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DEAF1 is a Pellino1-interacting protein required for interferon production by Sendai virus and double-stranded RNA.

Ordureau A, Enesa K, Nanda S, Le Francois B, Peggie M, Prescott A, Albert PR, Cohen P - J. Biol. Chem. (2013)

Bottom Line: The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1.DEAF1 is also needed for TLR3-dependent IFNβ production.Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

ABSTRACT
Double-stranded (ds) RNA of viral origin, a ligand for Melanoma Differentiation-associated gene 5 (MDA5) and Toll-Like Receptor 3 (TLR3), induces the TANK-Binding Kinase 1 (TBK1)-dependent phosphorylation and activation of Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1, which are required for interferon β (IFNβ) gene transcription. Here, we report that Pellino1 interacts with the transcription factor Deformed Epidermal Autoregulatory Factor 1 (DEAF1). The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1. We show that DEAF1 binds to the IFNβ promoter and to IRF3 and IRF7, that it is required for the transcription of the IFNβ gene and IFNβ secretion in MEFs infected with Sendai virus or transfected with poly(I:C). DEAF1 is also needed for TLR3-dependent IFNβ production. Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

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The activation and synthesis of component of the interferon pathway in MEFs infected with Sendai virus or transfected with poly(I:C).A and B, immortalized MEFs from wild type mice (DEAF1+/+) or DEAF1−/− mice were infected with Sendai virus (SeV) (100 HA units/ml) (A) or transfected with poly(I:C) (10 μg/ml) (B) for the times indicated. Cell lysates (30 μg protein) were denatured in SDS, subjected to SDS/PAGE, transferred to PVDF membranes and immunoblotted (IB) with the antibodies indicated. Panel P2 shows a separate experiment in which SDS was excluded from the sample and native gel electrophoresis performed to separate the monomeric (m) and dimeric (d) forms of IRF3, which were then detected by immunoblotting. C and D, same as A and B, except that DEAF1+/+ MEFs were incubated for 1 h without (−) or with (+) 1 μm Ruxolitinib prior to infection with Sendai virus (C) or transfection with poly(I:C) (D).
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Figure 7: The activation and synthesis of component of the interferon pathway in MEFs infected with Sendai virus or transfected with poly(I:C).A and B, immortalized MEFs from wild type mice (DEAF1+/+) or DEAF1−/− mice were infected with Sendai virus (SeV) (100 HA units/ml) (A) or transfected with poly(I:C) (10 μg/ml) (B) for the times indicated. Cell lysates (30 μg protein) were denatured in SDS, subjected to SDS/PAGE, transferred to PVDF membranes and immunoblotted (IB) with the antibodies indicated. Panel P2 shows a separate experiment in which SDS was excluded from the sample and native gel electrophoresis performed to separate the monomeric (m) and dimeric (d) forms of IRF3, which were then detected by immunoblotting. C and D, same as A and B, except that DEAF1+/+ MEFs were incubated for 1 h without (−) or with (+) 1 μm Ruxolitinib prior to infection with Sendai virus (C) or transfection with poly(I:C) (D).

Mentions: The results presented above indicated that DEAF1 exerted its effects by enhancing transcription of the gene encoding IFNβ. We were therefore initially surprised to observe that the phosphorylation and dimerization of IRF3 (Fig. 7, A and B, panels P1 and P2) and its translocation to the nucleus (supplemental Fig. S3), which are earlier events in the signaling network, were also reduced considerably in DEAF1−/− MEFs, whether they were infected with Sendai virus (Fig. 7A) or transfected with poly(I:C) (Fig. 7B). However, even though the secretion of IFNβ was barely detectable 2 h post-infection/transfection, this was sufficient to induce near maximal phosphorylation of STAT1 at Tyr-701 (Fig. 7, A and B, panel P4). STAT1 phosphorylation was barely detectable in DEAF1−/− MEFs, presumably due to the much lower level of IFNβ secreted (Fig. 2). The phosphorylation of STAT1 was clearly catalyzed by the JAK kinases, since it was blocked by Ruxolitinib (Fig. 7, C and D, panel P4) or Tofacitinib (supplemental Fig. S4), which are potent, specific, and structurally unrelated JAK inhibitors (9). Importantly, the JAK inhibitors also suppressed the phosphorylation and dimerization of IRF3 (Fig. 7, C and D, panels P1 and P2), indicating that the IFNβ-stimulated JAK-STAT pathway is required to detect robust activation of IRF3 2 h post-infection/transfection.


