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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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DEAF1 transcriptional activity and nuclear localization is needed to stimulate IFNβ production.A, immortalized MEFs from DEAF1−/− mice were stably reconstituted with either FLAG-tagged wild-type (WT) DEAF1, DEAF1[K251A], DEAF1[W253Q], DEAF1[K254A], DEAF1[H276S] and DEAF1[R303T/K305T]. These MEF cell lines, as well as the immortalized MEFs from wild type mice (+/+) and DEAF1−/− mice were infected with Sendai virus (SeV) (100 HA/ml) for the times indicated, and the concentration of IFNβ in the culture medium was measured by ELISA. The results are shown ± S.D. for triplicate determinations. B, the cell extract from A (25 μg protein) was denatured in SDS, subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted (IB) with anti-FLAG to show the expression of the different DEAF1 mutants in the reconstituted MEFs.
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Figure 5: DEAF1 transcriptional activity and nuclear localization is needed to stimulate IFNβ production.A, immortalized MEFs from DEAF1−/− mice were stably reconstituted with either FLAG-tagged wild-type (WT) DEAF1, DEAF1[K251A], DEAF1[W253Q], DEAF1[K254A], DEAF1[H276S] and DEAF1[R303T/K305T]. These MEF cell lines, as well as the immortalized MEFs from wild type mice (+/+) and DEAF1−/− mice were infected with Sendai virus (SeV) (100 HA/ml) for the times indicated, and the concentration of IFNβ in the culture medium was measured by ELISA. The results are shown ± S.D. for triplicate determinations. B, the cell extract from A (25 μg protein) was denatured in SDS, subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted (IB) with anti-FLAG to show the expression of the different DEAF1 mutants in the reconstituted MEFs.

Mentions: The finding that DEAF1 interacts with the IFNβ promoter and is required for IRF3 to interact with this promoter suggested that DEAF1 was likely to exert its effects by stimulating IFNβ gene transcription. We therefore stably transfected DEAF1−/− MEFs with either wild type or mutant forms of DEAF1 that are unable to stimulate gene transcription. These experiments showed that transfection with wild type mouse DEAF1 fully restored Sendai virus-induced IFNβ secretion to DEAF1−/− MEFs, but mutants of mouse DEAF1 lacking transcriptional activity could not (Fig. 5A). The mutations in the KDWK motif of the SAND domain (K251A, W253Q, K254A) prevent DEAF1 from binding to DNA (26), while the mutation of His-276 in the zinc binding motif to Ser also prevents DNA from binding to the SAND domain (27). The mutation of Arg-303 and Lys-305 to Thr, which prevents DEAF1 from entering the nucleus (28), has been shown to be transcriptionally inactive (26). Each mutant was expressed in MEFs at similar levels at similar levels to wild type DEAF1 (Fig. 5B). Thus the interaction of DEAF1 with DNA and its nuclear location are required to enhance the transcription of the IFNβ gene.


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)

DEAF1 transcriptional activity and nuclear localization is needed to stimulate IFNβ production.A, immortalized MEFs from DEAF1−/− mice were stably reconstituted with either FLAG-tagged wild-type (WT) DEAF1, DEAF1[K251A], DEAF1[W253Q], DEAF1[K254A], DEAF1[H276S] and DEAF1[R303T/K305T]. These MEF cell lines, as well as the immortalized MEFs from wild type mice (+/+) and DEAF1−/− mice were infected with Sendai virus (SeV) (100 HA/ml) for the times indicated, and the concentration of IFNβ in the culture medium was measured by ELISA. The results are shown ± S.D. for triplicate determinations. B, the cell extract from A (25 μg protein) was denatured in SDS, subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted (IB) with anti-FLAG to show the expression of the different DEAF1 mutants in the reconstituted MEFs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750155&req=5

Figure 5: DEAF1 transcriptional activity and nuclear localization is needed to stimulate IFNβ production.A, immortalized MEFs from DEAF1−/− mice were stably reconstituted with either FLAG-tagged wild-type (WT) DEAF1, DEAF1[K251A], DEAF1[W253Q], DEAF1[K254A], DEAF1[H276S] and DEAF1[R303T/K305T]. These MEF cell lines, as well as the immortalized MEFs from wild type mice (+/+) and DEAF1−/− mice were infected with Sendai virus (SeV) (100 HA/ml) for the times indicated, and the concentration of IFNβ in the culture medium was measured by ELISA. The results are shown ± S.D. for triplicate determinations. B, the cell extract from A (25 μg protein) was denatured in SDS, subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted (IB) with anti-FLAG to show the expression of the different DEAF1 mutants in the reconstituted MEFs.
Mentions: The finding that DEAF1 interacts with the IFNβ promoter and is required for IRF3 to interact with this promoter suggested that DEAF1 was likely to exert its effects by stimulating IFNβ gene transcription. We therefore stably transfected DEAF1−/− MEFs with either wild type or mutant forms of DEAF1 that are unable to stimulate gene transcription. These experiments showed that transfection with wild type mouse DEAF1 fully restored Sendai virus-induced IFNβ secretion to DEAF1−/− MEFs, but mutants of mouse DEAF1 lacking transcriptional activity could not (Fig. 5A). The mutations in the KDWK motif of the SAND domain (K251A, W253Q, K254A) prevent DEAF1 from binding to DNA (26), while the mutation of His-276 in the zinc binding motif to Ser also prevents DNA from binding to the SAND domain (27). The mutation of Arg-303 and Lys-305 to Thr, which prevents DEAF1 from entering the nucleus (28), has been shown to be transcriptionally inactive (26). Each mutant was expressed in MEFs at similar levels at similar levels to wild type DEAF1 (Fig. 5B). Thus the interaction of DEAF1 with DNA and its nuclear location are required to enhance the transcription of the IFNβ gene.

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