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研究生:楊宗翰
研究生(外文):Tsung-Han Yang
論文名稱:SAE1/SAE2之磷酸化促進in vitro Sumoylation
論文名稱(外文):Phosphorylation of SAE1/SAE2 Enhances Sumoylation in vitro
指導教授:黃昭蓮
指導教授(外文):Jaulang Hwang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:49
中文關鍵詞:小泛素化磷酸化調控
外文關鍵詞:sumoylationSUMOphosphorylationCdk1regulation
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對於許多細胞中的蛋白質作用而言,例如,細胞內的運輸、轉錄機制、訊息傳遞及細胞週期等,蛋白質的小泛素化(sumoylation)已經成為一種重要的轉譯後修飾機制。研究小泛素化的領域發展得很迅速,尤其是在探討小泛素化如何調控細胞週期的分子機制這方面更是引人注意。調控小泛素化這種動態的修飾作用主要是受到細胞內的刺激或一些侵入細胞所引起的反應,然而,研究如何調控小泛素化自己本身的過程仍在發展階段。依賴週期素的磷酸酶(Cyclin-dependent kinases, Cdks)會與不同類型的週期素(cyclins)結合並調控細胞週期的不同階段,在眾多的Cdks中,Cdk1是最主要的調控者。當Cdk1與週期素B結合(Cdk1/cyclin B)並活化之後,可以促使細胞進入分裂期。近期的文獻發現了許多調控小泛素化的機制,可分為基質層面的調控以及修飾酵素層面的調控兩種方式。對於細胞週期來說,小泛素化與Cdk1皆扮演了很重要的調控角色,因此我想要找出在這兩者之間是否有某種交互關係來幫助調控細胞週期。首先我將Cdk1/cyclin B直接加入活體外的小泛素化試驗中,我發現藉由Cdk1的磷酸化作用,在加入Cdk1/cyclin B之後可以促進蛋白質的小泛素化。利用放射性同位素標定的活體外磷酸化試驗,我發現除了小泛素的E1活化酵素(SAE1/SAE2)之外,其他的小泛素化蛋白都不會被Cdk1磷酸化。為了確認是否是因為磷酸化的E1而促進蛋白質的小泛素化,將E1先做磷酸化處理後,利用GST pull-down的方式將被磷酸化的E1與GST-Cdk1分離開來之後再做小泛素化試驗。與沒有磷酸化的E1相比,磷酸化的E1的確可以促進蛋白質的小泛素化,顯示出E1被Cdk1/cyclin B磷酸化之後可能提升了它的活性,因此可以促進蛋白質的小泛素化作用。這個結果提供了一個小泛素化與Cdk1磷酸化可能可以共同調控細胞週期的分子機制,更詳盡的調控機制有賴之後繼續的研究與發現。
Protein sumoylation has emerged as an important post-translational regulatory mechanism for several cellular events such as subcellular localization, transcriptional machinery, signal transduction and cell cycle progression. The field of protein sumoylation has advanced rapidly, especially in the area of understanding the molecular mechanisms of sumoylation in the control of cell cycle. Sumoylation is a highly dynamic process that is regulated in response to cellular stimuli or pathogenic challenges. However, how sumoylation is regulated itself has just started to be delineated. Cyclin-dependent kinases (Cdks) bind to different types of cyclins and control various phases of cell cycle. Among them, Cdk1 is the master regulator and the activation of Cdk1/cyclin B triggers the initiation of mitosis. Recent studies have uncovered several mechanisms for the regulation of sumoylation system, either at the substrates level or at the level of SUMO enzymes. Both sumoylation and Cdk1 are important regulators in cell cycle progression, therefore, I tried to find out whether there is an correlation between Cdk1 and sumoylation in the control of cell cycle. At first, Cdk1/cyclin B was directly added into the in vitro sumoylation assay. I found that Cdk1 promotes sumoylation by means of protein phosphorylation. To study in more detail, SUMO E1 activating enzyme (SAE1/SAE2), but not other SUMO components, was found to be phosphorylated by Cdk1/cyclin B. Besides, E1 was pretreated with Cdk1/cyclin B and the phosphorylated E1 was isolated by removal of GST-Cdk1. Compared to non-phosphorylated E1, addition of the purified phospho-E1 enhanced sumoylation in the in vitro assay. These results indicated that after phosphorylation by Cdk1, E1 activity was increased and was able to enhance global sumoylation level. This provided a possible regulatory mechanism that sumoylation and Cdk1 phosphorylation may have synergistic effect on cell cycle progression.
ACKNOWLEDGEMENT
中文摘要
ABSTRACT
TABLE OF CONTENTS
LIST OF FIGURES
CHAPTER I INTRODUCTION……………………………….01
1.SUMO and Sumoylation……………………………........01
2.Regulation of Sumoylation…………………….........07
3.SUMO and the Cell Cycle……………………….........10
CHAPTER II MATERIALS AND METHODS………………....13
1.Materials………………………………………….........13
2.Reagents……………………………………………........14
3.Methods…………………………………………….........15
CHAPTER III RESULTS…………………………………....20
1.Establishment of An in vitro Sumoylation System...20
2.Effect of Kinase on Sumoylation…………….........22
3.Cdk1 Phosphorylated E1 and Enhanced Sumoylation….24
CHAPTER IV DISCUSSION…………………………………..26
CHAPTER V CONCLUSIONS………………………………...30
CHAPTER VI FIGURES……………………………………….31
CHAPTER VII REFERENCES………………………………....38
CHAPTER VIII APPENDICES…………………………………..45
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