DEAF1 is a Pellino1-interacting protein required for interferon production by Sendai virus and double-stranded RNA.

Ordureau A, Enesa K, Nanda S, Le Francois B, Peggie M, Prescott A, Albert PR, Cohen P - J. Biol. Chem. (2013)

The activation and synthesis of component of the interferon pathway in MEFs infected with Sendai virus or transfected with poly(I:C).A and B, immortalized MEFs from wild type mice (DEAF1+/+) or DEAF1−/− mice were infected with Sendai virus (SeV) (100 HA units/ml) (A) or transfected with poly(I:C) (10 μg/ml) (B) for the times indicated. Cell lysates (30 μg protein) were denatured in SDS, subjected to SDS/PAGE, transferred to PVDF membranes and immunoblotted (IB) with the antibodies indicated. Panel P2 shows a separate experiment in which SDS was excluded from the sample and native gel electrophoresis performed to separate the monomeric (m) and dimeric (d) forms of IRF3, which were then detected by immunoblotting. C and D, same as A and B, except that DEAF1+/+ MEFs were incubated for 1 h without (−) or with (+) 1 μm Ruxolitinib prior to infection with Sendai virus (C) or transfection with poly(I:C) (D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: The activation and synthesis of component of the interferon pathway in MEFs infected with Sendai virus or transfected with poly(I:C).A and B, immortalized MEFs from wild type mice (DEAF1+/+) or DEAF1−/− mice were infected with Sendai virus (SeV) (100 HA units/ml) (A) or transfected with poly(I:C) (10 μg/ml) (B) for the times indicated. Cell lysates (30 μg protein) were denatured in SDS, subjected to SDS/PAGE, transferred to PVDF membranes and immunoblotted (IB) with the antibodies indicated. Panel P2 shows a separate experiment in which SDS was excluded from the sample and native gel electrophoresis performed to separate the monomeric (m) and dimeric (d) forms of IRF3, which were then detected by immunoblotting. C and D, same as A and B, except that DEAF1+/+ MEFs were incubated for 1 h without (−) or with (+) 1 μm Ruxolitinib prior to infection with Sendai virus (C) or transfection with poly(I:C) (D).
Mentions: The results presented above indicated that DEAF1 exerted its effects by enhancing transcription of the gene encoding IFNβ. We were therefore initially surprised to observe that the phosphorylation and dimerization of IRF3 (Fig. 7, A and B, panels P1 and P2) and its translocation to the nucleus (supplemental Fig. S3), which are earlier events in the signaling network, were also reduced considerably in DEAF1−/− MEFs, whether they were infected with Sendai virus (Fig. 7A) or transfected with poly(I:C) (Fig. 7B). However, even though the secretion of IFNβ was barely detectable 2 h post-infection/transfection, this was sufficient to induce near maximal phosphorylation of STAT1 at Tyr-701 (Fig. 7, A and B, panel P4). STAT1 phosphorylation was barely detectable in DEAF1−/− MEFs, presumably due to the much lower level of IFNβ secreted (Fig. 2). The phosphorylation of STAT1 was clearly catalyzed by the JAK kinases, since it was blocked by Ruxolitinib (Fig. 7, C and D, panel P4) or Tofacitinib (supplemental Fig. S4), which are potent, specific, and structurally unrelated JAK inhibitors (9). Importantly, the JAK inhibitors also suppressed the phosphorylation and dimerization of IRF3 (Fig. 7, C and D, panels P1 and P2), indicating that the IFNβ-stimulated JAK-STAT pathway is required to detect robust activation of IRF3 2 h post-infection/transfection.

Bottom Line: The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1.DEAF1 is also needed for TLR3-dependent IFNβ production.Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

ABSTRACT
Double-stranded (ds) RNA of viral origin, a ligand for Melanoma Differentiation-associated gene 5 (MDA5) and Toll-Like Receptor 3 (TLR3), induces the TANK-Binding Kinase 1 (TBK1)-dependent phosphorylation and activation of Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1, which are required for interferon β (IFNβ) gene transcription. Here, we report that Pellino1 interacts with the transcription factor Deformed Epidermal Autoregulatory Factor 1 (DEAF1). The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1. We show that DEAF1 binds to the IFNβ promoter and to IRF3 and IRF7, that it is required for the transcription of the IFNβ gene and IFNβ secretion in MEFs infected with Sendai virus or transfected with poly(I:C). DEAF1 is also needed for TLR3-dependent IFNβ production. Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

Show MeSH
Related in: MedlinePlus