臺灣博碩士論文加值系統

BGLF4是EB病毒的Serine/Throenine蛋白激酶，可以磷酸化數個哺乳動物細胞中的Cdc2受質。BGLF4的表現已知會造成細胞染色體濃縮、核板離散與細胞骨架重組等類似細胞周期M期之現象。透過類似Cdc2之作用機制，BGLF4對condensin與lamin的磷酸化，產生染色體濃縮與核板離散之現象，但對細胞骨架重組的機制仍不清楚。本篇研究第一部份是探討BGLF4與利用酵母菌雙雜交試驗找尋到與BGLF4有交互作用的細胞蛋白質LAMR1 (Laminin receptor 1) 間的交互作用，與BGLF4對LAMR1功能可能之影響。LAMR1是一個與細胞外基質蛋白質Laminin-1有高度親合性之細胞膜受器，在細胞貼附與移動上可能扮演重要角色，並且在許多癌細胞中有高量表現。因此在本研究中首先在HeLa細胞轉染表現BGLF4，以共同免疫沉澱方式證明細胞內生性的LAMR1可與BGLF4產生交互作用，同時以免疫螢光染色發現BGLF4會改變LAMR1的分佈位置，但BGLF4不影響LAMR1在細胞內的表現量。利用純化的GST-LAMR1與BGLF4進行試管內激酶試驗，發現BGLF4不會磷酸化LAMR1。為了探討BGLF4是否參與在LAMR1功能上面的調控，以可受tetracycline誘導之293 T-REx-BGLF4 (B22) 與293 T-RE-K102I (K9) 細胞進行貼附試驗與細胞移動試驗觀察，在初步實驗中未發現BGLF4對於LAMR1對於細胞貼附Laminin-1有明顯影響。另外先前已報導在酵母菌中的Cdk1會調節Rho GTPases中的Cdc42，對於酵母菌出芽生殖過程扮演重要角色，因此第二部份探討Rho GTPases是否參與在BGLF4所造成的細胞骨架重組過程中。將可表現顯性抑制型GFP Rho GTPases (dominant negative GFP RhoA, Rac1, Cdc42) 與BGLF4的質體轉染至細胞，初步實驗中發現顯性抑制型GFP Rho GTPases可以降低BGLF4造成細胞圓形化的比例。但這些初步觀察均有待未來實驗的進一步確認。

BGLF4 of Epstein-Barr virus (EBV) is a proline dependent Serine/throenine protein kinase which shares multiple protein substrates with cyclin-dependent kinase 1 (cdk1, or cdc2). Transient expression of BGLF4 induces multiple mitotic events, including premature chromosome condensation, nuclear lamina disassembly and cytoskeleton rearrangement. BGLF4 induces nuclear changes through the phosphorylation of condensin and nuclear lamin in a cdc2 mimicking manner, whereas the mechanism involved in cytoskeleton change remains unclear. The first part of this study is to characterize the interaction between BGLF4 and one of its interacting partner, Laminin receptor 1 (LAMR1), identified in yeast-two hybrid screening. LAMR1 is a membrane receptor that has a high affinity to extracellular matrix (ECM) Lminin-1. LAMR1 not only plays an important role in cell adhesion and migration but also correlates with metastastic potentials in multiple human malignancies. The interaction between LAMR1 and BGLF4 was first confirmed in co-immunoprecipitation. Immunofluorescence staining revealed that endogenous LAMR1 is redistributed and colocalized with BGLF4 or kinase dead K102I in transiently transfected HeLa cells. Phosphorylation signal was not observed in in vitro BGLF4 kinase assay using bacterially recombinant GST-LAMR1 as a substrate. Possible effects of BGLF4 on LAMR1 function were examined using tetracycline inducible 293 T-REx-BGLF4 (B22) and 293 T-REx-K102I (K9). The preliminary data indicate that BGLF4 seems not to affect cell binding to Laminin-1 coated surface significantly. Secondly, it was observed that yeast Rho GTPase cdc42 is regulated by cdk1 in yeast and Rho GTPases are important regulators for cell morphology and migration. Therefore it was addressed whether Rho GTPases are involved in BGLF4 induced cytoskeleton rearrangement by expressing GFP-tagged dominant negatives of Cdc42, RhoA and Rac1. In transiently transfected HeLa cells, BGLF4 mediated cell rounding was reduced in the presence of these dominant negative Rho GTPases. The biological significance of these observations will need further study in the future.

口試委員會審定書 …………………………………… I
中文摘要 …………………………………… 　II
英文摘要 ……………………………………　　 III
1．導論 …………………………………… 1
2. 材料與方法 …………………………………… 9
3. 結果 …………………………………… 16
4. 討論 …………………………………… 21
5. 圖表 …………………………………… 25
6. 參考文獻 …………………………………… 41

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