Department of Bioscience & Biotechnology/Institute of Bioscience, BK21 Graduate Program, Sejong University, Seoul 143-747, Republic of Korea.
Department of Molecular Biology, Dankook University, Gyeonggi-do 448-701, Republic of Korea.
Biochem Biophys Res Commun. 2014 Feb 21;444(4):605-10. doi: 10.1016/j.bbrc.2014.01.105. Epub 2014 Jan 31.
Transcriptional activity of the retinoic acid receptor (RAR) is regulated by diverse binding partners, including classical corepressors and coactivators, in response to its ligand retinoic acid (RA). Recently, we identified a novel corepressor of RAR called the retinoic acid resistance factor (RaRF) (manuscript submitted). Here, we report how adenovirus E1A stimulates RAR activity by associating with RaRF. Based on immunoprecipitation (IP) assays, E1A interacts with RaRF through the conserved region 2 (CR2), which is also responsible for pRb binding. The first coiled-coil domain of RaRF was sufficient for this interaction. An in vitro glutathione-S-transferase (GST) pull-down assay was used to confirm the direct interaction between E1A and RaRF. Further fluorescence microscopy indicated that E1A and RaRF were located in the nucleoplasm and nucleolus, respectively. However, RaRF overexpression promoted nucleolar translocation of E1A from the nucleoplasm. Both the RA-dependent interaction of RAR with RaRF and RAR translocation to the nucleolus were disrupted by E1A. RaRF-mediated RAR repression was impaired by wild-type E1A, but not by the RaRF binding-defective E1A mutant. Taken together, our data suggest that E1A is sequestered to the nucleolus by RaRF through a specific interaction, thereby leaving RAR in the nucleoplasm for transcriptional activation.
视黄酸受体（RAR）的转录活性受多种结合伴侣调节，包括经典的核心抑制剂和共激活剂，以响应其配体视黄酸（RA）。最近，我们鉴定了一种称为视黄酸抗性因子（RaRF）的新型 RAR 核心抑制剂（已提交的手稿）。在这里，我们报告了腺病毒 E1A 如何通过与 RaRF 结合来刺激 RAR 活性。基于免疫沉淀（IP）测定，E1A 通过保守区域 2（CR2）与 RaRF 相互作用，该区域也负责 pRb 结合。RaRF 的第一个卷曲螺旋结构域足以进行这种相互作用。体外谷胱甘肽 S-转移酶（GST）下拉测定用于确认 E1A 和 RaRF 之间的直接相互作用。进一步的荧光显微镜观察表明，E1A 和 RaRF 分别位于核质和核仁中。然而，RaRF 的过表达促进了 E1A 从核质向核仁的核仁易位。E1A 破坏了 RAR 与 RaRF 的 RA 依赖性相互作用和 RAR 向核仁的易位。RaRF 介导的 RAR 抑制被野生型 E1A 破坏，但不是由 RaRF 结合缺陷的 E1A 突变体破坏。总之，我们的数据表明，E1A 通过特定的相互作用被 RaRF 隔离到核仁中，从而使 RAR 留在核质中进行转录激活。
