1998 Fiscal Year Annual Research Report

CaMキナーゼIVをモデル分子とした神経細胞特異的核蛋白質輸送機構の解析

Research Project

Project/Area Number	10480200
Research Institution	Osaka University
Principal Investigator	米田悦啓大阪大学, 医学部, 教授 (80191667)
Co-Investigator(Kenkyū-buntansha)	今本尚子大阪大学, 医学部, 助手 (20202145)
Keywords	核蛋白質輸送 / 核局在化シグナル / 神経細胞 / CaMキナーゼ / セミインタクト細胞 / importin / 細胞核
Research Abstract	核-細胞質問蛋白質輸送機構の解明は、細胞増殖、細胞死、個体発生など、様々な生命現象に密接に関わる細胞生物学上の重要な研究テーマである。本研究では、核蛋白質輸送因子の1つであるimportin αファミリーに属する分子群の発現様式に極端な差違が見られる神経組織に着目し、神経細胞特異的な核蛋白質輸送機構の存在を明らかにし、その分子メカニズムを解明する目的で研究を進め、以下のような成果を得た。 1. HA tagの付いたCaMキナーゼIV融合遺伝子を構築し、大腸菌でレコンビナント蛋白質を調製した。そのレコンビナント蛋白質がカルモジュリンに結合する活性を有していることを確認した後、Hela細胞の細胞質にマイクロインジェクションしたが、予期していた通り、CaMキナーゼIVの核への移行はHela細胞では確認されなかった。また、QMキナーゼIVの一次配列上に、bipartite typeの核局在化シグナルに類似の配列が2カ所存在するので、その部分のアミノ酸をGST-GFPの間に挿入した融合遺伝子を構築した。そのレコンビナント蛋白質を、同じくHela細胞の細胞質にマイクロインジェクションしたが、核への移行は観察されなかった。さらに、Hela細胞をカリウム刺激し、電気的に活性化させた状態でもCaMキナーゼIVは核へ移行しなかった。 2. CaMキナーゼIVには、ATP結合領域、カルモジュリン結合領域、CaMキナーゼキナーゼによってリン酸化を受けるアミノ酸が既に同定されているので、その部分を欠失あるいは変異させた変異遺伝子を複数作成した。 3. 今後は、神経細胞のprimary cultureを用いて、CaMキナーゼIVが細胞質から核へ移行するかどうかを検討する。また、セミインタクト細胞ir vitro輸送系において、神経細胞から細胞抽出液を調製し、CaMキナーゼIVの核への移行が再現できるかを調べていく。

Research Products
(14 results)

All Other

All Publications (14 results)

[Publications] Togo Ikuta: "Nuclear Localization and Export Signals of the Human Aryl Hydrocarbon Receptor" THE JOURNAL OF BIOLOGICAL CHEMISTRY. 273・5. 2895-2904 (1998)
[Publications] N.Imamoto: "Nuclear Transport Factors:Function,Behavior and Interaction" Eur.J.Histochem.42. 9-20 (1998)
[Publications] Norihiko Nakamura: "Localization of the Apoptosis-inducing Activity of Lupus Anticoagulant in an Annexin V-binding Antibody Subset" J.Clin.Invest.101・9. 1951-1959 (1998)
[Publications] T.Murai,MD: "Temporal lobe epilepsy in a genius of natural history MRI volumetric study of postmortem brain" NEUROLOGY. 50. 1373-1376 (1998)
[Publications] Nobuaki Kudo: "Leptomycin B Inhibition of Signal-Mediated Nuclear Export by Direct Binding to CRM1" EXPERIMENTAL CELL RESEARCH. 242. 540-547 (1998)
[Publications] Takahiro Matsusaka: "Mutations in fission yeast Cut15,an importin α homolog,lead to mitotic progression without chromosome condensation" Current Biology. 8・18. 1031-1034 (1998)
[Publications] Chang-Jie Jiang: "Functional Characterization of a Plant Importin α Homologue" THE JOURNAL OF BIOLOGICAL CHEMISTRY. 273・37. 24083-24087 (1998)
[Publications] Chang-Jie Jiang: "In vitro characterization of rice importin β1:molecular interaction with nuclear transport factors and mediation of nuclear protein import" FEBS Letters. 437. 127-130 (1998)
[Publications] Masami Yamada: "Nuclear transport factor p10/NTF2 function as a Ran-GDP dissociation inhibitor(Ran-GDI)" Current Biology. 8. 1342 (1998)
[Publications] Yosuke Matsuoka: "Identificatin and Characterization of Nuclear Pore Subcomplexes in Mitotic Extract of Human Somatic Cells" Biochemical and Biophysical Research Communications. 254. 417-423 (1999)
[Publications] Taro Tachibana: "Up-regulation of nuclear protein import by nuclear localization signal sequences in living cells" FEBS Letters. 442. 235-240 (1999)
[Publications] Shingo Kose: "β-Subunit of Nuclear Pore-targeting Complex(Importin-β)Can Be Exported from the Nucleus in a Ran-independent Manner" THE JOURNAL OF BIOLOGICAL CHEMISTRY. 274・7. 3946-3952 (1999)
[Publications] Yasuhiro Kamei: "Three Distinct Classes of the α-Subunit of the Nuclear Pore-targeting Complex(Importin-α)Are Differentially Expressed in Adult Mouse Tissues" The Journal of Histochemistry & Cytochemistry. 47・3. 1-10 (1999)
[Publications] Fumihiko Yokoya: "β-Catenin Can Be Transported into the Nucleus in a Ran-unassisted Manner" Molecular biology of the Cell. (in press). (1999)

