Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.
Mol Immunol. 2010 Mar;47(6):1283-91. doi: 10.1016/j.molimm.2009.12.002. Epub 2010 Jan 4.
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.
利用酵母双杂交筛选，我们发现三个 TRAF 蛋白 TRAF1、2 和 6 与 TLR3/4 衔接子 TICAM-1（TRIF）的 N 端区域结合。TRAF2，一种新鉴定的 TICAM-1 结合蛋白，通过丙氨酸取代突变，结合 TICAM-1 的 PxQxS 基序（aa333-338）。TICAM-1 已知诱导 NF-kappaB 和 IRF-3 的激活，这导致干扰素（IFN）-β启动子的激活，该活性在 N+TIR 片段（aa1-533）中保守。通过突变 N+TIR TICAM-1 中两个不同的 TRAF2 和 TRAF6 结合位点，完全阻断了 IFN-β的诱导。尽管 TRAF2 位点的单突变仅轻微影响 TICAM-1 介导的 I 型 IFN 诱导，但它进一步削弱了 TRAF6 位点突变体的功能。此外，TICAM-1 N+TIR 中 TRAF2 和 TRAF6 结合基序的双点突变降低了 IRF-3 和 NF-kappaB 的激活，这是 IFN-β表达的关键转录因子。此外，TRAF2/6 作为 E3 连接酶，诱导 N+TIR 上 K63 介导的泛素化，在缺乏 TRAF2/6 结合位点的突变体中，与 IFN 诱导活性平行，这种泛素化被消除。共焦显微镜分析表明，TRAF2 和 TRAF6 与寡聚化（即激活）的 TICAM-1 N+TIR 融合。然而，TRAF3，作为 TLR3 介导的 I 型 IFN 信号传导所必需的另一种 TRAF 家族成员，仍然在缺乏 TRAF2/6 结合位点的突变体中组装。我们的数据表明，TRAF2 和 TRAF6 与 TICAM-1 的结合通过 TICAM-1 的泛素化协同激活 IFN 诱导途径，这种修饰与 TICAM-1 信号复合物中 TRAF3 的募集无关。TRAF2/6 可能参与 TICAM-1 介导的 IFN-β诱导，而不仅仅是 TRAF3。
