中文摘要

　　4E-BP3為一個分子量14.6 kDa之蛋白，屬於4E-BPs家族的一員。過去研究發現去磷酸化的4E-BPs會與eIF4G競爭eIF4E的結合位置，使得cap-mRNA無法帶入43S pre-initiation complex，因而抑制轉譯的速率，對於細胞生長有負向調節的功能。因此，4E-BP3可以說是轉譯作用的一個抑制因子。本實驗室先前利用Yeast-Two Hybrid之方法，成功地由人類乳腺細胞基因庫中，篩選出另一個為32-kDa的replication protein A ( RPA) 次單元體-RPA2，會與4E-BP3有交互作用。RPA為一單股DNA結合蛋白，普遍存於真核細胞之細胞核中，主要是由RPA1、RPA2及RPA3三個次體組成一穩定複合物，且其功能牽涉到DNA複製、修補以及基因重組等生理反應。主要與單股DNA產生結合能力的為RPA1，而RPA2則是當細胞進入S phase時，會有磷酸化的現象產生，直到M phase的晚期才去磷酸。若是當細胞遭受到紫外光 (UV) 、游離輻射 (IR) 的照射，而對細胞造成損傷時，RPA2則會有高度磷酸化現象的發生。在本實驗中, 首先利用免疫沈澱的方法，確認了RPA2和4E-BP3發生交互作用是在細胞核內。以m7-GTP sepharose沈澱法則觀察到RPA2/4E-BP3在細胞核內的交互作用也包含 eIF4E。接著，利用先前所建立在HEK293會持續穩定表現4E-BP3蛋白的細胞株(SBP3)來進行去除生長因子(starvation) 的實驗。根據實驗結果，當SBP3處於starvation時，4E-BP3在Thr-23的磷酸化會減少，但與eIF4E的結合能力卻也相對的下降; 這是與4E-BP1非常不同的。另一方面，當SBP3施予starvation時，4E-BP3和RPA2在細胞核內的交互作用則約有百分之九十的增加。由於4E-BP1只存在於細胞質，而4E-BP3則可存在於細胞質和細胞核。因此，我們推測4E-BP3的生理功能更為複雜，而其調控機制則有待更進一步的探討。在細胞受曝照UV的刺激模式實驗中，根據細胞曝射UV後不同時間點的觀察，在外送4E-BP3蛋白的HEK293細胞顯示，RPA2和4E-BP3在細胞核內失去結合關係則發生的相當快，經UV刺激後十五分鐘即產生變化，約在UV 照射二十分鐘內即完全失去結合能力; 然而，RPA2則在UV照射兩小時後開始有高度磷酸化現象。由上述的實驗結果，我們初步推測RPA2和4E-BP3的交互作用並非取決於RPA2磷酸化狀態。為了更進一步了解內生性4E-BP3的調控機制是否和超表現的4E-BP3類似，我們使用一株人類前列腺癌細胞-LNCaP來做實驗，而此細胞已被證實有大量內生性4E-BP3的表現。截至目前為止，我們發現LNCaP在正常培養狀況時，不論是在細胞質或細胞核，4E-BP3在Thr-23都呈現高度磷酸化，而當LNCaP處於starvation時，4E-BP3在Thr-23的磷酸化則也是減少的現象，與SBP3中觀察到的一樣。然而，不論LNCaP進行starvation 二十四小時或四十八小時，4E-BP3分別與eIF4E、RPA2的交互作用似乎沒有變化。因此，我們推測LNCaP是一株對於血清不敏感的癌細胞。但若施予介白素-六 (interlukin-6)刺激，我們觀察到STAT3會先被活化，伴隨著4E-BP3在Thr-23的磷酸化下降;而此時，RPA2和4E-BP3的交互作用則有顯著的提高。另外，對LNCaP 細胞使用PI3K的抑制劑-LY294002則發現: 4E-BP3在Thr-23位置的磷酸化只受到局部的抑制。除此之外，我們利用定點突變的方法來決定4E-BP3蛋白本身哪些磷酸化區域對於4E-BP3/eIF4E以及4E-BP3/RPA2交互作用是最重要的。關於4E-BP3在LNCaP細胞受到不同的刺激下所引發的磷酸化機制及訊號傳遞則有待更進一步的印證和探討。

