Joint study for the thyroid hormone receptor phosphorylation

细菌表达的人甲状腺激素受体β-1（hTRβ1）最近被证明在体外被HeLa细胞质提取物磷酸化。在本研究中，我们首次证明了hTRβ1也可以通过纯化的酪蛋白激酶II（CKII）在体外磷酸化。磷氨基酸分析显示，hTRβ1在丝氨酸和苏氨酸残基上均被磷酸化。体外CKII磷酸化谷胱甘肽s -转移酶（GST）hTRβ1融合蛋白的CKII磷酸化预测CKII磷酸化位点发生突变，表明位于铰链区（t-210）的苏氨酸残基可以被CKII磷酸化。为了阐明CKII磷酸化Thr-210在hTRβ1中的功能意义，我们接下来使用野生型或Thr-210突变的hTRβ1进行了瞬时转染研究。有趣的是，当使用Thr-210突变的hTRβ1时，在没有配体的情况下，基础抑制水平显著降低。由于Thr-210位于与核受体共抑制因子（N-CoR）的相互作用区域内，我们接下来进行了电泳迁移量转移试验（EMSA）来检测氨基末端截断的N-CoR （NCoRI）与野生型或Thr-210突变的hTRβ1之间的相互作用。有趣的是，与类视黄酮X受体β(RXRβ)与两种类型的hTRβ1形成相同的异源二聚体相反，NCoRI比Thr-210突变的hTRβ1更倾向于与野生型hTRβ1形成异源二聚体。综上所述，我们推测CKII对hTRβ1中Thr-210的磷酸化可能会调节与N-CoR的相互作用，并在没有配体的情况下介导基础抑制。

Bacterially-expressed human thyroid hormone receptor β-1(hTRβ1) recently has been shown to be phosphorylated in vitro by HeLa cytosolic extract. In the present study, we first demonstrated that hTRβ1 also could be phosphorylated in vitro by purified casein kinase II(CKII). Phosphoamino acid analysis revealed that hTRβ1 was phosphorylated on both serine and threonine residues. In vitro CKII phosphorylation of glutathione S-transferase (GST)hTRβ1 fusion proteins whose predicted CKII phosphorylation sites were mutated demonstrated that a threonine residue located in the hinge region (Tht-210) could be phosphorylated by CKII. In order to elucidate the functional significance of CKII phosphorylation of Thr-210 in hTRβ1, we next performed transient transfection studies using either wild type or Thr-210 mutated hTRβ1. Interestingly, the basal repression level in the absence of ligand was attenuated significantly when Thr-210 mutated hTRβ1 was used. Since Thr-210 is located within the interacting domain with nuclear receptor co-repressor (N-CoR), we next performed electrophoretic mobility shift assay (EMSA) to examine the interaction between amino terminal-truncated N-CoR (NCoRI) and wild type or Thr-210 mutated hTRβ1. Interestingly, in contrast to retinoid X receptor β(RXRβ) which equally formed heterodimers with both types of hTRβ1, NCoRI preferentially formed heterodimers with wild type hTRβ1 than Thr-210 mutated hTRβ1. Taken together, we speculate that CKII phosphorylation of Thr-210 in hTRβ1 might modulate interaction with N-CoR, and contribute to mediate basal repression in the absence of ligand.