Hirota Keiko, Daitoku Hiroaki, Matsuzaki Hitomi, Araya Natsumi, Yamagata Kazuyuki, Asada Sachie, Sugaya Takeshi, Fukamizu Akiyoshi
Center of Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
J Biol Chem. 2003 Apr 11;278(15):13056-60. doi: 10.1074/jbc.C200553200. Epub 2003 Jan 7.
Previous studies have shown that FKHR, a member of the forkhead family of transcription factors, acts as a DNA binding-independent cofactor of nuclear receptors, including estrogen, retinoid, and thyroid hormone receptors, in addition to the original function as a DNA binding transcription factor that redistributes from the nucleus to the cytoplasm by insulin-induced phosphorylation. Here, we demonstrated the physical interaction of FKHR with hepatocyte nuclear factor (HNF)-4, a member of steroid/thyroid nuclear receptor superfamily, and the repression of HNF-4 transactivation by FKHR. FKHR interacted with the DNA binding domain of HNF-4 and inhibited HNF-4 binding to the cognate DNA. Furthermore, the binding affinity of HNF-4 with phosphorylated FKHR significantly decreased in comparison to that with unphosphorylated FKHR. Therefore, a phosphorylation of FKHR by insulin followed by its dissociation from HNF-4 and the redistribution of FKHR from the nucleus to the cytoplasm would expect to induce the transcriptional activation of HNF-4 by facilitating to the access of HNF-4 to its DNA element. Indeed, most intriguingly, insulin stimulation reversed the repression of HNF-4 transcriptional activity by phosphorylation-sensitive (wild-type) FKHR, but not by phosphorylation-deficient FKHR. These results suggest that insulin regulates the transcriptional activity of HNF-4 via FKHR as a signal-regulated transcriptional inhibitor.
先前的研究表明，叉头转录因子家族成员FKHR，除了作为一种通过胰岛素诱导的磷酸化作用从细胞核重新分布到细胞质的DNA结合转录因子的原始功能外，还作为包括雌激素、视黄酸和甲状腺激素受体在内的核受体的一种不依赖于DNA结合的辅因子发挥作用。在此，我们证明了FKHR与类固醇/甲状腺核受体超家族成员肝细胞核因子（HNF）-4之间的物理相互作用，以及FKHR对HNF-4反式激活的抑制作用。FKHR与HNF-4的DNA结合结构域相互作用，并抑制HNF-4与同源DNA的结合。此外，与未磷酸化的FKHR相比，HNF-4与磷酸化的FKHR的结合亲和力显著降低。因此，胰岛素使FKHR磷酸化，随后FKHR与HNF-4解离，以及FKHR从细胞核重新分布到细胞质，有望通过促进HNF-4与它的DNA元件结合来诱导HNF-4的转录激活。的确，最有趣的是，胰岛素刺激逆转了磷酸化敏感型（野生型）FKHR对HNF-4转录活性的抑制，但未逆转磷酸化缺陷型FKHR对HNF-4转录活性的抑制。这些结果表明，胰岛素通过FKHR作为信号调节转录抑制剂来调节HNF-4的转录活性。