Abstract

　The eukaryotic initiation factor 4E-binding protein 3 (4E-BP3), a member of 4E-BPs family, is a 14.6-kDa protein that is able to compete with eIF4G for binding to eIF4E (eukaryotic initiation factor 4E), thereby stifling eIF4F complex-directed translation. Therefore, the function of 4E-BP3 is a repressor of cap-dependent translation. In our previous studies, a yeast two-hybrid system using a 32-kDa subunit of replication protein A (RPA2) as a bait was performed and the results showed that RPA2 is able to interact with the 4E-BP3. RPA is a heterotrimeric (70-, 32- & 14-kDa subunits) single-stranded DNA-binding protein that is required for DNA replication, recombination and repair. The primary ssDNA binding activity is localized to the 70-kDa subunit (RPA1). However, RPA2 is phosphorylated in a cell cycle dependent manner and is additionally phosphorylated in response to DNA damage. Hyperphosphorylation of RPA2 occurs in response to DNA-damaging agents, such as UV or IR treatment. In this study, in vitro immunoprecipitation assay was performed to confirm that RPA2-4E-BP3 complex was located in the nuclear fraction. M7-GTP sepharose pull down assay showed that the complex of RPA2 and 4E-BP3 isolated from nuclear fraction of the HEK293 cell containing overexpression of His/c-Myc- tagged 4E-BP3 (SBP3), also containing eIF4E. We also observed that serum-starvation of SBP3 for 24 hours resulted in a significant reduction in the phosphorylation of 4E-BP3 at Thr-23 phosphorylated site and this coincided with a decrease association of 4E-BP3 and eIF4E. However, a 90% increase in the amount of 4E-BP3 bound to RPA2 in the nuclear fraction of serum-starvated SBP3 was detected. The dephosphorylation of 4E-BP3 at Thr-23 and 4E-BP3 dissociated from eIF4E upon serum-starvation is different from that of 4E-BP1, in which dephosphorylation of the equivalent site (Thr-37) in 4E-BP1 leads to association of 4E-BP1 with eIF4E after serum-starvation. Consequently, we presume that the biological function of 4E-BP3 is more complicated, so its regulatory mechanism still needs further investigation. In UV-stimulated experiments, when HEK293 cell transiently transfected with 4E-BP3 was treated with 50 J/m2 of UV, the dissociation of RPA2 and 4E-BP3 was detected at 15 minutes, and no RPA2 - 4E-BP3 complex was detected at 20 minutes in the nuclear fraction after UV damage, but the phosphorylated form of RPA2 was detected after 2 hours. These data indicated that the phosphorylation of RPA2 did not cause 4E-BP3 losing the binding ability to RPA2 in the HEK293 cell. In order to study the behavior of endogenous 4E-BP3, we used a human prostate carcinoma cell line-LNCaP, which was proved to overexpress endogenous 4E-BP3. We found that 4E-BP3 was phosphorylated not only in the cytoplasmic but also in the nuclear fractions in LNCaP cells under normal culture condition. When LNCaP cells were under serum-starvation situation, the phosphorylation of 4E-BP3 at Thr-23 also decreased as detected in SBP3. However, the interactions of 4E-BP3/eIF4E and 4E-BP3/RPA2 didn’t change in serum-starvation and normal circumstances. These results implied that LNCaP was serum-insensitive. Additionally, we treated the LNCaP cells with interleukin (IL)-6 or the inhibitor of PI3K, LY294002. Our propaedeutic data indicated that upon IL-6 stimulation, the LNCaP cells exhibited a dramatic increase in STAT3 transcriptional activity, the phosphorylation of 4E-BP3 at Thr-23 decreased, and the association of 4E-BP3 and RPA2 increased. Whereas when treated the LNCaP cells with PI3K inhibitor, LY294002, only a limited level of phosphorylated form of 4E-BP3 was detected. Besides, we used site-directed mutagenesis to determine which phosphorylation sites on 4E-BP3 are important for eIF4E and RPA2 binding. To further address the phosphorylation mechanism and signal transduction of 4E-BP3 in the LNCaP cells under various conditions will be investigated.

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