体細胞の細胞周期とその制御機構-RCC1-RanGTPaseの解析を中心として-

• Project/Area Number: 12002011
• Research Category: Grant-in-Aid for Specially Promoted Research
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kyushu University
• Principal Investigator: 西本 毅治 九州大学, 大学院・医学研究院, 教授 (10037426)
• Co-Investigator(Kenkyū-buntansha): 西嶋 仁 九州大学, 大学院・医学研究院, 特別研究員(PD) ; 西谷 秀男 九州大学, 大学院・医学研究院, 助手 (40253455) ; 沖 昌也 九州大学, 大学院・医学研究院, 特別研究員(PD) ; 関口 猛 九州大学, 大学院・医学研究院, 助手 (60187846)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥400,350,000 (Direct Cost: ¥322,500,000、Indirect Cost: ¥77,850,000); Fiscal Year 2004: ¥81,900,000 (Direct Cost: ¥63,000,000、Indirect Cost: ¥18,900,000); Fiscal Year 2003: ¥81,900,000 (Direct Cost: ¥63,000,000、Indirect Cost: ¥18,900,000); Fiscal Year 2002: ¥90,350,000 (Direct Cost: ¥69,500,000、Indirect Cost: ¥20,850,000); Fiscal Year 2001: ¥83,200,000 (Direct Cost: ¥64,000,000、Indirect Cost: ¥19,200,000); Fiscal Year 2000: ¥63,000,000 (Direct Cost: ¥63,000,000)
• Keywords: RanGAP / Histone H3-K9 methyltransferase / ヘテロクロマチン / 温度感受性変異マウス / Mog1 / NTF2 / RCC1 / カフェイン / ATR / Adenine / H3-K9メチル転移酸素 / SNF2 / 細胞周期制御 / DBX / RanBPM / Gap1 / Gtr1 / RagA / Ran / Cell Cycle / MOG1 / Gtr1p / Gtr2p / RagA,B,C,D

Research Abstract

1)RanGAP蛋白質の機能解析:分裂酵母のhistone H3と動物のヒストンを用いてRanGAPとの結合性をしらべたところ、histone H3のC端とRanGAPは結合した。この結合によりRanGAPの活性は完全に阻害された。そして、RanGAP/Sprna1p温度感受性変異のサプレッサーとして分離された、Histone H3-K9 methyltransferase Clr4がこの阻害を完全に回復させる事が生化学的に証明された。In vivoにおいても、ヒストンH3の強発現はclr4遺伝子欠損株では致死となったが、野生株では致死とはならなかった。さらに、興味あることにはRanGAP蛋白質がそれ自体でRan非依存的にClr4のHistone H3-K9 methyltransferase活性を促進した。これらの結果はRanGAPがヘテロクロマチン形成に関わっている事を強く示唆する。2)SpSnf2複合体を酵母より抽出し、Ranによる機能制御の有無をしらべる。【○!3】Clr4とSrSnf2以外にSerine/threonine kinase SpKsp1pが分離されている。このキナーゼとRanとの関連を調べる。3)出芽酵母Ran/Gsp1温度感受性変異株においてはDNA障害チェックポイント機構が活性化されており、野生型で致死である、MEC遺伝子欠損がこの株では致死とならなかった。この結果はRan cycleの新たな機能を示唆する。4)Mog1蛋白質の温度感受性変異株を分裂酵母を用いて作成し、そのサプレッサーを分離した結果、多数のNTF2が分離された。この結果はMog1蛋白質とNTF2蛋白質の機能的相関を強く示唆する。5)RCC1遺伝子に温度感受性変異を持つマウスは発生初期の卵割期に染色体凝縮を起こして死んだ。この結果、培養細胞での表現型が実際の動物体においても証明された。

