Competition between TRAF2 and TRAF6 regulates NF-kappaB activation in human B lymphocytes.
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.
Zhang
Wen
W
National Institute of Arthritis and Musculoskeletal and Skin Diseases, Intramural Research Program, National Institutes of Health, Bethesda, MD 20892, USA.
Zhang
Xuan
X
Wu
Xiao-Li
XL
He
Liu-Sheng
LS
Zeng
Xiao-Feng
XF
Crammer
Amrie C
AC
Lipsky
Peter E
PE
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000502:physiology*; D019013:CD40 Antigens / Q000378:metabolism; D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D047992:TNF Receptor-Associated Factor 2 / Q000235:genetics / Q000378:metabolism*; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D019013:CD40 Antigens; D016328:NF-kappa B; D016760:Proto-Oncogene Proteins c-fos; D047992:TNF Receptor-Associated Factor 2; D048029:TNF Receptor-Associated Factor 6; D051996:Transcription Factor RelA; D051550:I-kappa B Kinase; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases