李玉梅臺灣大學：生化科學研究所張峰銓Chang, Feng-ChuanFeng-ChuanChang2007-11-262018-07-062007-11-262018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/52780RNF4 是一種在C端具有RING finger 的蛋白，目前已知可以藉由與細胞核中的不同蛋白作用來調控基因的轉錄表現；RNF4被認為是轉錄機制中的協同抑制因子或是活化因子。根據我們過去的研究，我們發現RNF4參與GCH此基因的轉錄表現，而且這樣的活化過程是藉由GCH基因上游啟動子中的CCAAT-box來達成。由於RNF4對於CCAAT-box 此區域並沒有專一性的辨認，所以我們認為RNF4是透過輔助CCAAT-box專一性蛋白的NF-Y來達成此轉錄機轉。綜合來說，我們認為RNF4在GCH基因上的啟動子扮演重要的角色。 在這次的研究中，我發現藉由蛋白質激酶PKA對RNF4的磷酸化可以促進含有CCAAT-box的GCH基因轉錄表現。從實驗上的結果，我們知道突變態的RNF4蛋白會影響蛋白質激酶PKA對RNF4的磷酸化，而進一步影響RNF4在GCH基因上的活化表現。我也發現S127、S166是蛋白質激酶PKA磷酸化的重要位置。除此之外，我還發現RNF4蛋白的C端鹼性區域對於其本身的活化與受到PKA的磷酸化相當重要。 總結來說，我們首次證明RNF4是受到訊息傳遞中的蛋白質激酶PKA磷酸化進而活化GCH基因的表現。The small nuclear RING finger protein 4 (RNF4) is a recently identified human RING finger protein that contains C3HC4-type zinc finger motif in the C-terminal region. RNF4 has been implicated in transcriptional regulation through interacting with a variety of nuclear proteins, and functions as a transcriptional corepressor or coactivator. According to our previous study, we found that coactivator RNF4 is involved in the GTP cyclohydrolase I (GCH) gene expression and we defined the RNF4-responsive element on GCH proximal promoter as a CCAAT-box. RNF4 did not possess specific DNA binding activity toward this CCAAT box, which suggests that RNF4 is a coactivator of the CCAAT box binding protein nuclear factor Y (NF-Y). Overall, we prove that RNF4 imposes functional importance on GCH promoter. In this study, I demonstrated that protein kinase A (PKA)-dependent phosphorylation of RNF4 activates the CCAAT-box containing GCH promoter. Results from biological and functional characterizations of RNF4 mutants defective in PKA phosphorylation sites suggest that PKA-dependent RNF4 phosphorylation is important for its role in GCH promoter activation. S127 and S166 of RNF4 are important phosphorylation sites in RNF4-mediated CAAT-box activation by PKA. Furthermore, I also found that the C-terminal basic region is essential for RNF4 phosphorylation and activation by PKA. In summary, we first illustrate a novel RNF4-mediated PKA signaling pathway that leads to the activation of GCH promoter.EnglishAbstract……………………………………………………………………...3 ChineseAbstract……………………………………………………………………..4 Abbreviations…………………………………………………………………………5 1. Introduction 1.1 RNF4 …...……….………………………………………………………………...6 1.2 The RING finger motif…………………………………………………………….8 1.3 GTP cyclohydrolaseⅠ…………………………………………………………..10 1.4 RNF4 and GCH promoter………………………………………………………..12 1.5 CCAAT-Box……………………………………………………………………...13 1.6 NF-Y……………………………………………………………………………..14 1.7 RNF4 and NF-Y………………………………………………………………….16 1.8 PKA pathway……………………………………………………………………..16 2. Material and Methods 2.1 Plasmid constructs………………………………………………………………..19 2.2 Cell culture……………………………………………………………………….19 2.3 Plasmid DNA purification………………………………………………………..20 2.4 Expression and Purification of Fusion Protein…………………………………...21 2.5 In vitro phosphorylation………………………………………………………….21 2.6 Transfection………………………………………………………………………22 2.7 Transient transfection…………………………………………………………….22 2.8 Transcriptional Activation and Luciferase Assay………………………………...23 2.9 Transformation…………………………………………………………………...23 2.10 In vitro translation………………………………………………………………23 3. Results 3.1 Forskolin up-regulates the activity of RNF4 through CCAAT-box……………...24 3.2 RNF4 is phosphorylated in cells…………………………………………………25 3.3 RNF4 is phosphorylated by PKA in vitro………………………………………..26 3.4 Identification of RNF4 residues phosphorylated by PKA……………………….26 3.5 PKA-mediated RNF4 activation of the CCAAT-box by phosphorylation on S127 and S166………………………………………………………………………….27 3.6 Cross-talk to MAPK signaling induction by PKA pathways…………………….28 3.7 The C-terminal basic region is essential for RNF4 phosphorylation and activation by PKA…………………………………………………………………………...29 3.8 Interaction with TBP is increased by RNF4 phosphorylation…………………....30 4. Discussion 4.1 Phosphorylation of RNF4 could contribute to CCAAT-box related transcription………………………………………………………………………31 4.2 Possible mechanisms for phospho-RNF4 mediated enhancement of CCAAT-box………………………………………………………………………32 4.3 MAPK signaling pathway may play a role in regulating the activity of RNF4….33 4.4 Other biological function of phosphrylated RNF4……………………………….34 5. Reference……………..........................................................................................35 6. Figure……………………………………………………………………………40 7. Appendix………………………………………………………………………..53986532 bytesapplication/pdfen-US協同抑制因子活化因子磷酸化RNF4PKAphosphorylationotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52780/1/ntu-95-R93b46008-1.pdf