Key roles for phosphorylation and the Coiled-coil domain in TRIM56-mediated positive regulation of TLR3-TRIF–dependent innate immunity

Abstract

Tripartite-motif protein-56 (TRIM56) positively regulates the induction of type I interferon (IFN) response via the TLR3 pathway by enhancing IRF3 activation and depends on its C-terminal residues 621-750 for interacting with the adaptor TRIF. However, the precise underlying mechanism and detailed TRIM56 determinants remain unclear. Herein, we show ectopic expression of murine TRIM56 also enhances TLR3-dependent IFN-β promoter activation, suggesting functional conservation. We found that endogenous TRIM56 and TRIF formed a complex early (0.5-2 h) after poly-I:C stimulation and that TRIM56 overexpression also promoted activation of NF-κB by poly-I:C but not that by TNF-α or IL-1β, consistent with a specific effect on TRIF prior to the bifurcation of NF-κB and IRF3. Using transient transfection and Tet-regulated cell lines expressing various TRIM56 mutants, we demonstrated the Coiled-coil domain and a segment spanning residues ∼434-610, but not the B-box or residues 355-433, were required for TRIM56 augmentation of TLR3 signaling. Moreover, alanine substitution at each putative phosphorylation site, Ser 471 , Ser 475 and Ser 710 , abrogated TRIM56 function. Concordantly, mutants bearing Ser 471 Ala, Ser 475 Ala, or Ser 710 Ala, or lacking the Coiled-coil domain, all lost the capacity to enhance poly-I:C-induced establishment of an antiviral state. Furthermore, the Ser 710 Ala mutation disrupted the TRIM56-TRIF association. Using phospho-specific antibodies, we detected biphasic phosphorylation of TRIM56 at Ser 471 and Ser 475 following TLR3 stimulation, with the early phase occurring at ∼0.5-1 h, prior to IRF3 phosphorylation. Together, these data reveal novel molecular details critical for the TRIM56 augmentation of TLR3-dependent antiviral response and highlight important roles for TRIM56 scaffolding and phosphorylation.

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