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.
Sasai
Miwa
M
Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.
Tatematsu
Megumi
M
Oshiumi
Hiroyuki
H
Funami
Kenji
K
Matsumoto
Misako
M
Hatakeyama
Shigetsugu
S
Seya
Tsukasa
T
D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D016899:Interferon-beta / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D025821:Polyubiquitin / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism*; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism*; D051196:Toll-Like Receptor 3 / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014157:Transcription Factors; D054875:Ubiquitination
D033942:Adaptor Proteins, Vesicular Transport; D000596:Amino Acids; C470409:TICAM1 protein, human; D047992:TNF Receptor-Associated Factor 2; D048029:TNF Receptor-Associated Factor 6; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D014157:Transcription Factors; D025821:Polyubiquitin; D016899:Interferon-beta