核糖体蛋白RPS3 Ser209影响NF-κB转录活性及其与DNA结合能力

摘要

目的：构建人核糖体蛋白RPS3及其Ser209突变体的真核表达载体，初步探讨RPS3Ser209突变体对NF－κB转录活性及其DNA结合能力的影响。方法采用高保真PCR扩增RPS3的CDS序列，克隆入pcDNA－3．1myc－HisB载体，构建RPS3－myc表达载体。以构建成功的RPS3－myc质粒为模板，采用点突变方法构建RPS3Ser209突变体RPS3S209A－myc表达载体。报告基因实验检测RPS3及RPS3S209A对NF－κB转录活性的影响；共聚焦显微镜观察RPS3及RPS3S209A的细胞定位；EMSA 实验检测 RPS3及RPS3S209A的 DNA结合能力。结果成功构建RPS3－myc及RPS3S209A－myc真核表达载体，与野生型RPS3相比，RPS3S290A入核显著减少，其对NF－κB的激活显著降低，对NF－κB的DNA结合能力的影响显著下降。结论核糖体蛋白RPS3在NF－κB信号通路中的作用依赖于其第209位丝氨酸。%Objective To construct expression vectors of human ribosomal protein S 3(RPS3) and RPS3Ser209 mutant in orcler to investigate the effect of RPS3Ser209 mutant on NF-κB signaling pathway and DNA binding capacity .Methods The vector RPS3-myc was amplified by polymerase chain reaction ( PCR) from the human liver cDNA and subcloned into pcDNA-3.1myc-HisB.RPS3S209A represented mutant RPS3 expression vectors, in which the designated amino acid was mutated to an alanine residue .Dual luciferase reporter gene assay was used to detect the NF-κB transcription activity in HEK293 cells,immunofluorescence to detect RPS3 location, and EMSA to examine NF-κB DNA-binding activity.Results The expression vectors of RPS3-myc and RPS3S209A-myc were constructed.Compared with wild-type RPS3,the nucleus translocation, transactivation activity of NF-κB and DNA binding ability of RPS3S290A were reduced significantly .Conclu-sion The impact of RPS3 on NF-κB signaling pathway depend on its serine 209.