Transcriptional Regulation of Nuclear Receptors by Ubc9

• Main Authors: Chang Yung-Lung, 張永龍
• Other Authors: Haung Shih-Ming
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/39887116071799810253

碩士 === 國防醫學院 === 生物化學研究所 === 93 === Abstract The covalent attachment of small ubiquitin-like modifier 1 (SUMO-1) is an important post-translational modification which involved in many cellular biological and developmental process. Ubc9, the sole E2-conjugating enzyme essential for sumoylation, forms a thioester intermediate with an active-site cysteine (Cys93), from which it is ligated to a lysine residue of the target protein. Ubc9 itself appears to be critical for substrate recognition. In previous study, it is also involved in the gene regulation of AR, GR, TEL, and COUP-TFI, independent of its SUMO-1 conjugating enzyme activity. In the human cervical carcinoma HeLa and C33A cells, we have showed that Ubc9 not only enhanced ligand-bound AR-mediated transcriptional activity, but also enhanced ER and TR function as well. It can act synergistically with some NR coactivators, GRIP1 and Zac1, in the enhancement of AR and ER function, but additively in TR. In mammalian two-hybrid assay, the physical interaction between Ubc9 and GRIP1/Zac1 was established. Furthermore, Ubc9 enhanced transcriptional activation of GRIP1 and Zac1, suggesting that it might serve as a secondary coactivator in NR-mediated transcription. We also elucidated that Ubc9 enhanced AR function through its both activation functions, AF-1 and AF-2. The transactivation activity of Ubc9 was not abolished by the C93S substitution that abrogates SUMO-1 conjugating enzyme activity, indicating that Ubc9 can function as a nuclear receptor coactivator distinct from its E2-conjugating enzyme activity. Most importantly, we have identified that the Ubc9 C-terminal region, the residues 150-158, may be crucial for its coactivator function and its physical and functional interactions with GRIP1. In addition, Ubc9 was found to be sumolated at Lys 153. Our Data suggested that the mutations in Cys 93 and Lys 153 did not alter its transactivation function and cellular localization of Ubc9 in HeLa cells. Taken together, these results suggest that the E2-conjugating enzyme activity and the sumoylation of ubc9 itself might be not required for its transcriptional activation in NR-mediated gene expression.