる可能性を示唆することができた。以上のように、研究計画に沿った、核蛋白質輸送の多様性やその制御機構に関する研究成果を挙げることができた。

Research Products

• [Publications] Tajima, Y.: "CRM1-depemdemt nuclear export of Smurf1 is essential for negative regulation of TGF-β signaling by Smad7."J.Biol.Chem.. 278. 10716-10721 (2003)
• [Publications] Lee, S.J.: "Crystallization and preliminary crystallographic analysis of the importin-β-SREBP-2 complex."Acta Crystallogr.. D59. 1866-1868 (2003)
• [Publications] Konishi, A.: "Involvement of histone H1.2 in apoptosis induced by DNA double-strand breaks"Cell. 114. 673-688 (2003)
• [Publications] Lee, S.J.: "The structure of importin β bound to SREBP-2 : Insights into nuclear import of a transcription factor."Science. 302. 1571-1575 (2003)
• [Publications] Sakaguchi, N.: "Generation of a rat monoclonal antibody specific for importin alpha3/Qip1."Hybridoma and Hybridomics. in press. (2003)
• [Publications] Furuta, M.: "Heat-shock induced nuclear retention and recycling inhibition of importin α."Genes Cells. in press. (2003)
• [Publications] Yasuhara, N.: "Importin α/β-mediated nuclear protein import is regulated in a cell cycle-dependent manner."Exp.Cell Res.. in press. (2004)
• [Publications] Miyamoto, Y.: "How proteins are transported into and out of the nucleus. (Biomolecular Chemistry : A Bridge for the Future (ed. Scientific Program Committee of ISBC2003))"Maruzen Co., Ltd. (Tokyo). 4 (2003)
• [Publications] Kobayashi, M.: "Nuclear localization of duplin, a β-catenin-binding protein, is essential for its inhibitory activity on the Wnt signaling pathway"J. Biol. Chem.. 277. 5816-5822 (2002)
• [Publications] Shibata, S.: "Nucleocytoplasmic transport of proteins and poly(A)+RNA at the reconstituted Tpr-less nuclei in living mammalian cells"Genes Cells. 7. 421-434 (2002)
• [Publications] Katahira, J.: "Complex formation between Tap and p 15 affects binding to FG-repeat nucleoporins and nucleocytoplasmic shuttling"J. Biol. Chem.. 277. 9242-9246 (2002)
• [Publications] Miyamoto, Y.: "Genomic organization and chromosomal localization of the importin α1 gene in the mouse"Gene. 288. 49-56 (2002)
• [Publications] Matsuoka, Yu.: "A chromodomain-containing nuclear protein, MRG15 is expressed as a novel type of dendritic mRNA in neurons"Neurosci. Res.. 42. 299-308 (2002)
• [Publications] Matsuoka, Yo.: "Bovine liver chromatin fraction contains actin polymerization activity inducing micronuclei formation when injected into prometaphase cultured cells"Cell Struct. Funct.. 27. 39-45 (2002)
• [Publications] Matsuoka, Yo.: "Identification of Ewing's sarcoma gene product as a glycoprotein using a monoclonal antibody which recognizes an immunodeterminant containing O-linked N-acetylglucosamine moiety"Hybridoma and Hybridomics. 21. 233-236 (2002)
• [Publications] Kondo, Y.: "Molecular cloning of one isotype of human lamina-associated polypeptide 1s and a topological analysis using its deletion mutants"Biochem. Biophys. Res. Commun.. 294. 770-778 (2002)
• [Publications] Sugiyama, T.: "Activation of mitochondrial voltage-dependent anion channel by a pro-apoptotic BH3-only protein Bim"Oncogene. 21. 4944-4956 (2002)
• [Publications] Miyamoto, Y.: "Importin α can migrate into the nucleus in an importin β-and Ran-independent manner"EMBO J.. 21. 5833-5842 (2002)
• [Publications] Kametaka, A.: "Interaction of the chromatin compaction-inducing domain (LR domain) of Ki-67 antigen with HP1 proteins"Genes Cells. 7. 1231-1242 (2002)
• [Publications] Yoneda, Y.: "How many pathways are available for nuclear protein import in cells"Acta Histochem. Cytochem.. 35. 435-440 (2002)
• [Publications] Tajima, Y.: "CRM1-depemdemt nuclear export of Smurf1 is essential for negative regulation of TGF-β signaling by Smad7"J. Biol. Chem.. (in press). (2003)
• [Publications] Yoneda, Y.: "Protein transport"Nature Encyclopedia of the Human Genome. (in press). (2003)
