ERα AF-1区磷酸化位点突变体的构建及对ERα活性的影响  

Construction of mutants of phosphorylation sites in ERα AF-1 and effects of mutants on transcriptional activity of ERα

作　　者：宁康[1] 刘雅娟[2] 杨怀宁[3] 范洪学[3] 杨智洪[1] 李熔[1] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]吉林大学公共卫生学院营养与食品卫生学教研室,吉林长春130021 [3]吉林大学公共卫生学院卫生毒理学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2007年第2期200-203,共4页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(305303203037073830428012)

摘　　要：目的:构建雌激素受体(ERα)AF1区丝氨酸(Ser)磷酸化位点104、106、118和167位氨基酸的4个突变体,并在哺乳动物细胞293T中检测突变后对其活性的影响。方法:以pcDNA3-ERα为模板,用重组PCR的方法扩增出编码这4种突变体的基因片段,再将这些片段以正确相位与pcDNA3-FLAG载体中的FLAG编码序列融合。用Western blotting检测融合蛋白的表达,并在哺乳动物细胞293T中检测这些突变体突变后对ERα活性的影响。结果:成功构建了ERαAF1区丝氨酸磷酸化位点104、106、118和167位氨基酸的4个突变体,并在293T细胞中得到表达。活性测定结果表明,在不加雌激素(E2)的情况下,ERα及其突变体的转录活性分别是空载体pcDNA3的3.40、3.21、3.02、3.00和3.54倍;在雌激素的作用下,104、106位氨基酸的突变体对ERα的活性无影响,而118和167位氨基酸的突变体的活性值是ERα的50%。结论:在104、106、118和167这4个ERαAF1区磷酸化位点中,只有118和167位点的丝氨酸的磷酸化是雌激素调节靶基因转录过程中所必需的。Objective To construct the mutants of the four serine phosphorylation sites （104, 106, 118 and 167） in ERα AF-1 and detect their effects on the transcriptional activity of Erα in 293T cells. Methods The coding sequences of the ERα mutants were amplified by recombinant PCR with pcDNA3-ERα as a template and fused in frame with the coding region of FLAG in the pcDNA3-FLAG vector. The fusion proteins were characterized by Western blotting. The effects of the mutants on the transcriptional activity of ERa were determined. Results ERa mutants were successfully constructed and expressed in 293T cells. The transcriptional activities of ERα and its mutants were 3.40, 3.21, 3.02, 3.00, 3.54 times of pcDNA3 in the absence of E2. Only the mutants of 118 and 167 phosphorylation sites made the transcriptional activity reduce to 50% of ERα in the presence of E2. Conclusion Only118 and 167 phosphorylation sites are necessary during the regulation of estrogen on target gene transcription among the four serine phosphorylation sites （104, 106, 118 and 167） in ERα AF1.

关 键 词：受体 雌激素 反式激活(遗传学) 点突变 

分 类 号：R737.9[医药卫生—肿瘤]