Key Roles for Phosphorylation and the Coiled-coil Domain in TRIM56-mediated Positive Regulation of TLR3-TRIF-dependent Innate Immunity

Benjamin M. Liu, Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN; Divisions of Pathology and Laboratory Medicine, Children's National Hospital, Washington, DC; Departments of Pediatrics, Immunology and Tropical Medicine, the George Washington University School of Medicine and Health Sciences, Washington, DC; Departments of Pathology, Immunology and Tropical Medicine, the George Washington University School of Medicine and Health Sciences, Washington, DC; Departments of Microbiology, Immunology and Tropical Medicine, the George Washington University School of Medicine and Health Sciences, Washington, DC; Children's National Research Institute, Washington, DC; The District of Columbia Center for AIDS Research, Washington, DC.
Nan L. Li, Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN.
Ruixue Wang, Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN.
Xiaofan Li, Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN.
Z Alex Li, Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN.
Tony N. Marion, Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN.
Kui Li, Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN. Electronic address: kli1@uthsc.edu.

Document Type

Journal Article

Publication Date

3-29-2024

Journal

The Journal of biological chemistry

DOI

10.1016/j.jbc.2024.107249

Keywords

Coiled-coil domain; IRF3; NF-kappaB; TRIF; TRIM56; Toll-like receptor-3; antiviral state; interferon; phosphorylation

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, Ser and Ser, abrogated TRIM56 function. Concordantly, mutants bearing SerAla, SerAla, or SerAla, or lacking the Coiled-coil domain, all lost the capacity to enhance poly-I:C-induced establishment of an antiviral state. Furthermore, the SerAla mutation disrupted the TRIM56-TRIF association. Using phospho-specific antibodies, we detected biphasic phosphorylation of TRIM56 at Ser and Ser 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.

Department

Pathology

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