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研究生:巫善福
研究生(外文):Shan-Fu Wu
論文名稱:p78能誘導Nrf1在Ser-54位置的磷酸化
論文名稱(外文):Induction of Nrf1 Phosphorylation at Ser-54 by p78
指導教授:許濤許濤引用關係林陽生
指導教授(外文):Todd HsuYoung-Sun Lin
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:46
中文關鍵詞:Nrf1p78磷酸化蛋白質累積
外文關鍵詞:Nrf1p78phosphorylationprotein accumulation
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Nrf1是屬於CNC-bZIP家族的一員,此家族可與small Maf蛋白質形成異複合體並結合到球蛋白基因的LCR及抗氧化基因的ARE啟動子上的NF-E2/AP-1 like區域。剔除nrf1基因會導致小鼠貧血和胚胎期致死,而缺乏nrf1基因的細胞中抗氧化基因表現量較正常細胞少。之前已被證實Nrf1與p78交互結合的關係,並且在同時表現Nrf1和p78的293T細胞中,我們觀察到Nrf1會有累積和被修飾的現象。在此研究中,我們證實:蛋白質受p78影響的累積是廣泛性效應,對Nrf1並不具有專一性。另外藉由磷酸水解酶處理得知,和p78同時出現的Nrf1修飾型態為磷酸化。這Nrf1的磷酸化可被抗Ser/Thr磷酸化抗體所辨識,以定點突變確認此磷酸化是在Nrf1 Ser-54的位置,而ERK1/2磷酸酶抑制劑能阻斷此磷酸化。LexA-Nrf1正常型和S54A突變都能活化報告基因,且此能力都會受到p78的抑制。Nrf1的磷酸化在p78的活性抑制調控中是非必需的。綜合以上結果我們知道,核仁蛋白p78會誘導Nrf1在Ser-54位置的磷酸化,我們並推測ERK1/2是參與在這磷酸化中。
Nrf1 belongs to the CNC-bZIP family of transcription factors. CNC family members heterodimerize with small Maf proteins and these heterocomplexes bind to the NF-E2/AP1-like motif. This motif is contained in the LCR of globin genes and ARE of antioxidant genes. Disruption of nrf1 gene in mouse leads to anemia and embryonic lethality and nrf1-deficient cells display reduced expression of antioxidant genes. Previous study in our laboratory has identified one Nrf1-interacting protein, p78. When Nrf1 and p78 are co-expressed in 293T cells, two unusual phenomena are observed. First, accumulation of Nrf1 was observed; second, a slow-mobility Nrf1 species was detected. It was demonstrated that the effect of p78 on protein accumulation was a general effect, not specific for Nrf1. Treatment of cell lysate with CIP resulted in the elimination of the slow-mobility Nrf1, indicating that it is a phosphorylated species of Nrf1. The phosphorylated form of Nrf1 was further confirmed by anti-phospho-Ser/Thr antibody. Moreover, substitution of Ser-54 to Ala suppressed this phosphorylation of Nrf1. ERK1/2 kinase inhibitor, apigenin, significantly blocked the phosphorylation of Nrf1. Both WT and S54A Nrf1-LexA fusion proteins transactivated LexA reporter gene and the transactivation abilities of both were repressed by p78. Phosphorylation of Nrf1 is non-essential for p78-mediated repression. In summary, phosphorylation of Nrf1 at Ser-54 coincides with the expression of p78 and we propose that ERK1/2 may be involved in the phosphorylation of Nrf1.
List of Abbreviations ...............3
Chinese Abstract ...............4
English Abstract ...............5
Introduction ...............6
Materials and Method ...............13
Results ...............20
Discussions ...............25
References ...............28
Figures ...............34
Appendixes ...............44
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