The RING Finger Protein, RNF8, Interacts with Retinoid X Receptor α and Enhances Its Transcription-stimulating Activity*
The RING Finger Protein, RNF8, Interacts with Retinoid X Receptor α and Enhances Its Transcription-stimulating Activity*
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DOI：
10.1074/jbc.m309148200
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发表时间：
2004-04
影响因子：
4.8
通讯作者：
Y. Takano;Seiji Adachi;M. Okuno;Y. Muto;Takashi Yoshioka;R. Matsushima-Nishiwaki;H. Tsurumi;Ken-ichi Ito;S. Friedman;H. Moriwaki;S. Kojima;Y. Okano
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文献类型：
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作者：
Y. Takano;Seiji Adachi;M. Okuno;Y. Muto;Takashi Yoshioka;R. Matsushima-Nishiwaki;H. Tsurumi;Ken-ichi Ito;S. Friedman;H. Moriwaki;S. Kojima;Y. Okano
维甲酸X受体α(RXRα)是类固醇激素受体超家族的成员。通过酵母双杂交筛选、β-半乳糖苷酶分析和下拉实验，我们发现RNF8是一种环指蛋白，最近被分离出来作为与泛素结合酶结合的蛋白，通过这两种蛋白的N-末端区域与RXRα结合。荧光共振能量转移结果显示，在COS7细胞中，过表达的RNF8与RXRα共定位并相互作用。RNF8Cys-403Ser(C403S)的点突变破坏了RNF8的环指结构，或者RNF8的N-末端区域(ΔN)的缺失阻止了RNF8的核定位，而不影响RXRα的核定位。尽管RNF8的瞬时过表达对RXRα泛素化影响不大，但在不添加其配体9-顺式维甲酸(RA)的情况下，RNF8可剂量依赖地增强RXRα介导的RXR反应元件(RXRE)基因启动子的反式激活，并上调RXRE下游基因和RA反应元件的表达。在RNF8的C403S点突变体和ΔN缺失突变体中都没有发现这种反式激活增强活性。这些结果表明，RNF8通过它们各自的N-末端区域之间的相互作用，作为RXRα介导的转录活性的调节因子，具有新的功能。
Retinoid X receptor α (RXRα) is a member of the steroid hormone receptor superfamily. Using yeast two-hybrid screening, β-galactosidase assays, and pull-down assays, we show that RNF8, a RING finger protein recently isolated as a protein binding to a ubiquitin-conjugating enzyme, binds to RXRα through the N-terminal regions of both proteins. In COS7 cells, overexpressed RNF8 colocalized and interacted with RXRα in the nucleus, as shown by fluorescence resonance energy transfer. A point mutation of RNF8, Cys-403 to Ser (C403S), which disrupts the RING finger structure, or deletion of the N-terminal region (ΔN) of RNF8 prevented localization of RNF8 to the nucleus without affecting nuclear localization of RXRα. Although transient overexpression of RNF8 had little effect on RXRα ubiquitination, RNF8 dose-dependently enhanced RXRα-mediated transactivation of the RXR-responsive element (RXRE)-bearing gene promoter without the addition of its ligand, 9-cis-retinoic acid (RA), and up-regulated the expression of the genes downstream of RXRE as well as an RA-response element. This transactivation-enhancing activity was not seen with either the C403S point mutant or the ΔN deletion mutant of RNF8. These results suggest a novel function of RNF8 as a regulator of RXRα-mediated transcriptional activity through interaction between their respective N-terminal regions.