Study on the biological function of 4E-BP3 in human cell lines

• Main Authors: Lee Po-Hsin, 李柏欣
• Other Authors: Ming-Chung Chang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/38568581703145658106

碩士 === 國立成功大學 === 生物化學研究所 === 94 === Abstract Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G to form the eIF4F complex to regulate protein synthesis. The 4E-BPs (eukaryotic initiation factor 4E-binding proteins) are able to compete with eIF4G for binding to eIF4E and repress cap-dependent translation. Previously, using the yeast-two hybrid method, we found that the RPA2 interacted with the 4E-BP3, a member of 4E-BPs family. RPA is replication protein A, and it is a single-stranded DNA binding protein. The RPA is a complex of three subunits including RPA1 (70 kDa), RPA2 (32 kDa) and RPA3 (14 kDa). The complex also functions in DNA repair and recombination. The primary ssDNA binding domain is localized to the RPA1. However, RPA2 is phosphorylated in a cell cycle dependent manner and is additionally phosphorylated in response to DNA damage. Hyper-phosphorylation of RPA2 occurs in response to DNA-damaging agents, such as UV or IR treatment. In this study, the endogenous 4E-BP3 could be detected in various cells and the 4E-BP3 associated with the RPA2 was confirmed in these cell lines by immuno-precipitation assay. The result shows that the RPA2/4E-BP3 complex was detected in both the nuclear and cytoplasm fractions. When cultivated cell was starvated for over 24hr, phosphorylation form of 4E-BP3 decreased and the protein levels of RPA2/4E-BP3 complex significantly increased. Increased the RPA2-4E-BP3 complex coincide with dissociation of eIF4E from 4E-BP3. m7-GTP sepharose pull down assay showed that the complex of RPA2 and 4E-BP3 contain eIF4E. In UV-stimulated experiments, when HEK293T cell was treated with 50 J/m2 of UV, the dissociation of RPA2 and 4E-BP3 was detected at 2 hours, and the phosphorylated forms of RPA2 and 4E-BP3 were also detected after 2 hours. Previously, the binding area of RPA2 with 4E-BP3 located within phosphorylation domain of RPA2, and the proliferation rate of the stable clone of 4E-BP3 cell decreased by 50％. To investigate the phosphorylated of RPA2 could be influenced or not by the 4E-BP3, the mutant forms and siRNA of 4E-BP3 were performed. The result shows that the 4E-BP3 siRNA specifically knockdown the endogenous 4E-BP3 and the RPA2-4E-BP3 complex significantly decreased. When the endogenous 4E-BP3 was knockdown by 4E-BP3 siRNA, the Hyper-phosphorylated form of RPA2 significantly increased after 50 J/m2 of UV. But the amounts of hyper-phosphoryaltion forms of RPA2 also increased in HEK293T cell containing exogenous mutant forms of 4E-BP3. With the treatment of aphidicolin, the RPA2 was hypo-phosphorylated after the complex of RPA2/4E-BP3 would be dissociated in HEK293T. The regulation of cell proliferation rate is contributed to the RPA2 phosphorylation by 4E-BP3.