Ishibe Y
Department of Otolaryngology, Hokkaido University School of Medicine, Sapporo, Japan.
Hokkaido Igaku Zasshi. 1993 Sep;68(5):728-35.
Estrogen receptor (ER) complex with estrogen interacts with estrogen responsive element (ERE) in target gene promoters and modulates transcription. ER phosphorylation process might be involved in the transcription regulation. To investigate the ER phosphorylation, the author have purified ER from porcine uterine cytosol through heparin-Sepharose column chromatography followed by immunoprecipitation with monoclonal anti ER antibody and protein A-Sepharose. The ER was phosphorylated with [gamma-32P] ATP without any protein kinase, showing that ER has autophosphorylation activity. 63kDa and 120kDa proteins were mainly detected by SDS-PAGE analysis following autoradiography. ER became phosphorylated in the presence of divalent cation such as Mg2+ or Ca2+ in a time-dependent manner and an ATP concentration-dependent manner. Phosphoamino acid analysis showed phosphorylation of serine residue. Phosphorylation was stimulated by estrogen (E2), and phosphorylated receptor was bound to DNA fragment that contained EREs. It is suggested that ER phosphorylation process might participate in transcription regulation.
雌激素受体（ER）与雌激素形成的复合物与靶基因启动子中的雌激素反应元件（ERE）相互作用并调节转录。ER磷酸化过程可能参与转录调控。为了研究ER磷酸化，作者通过肝素-琼脂糖柱色谱从猪子宫胞质溶胶中纯化了ER，随后用单克隆抗ER抗体和蛋白A-琼脂糖进行免疫沉淀。ER在没有任何蛋白激酶的情况下用[γ-32P]ATP进行磷酸化，表明ER具有自磷酸化活性。放射自显影后通过SDS-PAGE分析主要检测到63kDa和120kDa的蛋白质。ER在二价阳离子如Mg2+或Ca2+存在下以时间依赖性方式和ATP浓度依赖性方式发生磷酸化。磷酸氨基酸分析显示丝氨酸残基发生磷酸化。雌激素（E2）刺激磷酸化，并且磷酸化的受体与含有ERE的DNA片段结合。提示ER磷酸化过程可能参与转录调控。
