- 學位論文
EB 病毒 BGLF4 蛋白質激酶對 ART-27 穩定性及細胞分裂後期促進複合體活性影響之探討
Effects of Epstein-Barr virus BGLF4 kinase on cellular ART-27 protein stability and APC/CCdc20 activity
摘要
EB 病毒 (Epstein-Barr virus, EBV) 是一種與許多人類惡性腫瘤的發生有高度關聯性的疱疹病毒。在我們實驗室先前的研究中發現，若表現 EB 病毒中唯一的serine/threonine 蛋白質激酶 BGLF4 會造成細胞在尚未進入有絲分裂時期就產生染色體濃縮，核膜溶解及細胞骨架重組等類似有絲分裂時期的現象，顯示 BGLF4 可能以類似細胞蛋白質激酶 Cdk1 的作用影響細胞週期所發生的事件。Cdk1 的主要作用之一是調控 anaphase promoting complex/cyclosome (APC/C) 活性，APC/C 是一個調控細胞週期重要的 E3 ubiquitin ligase，APC/CCdc20 可造成 cyclin A、cyclin B及securin 等蛋白質的降解，促進細胞從 metaphase 進入 anaphase。先前的研究中發現 BGLF4 會透過 APC/CCdc20 促進一個位於中心體的 ART-27蛋白質的降解，暗示 BGLF4 可能會影響 APC/CCdc20 的E3 ubiquitin ligase活性。共同免疫沉澱實驗的結果證明在in vivo情況下BGLF4可以與 APC/C 的活化單位 Cdc20 產生交互作用，而in vitro IP-kinase assay則進一步證明在 in vitro 情況下 BGLF4 可以磷酸化 Cdc20。利用短暫轉染的實驗觀察到共同表現 BGLF4 會造成 Cdc20表現量增加，並證明BGLF4是藉由增加Cdc20蛋白質的穩定性來影響Cdc20 的表現量。文獻指出 BGLF4 的細胞同源蛋白 Cdk1 可以藉由磷酸化 APC/C 的次單位促進 APC/CCdc20 活性，但是表現BGLF4 的細胞並沒有發現主要次單位 Cdc27 有明顯磷酸化的情況。此外，BGLF4 不會造成 APC/CCdc20 已知的受質 cyclin B1 的降解。更進一步探討 BGLF4 對 ART-27 可能的調控機制，在免疫螢光染色實驗中觀察發現 BGLF4 會造成 ART-27 表現的位置改變，並且在 in vitro 情況下 BGLF4 可以磷酸化 ART-27。最後本篇研究提出 BGLF4 調控 APC/CCdc20 的可能作用模式，即 BGLF4 可以透過磷酸化 Cdc20 增加 Cdc20 蛋白質的穩定性，藉由提高Cdc20 的表現量增加 APC/CCdc20 的活性，但是此時 APC/CCdc20 活性仍不足以造成所有受質的降解，BGLF4 可以透過磷酸化 ART-27 並幫助其與 APC/CCdc20 交互作用，而促進 ART-27 蛋白質降解。
並列摘要
Epstein-Barr virus (EBV) is a gamma-herpesvirus closely associated with human malignant diseases. Our previous study has demonstrated that expression of BGLF4 kinase induces multiple premature mitotic events such as chromosome condensation, nuclear lamina disassembly, and cytoskeleton rearrangement through Cdk1 mimicry. One of the major functions of Cdk1 is to regulate anaphase-promoting complex/cyclosome (APC/C). APC/C is an E3 ubiquitin ligase which promotes metaphase to anaphase transition by targeting substrates such as cyclin A, cyclin B, and securin for proteasomal degradation. Previously, co-expression of BGLF4 kinase induced the degradation of a centrosomal protein ART-27 through APC/CCdc20 ubiquitination pathway, suggesting that BGLF4 may regulate the E3 ubiquitin ligase activity of APC/CCdc20. To search for possible mechanisms involved, the physical interaction of Cdc20 and BGLF4 in vivo was demonstrated by co-immunoprecipitation in this study. Furthermore, Cdc20 was phosphorylated by BGLF4 in in vitro IP-kinase assay. In co-transfection assays, Cdc20 protein was stabilized by BGLF4 in a dose dependent manner. Because Cdk1 is known to activate APC/C activity through phosphorylating APC/C subunits, Cdc27 was examined for possible phosphorylation induced by BGLF4, and no obvious molecular weight shift was observed. Additionally, we found that the localization of ART-27 was changed upon BGLF4 coexpression and ART-27 was phosphorylated by BGLF4 in vitro. Taken together, we postulate that BGLF4 might stabilize Cdc20 protein through phosphorylation, which may then result in the activation of APC/CCdc20 activity. However, cyclin B1 degradation was not obvious in the presence of BGLF4, suggesting not all APC/CCdc20 substrates are degraded. Thus BGLF4 may also play a role in recruiting ART-27 to APC/CCdc20 for promoting its degradation. Further investigation will be needed to reveal how ART-27 protein stability contributes to BGLF4 induced premature chromosome condensation.
參考文獻
延伸閱讀
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