臺灣博碩士論文加值系統

本論文所要探討的LMP2A(latent membrane protein)蛋白質，是與人類惡性腫瘤相關之EB病毒潛伏期中所表現的其中一種基因產物。LMP2A蛋白質的結構中，胺基端包含許多具功能性的特異區域(motif)，對於LMP2A與其它細胞內蛋白質交互作用是相當重要的。根據報導指出，LMP2A藉由與Src 族的酪胺酸激酶，如Lyn、Syk交互作用，進而阻斷B細胞受器所傳遞的訊息，而在基因轉殖小鼠中，LMP2A可提供B細胞存活的訊息。雖然LMP2A的胺基端對於傳遞下游訊息扮演重要角色，但對於可與LMP2A胺基端交互作用之細胞蛋白及其相關之分子機制的研究仍然相當有限，因此我們嘗試找尋可與LMP2A結合的其他細胞蛋白質，並研究這些結合蛋白在LMP2A傳遞的訊息路徑中扮演之角色。
利用酵母菌雙雜交法 (Yeast two hybrid) 初步篩選可能與LMP2A有交互作用的蛋白質。在篩選的結果中，其中兩個可能與LMP2A結合的蛋白質，分別是RACK1(Receptor for activated C kinase 1)及UbA80 (Ubiquitin A 80)。而利用GST沉澱法 (Glutathione-S-Transferase pull down assay) 及共同免疫沉澱法(co-immunopricipitation assay)可分別在體外( in vitro)或體內(in vivo)實驗中進一步證實LMP2A與RACK1有交互作用。除此之外，亦利用免疫螢光染色法(immunofluorescent assay)觀察到LMP2A和RACK1在細胞內之表現區域有重疊的現象。因此體外及體內的實驗結果皆證實LMP2A與RACK1有交互作用。
由實驗室以前結果得知，LMP2A的表現會增加JNK(c-Jun N-terminal kinase)和ERK(extracellular signal-regulated kinase)蛋白質的磷酸化。因為內生性RACK1在細胞內表現量已有相當量，為了探討RACK1對於LMP2A活化JNK及ERK的重要性，因此在穩定表現LMP2A的細胞中，利用針對RACK1的微型干擾RNA (siRNA) 降低細胞內RACK1基因的表現。相較於對照控制組，若降低RACK1基因表現，則會減弱LMP2A增加JNK及ERK磷酸化之能力。因此RACK1不只與LMP2A交互作用，對於LMP2A活化JNK及ERK的訊息路徑中也扮演重要的角色。
本論文的研究，乃首篇報導LMP2A可與RACK1結合，並可能藉此交互作用來增強JNK及ERK的磷酸化。往後的研究可承接此點再深入研究，以求了解更多有關LMP2A和RACK1之間的交互作用對細胞的生物意義。有關RACK1在LMP2A所造成的生物現象中扮演何種角色尚待後續的研究做更進一步完整的探討。

Latent membrane protein 2A (LMP2A) is one of the latent proteins of Epstein-Barr Virus (EBV), which is an oncogenic virus associated many human malignancies. The N-terminal of LMP2A possesses several functional motifs which are critical for its interactions with various cellular proteins. Through interactions with cellular proteins such as Lyn and Syc, LMP2A can block downstream signaling pathways triggering by B cell receptors. In the LMP2A transgenic mice model, LMP2A can provide pro-survival signal for B cell growth in peripheral center. Duce the scaffold protein structure of LMP2A, we believe that LMP2A has potential to interact with many kinds of celluar proteins. Based on this assumption, we would like to investigate these novel LMP2A-interacting proteins and reveal their roles in LMP2A-triggered signaling pathways.
In order to pursue this goal, yeast-two-hybrid assay was performed to screen possible cellular proteins interacting with LMP2A. Among 11 prelimary LMP2A-interacting proteins, the receptor for activated C kinase 1(RACK1) futher was chosen for the investigation. GST-pull-down assay and co-immunoprecipitation assay were utilized to confirm the interaction between LMP2A and RACK1 in vitro and in vivo, respectively. In addition, co-localization of LMP2A and RACK was also observed in the immunofluorescent assay. These results all proved LMP2A can interact with RACK1 both in vitro and in vivo.
According to our previous studies, LMP2A could enhance the phosphorylation of cellular c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In order to elucidate if RACK1 is involved in LMP2A-mediated JNK and ERK phosphorylation, RACK1-targeted siRNA was approached. The state of LMP2A-induced JNK and ERK phosphorylation was significantily reduced while the cellular RACK1 was dimished due to the transfection of siRACK1 into the LMP2A-expressing stable line.This result indicated that RACK1 was acting not only as an interacting protein but also as a signaling mediator required for LMP2A-induced JNK and ERK phosphorylation.
This is the first demostrate that cellular protein RACK1 can interact with LMP2A and RACK1 is involved in the signaling pathway of LMP2A-triggering JNK and ERK phosphorylation. However the molecular mechanism of involvement of RACK1 in LMP2A-triggering signaling needs more investigation.

中文摘要……………………………………………………………………Ⅰ

英文摘要……………………………………………………………………Ⅲ

序論………………………………………………………………………….1

研究目的…………………………………………………………………… 8

實驗材料…………………………………………………………………… 9

實驗方法……………………………………………………………………17

結果…………………………………………………………………………26

討論…………………………………………………………………………31

圖表…………………………………………………………………………35

參考文獻……………………………………………………………………54

附錄…………………………………………………………………………60

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