 |
| 系統識別號 |
U0026-0812200910433741 |
| 論文名稱(中文) |
c-Jun C端的磷酸化/去磷酸化對於c-Jun和 Sp1 之間交互作用的影響
|
| 論文名稱(英文) |
Effect of c-Jun C-terminus phosphorylation/
dephosphorylation on the interaction
between c-Jun and Sp1 |
| 校院名稱 |
成功大學 |
| 系所名稱(中) |
藥理學研究所 |
| 系所名稱(英) |
Department of Pharmacology |
| 學年度 |
91 |
| 學期 |
2 |
| 出版年 |
92 |
| 研究生(中文) |
黃祺真 |
| 研究生(英文) |
Chi-Chen Huang |
| 學號 |
S2690404 |
| 學位類別 |
碩士 |
| 語文別 |
中文 |
| 論文頁數 |
70頁 |
| 口試委員 |
口試委員-洪文俊
指導教授-張文昌
口試委員-呂增宏
|
| 中文關鍵字 |
交互作用
去磷酸化
磷酸化
|
| 英文關鍵字 |
GST-pull down assay
c-Jun
Sp1
casein kinase
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| 學科別分類 |
|
| 中文摘要 |
在細胞內，蛋白質受protein kinase 和protein phosphatase 磷酸化及去磷酸化，會正向或逆向調控此蛋白質的活性。在我們實驗室裡，利用PP2B 抑制劑cyclosporin A 可抑制PMA 及EGF 所誘導12( S)-lipoxygenase promoter 的活性及c-Jun/ Sp1 interaction，此暗示著c-Jun/ Sp1 之間的interaction 可能會受到蛋白質的磷酸化及
去磷酸化所調控。而先前的研究指出: c-Jun N 端的transactivation domain 受到JNK 磷酸化之後，會造成c-Jun 構型的改變，促使c-Jun 的 C 端去磷酸化，增加其與DNA 相結合的親合力。此外也有研究指出c-Jun
是透過C 端的leucine zipper domain 和Sp1 相結合，因此我們便有興趣再進一步探討c-Jun C 端的磷酸化是否會影響c-Jun/ Sp1 之間的interaction。為了確認此一機制，便利用了E. coli純化出含GST tag 的c-Jun，TAM-67，TAM-67-M3D 以及TAM-67-M3A 等融合蛋白，以casein kinase II ( CKII ) 將GST 融合蛋白的C 端磷酸化，再以GST-pull down 的分析方法，將處理或未處理CKII 的GST 融合蛋白與A431 細胞核中的 Sp1 反應，觀察c-Jun C 端的磷酸化是否會對c-Jun/ Sp1 之間的
interaction 有所影響? 初步的實驗結果發現受CKII 磷酸化後的 GST-c-Jun 以及GST-TAM-67 與細胞核中Sp1 的結合能力較未磷酸化的GST-c-Jun 以及GST-TAM-67 為弱，而GST-TAM-67-M3D 為模擬C 端磷酸化的TAM-67，其與細胞核中Sp1 結合能力較未磷酸化的GST-TAM-67 為
弱，另一方面，GST-TAM-67-M3A 為模擬C 端去磷酸化的TAM-67，其與細胞核中Sp1 的結合能力較磷酸化的GST-TAM-67 為強，顯示磷酸化後的c-Jun 的確會減弱c-Jun/ Sp1 之間的interaction。
|
| 英文摘要 |
In intact cells, the modification of protein
phosphorylation and dephosphorylation by protein kinase or protein phosphatases either positively or negatively regulate its activity. In our study, we have found that a specific calcineurin
(PP2B) inhibitor, cyclosporin A, could inhibit PMA- or EGF-induced promoter activity of 12( S)-lipoxygenase gene and interaction between Sp1 and c-Jun, which indicate that the interaction between Sp1 and c-Jun might be regulated by phosphorylation and dephosphorylation of c-Jun. Early studies show that the phosphorylation of the N-terminal transactivation domain of c-Jun
by JNK causes a conformational change in c-Jun that facilitates the dephosphorylation of the C-terminus residues. Furthermore,the documents previously reported that the leucine zipper domain on c-Jun C-terminus was essential for interaction between c-Jun and Sp1. So we studied the role of the modification at c-Jun C-terminal domain in c-Jun/ Sp1 interaction. This mechanism was studied in vitro by using GST of c-Jun, TAM-67, TAM-67-M3D and TAM-67-M3A produced from E. coli. The C-terminal phosphorylation of c-Jun was mediated by casein kinase II (CKII). The interaction between Sp1 and modified c-Jun was determined by GST-pull down assay. We found that c-Jun/ Sp1 interaction was negatively regulated by phosphorylation of c-Jun C-terminus. On the other hand, TAM-67-M3D, which is a mutant of N-terminal deletion of TAM-67 with three substitutive aspartic acid residues at Thr-231,
Ser-243, and Ser-249, interacted with Sp1 less than TAM-67.Therefore, the phosphorylation and dephosphorylation of the C-terminus of c-Jun is important for c-Jun/ Sp1 interaction。
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| 論文目次 |
目錄
中文摘要---------1
英文摘要---------2
縮寫檢索---------4
第一章 緒論
1-1 研究動機-------5
1-2 研究目的-------5
1-3 研究範圍-------6
1-4 研究方法與內容-6
第二章 文獻之回顧
