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Tob蛋白質によるCCR4-NOT脱アデニル化酵素複合体の形成と機能の制御

Publicly Offered Research

Project AreaStructural basis of cell-signalling complexes mediating signal perception, transduction and responses
Project/Area Number 23121506
Research Category

Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionOkinawa Institute of Science and Technology Graduate University (2012)
The University of Tokyo (2011)

Principal Investigator

山本 雅  沖縄科学技術大学院大学, その他の研究科, 教授 (40134621)

Project Period (FY) 2011-04-01 – 2013-03-31
Project Status Completed (Fiscal Year 2012)
Budget Amount *help
¥8,840,000 (Direct Cost: ¥6,800,000、Indirect Cost: ¥2,040,000)
Fiscal Year 2012: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2011: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Keywords蛋白質リン酸化 / Tob蛋白質 / 増殖シグナル / DNA損傷 / X線結晶構造解析 / CCR4-NOT複合体 / CNOT3蛋白質 / 細胞周期
Outline of Annual Research Achievements

TobやCCR4-NOTの構造解析を進めた。
(1)C端側を、43アミノ酸残基と53アミノ酸残基を欠失させた、野生型Tob蛋白質(それぞれTob301,Tob291)を調製した。Tob301に比べTob291は発現量が3倍程度よく、約4リッター培養から3 mgの精製蛋白質を得た。結晶化に成功したが解析に適した回折は得られなかったので会合蛋白質との共結晶構造解析の準備を開始した。
(2)UV等によるDNA損傷依存的にTobをリン酸化し安定化させるキナーゼが、CGK733阻害剤感受性であることを見出していた。しかし、既知のCGK733感受性キナーゼである、ATRやATMにはそのような作用は見られなかった。一方で2D-DIGE法やin vitro リン酸化法を用いた研究から、Cdc7キナーゼがTobと会合しリン酸化しうることを示した。Cdc7によるTobのin vitroリン酸化実験から被リン酸化アミノ酸残基としてSer151, 153、205、並びにThr204を見出した。このうち、少なくともSer151のリン酸化は、in vivoでのTobの安定性増大に寄与することを示した。
(3)CCR4-NOT複合体の活性サブユニット中、直接会合することが示されているCNOT6LとCNOT7について、それぞれ全長配列を調製し、in vitroで会合させて複合体を精製し、結晶を得た。CNOT2とCNOT3の会合に関わるNOT boxをGST融合蛋白質として調製したが、精製過程でNOT box配列がグルタチオンセファロースに特異的に吸着することがわかり、調製方法の改善を図った。
(4)X線結晶解析用に昆虫細胞系でCNOT3を大量発現させたが、この配列も樹脂に吸着することが分かった。現在別法で調製するとともに、CNOT3をドメイン構造に基づいて3分割して結晶化解析に十分な量を調製した。

Research Progress Status

24年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

24年度が最終年度であるため、記入しない。

Report

(2 results)
  • 2012 Annual Research Report
  • 2011 Annual Research Report
  • Research Products

    (19 results)

All 2013 2012 2011 Other

All Journal Article (12 results) (of which Peer Reviewed: 12 results) Presentation (5 results) Remarks (2 results)

  • [Journal Article] ASBEL, an ANA/BTG3 antisense transcript required for tumorigenicity of ovarian carcinoma2013

    • Author(s)
      Yanagida S, Taniue K, Yamamoto T. (9名)
    • Journal Title

      Sci Rep

      Volume: Vol.3 Issue: 1 Pages: 1305-1305

    • DOI

      10.1038/srep01305

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor2013

    • Author(s)
      Yoshikawa S, Kukimoto-Niino M, Yamamoto T. (20名)
    • Journal Title

      Oncogene

      Volume: Vol.32 Issue: 1 Pages: 27-38

    • DOI

      10.1038/onc.2012.21

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Structural flexibility regulates phosphopeptide-binding activity of the tyrosine kinase binding domain of Cbl-c.2012

    • Author(s)
      Takesita K
    • Journal Title

      J. Biochem.

      Volume: 152 Issue: 5 Pages: 487-495

    • DOI

      10.1093/jb/mvs085

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Inhibition of DNA damage-induced apoptosis through Cdc7-mediated stabilization of Tob.2012

    • Author(s)
      Suzuki T
    • Journal Title

      J Biol Chem

      Volume: Nov 23;287(48) Issue: 48 Pages: 40256-40265

    • DOI

      10.1074/jbc.m112.353805

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Tob2 Inhibits Peroxisome Proliferator-Activated Receptor γ2 expression by sequestering Smads and C/EBPα during adipocyte differentiation2012

    • Author(s)
      Takahashi A, Morita M, Yamamoto T. (5名)
    • Journal Title

      Mol and Cell Biol

      Volume: Vol.32 Issue: 24 Pages: 5067-5077

    • DOI

      10.1128/mcb.00610-12

    • Related Report
      2012 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Involvement of CNOT3 in mitotic progression through inhibition of MAD1 expression2012

    • Author(s)
      Takahashi A, et al
    • Journal Title

      Biochem.Biophys.Res.Commun.