Research Products

• [Journal Article] A hamster temperature-sensitive alanyl-tRNA synthetase mutant cause degradation of cell-cycle related proteins and apoptosis. (2004)
  • Author(s): Y.Wang et al.
  • Journal Title: J.Biochem 135 Pages: 7-16
• [Journal Article] A novel human nucleolar protein, NOP132,binds to the G proteins, RRAG A/C/D (2004)
  • Author(s): T.Sekiguchi et al.
  • Journal Title: J.Biol.Chem. 279 Pages: 8343-8350
• [Journal Article] S.pombe RanGAP homolog, SpRna1,is required for centromeric silencing and chromosome segregation. (2004)
  • Author(s): A.Kusano et al.
  • Journal Title: Mol.Biol. Cell 15 Pages: 4960-4970
• [Journal Article] Human DDX3Y, the Y-encoded isoform of RNA helicase DDX3,rescues a hamster temperature-sensitive ET24 mutant cell line with a DDX3X mutation. (2004)
  • Author(s): T.Sekiguchi et al.
  • Journal Title: Exp.Cell Res. 300 Pages: 213-222
• [Journal Article] Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of geminin through its N-terminal region (2004)
  • Author(s): H.Nishitani et al.
  • Journal Title: J.Biol.Chem 279 Pages: 30807-30816
• [Publications] Y.Wang et al.: "A hamster temperature-sensitive alany1-tRNA synthetase mutant cause degradation of cell-cycle related proteins and apoptosis."J.Biochem. (in press). (2003)
• [Publications] J.Fukumura et al.: "A temperature-sensitive mutant of the mammalian RNA helicase, DEAD-BOX isoform, DBX, defective in the transition from G1 to S."J.Biochem. 134. 71-82 (2003)
• [Publications] H.Nishijima et al.: "Caffeine mimics adenine and 2'-deoxyadenosine, both of which inhibit the guanine-nucleotide exchange activity of RCC1 and the kinase activity of ATR."GENES to CELLS. 8. 423-435 (2003)
• [Publications] M.Umeda et al.: "A novel nuclear protein, Twa1, and muskelin comprise a complex with RanBPM."Gene. 303. 47-54 (2003)
• [Publications] A.Otomo et al.: "ALS2, a novel guanine nucleotide exchange factor for the small GTPase Rab5, is implicated in endosomal dynamics."Human Molecular Genetics. 12. 1671-1687 (2003)
• [Publications] M.Funakoshi et al.: "Budding yeast Dsk2p is a polyubiquitin-binding protein that can interact with the proteasome"RNAS. 99. 745-750 (2002)
• [Publications] M.Umeda et al.: "A novel unclear protein, Twa1, and Muskelin comprise a complex with RanBPM"Gene. 303. 47-54 (2003)
• [Publications] H.Nishijima et al.: "Caffeine mimics adenine and 2'-deoxyadenosine, both of which inhibit the guanine-uncleotide exchange activity of RCC1 and the kinase activity of ATR"GENES TO CELLS. (in press). (2003)
• [Publications] H.Nishitani, et al.: "The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S phase"J. Biol. Chem.. 276. 44905-44911 (2001)
• [Publications] T.Sekiguchi et al.: "Novel G protein, Rag C and Rag D, interact with GTP-binding proteins, Rag A and Rag B"J. Biol. Chem.. 276. 7246-7257 (2001)
• [Publications] H.Nishitani, et al.: "Full-sized RanBPM cDNA encodes a protein possessing a long stretch of proline snd glutamine within the N-terminal region, comprising a large protein complex"GENE. 272. 25-33 (2001)
• [Publications] N.Suzuki et al.: "The Saccharomyces carevisiae small GTPase, Gsp1p/Ran, is involved in 3' processing of 7S-to-5.8S rRNA and in degradation of the excised 5'-AO fragment of 35S pre-rRNA, both of which are carried out by the exosome"GENETICS. 158. 613-625 (2001)
• [Publications] T.Goda et al.: "The N-terminal helix of Xenopus cyclins A and B contributes to binding specificity of the cyclin-CDK complex"J. Biol. Chem. 276. 15415-15422 (2001)
• [Publications] T.Nishimoto: "Upstream and downstream of RanGTPase."Biol Chem.. 381. 397-405 (2000)
• [Publications] T.Sekiguchi et al.: "Novel G protein, Rag C and Rag D, interact with GTP-binding proteins, RagA and RagB."J.Biol.Chem.. (in press). (2001)
• [Publications] M.Yoshimi et al.: "Inhibition of N-linked glycosylation causes apoptosis in hamster BHK21 cells."Biochemical Biophysical Research Communication. 276. 965-969 (2000)
• [Publications] T.Makishima et al.: "A subunit of the mammalian oligosaccharyltransferase, DAD1, interacts with Mcl-1, one of the bcl-2 protein family."J.Biochem.. 128. 399-405 (2000)
• [Publications] M.Oki and T.Nishimoto: "Yrblp interaction with the Gsplp C terminus blocks Moglp stimulation of release from Gsplp."J.Biol.Chem.. 275. 32894-32900 (2000)
• [Publications] H.Nishitani et al.: "The Cdt1 protein is required to license DNA for replication in fission yeast."Nature. 404. 625-628 (2000)