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

酪蛋白激酶 II 磷酸化人甲状腺激素受体 beta-1

基本信息

批准号：
09671018
负责人：
SUGAWARA Akira
金额：
$ 1.98万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671018/
关键词：
Thyroid Hormone Receptor Phosphorylation

项目摘要

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.

最近显示，细菌表达的人甲状腺激素受体 beta-1 (hTRbeta1) 可在体外被 HeLa 胞浆提取物磷酸化。在本研究中，我们首先证明 hTRbeta1 也可以在体外被纯化的酪蛋白激酶 II (CKII) 磷酸化。磷酸氨基酸分析显示 hTrbeta1 的丝氨酸和苏氨酸残基均被磷酸化。预测CKII磷酸化位点突变的谷胱甘肽S-转移酶(GST)-hTRbeta1融合蛋白的体外CKII磷酸化表明，位于铰链区(Thr-210)的苏氨酸残基可以被CKII磷酸化。为了阐明hTRbeta1中Thr-210的CKfl磷酸化的功能意义，我们接下来使用野生型或Thr-210突变的hTRbeta1进行瞬时转染研究。有趣的是，在没有配体的情况下，基础抑制eve显着减弱使用 Thr-210 突变的 hTRbeta1。由于 Thr-210 位于与核受体辅阻遏物 (N-CoR) 的相互作用域内，因此我们接下来进行了电泳迁移率 shifi 测定 (EMSA)，以检查氨基末端截短的 N-CoR (NCoRI) 和野生型之间的相互作用或 Thr-210 突变的 hTRbeta1。有趣的是，与类维生素A OMEGA受体β(RXRbeta)同样与两种类型的hTR beta1形成异二聚体相反，N CoRIp与野生型hTRbeta1形成异二聚体，而不是与Thr-210突变的hTRbeta1形成异二聚体。综上所述，我们推测 Thr-210 m hTRbeta1 的 CKII 磷酸化可能调节与 N-Co R 的相互作用，并有助于在没有配体的情况下介导基础抑制。