Phosphorylation of Human Thyroid Hormone Receptor beta-1 by Casein Kinase II

细菌表达的人甲状腺激素受体β 1（hTR β 1）在体外可被HeLa细胞胞质提取物磷酸化。在本研究中，我们首次证明了hTR β 1也可以在体外被纯化的酪蛋白激酶II（CKII）B磷酸化。磷酸化氨基酸分析显示hTrbeta 1在丝氨酸和苏氨酸残基上都被磷酸化。体外CKII磷酸化谷胱甘肽S-转移酶（GST）-hTR β 1融合蛋白，其预测的CKII磷酸化位点突变表明，位于铰链区（Thr-210）的苏氨酸残基可以被CKII磷酸化。为了阐明CKf 1磷酸化Thr-210在hTR β 1中的功能意义，我们用野生型和Thr-210突变型hTR β 1进行瞬时转染研究。有趣的是，当使用Thr-210突变型hTR β 1时，在缺乏配体的情况下基础抑制显著减弱。由于Thr-210位于与核受体辅阻遏物（N-CoR）相互作用的结构域内，我们接下来进行电泳迁移率移位测定（EMSA）以检查氨基末端截短的N-CoR（NCoRI）与野生型或Thr-210突变的hTR β 1之间的相互作用。有趣的是，与同样与两种类型的hTR β 1形成异源二聚体的类维生素A OMEGA受体β（RXR β）相反，N CoRIp与野生型hTR β 1形成异源二聚体，而不是Thr-210突变的hTR β 1。综上所述，我们推测Thr-210 m hTR β 1的CKII磷酸化可能调节与N-Co R的相互作用，并在配体缺乏的情况下介导基础抑制。

Bacterially-expressed human thyroid hormone receptor beta-1 (hTRbeta1) recently has been shown to be phosphorylated in vitro by HeLa cytoso1ic extract. In the present study, we first demonstrated that hTRbeta1 also could be phosphorylated in vitro b purified casein kinase II (CKII). Phosphoamino acid analysis revealed hTrbeta1 was phosphorylated on both serine and threonine residues. In vitro CKII phosphorylation of glutathione S-transferase (GST)-hTRbeta1 fusion proteins whose predicted CKII phosphorylation sites were mutated demonstrated that a threonine residue located in the hinge region(Thr-210) could be phosphorylated by CKII. In order to elucidate the functional significance of CKfl phosphorylation of Thr-210 in hTRbeta1, we next performed transient transfection studies using either wild type or Thr-210 mutated hTRbeta1.In terestingly, the basal repression eve in the absence of ligand was attenuated significantly when Thr-210 mutated hTRbeta1 was used. Since Thr-210 is located within the interacting domain with nudear receptor co-repressor (N-CoR), we next performed electrophoretic mobility shifi assay (EMSA)to examine the interaction between amino terminal-truncated N-CoR (NCoRI) and wild type or Thr-210 mutated hTRbeta1. Interestingly, in contrast to retinoid OMEGA receptor beta(RXRbeta) which equally formed heterodimers with both types of hTR beta1, N CoRIp referentially formed heterodimers with wild type hTRbeta1 than Thr-210mutated hTRbeta1. Taken together, we speculate that CKII phosphorylation ofThr-210 m hTRbeta1 might modulate interaction with N-Co R, and contribute tomediate basal repression in the absence of ligand.