2000 Fiscal Year Annual Research Report

分解耐性型p27kip1の分子デザインと癌治療への応用

Research Project

Project/Area Number	12217110
Research Institution	Hamamatsu University School of Medicine
Principal Investigator	北川雅敏浜松医科大学, 医学部, 教授 (50294971)
Co-Investigator(Kenkyū-buntansha)	内田千晴浜松医科大学, 医学部, 助手 (60223567) 小田敏明浜松医科大学, 医学部, 助教授 (90126805)
Keywords	p27^<Kip1> / ユビキチン-プロテアソーム / 遺伝子治療 / 予後 / リン酸化 / 細胞周期
Research Abstract	1.p27^<Kip1>の分解機構の解析我々はin vitroおよびin vivoにおけるp27^<Kip1>の分解系を確立した。この系を用いた解析によりp27^<Kip1>がSkp2をユビキチンリガーゼとしたユビキチン-プロテアソーム系と限定分解という二つの経路で分解されることを見出した。限定分解酵素の精製方法もほぼ確立し、マイクロシークエンスによりCDK結合部位とサイクリン結合部位の二つの限定分解部位の決定に成功した。また、p27^<Kip1>のリン酸化部位の解析を行い、新規部位Ser10を見出した(Ishida et al.JBC,2000)。 2.p27^<Kip1>の機能と分解の個体レベルの解析 p27^<Kip1>のユビキチンリガーゼSkp2のノックアウトマウスを作成して解析した。マウスは正常に発生するが、矮小化、肝臓、腎臓、肺胞細胞の多倍数体化、中心体の過剰複製、p27^<Kip1>およびサイクリンEの蓄積が見られた(Nakayama et al.EMBO J,2000)。また、p27のトランスジェニックマウスの解析結果からT細胞の分化にp27^<Kip1>の発現量の低下が必要であることが証明された(Tsukiyama et al.Jimmunol,2001)。 3.分解耐性p27^<Kip1>の作成 Ser10のリン酸化あるいはそれをミミックしたGluへの変換によりp27^<Kip1>は分解耐性になることを見出した。一方で上記二つの限定分解部位に変異を導入することで限定分解を阻害することが可能になった(投稿準備中)。 Skp2ノックアウトマウスでp27^<Kip1>の分解は低下していたが確実に分解しており、もう一つの機構である限定分解機構の存在が強く指示された。一方でSer10のGluへの変換、Thr187のAlaへの変換および限定分解部位への変異の導入により、より高い分解抵抗性を持つp27^<Kip1>の創製が可能であることを見いだした。今後は本研究の成果をもとに、分解抵抗性p27^<Kip1>を用いた遺伝子治療実験を行い、予後不良の大腸癌、乳癌、肺癌、胃癌などの克服を目指す。

Research Products

(13 results)

All Other

All Publications (13 results)

[Publications] Tsukiyama,T. et al.: "Down-regulation of p27^<Kip1> expression is required for development and function of T cells."Journal of Immunology. 116. 304-312 (2001)
[Publications] Yamanaka,A. et al.: "Cell-cycle dependent expression of mammalian E2-C regulated by the anaphase promoting complex/cyclosome."Molecular Biology of the Cell. 11. 2821-2831 (2000)
[Publications] Ishida,N. et al.: "Phosphorylation at Ser10 a major phosphorylation site of p27^<Kip1>, increases its protein stability"Journal of Biological Chemistry. 275. 25146-25154 (2000)
[Publications] Ishimi,Y. et al.: "Inhibition of Mcm4,6,7 helicase activity by phosphorylation with cyclin A-Cdk2"Journal of Biological Chemistry. 275. 16235-16241 (2000)
[Publications] Nakayama,K. et al.: "Targeted disruption of Skp2 result in accumulation of Cyclin E and p27^<Kip1>, polyploidy, and centrosome overdeplication"EMBO Journal. 19. 2069-2081 (2000)
[Publications] Dobashi,Y. et al.: "Simultaneous suppression of Cdc2/Cdk2 activity is necessary and sufficient for neuronal differentiation of PC12."Journal of Biological Chemistry. 275. 12572-12580 (2000)
[Publications] Tanaka,T. et al.: "Geranylgeranyl-pyrophosphate (GGPP), an isoprenoid of mevalonate cascade, is a critical compound for rat primary cultured cortical neurons to protect the cell death induced by HMG-CoA reductase inhibition."Journal of Neuroscience. 20. 2852-2859 (2000)
[Publications] Shimohata,T. et al.: "Expanded polyglutamine stretches associated with CAG repeat diseases interact with hTAFII130, interfering with CREB-dependent transcription"Nature Genetics. 26. 29-36 (2001)
[Publications] Xue,H-H. et al.: "Store depletion by caffeine/ryanodine activates capacitative Ca2+ entry in nonexcitable A549 cells."Journal of Biochemistry. 128. 329-336 (2000)
[Publications] Zhao,D-M. et al.: "Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells"Journal of Physiology. 278. L726-L736 (2000)
[Publications] Oda,T. et al.: "Mitochondrial targeting signal-induced conformational change and repression of the peroxisomal targeting signal of the precursor for rat liver serine : pyruvate/alanine : glyoxylate aminotransferase."Journal of Biochemistry. 127. 665-671 (2000)
[Publications] Yokota,S. et al.: "Aggregate formation and degradation of overexpressed wild-type and mutant urate oxidase proteins.quality control af organelle-destined proteins by the endoplasmic reticulum"Histochemstry and Cell Biology. 114. 433-446 (2000)
[Publications] 北川雅敏(分担執筆): "わかる細胞周期と癌"田矢洋一編羊土社. 118 (2000)

