呂勝春Lee, Sheng-Chung臺灣大學:分子醫學研究所鄭雅云Cheng, Ya-YunYa-YunCheng2010-05-042018-07-092010-05-042018-07-092009U0001-2707200913154600http://ntur.lib.ntu.edu.tw//handle/246246/178740細胞面臨 stress 時，會啟動unfolded protein respons機制以協助細胞度過困厄的環境。此機制主要是藉由轉錄和轉譯兩個層面來調控下游基因的表現，這些基因的功能著重於協助蛋白質結構的折疊、失活蛋白的降解以及細胞的凋亡三方面。 CHOP 即為其中一員，其功能與細胞凋亡有關，CHOP 本身除了受轉錄調控之外，亦受到其基因上游 uORF 的轉譯調控。據本實驗室未發表的實驗成果得知：細胞在處理 anisomycin 的情況下，能誘導受uORFchop 轉譯調控的下游基因大量表現，而在這樣的轉譯調控機制中，轉譯啟始因子eIF4E、eIF2α 和 4E-BP1的磷酸化顯得十分重要。本篇論文討論的重點在於發現— AMPK，一個參與眾多環境壓力相關的重要蛋白，也參與 anisomycin 所引發的訊息傳遞途徑中，並深切影響著 uORFchop 的轉譯調控。在 anisomycin 的處理下，適度活化的AMPK會透過抑制 PP2C β1（去磷酸酶）來增強 p38 MAPK-Mnk-eIF4E 的訊息傳遞途徑，進而影響uORFchop所調控的轉譯表現。此外，在 AICAR 的刺激下，高度活化的AMPK同時也會經由 mTOR 訊息傳遞途徑去抑制此轉譯表現機制，故依我們的實驗結果推測得知：在 anisomycin 刺激下，AMPK 在 uORFchop 的轉譯表現中扮演著一個正向調控的角色。Unfolded protein response (UPR) regulates gene expression through transcriptional and translational control and results in ER stress recovery or cell apoptosis. CHOP is one of the components that involves in the ER stress-mediated pathway. Upstream open reading frame (uORF) of the CHOP plays an essential role in controlling the protein expression via translation. We have shown that anisomycin-induced uORF-mediated CHOP translation depends on the phosphorylated eIF4E/S209, eIF2α and 4E-BP1. In this report, we uncovered that AMPK is involved in the induction of uORF-mediated CHOP translation under anisomycin treatment. When moderately activated by anisomycin, AMPK maintains the phosphorylated level of p38 MAPK, Mnk, and eIF4E/S209 by negatively regulate phosphatase PP2Cβ1 activity. When fully activated by AICAR, AMPK inhibits both PP2C β1 and mTOR activities, leading to inability of dissociation of the eIF4E-4EBP1 complex and repression of translation initiation complex formation. Taken together, the present results indicate that anisomycin-activated AMPK plays positive regulatory roles in uORF-mediated CHOP translation.致謝 II要 IIIbstract IVontents Vntroduction 1aterial and methods 7lasmids and Constructs 7ell culture and transfection 9hemicals Treatment 9reparation of Whole Cell Extraction 10mmunopreciptation assay 10DS-PAGE 11estern blot analysis 12ntibodies 13uciferase assay 13esults 14he AMPK inhibitor, compound C, represses anisomycin induced uORF-mediated luciferase expression and reduces the phosphorylation levels of p38 MAPK, Mnk, eIF4E/S209. 14nisomycin and AICAR induced AMPK regulates uORFchop-mediated luciferase expression through mTOR pathway. 15ICAR treatment results in decrease of eIF4F formation (i.e., the association of eIF4G and eIF4E). 16he functions of AMPK in anisomycin-induced CHOP expression. 17P1 and PP2A are not AMPK downstream phosphatases that regulate p38 MAPK-Mnk-eIF4E pathway by anisomycin treatment. 17P2C β1 may be the downstream phosphatase of AMPK negatively regulates anisomycin stimulated p38 MAPK-Mnk-eIF4E pathway. 18iscussion 20eferences 24ist of Figures 31igure 1. Compound C represses anisomycin-induced uORFchop-regulated luciferase expression. 31igure 2. Anisomycin treatment activates AMPK activity, while compound C represses anisomycin-induced p38 MAPK, Mnk, eIF4E phosphorylation. 32igure 3. The AMPK activator, AICAR, represses anisomycin induced CHOP uORFchop-regulated luciferase expression but increases the phosphorylation of p38 MAPK, Mnk, and eIF4E. 35igure 4. AMPK influences uORFchop-regulated luciferase expression through mTOR pathway under anisomycin and AICAR co-treatment. 37igure 5. Decreased association of eIF4G to eIF4E by AICAR treatment. 38igure 6. AMPK mutants mimics anisomycin-induced CHOP expression. 40igure 7. PP1 and PP2A are not the AMPK downstream targets that regulate the p38 MAPK-Mnk-eIF4E pathway under anisomycin treatment. 42igure 8. PP2C β1 may be the downstream phosphatase of AMPK. 44igure 9. Proposed model for anisomycin-induced AMPK signaling pathways for activation of uORFchop-mediated translation. 45application/pdf2847475 bytesapplication/pdfen-US轉譯調控壓力AMPKanisomycinuORFhttp://ntur.lib.ntu.edu.tw/bitstream/246246/178740/1/ntu-98-R96448007-1.pdf