Limits...
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 dispensible for MAVS-mediated IRF3 activation. (A) Role of TRIM25 in polyI:C-induced IRF3 nuclear translocation. HEK293T cells were transfected with eGFP-IRF3 together with vector or TRIM25 expression plasmids. Cells were re-transfected with polyI:C 24 h after the first transfection. Localization of eGFP-IRF3 was examined under fluorescent microscopy (upper left panel). To see the effect of TRIM25 depletion, HEK293T cells were transfected with eGFP-IRF3 together with control shRNA (Ctrl) or shRNAs against TRIM25 (Right panel; TRIM25 KD). Three randomly chosen fields were examined for each group. Lower panel shows representative images from the shRNA experiment. NS. Not significant. (B) TRIM25 ectopic expression does not affect MAVS-induced IRF3 phosphorylation. HEK293T cells were transfected with indicated plasmids. Cell lysates were subjected to immunoblotting using indicated antibodies. (C) TRIM25 does not enhance TBK1-induced ISRE promoter activation. HEK293T cells were transfected with indicated plamids and ISRE reporter plasmid. ISRE promoter activity was analyzed by dual-luciferase assay. (D) Interaction between TRIM25 and IKK complex proteins. TRIM25 physically interacts with the IKK complex. HEK293T cells were transfected with Flag-TRIM25 and vector, HA-IKKα, HA-IKKβ, or HA-IKKγ. The 24 h after transfection, cell lysates were subjected to immunoprecipitation using an anti-HA antibody and analyzed by immunoblotting using the indicated antibodies. (E) TRIM25 does not affect the NF-κB (p65) phosphorylation upon IKKα or IKKβ expression. HEK293T cells were transfected with indicated plasmids and shRNAs. Knock-down efficiency was confirmed by measuring TRIM25 mRNA using RT-qPCR. Cell lysates were subjected to immunoblotting using indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4588718&req=5

f3-molce-38-9-759: TRIM25 is dispensible for MAVS-mediated IRF3 activation. (A) Role of TRIM25 in polyI:C-induced IRF3 nuclear translocation. HEK293T cells were transfected with eGFP-IRF3 together with vector or TRIM25 expression plasmids. Cells were re-transfected with polyI:C 24 h after the first transfection. Localization of eGFP-IRF3 was examined under fluorescent microscopy (upper left panel). To see the effect of TRIM25 depletion, HEK293T cells were transfected with eGFP-IRF3 together with control shRNA (Ctrl) or shRNAs against TRIM25 (Right panel; TRIM25 KD). Three randomly chosen fields were examined for each group. Lower panel shows representative images from the shRNA experiment. NS. Not significant. (B) TRIM25 ectopic expression does not affect MAVS-induced IRF3 phosphorylation. HEK293T cells were transfected with indicated plasmids. Cell lysates were subjected to immunoblotting using indicated antibodies. (C) TRIM25 does not enhance TBK1-induced ISRE promoter activation. HEK293T cells were transfected with indicated plamids and ISRE reporter plasmid. ISRE promoter activity was analyzed by dual-luciferase assay. (D) Interaction between TRIM25 and IKK complex proteins. TRIM25 physically interacts with the IKK complex. HEK293T cells were transfected with Flag-TRIM25 and vector, HA-IKKα, HA-IKKβ, or HA-IKKγ. The 24 h after transfection, cell lysates were subjected to immunoprecipitation using an anti-HA antibody and analyzed by immunoblotting using the indicated antibodies. (E) TRIM25 does not affect the NF-κB (p65) phosphorylation upon IKKα or IKKβ expression. HEK293T cells were transfected with indicated plasmids and shRNAs. Knock-down efficiency was confirmed by measuring TRIM25 mRNA using RT-qPCR. Cell lysates were subjected to immunoblotting using indicated antibodies.

Mentions: Upon activation of RIG-I or MDA5 signaling pathways, IKK and TBK complexes are recruited to MAVS to activate NF-κB and IRF3/7 transcriptional factors, respectively. To further elucidate the action of TRIM25 in the MDA5-MAVS signaling pathway, we examined the effect of TRIM25 on IRF3 nuclear localization and NF-κB activation. As depicted in Fig. 3A, ectopic expression or depletion of TRIM25 did not significantly change GFP-IRF3 nuclear translocation upon polyI:C transfection. Furthermore, ectopic expression of TRIM25 or TRIM25 RINGCS mutant which lose its E3-ligase activity did not affect the MAVS-induced IRF3 phosphorylation (Fig. 3B). Also, TBK1-induced activation of ISRE was not affected by ectopic expression of TRIM25 (Fig. 3C). Since we have shown that MDA5-MAVS induced NF-κB activation was enhanced by the ectopic expression of TRIM25 (Figs. 1D and 2D) and TRIM25 is required for efficient MDA5-MAVS-mediated NF-κB activation (Fig. 2B), it is likely that TRIM25 is involved in the activation of NF-κB via the MDA5-MAVS signaling pathway. Furthermore, a physical interaction between TRIM25 and IKK complex component proteins (IKKα, IKKβ, and IKKγ) was detected by coimmunoprecipitation assay in an overexpression system (Fig. 3D). Interestingly, phosphorylation of NF-κB (p65) induced by ectopic expression of IKKα or IKKβ was not decreased by depletion of TRIM25, suggesting that TRIM25 working at the level between MAVS and IKK complex to regulate NF-κB signaling (Fig. 3E).


