Journal
MOLECULAR IMMUNOLOGY
Volume 47, Issue 6, Pages 1283-1291Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.molimm.2009.12.002
Keywords
Toll-like receptor 3; TICAM-1 (TRIF); TRAF2; TRAF6; Interferon-beta
Categories
Funding
- Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases
- MEXT
- Sapporo Biocluster Bio-S
- Ministry of Education, Science, and Culture
- Ministry of Health. Labor, and Welfare of Japan
- Mitsubishi Foundation
- Mochida Foundation
- NorthTec Foundation
- Yakult Foundation
- Japan Society for the Promotion of Science
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Using yeast two-hybrid screening, we found three TRAF proteins TRAF1,2 and 6, bound the N-terminal region of the TLR3/4 adaptor TICAM-1 (TRIF). TRAF2, a newly identified TICAM-1-binding protein, bound the PxQxS motif (aa 333-338) of TICAM-1 using mutagenesis by alanine substitutions. TICAM-1 is known to induce the activation of NF-kappa B and IRF-3, which leads to activation of the interferon (IFN)-beta promoter, an activity that is conserved in the N + TIR fragment (aa 1-533). By mutation of the two distinct binding sites for TRAF2 and TRAF6 in N + TIR TICAM-1, the induction of IFN-beta was completely abrogated. Although the TRAF2 site single mutation only marginally affected TICAM-1-mediated type I IFN induction, it further impaired the function of the TRAF6 site mutant. Moreover, double point mutations of the TRAF2 and TRAF6 binding motifs in TICAM-1 N + TIR reduced the activation of IRF-3 and NF-kappa B, the critical transcription factors for IFN-beta expression. Furthermore, TRAF2/6 functioned as an E3 ligase to induce K63-mediated ubiquitination on N + TIR which was abrogated in the mutant lacking the TRAF2/6 sites in parallel with IFN-inducing activity. Confocal microscopy analysis indicated that TRAF2 and TRAF6 merged with oligomerized (i.e. activated) TICAM-1 N + TIR. However, TRAF3, which is another TRAF family member essential for TLR3-mediated type-IIFN signaling, still assembled in the mutant lacking the TRAF2/6 sites. Our data suggest that the binding of TRAF2 and TRAF6 to TICAM-1 cooperatively activates the IFN-inducing pathway through ubiquitination of TICAM-1, a modification which occurs unrelated to TRAF3 recruitment in the TICAM-1 signaling complex. TRAF2/6 may participate in TICAM-1-mediated IFN-beta induction besides TRAF3. (C) 2009 Elsevier Ltd. All rights reserved.
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