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Regulation of Binding of Lamin B Receptor to Chromatin by SR

Regulation of Binding of Lamin B Receptor to Chromatin by SR Protein Kinase and cdc2 Kinase in Xenopus Egg Extracts. Takano et cl.,(2004) J. Bio. Chem. 279 13265-13271. 生科四甲 91390410 謝宗文. 名詞解釋. Xenopus egg : 成熟的 cell cycle 研究體系 , 在加入 ATP 再生系統 , 可誘導不同的細胞週期的發生 .

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Regulation of Binding of Lamin B Receptor to Chromatin by SR

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  1. Regulation of Binding of Lamin B Receptor to Chromatin by SR Protein Kinase and cdc2 Kinase in Xenopus Egg Extracts Takano et cl.,(2004) J. Bio. Chem. 279 13265-13271 生科四甲 91390410 謝宗文

  2. 名詞解釋 • Xenopus egg : 成熟的cell cycle研究體系,在加入ATP再生系統,可誘導不同的細胞週期的發生. • Lamin b receptor (LBR): 在interphase時.負責連接到chromatin的內核膜protein 內核膜上的protein

  3. cdc2 kinase : 和cell cycle protein結合才有kinase的活性.亦被稱作CdK1.活化後可將protein磷酸化而產生相應的生理效應

  4. SR protein kinase 1: SR protein 的激酶.在此實驗中扮演著刺激the binding of LBR to chromatin的角色.

  5. Cell cycle SC (S-phase cytosol fraction) MC (M-phase cytosol fraction)

  6. Cell cycle轉動時. 核膜會有消失的時候.而LBR位於內核膜上.故作者認為the binding of LBR to chromatin可能受到cell cycle的調控. • 而在內核膜上主要的protein是負責bind to chromatin, ex : LBR、emerin、 lamina associated polypeptide 2(LAP2) family等. • 在這篇研究中,作者主要研究的是LBR,藉由cell cycle-specific Xenopus egg cytosol fractions來分析kinase負責調控the binding of LBR to chromatin.

  7. 作者利用GST fusion protein來bind LBR,即是GST-NK和GST-RS • glutathione S-transferase (GST) : 用來bind所需protein.此實驗中的protein是LBR. • NK :LBR的nucleoplasmic區域(1-211) • RS : 在NK之中的arginine-serine重複片段(53-89) LBR

  8. 圖1 SRPK1 in SC是負責刺激此一binding的kinase

  9. Dep : deplete SRPK 圖2 SRPK耗盡會使SC失去刺激binding能力.重新加入SRPK後又恢復能力

  10. 透過此實驗結果可驗證SRPK耗盡會使SC失去刺激(LBR磷酸化)binding的能力透過此實驗結果可驗證SRPK耗盡會使SC失去刺激(LBR磷酸化)binding的能力 圖3

  11. 接下來.我們檢查在S-phase到M-phase中間這段過渡時期對於此binding的影響.接下來.我們檢查在S-phase到M-phase中間這段過渡時期對於此binding的影響. • kinase有負責刺激.也有負責抑制此binding的 • 為了區分出何種kinase參與抑制.作者用各種的kinase inhibtitor來加入GST-NK和GST-NK(S71A).

  12. 圖4 包含cdc2 kinase在內有多種kinase參與抑制

  13. 對於此binding而言.cdc2 kinase負責一部分的抑制 圖5

  14. 圖6

  15. cdc2 kinase/S71A中消失的.c.e兩點.其中必定含有serine71 兩相重疊後.位置相同的spot上必有相同的phosphopeptide.

  16. 和SC相較之下.另兩個結果的spot 10.12有明顯的磷酸化

  17. 相較之下.MC+Rosco上的spot 12.10因cdc2 kinase被抑制而消失了 相較之下.MC/S71A上的spot 12消失了.而spot 10被磷酸化. • 由以上結果可推知spot 10.12含有serine71.並且在MC時被cdc2 kinase磷酸化.

  18. 圖7

  19. 在圖7中.由2.3可知.以SRPK1取代SC.對於此binding有相似的增強.在圖7中.由2.3可知.以SRPK1取代SC.對於此binding有相似的增強. • 由5.6可知.同樣用cdc2 kinase處理的SRPK1和SC.對於此binding有相似的減弱. • 由此可知.刺激是由SRPK1所負責.而抑制則是由cdc2 kinase負責一部分.

  20. 圖8 cdc2 kinase在MC刺激serine71磷酸化是負責一部分的抑制the binding of GST-NK to DNA-cellulose.

  21. Regulation of Binding of Lamin B Receptor to Chromatin by SR Protein Kinase and cdc2 Kinase: • Phosphorylation of GST-NK by cdc2 kinase in MC導致一部份的抑制the binding to chromatin. • 抑制the binding to chromatin的kinase不只cdc2一種.還有其他kinase可執行此功能. • SRPK in SC 刺激the binding of GST-NK to chromatin .

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