TRAF2与TRAF6之间的竞争调节人B淋巴细胞中的NF-κB激活。

Competition between TRAF2 and TRAF6 regulates NF-kappaB activation in human B lymphocytes.

作者信息

Zhang Wen, Zhang Xuan, Wu Xiao-Li, He Liu-Sheng, Zeng Xiao-Feng, Crammer Amrie C, Lipsky Peter E

机构信息

National Institute of Arthritis and Musculoskeletal and Skin Diseases, Intramural Research Program, National Institutes of Health, Bethesda, MD 20892, USA.

出版信息

Chin Med Sci J. 2010 Mar;25(1):1-12. doi: 10.1016/s1001-9294(10)60013-2.

DOI:10.1016/s1001-9294(10)60013-2

PMID:20449947

Abstract

OBJECTIVE

To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-kappaB (NF-kappaB) signaling pathway and whether CD40 signaling requires TRAF2.

METHODS

Human B cell lines were transfected with plasmids expressing wild type TRAF2 or dominant negative TRAF2, TRAF2-shRNA, or TRAF6-shRNA. The activation of NF-kappaB was detected by Western blot, kinase assay, transfactor enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET). Analysis of the role of TRAF-2 and TRAF-6 in CD40-mediated NF-kappaB activity was examined following stimulation with recombinant CD154.

RESULTS

TRAF2 induced activity of IkappaB-kinases (IKKalpha, IKKi/epsilon), phosphorylation of IkappaBalpha, as well as nuclear translocation and phosphorylation of p65/RelA. In contrast, TRAF6 strongly induced NF-kappaB activation and nuclear translocation of p65 as well as p50 and c-Rel. Engagement of CD154-induced nuclear translocation of p65 was inhibited by a TRAF6-shRNA, but conversely was enhanced by a TRAF2-shRNA. Examination of direct interactions between CD40 and TRAFs by FRET documented that both TRAF2 and TRAF6 directly interacted with CD40. However, the two TRAFs competed for CD40 binding.

CONCLUSIONS

These results indicate that TRAF2 can signal in human B cells, but it is not essential for CD40-mediated NF-kappaB activation. Moreover, TRAF2 can compete with TRAF6 for CD40 binding, and thereby limit the capacity of CD40 engagement to induce NF-kappaB activation.

摘要

目的

研究肿瘤坏死因子受体相关因子2（TRAF-2）和TRAF6在CD40诱导的核因子-κB（NF-κB）信号通路中的作用，以及CD40信号传导是否需要TRAF2。

方法

用人B细胞系转染表达野生型TRAF2或显性负性TRAF2、TRAF2-shRNA或TRAF6-shRNA的质粒。通过蛋白质免疫印迹法、激酶测定、转录因子酶联免疫吸附测定（ELISA）和荧光共振能量转移（FRET）检测NF-κB的激活情况。在用重组CD154刺激后，分析TRAF-2和TRAF-6在CD40介导的NF-κB活性中的作用。

结果

TRAF2诱导IκB激酶（IKKα、IKKi/ε）的活性、IκBα的磷酸化，以及p65/RelA的核转位和磷酸化。相比之下，TRAF6强烈诱导NF-κB激活以及p65、p50和c-Rel的核转位。TRAF6-shRNA抑制了CD154诱导的p65核转位，但相反，TRAF2-shRNA增强了这种核转位。通过FRET检测CD40与TRAFs之间的直接相互作用，结果表明TRAF2和TRAF6均与CD40直接相互作用。然而，这两种TRAFs竞争与CD40的结合。