Functional analysis of a novel tumor-suppressing protein kinase interactive protein

• Main Authors: Chia-Yi Chien, 簡佳怡
• Other Authors: Chen-Kung Chou
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/88896940398387638257

碩士 === 國立陽明大學 === 微生物暨免疫學研究所 === 90 === FLIP1 was previously identified as an interacting protein of a human tumor-suppressing protein kinase, LKB1, through yeast two-hybrid screening in our laboratory. Ectopically expressed FLIP1 in mammalian cell was shown, subsequently, to block NFκB activation induced by various cytokines. Interestingly, it was later found that the Gal4BD-FLIP1 fusion protein was able to drive the expression of GAL4-responsive reporter gene in yeast efficiently without the help of any transcription factor. Since FLIP1 by itself does not resemble any of known transcription factors in every aspect, it seems that FLIP1 possesses a function of transcriptional coactivator. These results described above readily hinted that FLIP1 should have a role in the regulation of gene expression. In this study, I have used yeast as a model system to explore the transcriptional regulatory mechanism of FLIP1. I have confirmed that FLIP1 does indeed activate transcription in both yeast and mammalian cells. Although only the N-terminally truncated version of FLIP1 has the activity in mammalian cells, the full-length FLIP1 strongly activates the transcription of reporter gene in yeast. It was later determined that the C-terminal region of FLIP1 is essential for its transcriptional activity in these two organisms. In addition, the discrepancy of FLIP1’s role in yeast and mammalian cells has led to the identification of distinct regulatory mechanisms of FLIP1 in these two organisms. Unlike its function in yeast, the transcriptional activity of FLIP1 strongly correlates with its subcellular localization in mammalian cells. It turns out that the N-terminal 195 amino acids are required to retain FLIP1 in the cytoplasm and, hence, disable its ability to activate transcription. In summary, FLIP1 is a novel transcriptional coactivator and its cellular function is dependent on a spatial regulation of the protein, possibly, through its N-terminus.