突变雄激素受体第651位丝氨酸磷酸化对致病蛋白功能的影响

Phosphorylation at Androgen Receptor Ser 651 Affects the Nuclear Translocation of Mutant AR Protein

目的定点改变突变雄激素受体(AR)的第651位丝氨酸,建立脊髓延髓肌肉萎缩症细胞模型,探究该位点不同磷酸化状况对致病AR蛋白核转位的影响。方法体外培养小鼠神经母细胞瘤Neuro 2a细胞系,以AR-65Q质粒作为模板,将AR-65Q第651位丝氨酸分别定点突变为丙氨酸和天冬氨酸,构建AR-65Q的去磷酸化S651A质粒和拟磷酸化S651D质粒,以及带绿色荧光蛋白的重组质粒EGFP AR-65Q、EGFP AR S651A和EGFP AR S651D。将上述质粒测序鉴定后转入细胞系,应用激光共聚焦显微镜观察不同磷酸化状况致病AR蛋白在胞内的分布,采用实时定量PCR验证磷酸化对致病AR m RNA水平的影响,用Western blot分析验证致病AR蛋白的胞质胞核分布情况以及稳定性。结果 (1)以AR-65Q质粒构建的致病AR蛋白第651位丝氨酸拟磷酸化AR S651D质粒和去磷酸化AR S651A质粒,相应的EGFP AR-65Q、EGFP AR S651D和EGFP AR S651A质粒测序正确;(2)第651位丝氨酸磷酸化可使致病AR蛋白在细胞核的聚集减少(P<0.05);(3)致病AR蛋白第651位丝氨酸磷酸化不影响致病AR m RNA表达水平(P>0.05)和蛋白表达水平(P>0.05)。结论致病AR蛋白第651位丝氨酸的磷酸化可以减少致病AR蛋白在细胞核内聚集,调控该位点磷酸化有望成为治疗脊髓延髓肌肉萎缩症的药物新靶点。

Aim To construct phosphorylation mutant androgen receptor plasmids at Ser 651 and explore its influence on mutant androgen receptor（AR） protein nuclear translocation in cell models of SBMA. Methods The coding sequence of mutant AR was amplified by PCR from plasmid AR-65 Q. After site-directed mutagenesis in AR-65 Q in which serine 651 was substituted with either alanine, which cannot be phosphorylated, or aspartate, which mimics constitutive phosphorylation, DNA sequencing was performed to confirm them. So did the plasmids fused with EGFP. These recombinant plasmids were transfected into Neuro2 a cells and a confocal microscope was used to observe how phosphorylation affect the mutant AR protein subcellular translocation. The influence of phosphorylation on the m RNA level of mutant AR was examined by quantitative real-time PCR and the nuclear/cytoplasmic distribution and protein stability of mutant AR were detected by Western blot assay in transfected cells. Results（1） The recombinant plasmids which were site-directed phosphorylated at AR Ser 651 were confirmed right by DNA sequencing;（2） Phosphorylated mutant AR protein at Ser 651 had less nuclear aggregations（P〈0.05）.（3） Phosphorylation on the AR Ser 651 have no influence on m RNA expression of AR（P〈0.05） and the protein stability（P〈0.05）. Conclusion Our study demonstrated that phosphorylation on the AR Ser 651 reduced the nuclear accumulation of mutant AR protein and regulation on the phosphorylation at AR Ser 651 would be an novel drug target for Kennedy disease.