NUAK1—NFkB通路新转录调控或共调控因子.doc

NUAK1—NFkB通路新转录调控或共调控因子？ 杨兰兰， 刘先俊*（重庆医科大学：分子医学与肿瘤研究中心，重庆 400016） 摘要：目的 确定蛋白激酶NUAK1的定位，检测其是否参与NFkB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NUAK1( D178N )-pcDNA 3.1，以空载pcDNA3.1做对照，分别过表达于带稳转有NFkB荧光酶素报告基因质粒的293细胞株中，检测在有或没有TNFα刺激下的NFkB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 NUAK1定位于细胞核中，在293细胞中NUAK1能够抑制NFkB的活性，且和其表达量成正相关。结论 NUAK1有可能是NFkB的转录调控或共调控因子。 关键词： NUAK1；NFKB；239细胞；核定位；转录调控 NUAK1---a new transcriptional regulated or co-regulated factor of NFkB pathway? YANG Lanlan， LIU Xianjun (Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016) Abstract: Objective To make sure the subcellular location of NUAK1 and that whether NUAK1 is involved in NFkB 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 test the NFkB luciferase activation no or under the stimulation of TNFα , with the control of pcDNA3.1, NUAK1-pcDNA3.1 and NUAK1( D178N )-pcDNA 3.1 were overexpressed in a 293 cell line which was stably expressing NFkB luciferase promoter reporter, and a plasmid dose response experiment was performed for further study. Results NUAK1 was constitutively distributed in the nucleus. In 293 cells , NUAK1 inhibited the NFkB luciferase activation and had a significantly positive correlation with the expression of NUAK1. Conclusion NUAK1 might be one regulated or co-regulated transcriptional factor of the NFkB pathway . Keywords: NUAK1; NFkB; 293 cell; Nucleus Location; Transcriptional Regulation AMP-激活蛋白激酶（AMPK）是真核生物能量代谢信号转导中一个高保守的关键因子，有着能量传感器之称，它不仅调控了细胞的能量代谢，更控制着整个生命个体的能量平衡。目前它定位于日益增长的代谢紊乱病中肥胖症和Ⅱ型糖尿病的可能药物治疗靶点（1，2）。它是一个异源三聚体，包括一个催化亚基（α1 、 α2 ）和 两个调节亚基 （β1、β2和γ1、γ2、γ3），不同物种的这些亚基的亚型和定位存在区别（3-5），其中催化亚基都含有一个激酶区（KD）和紧跟其后的一个自抑制区（与UBA（ubiquitin -associated domain）具有低同源性）