• [Publications] Shimizu, S.: "Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells"J.Cell Biol.. 152. 237-250 (2001)
• [Publications] Nagoshi, E.: "Dimerization of the sterol regulatory element-binding protein-2 via the helix-loop-helix-leucine zipper domain is prerequisite for its nuclear localization mediated by importin β"Mol.Cell.Biol.. 21. 2779-2789 (2001)
• [Publications] Jiang, C.-J.: "Molecular cloning of a novel importin α homologue from rice, by which constitutive photomorphogenic 1 (COP1) nuclear localization signal (NLS)-protein is preferentially nuclear imported"J.Biol.Chem.. 276. 9322-9329 (2001)
• [Publications] Akakura, S.: "A role for Hsc70 in regulating nucleo-cytoplasmic transport of a temperature-sensitive p53 (p53Val135)"J.Biol.Chem.. 276. 14649-14657 (2001)
• [Publications] Kurisaki, A.: "Transforming growth factor-b induces nuclear import of Smad3 in an importin-b1 and Ran-dependent manner"Mol.Biol.Cell. 12. 1079-1091 (2001)
• [Publications] Hieda, M.: "Nuclear import of the U1A splicesome protein is mediated by importin α/β and Ran in living mammalian cells"J.Biol.Chem.. 276. 16824-16832 (2001)
• [Publications] Takagi, M.: "A novel nucleolar protein, NIFK, interacts with the forkhead associated domain of Ki-67 antigen in mitosis"J.Biol.Chem.. 276. 25386-25391 (2001)
• [Publications] Haraguchi, T.: "BAF is required for emerin assembly into the reforming nuclear envelope"J.Cell Sci.. 114. 4575-4585 (2001)
• [Publications] Kobayashi, M.: "Nuclear localization of duplin, a β-catenin-binding protein, is essential for its inhibitory activity on the Wnt signaling pathway"J.Biol.Chem.. (in press). (2002)
• [Publications] Shibata, S.: "Nucleocytoplasmic transport of proteins and poly(A)+RNA at the reconstituted Tpr-less nuclei in living mammalian cells"Genes Cells. (in press). (2002)
• [Publications] Katahira, J.: "Complex formation between Tap and p15 affects binding to FG-repeat nucleoporins and nucleocytoplasmic shuttling"J.Biol.Chem.. (in press). (2002)
• [Publications] Miyamoto, Y.: "Genomic organization and chromosomal localization of the importin a1 gene in the mouse"Gene. (in press). (2002)
• [Publications] Matsuoka, Yu.: "A chromodomain-containing nuclear protein, MRG15 is expressed as a novel type of dendritic mRNA in neurons"Neurosci.Res.. (in press). (2002)
• [Publications] Matsuoka, Yo.: "Bovine liver chromatin fraction contains actin polymerization activity inducing micronuclei formation when injected into prometaphase cultured cells"Cell Struct.Funct.. (in press). (2002)
• [Publications] Haraguchi,T.: "Live fluorescence imaging reveals early recruitment of emerin, LBR, HP1β and RanBP2 to reforming functional nuclear envelope."J.Cell Sci.. 113. 779-794 (2000)
• [Publications] Matsubae,M.: "Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein."FEBS Lett.. 468. 171-175 (2000)
• [Publications] Tachibana,T.: "Recycling of importin α from the nucleus is suppressed by loss of RC1 function in living mammalian cells."Cell Struct.Funct.. 25. 95-103 (2000)
• [Publications] Ikuta,T.: "Nucleocytoplasmic shuttling of the aryl hydrocarbon receptor."J.Biochem.. 127. 503-509 (2000)
• [Publications] Tanabe,K.: "The small GTP-binding protein TC10 promotes growth cone formation and nerve elongation in neuronal cells, and its transcription is induced during nerve regeneration in rat."J.Neurosci.. 20. 4138-4144 (2000)
• [Publications] Takeda,K.: "Characterization of human herpesvirus 7 U27 gene product and identification of its nuclear localization signal."Virol.. 272. 394-401 (2000)
• [Publications] Lee,S.J.: "The adoption of a twisted structure of importin-β is essential for the protein-protein interaction required for unclear transport."J.Mol.Biol.. 302. 251-264 (2000)
• [Publications] Sueishi,M.: "The FHA domain of Ki-67 antigen interacts with a novel kinesin-like protein Hklp2."J.Biol.Chem.. 275. 28888-28892 (2000)
