The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL.
TRAF6, a crucial adaptor molecule in innate and adaptive immunity, contains three distinct functional domains. The C-terminal TRAF domain facilitates oligomerization and sequence-specific interaction with receptors or other adaptor proteins. In conjunction with the dimeric E2 enzyme Ubc13-Uev1A, the N-terminal RING domain of TRAF6 functions as an E3 ubiquitin (Ub) ligase that facilitates its own site-specific ubiquitination through the generation of a Lys-63-linked poly-Ub chain. This modification does not cause its proteasomal degradation but rather serves as a scaffold to activate both the IKK and stress kinase pathways. Connecting the N-and C-terminal regions, the four internal zinc finger (ZF) motifs have yet to be functionally defined. In this study, we examined the role of the ZF domains in interleukin-1, lipopolysaccharide, and RANKL signaling by reconstitution of TRAF6-deficient cells with point mutations or deletions of these ZF motifs. Although ZF domains 2-4 are dispensable for activating IKK, p38, and JNK by interleukin-1 and lipopolysaccharide, the first ZF domain together with an intact RING domain of TRAF6 is essential for activating these pathways. Furthermore, TRAF6 autoubiquitination and its interaction with Ubc13 are dependent on ZF1 and an intact RING domain. Additionally, expression of TRAF6 lacking ZF2-4 in TRAF6-deficient monocytes rescues RANKL-mediated osteoclast differentiation and LPS-stimulated interleukin-6 production. These data provide evidence for the critical role of the Ub ligase activity of TRAF6, which is coordinated via the RING domain and ZF1 to supply the necessary elements in signaling by cytokines dependent upon TRAF6.
Lamothe
Betty
B
Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Campos
Alejandro D
AD
Webster
William K
WK
Gopinathan
Ambily
A
Hur
Lana
L
Darnay
Bryant G
BG
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D007113:Immunity, Innate / Q000502:physiology; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology; D016335:Zinc Fingers / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
C508600:IL6 protein, human; D007375:Interleukin-1; D015850:Interleukin-6; D008070:Lipopolysaccharides; D053245:RANK Ligand; D048029:TNF Receptor-Associated Factor 6; C505688:TNFSF11 protein, human; C505689:Tnfsf11 protein, mouse; D014157:Transcription Factors; C471607:UBE2N protein, human; C471624:UBE2V1 protein, human; C524395:UBE2V1 protein, mouse; C514982:Ube2n protein, mouse; D044763:Ubiquitin-Conjugating Enzymes; D051550:I-kappa B Kinase; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D046988:Proteasome Endopeptidase Complex