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Direct binding of TRAF2 and TRAF6 to TICAM-1/TRIF adaptor participates in activation of the Toll-like receptor 3/4 pathway.

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-kappaB 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-kappaB, 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-I IFN 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.

Pubmed ID: 20047764

Authors

  • Sasai M
  • Tatematsu M
  • Oshiumi H
  • Funami K
  • Matsumoto M
  • Hatakeyama S
  • Seya T

Journal

Molecular immunology

Publication Data

March 1, 2010

Associated Grants

None

Mesh Terms

  • Adaptor Proteins, Vesicular Transport
  • Amino Acid Sequence
  • Amino Acids
  • Animals
  • Binding Sites
  • Cell Line
  • Humans
  • Interferon-beta
  • Mice
  • Microscopy, Confocal
  • Molecular Sequence Data
  • Polyubiquitin
  • Protein Binding
  • Protein Transport
  • Signal Transduction
  • Structure-Activity Relationship
  • TNF Receptor-Associated Factor 2
  • TNF Receptor-Associated Factor 6
  • Toll-Like Receptor 3
  • Toll-Like Receptor 4
  • Transcription Factors
  • Ubiquitination