1999 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, 医学系研究科, 教授 (80191667)
Co-Investigator(Kenkyū-buntansha)	今本尚子大阪大学, 医学系研究科, 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	Calcium/calmodulin-dependent protein kinase type IV(CaM kinase IV)は、細胞内シグナル伝達に重要な役割を果たすリン酸化酵素であるが、免疫組織化学法による解析から、脳などの神経細胞では主として核に局在するのに対し、甲状腺傍濾胞細胞などの非神経系細胞では細胞質中心であることが知られている。そこで、本研究では、CaM kinase IVを指標にすることにより、組織あるいは細胞種特異的な核蛋白質輸送機構を明らかにすることができるのではないかと考え、研究を進めた。大腸菌で発現させたレコンビナントGFP-CaM kinaseIVを培養細胞の細胞質にマイクロインジェクションしたところ、COS7細胞では速やかに核に移行したのに対し、HeLa細胞では核への集積がほとんど見られなかった。次に、ジギトニンで処理したCOS7セミインタクト細胞を用いたin vitro 核蛋白質輸送実験系を利用して解析を進めたところ、Ehrlich腹水癌細胞およびマウス脳からの細胞抽出液に依存してCaM kinaseIVの核への移行が見られたが、脳からの抽出液の方がより強い輸送活性が認められた。CaM kinaseIVの核への移行は、低分子量G蛋白質Ranの変異体であるQ69LRan-GTPによって阻害されることから、Ranに依存した経路によって輸送されると推測されるが、importinαのIBB(importin β binding)domainやimportinβの核膜孔複合体結合領域によっては阻害されないことから、既知の因子であるimportin α/βが関与する輸送経路ではない新規の経路で輸送されていることが示唆された。以上の結果を考え合わせると、CaM kinaseIVは、importinβファミリー分子に依存しない、未知の輸送因子によって核内へ輸送されており、その輸送活性が細胞種によって何らかの形で調節されるという、細胞種特異性のある新規輸送系が存在する可能性が示唆された。

Research Products

(16 results)

All Other

All Publications (16 results)

[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 characterization 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. 3946-3952 (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(importune-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 throough 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).

1999 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, 医学系研究科, 教授 (80191667)

Research Products

[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 characterization 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. 3946-3952 (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(importune-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 throough 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).

米田悦啓大阪大学, 医学系研究科, 教授 (80191667)