2000 Fiscal Year Annual Research Report

分解耐性型p27kip1の分子デザインと癌治療への応用

Principal Investigator

北川 雅敏 浜松医科大学, 医学部, 教授 (50294971)

Research Products

[Publications] Tsukiyama,T. et al.: "Down-regulation of p27^<Kip1> expression is required for development and function of T cells."Journal of Immunology. 116. 304-312 (2001)

[Publications] Yamanaka,A. et al.: "Cell-cycle dependent expression of mammalian E2-C regulated by the anaphase promoting complex/cyclosome."Molecular Biology of the Cell. 11. 2821-2831 (2000)

[Publications] Ishida,N. et al.: "Phosphorylation at Ser10 a major phosphorylation site of p27^<Kip1>, increases its protein stability"Journal of Biological Chemistry. 275. 25146-25154 (2000)

[Publications] Ishimi,Y. et al.: "Inhibition of Mcm4,6,7 helicase activity by phosphorylation with cyclin A-Cdk2"Journal of Biological Chemistry. 275. 16235-16241 (2000)

[Publications] Nakayama,K. et al.: "Targeted disruption of Skp2 result in accumulation of Cyclin E and p27^<Kip1>, polyploidy, and centrosome overdeplication"EMBO Journal. 19. 2069-2081 (2000)

[Publications] Dobashi,Y. et al.: "Simultaneous suppression of Cdc2/Cdk2 activity is necessary and sufficient for neuronal differentiation of PC12."Journal of Biological Chemistry. 275. 12572-12580 (2000)

[Publications] Tanaka,T. et al.: "Geranylgeranyl-pyrophosphate (GGPP), an isoprenoid of mevalonate cascade, is a critical compound for rat primary cultured cortical neurons to protect the cell death induced by HMG-CoA reductase inhibition."Journal of Neuroscience. 20. 2852-2859 (2000)

[Publications] Shimohata,T. et al.: "Expanded polyglutamine stretches associated with CAG repeat diseases interact with hTAFII130, interfering with CREB-dependent transcription"Nature Genetics. 26. 29-36 (2001)

[Publications] Xue,H-H. et al.: "Store depletion by caffeine/ryanodine activates capacitative Ca2+ entry in nonexcitable A549 cells."Journal of Biochemistry. 128. 329-336 (2000)

[Publications] Zhao,D-M. et al.: "Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells"Journal of Physiology. 278. L726-L736 (2000)

[Publications] Oda,T. et al.: "Mitochondrial targeting signal-induced conformational change and repression of the peroxisomal targeting signal of the precursor for rat liver serine : pyruvate/alanine : glyoxylate aminotransferase."Journal of Biochemistry. 127. 665-671 (2000)

[Publications] Yokota,S. et al.: "Aggregate formation and degradation of overexpressed wild-type and mutant urate oxidase proteins.quality control af organelle-destined proteins by the endoplasmic reticulum"Histochemstry and Cell Biology. 114. 433-446 (2000)

[Publications] 北川雅敏(分担執筆): "わかる細胞周期と癌"田矢洋一 編 羊土社. 118 (2000)

北川雅敏浜松医科大学, 医学部, 教授 (50294971)

[Publications] 北川雅敏(分担執筆): "わかる細胞周期と癌"田矢洋一編羊土社. 118 (2000)