• [Publications] Yoneda,Y.: "Nucleocytoplasmic protein traffic and its significance to cell function."Genes Cells. 5. 777-787 (2000)
• [Publications] Yoneda,Y.: "New steps toward the nucleocytoplasmic traffic of macromolecules."Cell Struct.Funct.. 25. 241-242 (2000)
• [Publications] Shimizu,S.: "Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells."J.Cell Biol.. 152. 237-250 (2001)
• [Publications] Nagoshi,E.: "Dimerization of the sterol regulatory element-binding protein-2 via the helix-loop-helix-leucine zipper domain is prerequisite for its nuclear localization mediated by importin β."Mol.Cell.Biol.. (in press). (2001)
• [Publications] Akakura,S.: "A role for Hsc70 in regulating nucleo-cytoplasmic transport of a temperature-sensitive p53 (p53Val135)."J.Biol.Chem.. (in press). (2001)
• [Publications] Kurisaki,a.: "Transforming growth factor-β induces nuclear import of Smad3 in an importin-β1 and Ran-dependent manner."Mol.Biol.Cell. (in press). (2001)
• [Publications] Hieda,M.: "Nuclear import of the U1A splicesome protein is mediated by importin α/β and Ran in living mammalian cells."J.Biol.Chem.. (in press). (2001)
• [Publications] Tachibana, T.: "Up-regulation of nuclear protein import by nuclear localization signal sequences in living cells"FEBS Lett.. 442. 235-240 (1999)
• [Publications] Matsuoka, Y.: "Identification and characterrization of nuclear pore subcomplexes in mitotic extract of human somatic cells"Biochem. Biophys. Res. Commun.. 254. 417-423 (1999)
• [Publications] Kose, S.: "β-Subunit of nuclear pore-targeting complex (importin-β) can be exported from the nucleus in a Ran-independent manner"J. Biol. Chem.. 274. 2946-3952 (1999)
• [Publications] Hieda, M.: "A monoclonal antibody to the C-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells"J. Cell. Biol.. 144. 645-655 (1999)
• [Publications] Kamei, Y.: "Three distinct classes of the α subunit of the nuclear pore-targeting complex (importin-α) are differentially expressed in adult mouse tissues"J. Histochem. Cytochem.. 47. 363-372 (1999)
• [Publications] Kikuchi, N.: "Molecular shape and ATP binding activity of rat p50, a putative mammalian homologue of RuvB DNA helicase"J. Biochem.. 125. 487-494 (1999)
• [Publications] Yokoya, F.: "β-Catenin can be transported into the nucleus in a Ran-unassisted manner"Mol. Biol. Cell. 10. 1119-1131 (1999)
• [Publications] Nagoshi, E.: "Nuclear import of sterol regulatory element-binding protein-2, a basic helix-loop-helix-leucine zipper (bHLH-Zip)-containing transcription factor occurs through the direct interaction of importin β with HTH-Zip"Mol. Biol. cell. 10. 2221-2233 (1999)
• [Publications] Takagi, M.: "Chmadrin : a novel Ki-67 antigen-related perichromosomal protein possibly implicated in higher order chromatin structure"J. Cell Sci.. 112. 2463-2472 (1999)
• [Publications] Sahara, S.: "Acinus, a caspase-3-activated protein responsible for apoptotic chromatin condensation"Nature. 401. 168-173 (1999)
• [Publications] Ohshima, T.: "CRM1 mediates nuclear export of nonstructural protein 2 from parvovirus minute virus of mine"Biochem. Biophys. Res. Commun.. 264. 144-150 (1999)
• [Publications] Kose, S.: "Distinct energy requirement for nuclear import and export of importin β in living cells"FEBS Lett.]. 463. 327-330 (1999)
• [Publications] Yoneda, Y.: "Nucleocytoplasmic protein transport and recycling of Ran"Cell Struct. Funct.. 24. 425-433 (1999)
• [Publications] Haraguchi, T.: "Live fluorescence imaging reveals early recruitment of emerin, LBR, HP1β and RanBP2 to reforming functional nuclear envelope"J. Cell Sci.. 113. 779-794 (2000)
• [Publications] Matsubae, M.: "Characterization of the nuclear transport of a novel leucine-rich acidic nuclear protein-like protein"FEBS Lett.. (in press).
• [Publications] Tachibana, T.: "Recycling of importin α from the nucleus is suppressed by loss of RCC1 fuction in living mammalian cells"Cell Struct Funct.. (in press).