Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation.
Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is a key mediator in proximal signaling of the interleukin-1/Toll-like receptor and the TNF receptor superfamily. Analysis of TRAF6-deficient mice revealed a fundamental role of TRAF6 in osteoclastogenesis; however, the molecular mechanism underlying TRAF6 signaling in this biological process is not understood. Recent biochemical evidence has indicated that TRAF6 possesses ubiquitin ligase activity that controls the activation of IKK and NF-kappaB. Because these studies are primarily based on cell-free systems, the role of the ubiquitin ligase activity of TRAF6 and its auto-ubiquitination to initiate the NF-kappaB pathway in vivo remain elusive. Here we show that an intact RING domain of TRAF6 in conjunction with the E2 enzyme Ubc13/Uev1A is necessary for Lys-63-linked auto-ubiquitination of TRAF6 and for its ability to activate IKK and NF-kappaB. Furthermore, a RING mutant of TRAF6 abolishes its ability to induce receptor activator of NF-kappaB-independent osteoclast differentiation and nuclear accumulation of the transcription factor NFATc1. Notably, we map the auto-ubiquitination site of TRAF6 to a single Lys residue, which if mutated renders TRAF6 unable to activate transforming growth factor-beta-activated kinase 1 and IKK and to cause spontaneous osteoclast differentiation. Additionally, we provide biochemical and in vivo evidence that TRAF6 serves as an E3 to directly ubiquitinate NEMO. Reconstituting TRAF6-deficent cells with various TRAF6 mutants, we clearly demonstrate the requirement for the TRAF6 RING domain and site-specific auto-ubiquitination of TRAF6 to activate IKK in response to interleukin-1. These data establish a signaling cascade in which regulated site-specific Lys-63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of IKK.
Lamothe
Betty
B
Department of Experimental Therapeutics, The University of Texas M D Anderson Cancer Center, Houston, Texas 77030, USA.
Besse
Arnaud
A
Campos
Alejandro D
AD
Webster
William K
WK
Wu
Hao
H
Darnay
Bryant G
BG
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D048029:TNF Receptor-Associated Factor 6 / Q000737:chemistry* / Q000172:deficiency / Q000378:metabolism* / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism / Q000502:physiology
D016328:NF-kappa B; D048029:TNF Receptor-Associated Factor 6; D014157:Transcription Factors; D025801:Ubiquitin; C471607:UBE2N protein, human; C471624:UBE2V1 protein, human; C514982:Ube2n protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; D051550:I-kappa B Kinase; D008239:Lysine