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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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IFNβ-stimulated signal transduction does not require DEAF1.A–D, immortalized MEFs from wild type (black bars) and DEAF1−/− (white bars) mice were stimulated with 500 units/ml IFNβ for the times indicated. Total RNA was extracted and analyzed by qRT-PCR to measure the mRNA encoding CXCL10 (A), ISG15 (B), MX1 (C), and IRF7 (D). The results show fold increase in mRNA relative to the values measured in unstimulated MEFs (± S.D. for triplicate determinations). Similar results were obtained in two independent experiments. E, MEFs from wild type (DEAF1+/+) and DEAF1−/−) mice were stimulated with IFNβ as in A–D, and then lysed in the presence of phosphatase inhibitors. Aliquots of the cell extract (20 μg protein) were subjected to SDS-PAGE and, after transfer to PVDF membranes, immunoblotted with an antibody that recognizes STAT1 phosphorylated at Tyr-701 (p-STAT1) or that recognize MDA5 or TBK1.
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Figure 3: IFNβ-stimulated signal transduction does not require DEAF1.A–D, immortalized MEFs from wild type (black bars) and DEAF1−/− (white bars) mice were stimulated with 500 units/ml IFNβ for the times indicated. Total RNA was extracted and analyzed by qRT-PCR to measure the mRNA encoding CXCL10 (A), ISG15 (B), MX1 (C), and IRF7 (D). The results show fold increase in mRNA relative to the values measured in unstimulated MEFs (± S.D. for triplicate determinations). Similar results were obtained in two independent experiments. E, MEFs from wild type (DEAF1+/+) and DEAF1−/−) mice were stimulated with IFNβ as in A–D, and then lysed in the presence of phosphatase inhibitors. Aliquots of the cell extract (20 μg protein) were subjected to SDS-PAGE and, after transfer to PVDF membranes, immunoblotted with an antibody that recognizes STAT1 phosphorylated at Tyr-701 (p-STAT1) or that recognize MDA5 or TBK1.

Mentions: The production of IFNβ triggered by dsRNA of viral origin can be divided into two phases, an early phase in which IRF3 is activated and IFNβ gene transcription is initiated, and a second phase in which IFNβ activates the JAK-STAT1/2 signaling pathway, driving a positive feedback loop that gradually accelerates the rate of IFNβ production (see Introduction). To investigate whether DEAF1 exerted its effects at the second phase, we stimulated MEFs with IFNβ. These experiments revealed that the transcription of IFNβ-stimulated genes, was similar in MEFs from DEAF1−/− and DEAF1+/+ mice (Fig. 3, A–D). These genes included IRF7 (Fig. 3D), which is required for the positive feedback loop. The IFNβ-stimulated synthesis of MDA5 was also similar in MEFs from DEAF1−/− mice (Fig. 3E).


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)

IFNβ-stimulated signal transduction does not require DEAF1.A–D, immortalized MEFs from wild type (black bars) and DEAF1−/− (white bars) mice were stimulated with 500 units/ml IFNβ for the times indicated. Total RNA was extracted and analyzed by qRT-PCR to measure the mRNA encoding CXCL10 (A), ISG15 (B), MX1 (C), and IRF7 (D). The results show fold increase in mRNA relative to the values measured in unstimulated MEFs (± S.D. for triplicate determinations). Similar results were obtained in two independent experiments. E, MEFs from wild type (DEAF1+/+) and DEAF1−/−) mice were stimulated with IFNβ as in A–D, and then lysed in the presence of phosphatase inhibitors. Aliquots of the cell extract (20 μg protein) were subjected to SDS-PAGE and, after transfer to PVDF membranes, immunoblotted with an antibody that recognizes STAT1 phosphorylated at Tyr-701 (p-STAT1) or that recognize MDA5 or TBK1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: IFNβ-stimulated signal transduction does not require DEAF1.A–D, immortalized MEFs from wild type (black bars) and DEAF1−/− (white bars) mice were stimulated with 500 units/ml IFNβ for the times indicated. Total RNA was extracted and analyzed by qRT-PCR to measure the mRNA encoding CXCL10 (A), ISG15 (B), MX1 (C), and IRF7 (D). The results show fold increase in mRNA relative to the values measured in unstimulated MEFs (± S.D. for triplicate determinations). Similar results were obtained in two independent experiments. E, MEFs from wild type (DEAF1+/+) and DEAF1−/−) mice were stimulated with IFNβ as in A–D, and then lysed in the presence of phosphatase inhibitors. Aliquots of the cell extract (20 μg protein) were subjected to SDS-PAGE and, after transfer to PVDF membranes, immunoblotted with an antibody that recognizes STAT1 phosphorylated at Tyr-701 (p-STAT1) or that recognize MDA5 or TBK1.
Mentions: The production of IFNβ triggered by dsRNA of viral origin can be divided into two phases, an early phase in which IRF3 is activated and IFNβ gene transcription is initiated, and a second phase in which IFNβ activates the JAK-STAT1/2 signaling pathway, driving a positive feedback loop that gradually accelerates the rate of IFNβ production (see Introduction). To investigate whether DEAF1 exerted its effects at the second phase, we stimulated MEFs with IFNβ. These experiments revealed that the transcription of IFNβ-stimulated genes, was similar in MEFs from DEAF1−/− and DEAF1+/+ mice (Fig. 3, A–D). These genes included IRF7 (Fig. 3D), which is required for the positive feedback loop. The IFNβ-stimulated synthesis of MDA5 was also similar in MEFs from DEAF1−/− mice (Fig. 3E).

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