Interactions of the retinoid X receptor with a RING protein and its effects on ubiquitylation and transcription

类视黄醇 X 受体与 RING 蛋白的相互作用及其对泛素化和转录的影响

基本信息

批准号：
16570093
负责人：
OKANO Yukio
金额：
$ 2.37万
依托单位：
Gifu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16570093/
关键词：
RING-finger retinoid X receptor (RXR)Ubc13 transactivation ubiquitylation Lys63 dominant negative FRET RNF8 UBC13 RING-finger Ubiquitinylation retinoid X receptor(RXR)Yeast two-hybrid FHA Transactivation C403S

项目摘要

We previously reported the interaction of the RING-finger protein, RNF8, a binding partner of UBE2E2 (a ubiquitin-conjugating enzyme), with retinoid X receptor (RXR). Using the deletion mutants of RNF8 and RXR, the important domains in these proteins were examined by two-hybrid assay or in vitro pull-down assay. Wild-type RNF8 were distributed in nucleus, whereas an N-terminal deletion mutant (deltaN) and the RING-disrupted mutant (dominant negative) of RNF8 were observed in cytosole. Although RNF8 did not seem to act as an E3 for RXR ubiquitylation, it enhanced RXR transcriptional activity independently of a selective ligand, 9-cis retinoic acid. This transactivation effects were abolished with deltaN and dominant negative mutants of RNF8.Furthermore, in collaboration with Dr.Timothy Thomson's group in Spain, following observations were made. Two-hybrid screening using UBC13 as a bait, we cloned the variant E2, UEV, and several RING-finger proteins including RNF8, KIAA00675, CHFR, KF1, and ZNRF2. UEV is known to form multi-ubiquitin chains via K63 but not ordinary K48. Autoubiquitylation of RNF8 was examined using wild-type ubiquitin (UbWT), and 3 types of K to R mutants of ubiquitin (UbK48,63R; UbK29,63R; UbK29,48R). In vitro ubiquitylation experiments in combination with the expressions of dominant negative UBC13 mutant (UBC13-C87A) and UbK29,48R, revealed ubiquitylation of RNF8 via K63 but not K48.

我们之前报道了环指蛋白 RNF8（UBE2E2（一种泛素缀合酶）的结合伴侣）与类视黄醇 X 受体 (RXR) 的相互作用。使用 RNF8 和 RXR 的缺失突变体，通过双杂交测定或体外 Pull-down 测定检查这些蛋白质中的重要结构域。野生型RNF8分布在细胞核中，而RNF8的N末端缺失突变体(deltaN)和RING破坏突变体(显性失活)则在胞质中观察到。尽管 RNF8 似乎并不充当 RXR 泛素化的 E3，但它独立于选择性配体 9-顺式视黄酸而增强 RXR 转录活性。这种反式激活效应被 deltaN 和 RNF8 的显性失活突变体所消除。此外，与西班牙 Timothy Thomson 博士的小组合作，进行了以下观察。使用UBC13作为诱饵进行二杂交筛选，我们克隆了变体E2、UEV和几个环指蛋白，包括RNF8、KIAA00675、CHFR、KF1和ZNRF2。已知 UEV 通过 K63 形成多泛素链，但不是普通的 K48。使用野生型泛素 (UbWT) 和 3 种类型的泛素 K 到 R 突变体 (UbK48,63R; UbK29,63R; UbK29,48R) 检查 RNF8 的自身泛素化。体外泛素化实验结合显性失活 UBC13 突变体 (UBC13-C87A) 和 UbK29,48R 的表达，揭示了 RNF8 通过 K63 而不是 K48 进行泛素化。