Bhat M K, Ashizawa K, Cheng S Y
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):7927-31. doi: 10.1073/pnas.91.17.7927.
To understand the molecular basis of the phosphorylation-enhanced transcriptional activity of human thyroid hormone nuclear receptor subtype beta 1 (hTR beta 1), we studied the effect of phosphorylation on the interaction of hTR beta 1 with the retinoid X receptor beta (RXR beta), we studied the effect of phosphorylation on the interaction of hTR beta 1 with the retinoid X receptor beta (RXR beta). In vitro, the extent of hTR beta 1.RXR beta heterodimer bound to various thyroid hormone response elements (TREs) was compared before and after phosphorylation of hTR beta 1. Without phosphorylation, hTR beta 1.RXR beta heterodimer was barely detectable under the experimental conditions. After phosphorylation of hTR beta 1, heterodimer bound to (i) the chicken lysozyme gene TRE, (ii) a TRE consisting of direct repeats of half-site binding motifs separated by four gaps, and (iii) a palindromic TRE was enhanced by approximately 10-, 7-, and 6-fold, respectively. The effect of phosphorylation on hTR beta 1.RXR beta heterodimerization was reversible. Dephosphorylation of the phosphorylated hTR beta 1 by alkaline phosphatase led to loss of the ability of hTR beta 1 to form a heterodimer with RXR beta in either the absence or the presence of DNA. These results indicate that the heterodimerization is enhanced by phosphorylation. To evaluate the effect of phosphorylation on the interaction of hTR beta 1 with RXR beta in vivo, we cotransfected hTR beta 1, RXR beta and TRE-chloramphenicol acetyltransferase (CAT) expression plasmids into CV-1 cells. CAT activity was assessed in the presence or absence of okadaic acid. Okadaic acid is a potent inhibitor of phosphatases 1 and 2A and increases the in vivo phosphorylation of hTR beta 1 by approximately 10-fold. Using the CAT reporter gene under control of the TRE from the malic enzyme gene, we found that RXR beta increased the okadaic acid-enhanced hTR beta 1-mediated CAT activity by 2- to 3-fold in the presence of 3,3',5-triiodo-L-thyronine. However, 9-cis-retinoic acid did not enhance the effect of okadaic acid. Our results indicate that phosphorylation is essential for the interaction of hTR beta 1 with RXR beta. Thus, phosphorylation plays a pivotal role in the gene-regulating activity of hTR beta 1.
为了解人甲状腺激素核受体β1亚型（hTRβ1）磷酸化增强转录活性的分子基础，我们研究了磷酸化对hTRβ1与视黄酸X受体β（RXRβ）相互作用的影响。在体外，比较了hTRβ1磷酸化前后hTRβ1.RXRβ异二聚体与各种甲状腺激素反应元件（TRE）结合的程度。未磷酸化时，在实验条件下几乎检测不到hTRβ1.RXRβ异二聚体。hTRβ1磷酸化后，与（i）鸡溶菌酶基因TRE、（ii）由四个间隔隔开的半位点结合基序直接重复组成的TRE以及（iii）回文TRE结合的异二聚体分别增强了约10倍、7倍和6倍。磷酸化对hTRβ1.RXRβ异二聚化的影响是可逆的。用碱性磷酸酶使磷酸化的hTRβ1去磷酸化，导致hTRβ1在有无DNA的情况下均丧失与RXRβ形成异二聚体的能力。这些结果表明磷酸化增强了异二聚化。为了评估磷酸化在体内对hTRβ1与RXRβ相互作用的影响，我们将hTRβ1、RXRβ和TRE - 氯霉素乙酰转移酶（CAT）表达质粒共转染到CV - 1细胞中。在有或无冈田酸的情况下评估CAT活性。冈田酸是磷酸酶1和2A的有效抑制剂，可使hTRβ1的体内磷酸化增加约10倍。使用苹果酸酶基因TRE控制下的CAT报告基因，我们发现，在3,3',5 - 三碘 - L - 甲状腺原氨酸存在的情况下，RXRβ使冈田酸增强的hTRβ1介导的CAT活性提高了2至3倍。然而，9 - 顺式视黄酸并未增强冈田酸的作用。我们的结果表明磷酸化对于hTRβ1与RXRβ的相互作用至关重要。因此，磷酸化在hTRβ1的基因调控活性中起关键作用。
