SUMO-2/3与孕激素受体的共价结合及其对该受体转录活性的调节

SUMO-2/3 can covalently bind to progesterone receptor B to regulate its transcriptional activity

目的研究B型孕激素受体(progesterone receptor B type;PRB)是否能被SUMO-2、SUMO-3类泛素化修饰,并探讨这种修饰对孕激素受体转录活性的影响。方法以人的MCF-7 cDNA为模板进行PCR反应,扩增出SUMO-2、SUMO-3的cDNA,构建真核表达载体pcDNA3FLAG-SUMO2与pcDNA3FLAG-SUMO3;将质粒pXJ40-myc-PRB分别与pcDNA3-FLAG、pcDNA3FLAG-SUMO2、pcDNA3FLAG-SUMO3共转染人胚肾细胞293T,运用免疫共沉淀及western印迹的方法检测其是否发生类泛素化修饰;运用测定荧光素酶报告基因的方法检测类泛素化修饰对孕激素受体转录活性的影响。结果构建成功表达载体pcDNA3FLAG-SUMO2与pcDNA3FLAG-SUMO3;免疫共沉淀实验证实sumo2/3均可以与PRB共价结合;SUMO-2、SUMO-3能以孕激素依赖的方式增强PRB的转录活性。结论 SUMO-2、SUMO-3分子能够对孕激素受体PRB进行类泛素化修饰,并调节其分子功能。

Objective To investigate whether progesterone receptor B（PRB） can be sumoylated by SUMO-2/3 and the effect of sumoylation on PRB transcriptional activity.Methods SUMO-2/3 cDNA was amplified from MCF-7 cDNA and cloned into the eukaryotic expression vector pcDNA3-FLAG.The plasmid pXJ40-myc-PRB was cotransfected with pcDNA3FLAG-SUMO2,pcDNA3FLAG-SUMO3 or the mock control into 293T cells,and PRB sumoylation was detected by immunoprecipitation and Western blotting.The effect of PRB sumoylation on its transcriptional activity was determined using reporter luciferase assay.Results pcDNA3FLAG-SUMO2 and pcDNA3FLAG-SUMO3 vectors were successfully constructed.SUMO-2/3 could bind covalently to PRB and increase its transcriptional dependent on the presence of progesterone.Conclusion PRB can be sumoylated by SUMO-2/3 and its function is regulated by this modification.