1998 Fiscal Year Annual Research Report

CaMキナーゼIVをモデル分子とした神経細胞特異的核蛋白質輸送機構の解析

Principal Investigator

米田 悦啓 大阪大学, 医学部, 教授 (80191667)

Research Products

[Publications] Togo Ikuta: "Nuclear Localization and Export Signals of the Human Aryl Hydrocarbon Receptor" THE JOURNAL OF BIOLOGICAL CHEMISTRY. 273・5. 2895-2904 (1998)

[Publications] N.Imamoto: "Nuclear Transport Factors:Function,Behavior and Interaction" Eur.J.Histochem.42. 9-20 (1998)

[Publications] Norihiko Nakamura: "Localization of the Apoptosis-inducing Activity of Lupus Anticoagulant in an Annexin V-binding Antibody Subset" J.Clin.Invest.101・9. 1951-1959 (1998)

[Publications] T.Murai,MD: "Temporal lobe epilepsy in a genius of natural history MRI volumetric study of postmortem brain" NEUROLOGY. 50. 1373-1376 (1998)

[Publications] Nobuaki Kudo: "Leptomycin B Inhibition of Signal-Mediated Nuclear Export by Direct Binding to CRM1" EXPERIMENTAL CELL RESEARCH. 242. 540-547 (1998)

[Publications] Takahiro Matsusaka: "Mutations in fission yeast Cut15,an importin α homolog,lead to mitotic progression without chromosome condensation" Current Biology. 8・18. 1031-1034 (1998)

[Publications] Chang-Jie Jiang: "Functional Characterization of a Plant Importin α Homologue" THE JOURNAL OF BIOLOGICAL CHEMISTRY. 273・37. 24083-24087 (1998)

[Publications] Chang-Jie Jiang: "In vitro characterization of rice importin β1:molecular interaction with nuclear transport factors and mediation of nuclear protein import" FEBS Letters. 437. 127-130 (1998)

[Publications] Masami Yamada: "Nuclear transport factor p10/NTF2 function as a Ran-GDP dissociation inhibitor(Ran-GDI)" Current Biology. 8. 1342 (1998)

[Publications] Yosuke Matsuoka: "Identificatin and Characterization of Nuclear Pore Subcomplexes in Mitotic Extract of Human Somatic Cells" Biochemical and Biophysical Research Communications. 254. 417-423 (1999)

[Publications] Taro Tachibana: "Up-regulation of nuclear protein import by nuclear localization signal sequences in living cells" FEBS Letters. 442. 235-240 (1999)

[Publications] Shingo Kose: "β-Subunit of Nuclear Pore-targeting Complex(Importin-β)Can Be Exported from the Nucleus in a Ran-independent Manner" THE JOURNAL OF BIOLOGICAL CHEMISTRY. 274・7. 3946-3952 (1999)

[Publications] Yasuhiro Kamei: "Three Distinct Classes of the α-Subunit of the Nuclear Pore-targeting Complex(Importin-α)Are Differentially Expressed in Adult Mouse Tissues" The Journal of Histochemistry & Cytochemistry. 47・3. 1-10 (1999)

[Publications] Fumihiko Yokoya: "β-Catenin Can Be Transported into the Nucleus in a Ran-unassisted Manner" Molecular biology of the Cell. (in press). (1999)

米田悦啓大阪大学, 医学部, 教授 (80191667)