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系統識別號 U0026-0812200915033034
論文名稱(中文) 4E-BP3蛋白在人類細胞內扮演的生物功能之研究
論文名稱(英文) Biological Function of 4E-BP3 in Human Cell Lines
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 97
學期 1
出版年 98
研究生(中文) 王亭凱
研究生(英文) Ting-Kai Wang
電子信箱 s1695501@ccmail.ncku.edu.tw
學號 s1695501
學位類別 碩士
語文別 中文
論文頁數 62頁
口試委員 指導教授-張敏政
口試委員-賴明德
口試委員-鄭宏祺
中文關鍵字 mRNA 運送  4E-BP3  eIF4E  cyclin D1 
英文關鍵字 cyclin D1  eIF4E  4E-BP3  mRNA transport 
學科別分類
中文摘要 4E-BP3 是4EBP家族中最晚被發現的成員,此家族成員的共同特性是會與eIF4G競爭eIF4E的結合為致使其無法形成eIF4F轉譯複合體進行細胞內的cap-dependent轉譯機制。4E-BP3跟4E-BPs家族中研究最為透徹的4E-BP1的不同,為4E-BP3除了細胞質也會存在於細胞核中。由於4E-BP3具有與eif4E結合的能力,我們認為4E-BP3在核內可以去抑制eIF4E幫助運送特定mRNA的功能,會被eIF4E調控的mRNA大多與細胞生長週期相關,例如cyclin D1,我們驗證了在U2OS, HEK293T, 以及MCF7中大量表現4E-BP3會使cyclin D1的蛋白濃度降低,而mRNA 的濃度分析顯示cyclin D1的mRNA 濃度並無改變。另外我們也有興趣知道4E-BP3的功能是否會受到本身的磷酸化狀態所調控。利用定位點突變技術在4E-BP3第23 與32個位置修改4E-BP3模擬其磷酸化(4E-BP3 2D)與去磷酸化(4E-BP3 2A)的狀態。利用免疫沈澱法,我們觀察到2D對於eIF4E有較強的結合力,而2A與eIF4E的結合力則有下降的趨勢。將2A與2D送入細胞中可以觀察到,表現2D的細胞cyclin D1蛋白的濃度明顯低於表現2A細胞的cyclin D1濃度,顯示4E-BP3的生理功能很有可能透過磷酸化來調控,但目前對於其磷酸化的機制仍然不明。最後,我們利用了GST pull down的方法證實了4E-BP3與RPA2蛋白有結合的能力,並且此交互作用不需透過eIF4E的參與
英文摘要 The protein 4E-BP3 is the last identified member of 4E-BP (eIF4E[eukaryotic initiatioln factor 4E]-binding proteins) family. A common property of 4E-BP family is the ability to compete with eIF4G for eIF4E binding which hinders the forming of eIF4F translation complex. The eIF4F translation complex is a critical machinery for cap-dependent translation and the binding of 4E-BPs would repress its role in translation. Unlike 4E-BP1, which only exists in cytoplasm fraction, 4E-BP3 can be found in both nucleus and cytoplasmic fractions and bound to eIF4E in both fractions. A recent study has shown that nuclear eIF4E could promote the nucleo-cytoplasmic export of a subset of growth-promoting mRNA including cyclinD1. In our study we found out that both transient and stable overexpression of 4EBP3 in U2OS cell resulted in decreased in cyclinD1 protein level where its total mRNA level remained constant. Similar result also observed in HEK293T and MCF 7 cell line. 4E-BP3 is also a phospho-protein and supposed to have the similar phosphorylation sites as 4E-BP1. However, unlike 4E-BP1 and 4E-BP2 whose phosphorylations are induced by insulin, the phosphorylation of 4EBP3 is insensitive to insulin. In comparison to hypo phorylated form of 4E-BP3, the hyper-phosphorylated 4E-BP3 has a higher binding affinity with eIF4E. In constrast, hyper-phosphorylated form of 4E-BP1 showed a lower binding affinity with eIF4E compared to dephosphorulated form of 4E-BP1. In this study we found that ectopic expression of T23A/T32A mutant form of 4EBP3 resulted in increased in cyclin D1 protein level, where as ectopic expression of T23D/32D mutant form of 4EBP3 resulted in decreased cyclin D1 protein level. The result suggested the phosphorylation of 4EBP3 is critical for the formation of 4EBP3-eIF4E complex and 4EBP3 mediated the cyclinD1mRNA transcription. The earlier yeast-two-hybrid result also suggested that 4EBP3 binds with RPA2 (replication protein A2). This interaction between RPA2 & 4EBP3 was re-confirmed by in-vitro GST pull down.
論文目次 中文摘要 i
Abstract ii
致謝 iv
圖目錄 vii
附錄目錄 viii
縮寫檢索表 ix
第一章 序論 1
1-1 Eukaryotic translation initiation factor 4E (eIF4E) 1
1-1-2 細胞核內eIF4E調控RNA機制 2
1-2 eIF4E Binding Protein (4E-BPs) 3
1-2-1 4E-BPs的磷酸化機制 5
1-2-2 4E-BP3 與 4E-BP1, 4E-BP2的不同處 6
1-2-3 4E-BP3 與RPA2 的交互作用 7
1-3 研究動機 9
第二章 材料與方法 10
2-1 使用之菌株、載體及培養基 10
1-1-1 菌株 10
1-1-2 載體 10
1-1-3 培養基 10
2-2 質體 DNA 在大腸桿菌的轉形作用 (Transformation) 11
2-3 少量質體 DNA 的抽取 13
2-4 細胞解凍與保存 14
2-5 細胞繼代培養 材料與試劑 15
2-6 細胞數目測定 17
2-7 轉染 (transfection) 質體至細胞中 18
2-8 收取細胞 (Harvesting cell lysate) 18
2-9 細胞核及細胞質內蛋白質萃取 19
2-10 蛋白質濃度的定量 (Bradford, 1976) 21
2-11 SDS-PAGE 之蛋白質分子量分析 21
2-12 西方墨點法 (Western blotting) 23
2-13 免疫沈澱 (immunoprecipitation) 26
2-14 GST pull down 27
2-15 使用之細胞株 28
2-16 細胞 RNA 的萃取 29
2-17 RT-PCR 30
第三章 實驗結果 31
3-1 細胞內4E-BP3對cyclin D1的影響 31
3-2 對4E-BP3做點突變改造模擬其磷酸化狀態 32
3-3 定位點突變4E-BP3對與eIF4E的交互作用情形 32
3-4 定位點突變4E-BP3對細胞內cyclin D1 蛋白的影響 33
3-5 定位點突變4E-BP3對cyclin D1的影響非透過對轉錄的抑制 34
3-6 外送 4E-BP3在實驗結果上的不穩定性 34
3-7 4E-BP3接上入核訊號(NLS)穩定實驗結果 35
3-8 使用GST pull down 證實4E-BP3與RPA2的in vitro交互作用 36
第四章 討論 37
參考文獻 41
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