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 dispensible for MAVS-mediated IRF3 activation. (A) Role of TRIM25 in polyI:C-induced IRF3 nuclear translocation. HEK293T cells were transfected with eGFP-IRF3 together with vector or TRIM25 expression plasmids. Cells were re-transfected with polyI:C 24 h after the first transfection. Localization of eGFP-IRF3 was examined under fluorescent microscopy (upper left panel). To see the effect of TRIM25 depletion, HEK293T cells were transfected with eGFP-IRF3 together with control shRNA (Ctrl) or shRNAs against TRIM25 (Right panel; TRIM25 KD). Three randomly chosen fields were examined for each group. Lower panel shows representative images from the shRNA experiment. NS. Not significant. (B) TRIM25 ectopic expression does not affect MAVS-induced IRF3 phosphorylation. HEK293T cells were transfected with indicated plasmids. Cell lysates were subjected to immunoblotting using indicated antibodies. (C) TRIM25 does not enhance TBK1-induced ISRE promoter activation. HEK293T cells were transfected with indicated plamids and ISRE reporter plasmid. ISRE promoter activity was analyzed by dual-luciferase assay. (D) Interaction between TRIM25 and IKK complex proteins. TRIM25 physically interacts with the IKK complex. HEK293T cells were transfected with Flag-TRIM25 and vector, HA-IKKα, HA-IKKβ, or HA-IKKγ. The 24 h after transfection, cell lysates were subjected to immunoprecipitation using an anti-HA antibody and analyzed by immunoblotting using the indicated antibodies. (E) TRIM25 does not affect the NF-κB (p65) phosphorylation upon IKKα or IKKβ expression. HEK293T cells were transfected with indicated plasmids and shRNAs. Knock-down efficiency was confirmed by measuring TRIM25 mRNA using RT-qPCR. Cell lysates were subjected to immunoblotting using indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588718&req=5

f3-molce-38-9-759: TRIM25 is dispensible for MAVS-mediated IRF3 activation. (A) Role of TRIM25 in polyI:C-induced IRF3 nuclear translocation. HEK293T cells were transfected with eGFP-IRF3 together with vector or TRIM25 expression plasmids. Cells were re-transfected with polyI:C 24 h after the first transfection. Localization of eGFP-IRF3 was examined under fluorescent microscopy (upper left panel). To see the effect of TRIM25 depletion, HEK293T cells were transfected with eGFP-IRF3 together with control shRNA (Ctrl) or shRNAs against TRIM25 (Right panel; TRIM25 KD). Three randomly chosen fields were examined for each group. Lower panel shows representative images from the shRNA experiment. NS. Not significant. (B) TRIM25 ectopic expression does not affect MAVS-induced IRF3 phosphorylation. HEK293T cells were transfected with indicated plasmids. Cell lysates were subjected to immunoblotting using indicated antibodies. (C) TRIM25 does not enhance TBK1-induced ISRE promoter activation. HEK293T cells were transfected with indicated plamids and ISRE reporter plasmid. ISRE promoter activity was analyzed by dual-luciferase assay. (D) Interaction between TRIM25 and IKK complex proteins. TRIM25 physically interacts with the IKK complex. HEK293T cells were transfected with Flag-TRIM25 and vector, HA-IKKα, HA-IKKβ, or HA-IKKγ. The 24 h after transfection, cell lysates were subjected to immunoprecipitation using an anti-HA antibody and analyzed by immunoblotting using the indicated antibodies. (E) TRIM25 does not affect the NF-κB (p65) phosphorylation upon IKKα or IKKβ expression. HEK293T cells were transfected with indicated plasmids and shRNAs. Knock-down efficiency was confirmed by measuring TRIM25 mRNA using RT-qPCR. Cell lysates were subjected to immunoblotting using indicated antibodies.
Mentions: Upon activation of RIG-I or MDA5 signaling pathways, IKK and TBK complexes are recruited to MAVS to activate NF-κB and IRF3/7 transcriptional factors, respectively. To further elucidate the action of TRIM25 in the MDA5-MAVS signaling pathway, we examined the effect of TRIM25 on IRF3 nuclear localization and NF-κB activation. As depicted in Fig. 3A, ectopic expression or depletion of TRIM25 did not significantly change GFP-IRF3 nuclear translocation upon polyI:C transfection. Furthermore, ectopic expression of TRIM25 or TRIM25 RINGCS mutant which lose its E3-ligase activity did not affect the MAVS-induced IRF3 phosphorylation (Fig. 3B). Also, TBK1-induced activation of ISRE was not affected by ectopic expression of TRIM25 (Fig. 3C). Since we have shown that MDA5-MAVS induced NF-κB activation was enhanced by the ectopic expression of TRIM25 (Figs. 1D and 2D) and TRIM25 is required for efficient MDA5-MAVS-mediated NF-κB activation (Fig. 2B), it is likely that TRIM25 is involved in the activation of NF-κB via the MDA5-MAVS signaling pathway. Furthermore, a physical interaction between TRIM25 and IKK complex component proteins (IKKα, IKKβ, and IKKγ) was detected by coimmunoprecipitation assay in an overexpression system (Fig. 3D). Interestingly, phosphorylation of NF-κB (p65) induced by ectopic expression of IKKα or IKKβ was not decreased by depletion of TRIM25, suggesting that TRIM25 working at the level between MAVS and IKK complex to regulate NF-κB signaling (Fig. 3E).

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