2-1. Lipoxygenase 的生理功能及分類--------------7
2-2. EGF 和PMA 於子宮頸上皮癌細胞的訊息傳導途徑-8
2-3. 磷酸化/去磷酸化對c-Jun 及Sp1 的調控--------9
2-4. 磷酸化/去磷酸化對c-Jun/Sp1 interaction 的影
響---------------------------------------------10
2-5. Casein kinase II 之相關研究文獻-----------11
第三章 實驗材料--------------------------------13
第四章 實驗方法
第一節 細胞培養及組織蛋白萃取------------------18
第二節 Glutathione S-transferase 融合蛋白之表現基因建構工程-------------------------------------24
第三節 Glutathione S-transferase 融合蛋白之表現與純化-------------------------------------------29
第四節 利用casein kinaseII assay 作in vitro 磷酸化---------------------------------------------32
第五節 GST-pull down assay --------------------33
第六節 Casein kinase II / GST-pull down assay -33
第五章 實驗結果
第一節 探討利用E.coli表現出的GST-c-Jun、GST-TAM-67、GST-TAM-67-M3A和GST-TAM-67-M3D 與細胞核中的Sp1 結合關係
1-1. GST-fusion protein 的純化-----------------35
1-2. GST-fusion protein 與Sp1 的結合-----------36
第二節 探討GST 融合蛋白在in vitro受casein kinaseII 磷酸化的情形--------------------------37
第三節 探討c-Jun C 端的磷酸化對c-Jun /Sp1 interaction的影響
3-1. GST-c-Jun 與GST-TAM-67 受casein kinase II 磷酸化之後與Sp1結合的影響------------------------39
3-2. 模擬磷酸化狀態的GST-TAM-67-M3D 及去磷酸化狀態GST-TAM-67-M3A 與Sp1 結合的探討--------------39
第六章 總結與討論------------------------------41
參考文獻---------------------------------------44
附圖-------------------------------------------52
圖目錄
Table 1. Expression condition of wt and mutated GST-c-Jun proteins-----------------------------52
Fig.1 Mammalianlipoxygenase--------------------53
Fig.2 Signal transduction of EGF and PMA-induced gene expression of 12(S)-lipoxygenase----------54
Fig.3 Physical interaction between c-Jun and Sp1 is mediated via the bZip region of c-Jun and one of the two homologous Gln- and Ser/Thr-region and/or the DNAbinding domain of Sp1------------55
Fig.4 Effect of cyclosporin A on PMA-induced expression of 12(S)-lipoxygenase in A431 cells. GST-pull down assay----------------------------56
Fig.5 Effect of cyclosporin A on the interaction between c-Jun and Sp1 in cells-----------------57
Fig.6 GST-pull down assay----------------------58
Fig.7 Schematic representation of the wt or mutated c-Jun proteins ------------------------59
Fig.8 Expression profile of the wt and mutant GST-c-Jun forms in bacterial strain DH-5αand BL-21---------------------------------------------60
Fig.9 Analysis of physical interactions between Sp1 with wt and mutated c-Jun proteins---------61
Fig.10 Analysis of physical interactions between Sp1 with mutated c-Jun proteins----------------62
Fig.11 Phosphorylation of GST-c-Jun and GST-TAM-67 by casein kinase II in cell free system-----63
Fig.12. Phosphorylation of mutated GST-c-Jun proteins by casein kinase II in cell free system------------------------------------------------64
Fig.13 CKII / GST-pull down assay--------------65
Fig.14 Interaction between phosphorylation/
nonphosphorylation wt or mutated c-Jun proteins with nuclear Sp1 protein-----------------------66
Fig.15 The dephosphorylation of c-Jun in C – terminus enhanced its interaction with Sp1-----67
Fig.16 Analysis of the effect of c-Jun C-terminus three phosphorylation sites on the interaction between c-Jun and Sp1.-------------68
Fig.17 Analysis of the effect of c-Jun C-terminus three phosphorylation sites on the interaction between c-Jun and Sp1--------------69
Fig.18 Phosphorylation at the three phosphorylation sites of c-Jun C-terminus decreased its interaction with Sp1-------------70
|
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