Joint study for the thyroid hormone receptor phosphorylation

最近，细菌表达的人甲状腺激素受体β-1(HTRβ1)在体外被HeLa胞浆提取物磷酸化。在本研究中，我们首次证明hTRβ1在体外也可以被纯化的酪蛋白激酶II(CKII)磷酸化。磷酸氨基酸分析表明，HTRβ1在丝氨酸和苏氨酸残基上均被磷酸化。体外细胞角蛋白II对谷胱甘肽S转移酶HTRβ1融合蛋白的磷酸化作用表明，位于铰链区的苏氨酸残基(THT-2 10)可以被细胞角蛋白II磷酸化。为了阐明hTrβ1中Thr-2 10的CKII磷酸化的功能意义，我们接下来进行了野生型和突变的hTrβ1的瞬时转染研究。有趣的是，当使用Thr-2 10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物(N-COR)相互作用的区域内，我们接下来进行了凝胶迁移率改变分析，以检测氨基端截短的N-COR(N-COR)与野生型或突变的HTRβ1之间的相互作用。有趣的是，与维甲酸X受体β(RXRβ)与两种类型的HTRβ1形成相同的异源二聚体不同，NCoRI优先与野生型HTRβ1形成异二聚体，而不是Thr-210突变的HTRβ1。综上所述，我们推测在缺乏配体的情况下，β1中Thr-210的CKII磷酸化可能调节与N-COR的相互作用，并有助于介导基础抑制。

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.