JAB1与糖皮质激素受体的相互作用及对其转录活性的影响

JAB1 Interacts With GR and Enhances GR-mediated Transcription

应用酵母双杂交方法筛选到与糖皮质激素受体 (GR)结合的蛋白JAB1 ,进一步验证JAB1与GR的结合作用并证明其对GR的影响 .构建与Gal4 BD融合表达的载体pGBKT7 GRLBD ,与构建于pACT2载体上的人骨髓cDNA文库杂交 ,在SD/ Ade/ His/ Leu/ Trp选择培养板上培养 ,经X α gal检测 ,阳性克隆片段插入pGEM TVector载体 ,测序 ,再经酵母双杂交和GSTpulldown蛋白质结合实验验证其结合作用 ,应用反映GR转录活性的CAT报告基因检测JAB1对GR的调节活性 .结果在人骨髓cDNA文库中 ,筛选到 4 2个X α gal检测变蓝且含有pACT2质粒序列的克隆 ,其中有 5个克隆的序列皆为Jun活性区结合蛋白JAB1的一部分 .酵母双杂交和蛋白质结合实验表明 ,JAB1与COS7真核表达的GR LBD在体外有结合作用 .

A glucocorticoid receptor(GR) interacting protein JAB1 was isolated from human marrow cDNA library by two hybrid screening in yeast using the GR ligand binding domains (GR LBD) as bait. To further demonstrate the interaction between GR and JAB1 and the effect of JAB1 on GR, PCR was performed to amplify GR LBD fragments and it was cloned into the bait vector pGBKT7 to create the plasmid pGBKT7 GR LBD. The plasmid was used as bait to screen a cDNA library constructed in the pACT2 vector. Transformants were plated on SD/-Ade/-His/-Leu/-Trp. Yeast colonies were assayed for α galactosidase activity. The positive colonies were sequenced in which JAB1 cDNA fragments were cloned into the vector pGEX 4T 1 for GST pull analysis. A GRE driven reporter gene was used for CAT activity assay. The results were as follows: 42 clones turned blue in the yeast α galactosidase assay and contained pACT2 sequence in which 5 clones corresponded to a fragment of JAB1 as shown by DNA sequencing. Yeast two hybrid and GST pull down assay verified that JAB1 interacted with GR LBD. GR, a GRE driven reporter gene cotransfected with JAB1, strongly potentiated the transactivation properties of GR. The studies suggest that JAB1 interacts with GR LBD expressed in COS7 cells in vitro and it potentiates the transactivation properties of GR.