Abstract
Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM-1, also named TIR domain-containing adaptor-inducing interferon (IFN)-beta or TRIF)) is a signaling adaptor of Toll-like receptor (TLR) 3/4 that activates the transcription factors, interferon regulatory factor-3 (IRF-3) and NF-kappaB leading to inducing IFN-beta production. The mechanisms by which TICAM-1 is activated by TLR3/4 to serve as a signaling platform are unknown. In this study, we show that homo-oligomerization of TICAM-1 is critical for TICAM-1-mediated activation of NF-kappaB and IRF-3. Both TIR and C-terminal domain of TICAM-1 mediated TICAM-1 oligomerization. Pro(434) located in the TIR domain and the C-terminal region, with the exception of the RIP homotypic-interacting motif, were determinants of TICAM-1 oligomerization. Mutation of TIR domain (P434H) or deletion of C-terminal domain greatly reduced TICAM-1-mediated NF-kappaB and IFN-beta promoter activation. TICAM-1 oligomerization at either the TIR domain or the C-terminal region resulted in recruitment of tumor necrosis factor receptor-associated factor 3, a downstream signaling molecule essential for TICAM-1-mediated IRF-3 activation, but not recruitment of the IRF-3 kinase complex, NF-kappaB-activating kinase-associated protein 1 and TANK-binding kinase 1. In addition, RIP homotypic-interacting motif mutant, which possesses two oligomerization motifs but not the RIP1 binding motif, also failed to recruit NF-kappaB-activating kinase-associated protein 1 and TANK-binding kinase 1. Thus, full activation and formation of TICAM-1 signalosomes requires oligomerization induced at two different sites and RIP1 binding.
Highlights
Among these receptors, TLR3 has distinct properties that allow recognition of extracellular virus-derived doublestranded RNA and induction of type I IFN/cytokine production and dendritic cell maturation that results in activation of natural killer cells and cytotoxic T lymphocytes [3,4,5,6]
TLR3 signaling is mediated via an adaptor molecule, Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM-1, named TIR domain-containing adaptor inducing IFN- (TRIF)), which activates the transcription factors interferon regulatory factor-3 (IRF-3), NF-B, and AP-1 [7, 8]
Toll-like receptors (TLRs)- and retinoic acid-inducible gene-I (RIG-I)/MDA5mediated signaling is transmitted via interaction of unique domain structures, TIR and caspase recruitment domain (CARD), respectively, the detailed mechanisms of signal transduction regulated by these domains as well as the spatiotemporal regulation of signaling have not been studied
Summary
Cell Culture and Reagents—HEK293 cells and HEK293FT cells were maintained in Dulbecco’s Modified Eagle’s medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen) and antibiotics. The various BD- and AD-TICAM-1 mutants were constructed by inserting each cDNA fragment into the pGBKT7 (bait) or pGADT7 (prey) plasmids (Clontech). The following day, cells were transfected with the indicated plasmids using FuGENE HD (Roche Diagnostics). In the cells transfected with wild-type TICAM-1 and the P434H mutant, z-VAD-fmk (20 M) was added to the cells before transfection to inhibit apoptosis. Reporter Gene Assay—HEK293 cells (4 ϫ 104 cells/well) cultured in 96-well plates were transfected with the expression vectors for wild-type TICAM-1, TICAM-1 mutants, or empty vector together with the reporter plasmid (30 ng/well) and an internal control vector, phRL-TK (Promega, Madison, WI) (1 ng/well) using the calcium phosphate method as described previously [26]. For the wild-type TICAM-1 and the TICAM-1 P434H mutant, z-VAD-fmk (20 M) was added to the cells before transfection to inhibit apoptosis. Samples were analyzed by SDS-PAGE (7.5–10%) under reducing conditions followed by immunoblotting with anti-tag Abs
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