Oligomerized TICAM‐1 (TRIF) in the cytoplasm recruits nuclear BS69 to enhance NF‐κB activation and type I IFN induction

Abstract

Although adenovirus 5 E1A-binding protein (BS69) is a nuclear protein acting as a transcriptional repressor, we found by an yeast two-hybrid and human cell immunoprecipitation another cytoplasmic function for this protein. BS69 bound Toll-interleukin 1 receptor domain (TIR)-containing adaptor molecule-1 (TICAM-1) (also named TRIF), an adaptor protein that couples with TLR3 around the endosome. BS69 translocated from the nucleus to the cytoplasm when cells were stimulated with dsRNA or transfected with TICAM-1. Confocal analysis of cells with over-expressed TICAM-1 or those stimulated with dsRNA revealed the characteristic ''TICAM-1 speckle'', which reflects signalosome formation necessary for the activation of NF-jB and IFN-regulatory factor (IRF)-3. BS69 was involved in the TICAM-1 complex, and the activation of NF-jB/IRF-3 followed by cytokine production was augmented in the presence of BS69 overexpression. Knockdown of endogenous BS69 resulted in a decrease of IFN-b induction, suggesting that BS69 is a positive regulator for the TLR3-TICAM-1 pathway. These results, together with a recent report showing the negative regulatory properties of BS69 in NF-jB activation by EBVderived latent membrane protein 1, suggest that BS69 harbors dual modes of cytoplasmic NF-jB regulation, positively in the TICAM-1 pathway and negatively in the latent membrane protein 1 pathway.Key words: BS69 . IFN-b . NF-jB . TICAM-1/TRIF . TLR3 Supporting Information available online IntroductionToll-interleukin 1 receptor domain (TIR)-containing adaptor molecule-1 (TICAM-1) acts as an adaptor for TLR3 and activates both the IFN-regulatory factor (IRF)-3 and the IFN-b promoter [1]. TLR3 is localized to the endosome in immature myeloid dendritic cells (mDC) and resting macrophages [2]. Recent imaging analyses revealed that TICAM-1 merges with endosomal TLR3 within 20 min in response to dsRNA stimuli, and after 60 min translocates to form speckles in the cytoplasm which represent the TICAM-1 signalosome [3]. NAP1 and RIP1 are recruited to the TICAM-1 complex, both of which are known to be important factors for activating downstream elements of the TICAM-1 pathway [3,4]. The forced expression of TICAM-1 leads to the formation of multimers in the signalosome complex [4]. To elucidate what molecules constitute the TICAM-1 complex, we screened TICAM-1-binding proteins by à These authors contributed equally to this work. [6,7]. The C-terminal MYND domain of BS69 was shown to bind to the PxLxP motif existing on E1A, EBVencoded EBNA2 and a Myc-related cellular protein MGA [8]. Although BS69 is unequivocally a nuclear protein, it has been shown that BS69 interacts with EBV-encoded latent membrane protein 1 (LMP1) in the cytoplasm through its MYND domain and acts as a scaffold protein in the LMP1-mediated JNK pathway by interacting with TRAF6 [9]. Furthermore, a recent report speculated that nuclear BS69 colocalizes with LMP1 in the cytoplasm proximal to the nucleus [10]. The stimulus which induces BS69 protein trafficking, however, remains undeterm...

Simulated Microgravity Promotes Cell Apoptosis Through Suppressing Uev1A/TICAM/TRAF/NF‐κB‐Regulated Anti‐Apoptosis and p53/PCNA‐ and ATM/ATR‐Chk1/2‐Controlled DNA‐Damage Response Pathways

“…TRAF6-binding motifs in the N-terminal region [Sato et al, 2003] is a regulatory molecule, the downstream pathway of which initiates NF-kB activation [Takaki et al, 2009]. Interestingly, our Western blot analysis demonstrates that microgravity down-regulates expression of the Uev1A, TRAF2, and TICAM1 activators of the NF-kB pathway.…”

“…Uev1A is a co‐factor functioning in Ubc13‐catalyzed polyubiquitination of NEMO/IKKγ through K63‐linked chains [Zhou et al, ], which is a regulatory subunit of IκB kinase in the NF‐κB signaling pathway [Syed et al, ]. TRAF2 has been found to activate the NF‐κB signaling pathway [Shi and Kehrl, ] via catalyzing non‐degradative K63‐linked polyubiquitination by its RING domains [Fotin‐Mleczek et al, ], while TICAM1 activating TRAF6 via its TRAF6‐binding motifs in the N‐terminal region [Sato et al, ] is a regulatory molecule, the downstream pathway of which initiates NF‐κB activation [Takaki et al, ]. Interestingly, our Western blot analysis demonstrates that microgravity down‐regulates expression of the Uev1A, TRAF2, and TICAM1 activators of the NF‐κB pathway.…”

Unabated Adenovirus Replication following Activation of the cGAS/STING-Dependent Antiviral Response in Human Cells

“…E1A suppresses signaling downstream of IFNR activation at STAT1 (51,81), compromises IFN-directed ISG activation by disrupting IFN-induced histone 2B monoubiquitination required for ISG induction (82), and blocks RNA activation of a TRIF/BS69 signalosome, which leads to NF-B and IRF3 activation (83). E1B functions to effectively repress IFN-induced antiviral strategies that impact replication (53,54).…”

TLR3 immunity to infection in mice and humans

“…Other molecules appear to be involved in TLR3-IRF3/NF-κB-IFN pathways (Figure 1, Table 1), either as ‘positive regulators’, such as BS69, TAPE, TRAF2, TANK, TRIL, SINTBAD, TRADD, DDX3X, CaMKII, RAFLIN, GAB1, TRIM21, TRIM56, HMGB, ELMOD2, PELLINO1, SCARA1 and TRIS, which is a splice variant of TRIF [86-106], or as ‘negative regulators’, such as SHP-2, RIP3, TRAF1, TRAF4, A20, SARM1, PIASy, SIKE, TRIP, TIPE2 and DUBA [65,80,107-116]. Some molecules, such as NEMO [117] and TANK [90,91], have been shown to link the TLR3-IRF3 and TLR3-NF-κB pathways.…”