RB1-C磷酸化位点突变体与Sedlin的共定位研究

Co-localization of RB1-C-terminal phosphorylation site mutants and Sedlin

目的研究人视网膜母细胞瘤蛋白1羧基端(RB1-C)磷酸化位点突变体的蛋白表达和细胞定位情况及其与迟发性脊椎骨骺发育不良病蛋白(Sedlin)共定位的变化情况。方法设计RB1-C磷酸化位点突变体的引物,以质粒pcDNA3.1-FLAG-RB1-C为模板,利用PCR技术分别扩增出pcDNA3.1-FLAG-RB1-C-S788A、S795A、S807A、T811A、T821A、T826A共6个突变体,并对各位点进行测序。分别将以上突变体单独转染至HEK 293T、COS7细胞中,利用Western blot和免疫荧光(IF)技术观察其蛋白表达和细胞定位情况;分别将以上突变体及Sedlin共同转染至COS7细胞中,利用IF技术观察二者在细胞内的共定位情况,并与野生型RB1-C的结果进行比较分析。结果测序结果显示RB1-C的6个磷酸化位点均突变成功,并能够在真核细胞内正常表达,且与野生型RB1-C的细胞定位基本一致,即主要定位在细胞核内。与野生型RB1-C相比,RB1-C-S788A、S795A、T821A、T826A与Sedlin的共定位未发生改变,即主要共定位在细胞核内;但RB1-C-S807A、T811A与Sedlin的共定位发生了显著变化,除了共定位于细胞核外,在细胞质中也存在共定位现象。结论RB1-C磷酸化位点突变体成功构建并表达,其中S807A、T811A两个磷酸化位点的突变能够显著改变RB1-C与Sedlin在细胞中共定位区域,表明它们是RB1-C和Sedlin结合的关键位点之一,为进一步研究RB1-C和Sedlin的相互作用奠定了基础。

Objective To study the protein expression and cell localization of RB1-C-terminal phosphorylation site mutants,and to investigate the change of co-localization with Sedlin.Methods The primers of RB1-C-terminal phosphorylation site mutants were designed,and pcDNA3.1-FLAG-RB1-C was used as the template to amplify pcDNA3.1-FLAG-RB1-C-S788A,S795A,S807A,T811A,T821A,T826A.The above mutants were separately transfected into HEK 293T and COS7 cells,and their protein expression and cell localization were observed by Western blot and immunofluorescence technology;the co-localization of the above mutants and Sedlin were observed by IF technology,and compared with the results of wild type RB1-C.Results The sequencing results showed that the six phosphorylation sites of RB1-C were mutated successfully and expressed normally in eukaryotic cells,which was basically consistent with the cell localization of wild type RB1-C,and they were mainly located in the nucleus.In addition,compared with the wild-type RB1-C,the co-localization of RB1-C-S788A,S795A,T821A,T826A and Sedlin did not change,that is,they were mainly co-located in the nucleus,but the co-localization of RB1-C-S807A,T811A and Sedlin changed significantly,in addition to co-localization and nucleus,there was also significant co-localization in the cytoplasm.Conclusion RB1-C phosphorylation site mutants were successfully constructed and expressed.The mutations of two phosphorylation sites S807A or T811A can significantly change the co-localization region of RB1-C and Sedlin in cells,indicating that they are one of the key binding sites of RB1-C and Sedlin,which lays a foundation for further study of the interaction between RB1-C and Sedlin.