Objective To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-κB (NF-κB) 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-κB 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-κB activity was examined following stimulation with recombinant CD154. Results TRAF2 induced activity of IκB-kinases (IKKα

IKKi/ε)

phosphorylation of IκBα

as well as nuclear translocation and phosphorylation of p65/RelA. In contrast

TRAF6 strongly induced NF-κB 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-κB activation. Moreover

TRAF2 can compete with TRAF6 for CD40 binding

and thereby limit the capacity of CD40 engagement to induce NF-κB activation. Supported by Key Projects of the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (2008-BAI59B02)