      Volume: 419 Issue: 2 Pages: 268-273

    • DOI

      10.1016/j.bbrc.2012.02.007

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4-NOT2011

    • Author(s)
      Fabian MR, Cieplak MK, Yamamoto T (11名)
    • Journal Title

      Nat Struct and Mol Biol

      Volume: Vol.18 Issue: 11 Pages: 1211-1217

    • DOI

      10.1038/nsmb.2149

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability2011

    • Author(s)
      Ito K, et al
    • Journal Title

      Protein Cell

      Volume: 2 Issue: 4 Pages: 755-763

    • DOI

      10.1111/j.1365-2443.2011.01492.x

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Obesity resistance and increased hepatic expression of catabolism- related mRNAs in Cnot3+/- mice2011

    • Author(s)
      Morita M, Oike Y, Yamamoto T (10名)
    • Journal Title

      EMBO J

      Volume: Vol.30 Issue: 22 Pages: 4678-4691

    • DOI

      10.1038/emboj.2011.320

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Distinct expression patterns of the subunits of the CCR4-NOT deadenylase complex during neural development2011

    • Author(s)
      Chen C, et al
    • Journal Title

      Biochem.Biophys.Res.Commun.

      Volume: 411 Issue: 2 Pages: 360-364

    • DOI

      10.1016/j.bbrc.2011.06.148

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Monoubiquitination of Tob/BTG family proteins competes with degradation-targeting polyubiquitination.2011

    • Author(s)
      Suzuki T
    • Journal Title

      Biochem Biophys Res Commun

      Volume: 409 Issue: 1 Pages: 70-4

    • DOI

      10.1016/j.bbrc.2011.04.107

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Genetic incorporation of a photo-crosslinkable amino acid reveals novel protein complexes with GRB2 in mammalian cells2011

    • Author(s)
      Hino N., Oyama M., Sato A., Mukai T., Iraha F., Hayashi A., Kozuka-Hata H., Yamamoyo T., Yokoyama S., Sakamoto K
    • Journal Title

      J. Mol. Biol.

      Volume: 406 Issue: 2 Pages: 343-353

    • DOI

      10.1016/j.jmb.2010.12.022

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Presentation] Effect of Tob deficiency in hepatocellular carcinoma development.2012

    • Author(s)
      Suzuki, T
    • Organizer
      第71回日本癌学会学術総会
    • Place of Presentation
      ロイトン札幌、さっぽろ芸文館、札幌市教育文化会館
    • Related Report
      2012 Annual Research Report
  • [Presentation] Possible link between the CCR4-NOT deadenylase and enhanced expression of specific mRNAs that are related to energy metabolism2011

    • Author(s)
      Miura A, et al
    • Organizer
      第34回日本分子生物学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2011-12-14
    • Related Report
      2011 Annual Research Report
  • [Presentation] Competition of Cdc7/Tob with Cul4-DDB1-Cdt2 E3 complex in DNA damage-induced apoptosis2011

    • Author(s)
      Suzuki T, et al
    • Organizer
      第34回日本分子生物学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2011-12-13
    • Related Report
      2011 Annual Research Report
  • [Presentation] Tob2 inhibits adipocyte differentiation2011

    • Author(s)
      Takahashi A, et al
    • Organizer
      第34回日本分子生物学会年会
    • Place of Presentation
      横浜
    • Year and Date
      2011-12-13
    • Related Report
      2011 Annual Research Report
  • [Presentation] CDC7-dependent inhibition of Tob degradation Mintains cell survival after DNA damage2011

    • Author(s)
      Suzuki T, Masai H, Yamamoto T
    • Organizer
      第70回日本癌学会学術総会
    • Place of Presentation
      名古屋
    • Year and Date
      2011-10-03
    • Related Report
      2011 Annual Research Report
  • [Remarks] OIST 細胞シグナルユニットホームページ

    • URL

      https://groups.oist.jp/ja/csu

    • Related Report
      2012 Annual Research Report
  • [Remarks]

    • URL

      http://groups.oist.jp/csu

    • Related Report
      2011 Annual Research Report

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Published: 2011-04-06   Modified: 2018-03-28  

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