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研究生：	黃祺真 Huang, Chi-Chen
論文名稱：	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 Effect of c-Jun C-terminus phosphorylation/ dephosphorylation on the interaction between c-Jun and Sp1
指導教授：	張文昌 Chang, Wen-Chang
學位類別：	碩士 Master
系所名稱：	醫學院 - 藥理學研究所 Department of Pharmacology
論文出版年：	2003
畢業學年度：	91
語文別：	中文
論文頁數：	70
中文關鍵詞：	交互作用、去磷酸化、磷酸化
外文關鍵詞：	GST-pull down assay, c-Jun, Sp1, casein kinase
相關次數：	點閱：104 下載：2
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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。

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。

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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校內：2004-08-08公開
校外：2004-08-08公開

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