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Regulation of MDA5-MAVS Antiviral Signaling Axis by TRIM25 through TRAF6-Mediated NF-κB Activation.

Lee NR, Kim HI, Choi MS, Yi CM, Inn KS - Mol. Cells (2015)

Bottom Line: Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling.Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation.These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Korea.

ABSTRACT
Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling. Here, we demonstrate that TRIM25 is required for melanoma differentiation-associated gene 5 (MDA5) and MAVS mediated activation of NF-κB and interferon production. TRIM25 is required for the full activation of NF-κB at the downstream of MAVS, while it is not involved in IRF3 nuclear translocation. Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation. These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.

No MeSH data available.


Related in: MedlinePlus

TRIM25 is involved in TRAF6-mediated NF-κB signaling. (A) Enhanced TRAF6-induced NF-κB activation by ectopic expression of TRIM25. HEK293T cells were transfected with plasmids as indicated. Cells were cotransfected with NF-κB luciferase reporter and TK-Renilla reporter plasmids. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection. (B) Suppression of TRAF6-induced NF-κB activation by depletion of TRIM25. HEK293T cells were transfected with control shRNA (Ctrl) or shRNAs targeting TRIM25 as indicated together with reporter plasmids. Promoter activities were determined using procedures similar to those in (A). Knock-down efficiency was confirmed by measuring TRIM25 mRNA using RT-qPCR. (C) Role of TRIM25 in TRAF6 ubiquitination. HEK293T cells were transfected with flag-TRAF6 together with control shRNA (Ctrl) or shRNAs targeting TRIM25. Cell lysates were subjected to immunoprecipitation using an anti-flag antibody and analyzed by immunoblotting using the indicated antibodies to examine the level of TRAF6 ubiquitination. Suppression of TRIM25 mRNA synthesis was confirmed by RT-qPCR. (D) E3-ligase activity of TRIM25 is required for TRAF6-induced NF-κB activation. HEK293T cells were transfected with wild-type (WT) and E3-ligase activity dead C50S/C53S mutant (CS) TRIM25 together with vector or TRAF6 plasmid. NF-κB luciferase reporter and TK-Renilla reporter plasmids were co-transfected. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection.
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f4-molce-38-9-759: TRIM25 is involved in TRAF6-mediated NF-κB signaling. (A) Enhanced TRAF6-induced NF-κB activation by ectopic expression of TRIM25. HEK293T cells were transfected with plasmids as indicated. Cells were cotransfected with NF-κB luciferase reporter and TK-Renilla reporter plasmids. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection. (B) Suppression of TRAF6-induced NF-κB activation by depletion of TRIM25. HEK293T cells were transfected with control shRNA (Ctrl) or shRNAs targeting TRIM25 as indicated together with reporter plasmids. Promoter activities were determined using procedures similar to those in (A). Knock-down efficiency was confirmed by measuring TRIM25 mRNA using RT-qPCR. (C) Role of TRIM25 in TRAF6 ubiquitination. HEK293T cells were transfected with flag-TRAF6 together with control shRNA (Ctrl) or shRNAs targeting TRIM25. Cell lysates were subjected to immunoprecipitation using an anti-flag antibody and analyzed by immunoblotting using the indicated antibodies to examine the level of TRAF6 ubiquitination. Suppression of TRIM25 mRNA synthesis was confirmed by RT-qPCR. (D) E3-ligase activity of TRIM25 is required for TRAF6-induced NF-κB activation. HEK293T cells were transfected with wild-type (WT) and E3-ligase activity dead C50S/C53S mutant (CS) TRIM25 together with vector or TRAF6 plasmid. NF-κB luciferase reporter and TK-Renilla reporter plasmids were co-transfected. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection.

Mentions: Previously, it has been shown that TRAF6 is crucial for RIG-I-mediated NF-κB activation (Yoboua et al., 2010; Yoshida et al., 2008). To further delineate the role of TRIM25 in MAVS-mediated NF-κB activation, we analyzed the role of TRIM25 in TRAF6-mediated NF-κB activation. As depicted in Figs. 4A and 4B, ectopic expression of TRIM25 enhanced TRAF6-mediated NF-κB activation, whereas depletion of TRIM25 resulted in suppression of TRAF6-mediated NF-κB activation. Furthermore, ubiquitination of TRAF6, which is critical for its activity, was reduced by depletion of TRIM25, further supporting the role of TRIM25 in TRAF6-mediated downstream NF-κB activation (Fig. 4C). To examine whether the E3-ubiqutin ligase enzyme activity of TRIM25 is required for its role in TRAF6-mediated NF-κB signaling, we compared the activities of wild-type and TRIM25CS (C50S/C53S), a mutant TRIM25 that does not carry E3-ubiquitin ligase activity. TRIM25CS was unable to enhance TRAF6-mediated NF-κB activation, suggesting that E3-ubiquitin ligase activity is required for its downstream NF-κB activating function (Fig. 4D).


Regulation of MDA5-MAVS Antiviral Signaling Axis by TRIM25 through TRAF6-Mediated NF-κB Activation.

