蛋白激酶NUAK1的定位及其与NF-κB信号通路关系的探讨

Location of Kinase NUAK1 and Research into NUAK1 in NF-κB Pathway

目的:确定蛋白激酶NUAK1的定位,检测其是否参与NF-κB信号通路。方法:构建质粒NUAK1-pEGFP顺转于293细胞,然后用DAPY染核,荧光显微镜下观察NUAK1的定位。构建质粒NUAK1-pcDNA3.1和显性失活突变体NU-AK1(D178N)-pcDNA3.1,以空载pcDNA3.1做对照,分别过表达于带稳转有NF-κB荧光酶素报告基因质粒的293细胞株中,检测在有或没有TNF-α刺激下的NF-κB荧光酶素报告基因的活性,为进一步确认,对NUAK1-pcDNA3.1设了个浓度梯度进行检测。结果:293细胞中,NUAK1定位于细胞核。在TNF-α的刺激下,以空载和显性失活突变体为对照,NU-AK1显著抑制NF-κB的活性,且与其表达量成正相关。结论:由于NUAK1定位于细胞核中,且显著抑制TNFα刺激下NF-κB的活性,所以NUAK1可能参与NF-κB的转录调控。

AIM：To make sure the subcellular location of NUAK1 and whether NUAK1 is involved in NF-κB pathway.METHODS：First prepared plasmids NUAK1-pEGFP,NUAK1-pcDNA3.1 and NUAK1（ D178N ）-pcDNA 3.1.Then after NUAK1-pEGFP was transiently expressed in transfected 293 cells,nuclear DNA was counterstained with DAPY and the cells were observed under fluorescence microscope system.To detect the NF-κB luciferase activation under or without the stimulation of TNF-α,with pcDNA3.1 as the control,NUAK1-pcDNA3.1 and the dominant negative NUAK1（ D178N ）-pcDNA 3.1 were overexpressed in a 293 cell line which was stably expressing NF-κB luciferase promoter reporter,and a plasmid dose response experiment was performed for further study.RESULTS：In 293 cells,NUAK1 was constitutively distributed in the nucleus.And under the stimulation of TNF-α,NUAK1 inhibited the NF-κB luciferase activation and had a significantly positive correlation with the expression of NUAK1.CONCLUSION：Due to both of nuclear localization and the remarkable inhibition in the detection of the NF-κB luciferase activation under the stimulation of TNF-α,NUAK1 might be involved in the transcriptional regulation of NF-κB pathway.