Lee NR, Kim HI, Choi MS, Yi CM, Inn KS - Mol. Cells (2015)

TRIM25 is involved in TRAF6-mediated NF-κB signaling. (A) Enhanced TRAF6-induced NF-κB activation by ectopic expression of TRIM25. HEK293T cells were transfected with plasmids as indicated. Cells were cotransfected with NF-κB luciferase reporter and TK-Renilla reporter plasmids. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection. (B) Suppression of TRAF6-induced NF-κB activation by depletion of TRIM25. HEK293T cells were transfected with control shRNA (Ctrl) or shRNAs targeting TRIM25 as indicated together with reporter plasmids. Promoter activities were determined using procedures similar to those in (A). Knock-down efficiency was confirmed by measuring TRIM25 mRNA using RT-qPCR. (C) Role of TRIM25 in TRAF6 ubiquitination. HEK293T cells were transfected with flag-TRAF6 together with control shRNA (Ctrl) or shRNAs targeting TRIM25. Cell lysates were subjected to immunoprecipitation using an anti-flag antibody and analyzed by immunoblotting using the indicated antibodies to examine the level of TRAF6 ubiquitination. Suppression of TRIM25 mRNA synthesis was confirmed by RT-qPCR. (D) E3-ligase activity of TRIM25 is required for TRAF6-induced NF-κB activation. HEK293T cells were transfected with wild-type (WT) and E3-ligase activity dead C50S/C53S mutant (CS) TRIM25 together with vector or TRAF6 plasmid. NF-κB luciferase reporter and TK-Renilla reporter plasmids were co-transfected. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4588718&req=5

f4-molce-38-9-759: TRIM25 is involved in TRAF6-mediated NF-κB signaling. (A) Enhanced TRAF6-induced NF-κB activation by ectopic expression of TRIM25. HEK293T cells were transfected with plasmids as indicated. Cells were cotransfected with NF-κB luciferase reporter and TK-Renilla reporter plasmids. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection. (B) Suppression of TRAF6-induced NF-κB activation by depletion of TRIM25. HEK293T cells were transfected with control shRNA (Ctrl) or shRNAs targeting TRIM25 as indicated together with reporter plasmids. Promoter activities were determined using procedures similar to those in (A). Knock-down efficiency was confirmed by measuring TRIM25 mRNA using RT-qPCR. (C) Role of TRIM25 in TRAF6 ubiquitination. HEK293T cells were transfected with flag-TRAF6 together with control shRNA (Ctrl) or shRNAs targeting TRIM25. Cell lysates were subjected to immunoprecipitation using an anti-flag antibody and analyzed by immunoblotting using the indicated antibodies to examine the level of TRAF6 ubiquitination. Suppression of TRIM25 mRNA synthesis was confirmed by RT-qPCR. (D) E3-ligase activity of TRIM25 is required for TRAF6-induced NF-κB activation. HEK293T cells were transfected with wild-type (WT) and E3-ligase activity dead C50S/C53S mutant (CS) TRIM25 together with vector or TRAF6 plasmid. NF-κB luciferase reporter and TK-Renilla reporter plasmids were co-transfected. Promoter activities were determined by Dual-Luciferase assay 16 h after transfection.
Mentions: Previously, it has been shown that TRAF6 is crucial for RIG-I-mediated NF-κB activation (Yoboua et al., 2010; Yoshida et al., 2008). To further delineate the role of TRIM25 in MAVS-mediated NF-κB activation, we analyzed the role of TRIM25 in TRAF6-mediated NF-κB activation. As depicted in Figs. 4A and 4B, ectopic expression of TRIM25 enhanced TRAF6-mediated NF-κB activation, whereas depletion of TRIM25 resulted in suppression of TRAF6-mediated NF-κB activation. Furthermore, ubiquitination of TRAF6, which is critical for its activity, was reduced by depletion of TRIM25, further supporting the role of TRIM25 in TRAF6-mediated downstream NF-κB activation (Fig. 4C). To examine whether the E3-ubiqutin ligase enzyme activity of TRIM25 is required for its role in TRAF6-mediated NF-κB signaling, we compared the activities of wild-type and TRIM25CS (C50S/C53S), a mutant TRIM25 that does not carry E3-ubiquitin ligase activity. TRIM25CS was unable to enhance TRAF6-mediated NF-κB activation, suggesting that E3-ubiquitin ligase activity is required for its downstream NF-κB activating function (Fig. 4D).

Bottom Line: Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling.Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation.These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Korea.

ABSTRACT
Tripartite motif protein 25 (TRIM25), mediates K63-linked polyubiquitination of Retinoic acid inducible gene I (RIG-I) that is crucial for downstream antiviral interferon signaling. Here, we demonstrate that TRIM25 is required for melanoma differentiation-associated gene 5 (MDA5) and MAVS mediated activation of NF-κB and interferon production. TRIM25 is required for the full activation of NF-κB at the downstream of MAVS, while it is not involved in IRF3 nuclear translocation. Mechanical studies showed that TRIM25 is involved in TRAF6-mediated NF-κB activation. These collectively indicate that TRIM25 plays an additional role in RIG-I/MDA5 signaling other than RIG-I ubiquitination via activation of NF-κB.

No MeSH data available.


Related in: MedlinePlus