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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 and Pellino1 interact in overexpression studies. HEK293FT cells were transfected with DNA encoding HA-DEAF1 and/or GFP-Pellino1. After 36 h, the cells were lysed in buffer containing phosphatase inhibitors (see “Experimental Procedures”) plus 50 mm iodoacetamide. A, cell extracts (3 μg of protein) were denatured in SDS, subjected to SDS-PAGE, transferred on to a PVDF membrane, and immunoblotted (IB) with anti-HA and or anti-GFP to monitor the expression of the DEAF1 and Pellino1 fusion proteins. B, Pellino1 was immunoprecipitated (IP) from 0.1 mg of cell extract protein with the GFP antibody (GFP-Trap beads). The bound proteins were denatured in SDS, and immunoblotted with anti-HA and anti-GFP to detect DEAF1 and Pellino1, respectively. C, as in B except that DEAF1 was immunoprecipitated from 0.1 mg of cell lysate protein with an HA antibody. D–F, same as A–C, except that HA-DEAF1 was transfected with either wild-type (WT) or the F397A mutant (F/A) of Pellino1. G and H, same as D, E except that the cells were also transfected with FLAG-tagged wild-type (WT) IRAK1 or the catalytically inactive IRAK1[D358A] mutant (D/A). I, same as A, except that the cells were transfected with HA-DEAF1 and either wild-type FLAG-IKKϵ (WT) or the catalytically inactive FLAG-IKKϵ[D157A] mutant (D/A). The cell extracts were immunoblotted with anti-HA and anti-FLAG.
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Figure 1: DEAF1 and Pellino1 interact in overexpression studies. HEK293FT cells were transfected with DNA encoding HA-DEAF1 and/or GFP-Pellino1. After 36 h, the cells were lysed in buffer containing phosphatase inhibitors (see “Experimental Procedures”) plus 50 mm iodoacetamide. A, cell extracts (3 μg of protein) were denatured in SDS, subjected to SDS-PAGE, transferred on to a PVDF membrane, and immunoblotted (IB) with anti-HA and or anti-GFP to monitor the expression of the DEAF1 and Pellino1 fusion proteins. B, Pellino1 was immunoprecipitated (IP) from 0.1 mg of cell extract protein with the GFP antibody (GFP-Trap beads). The bound proteins were denatured in SDS, and immunoblotted with anti-HA and anti-GFP to detect DEAF1 and Pellino1, respectively. C, as in B except that DEAF1 was immunoprecipitated from 0.1 mg of cell lysate protein with an HA antibody. D–F, same as A–C, except that HA-DEAF1 was transfected with either wild-type (WT) or the F397A mutant (F/A) of Pellino1. G and H, same as D, E except that the cells were also transfected with FLAG-tagged wild-type (WT) IRAK1 or the catalytically inactive IRAK1[D358A] mutant (D/A). I, same as A, except that the cells were transfected with HA-DEAF1 and either wild-type FLAG-IKKϵ (WT) or the catalytically inactive FLAG-IKKϵ[D157A] mutant (D/A). The cell extracts were immunoblotted with anti-HA and anti-FLAG.

Mentions: Another five positive clones identified in the screen encoded the C-terminal region of the transcription factor DEAF1, the smallest comprising residues 302–565. This finding was intriguing because Pellino1 and the Drosophila orthologue of DEAF1 are both required for the production of the anti-bacterial peptides Drosomycin and Metchnikowin in the fruit fly D. melanogaster (see Introduction), but were not considered to interact, since Pellino was reported to interact at the plasma membrane with Pelle, the Drosophila orthologue of IRAK, while DEAF1 functions in the nucleus. To investigate further whether Pellino1 and DEAF1 bind to one another, we co-expressed DNA vectors encoding hemagglutinin (HA)-tagged DEAF1 and either GFP-tagged Pellino1 (Fig. 1A) or FLAG-tagged Pellino1 (supplemental Fig. S1A) in HEK293FT cells. We found that the expressed HA-DEAF1 could be co-immunoprecipitated with GFP-Pellino1 using anti-GFP (Fig. 1B) or with FLAG-Pellino1 using anti-FLAG (supplemental Fig. S1B). Conversely, GFP-Pellino1 (Fig. 1C) or FLAG-Pellino1 (supplemental Fig. S1C) could both be immunoprecipitated with HA-DEAF1, using anti-HA. The results confirmed that these two proteins do indeed interact with one another.


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 and Pellino1 interact in overexpression studies. HEK293FT cells were transfected with DNA encoding HA-DEAF1 and/or GFP-Pellino1. After 36 h, the cells were lysed in buffer containing phosphatase inhibitors (see “Experimental Procedures”) plus 50 mm iodoacetamide. A, cell extracts (3 μg of protein) were denatured in SDS, subjected to SDS-PAGE, transferred on to a PVDF membrane, and immunoblotted (IB) with anti-HA and or anti-GFP to monitor the expression of the DEAF1 and Pellino1 fusion proteins. B, Pellino1 was immunoprecipitated (IP) from 0.1 mg of cell extract protein with the GFP antibody (GFP-Trap beads). The bound proteins were denatured in SDS, and immunoblotted with anti-HA and anti-GFP to detect DEAF1 and Pellino1, respectively. C, as in B except that DEAF1 was immunoprecipitated from 0.1 mg of cell lysate protein with an HA antibody. D–F, same as A–C, except that HA-DEAF1 was transfected with either wild-type (WT) or the F397A mutant (F/A) of Pellino1. G and H, same as D, E except that the cells were also transfected with FLAG-tagged wild-type (WT) IRAK1 or the catalytically inactive IRAK1[D358A] mutant (D/A). I, same as A, except that the cells were transfected with HA-DEAF1 and either wild-type FLAG-IKKϵ (WT) or the catalytically inactive FLAG-IKKϵ[D157A] mutant (D/A). The cell extracts were immunoblotted with anti-HA and anti-FLAG.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: DEAF1 and Pellino1 interact in overexpression studies. HEK293FT cells were transfected with DNA encoding HA-DEAF1 and/or GFP-Pellino1. After 36 h, the cells were lysed in buffer containing phosphatase inhibitors (see “Experimental Procedures”) plus 50 mm iodoacetamide. A, cell extracts (3 μg of protein) were denatured in SDS, subjected to SDS-PAGE, transferred on to a PVDF membrane, and immunoblotted (IB) with anti-HA and or anti-GFP to monitor the expression of the DEAF1 and Pellino1 fusion proteins. B, Pellino1 was immunoprecipitated (IP) from 0.1 mg of cell extract protein with the GFP antibody (GFP-Trap beads). The bound proteins were denatured in SDS, and immunoblotted with anti-HA and anti-GFP to detect DEAF1 and Pellino1, respectively. C, as in B except that DEAF1 was immunoprecipitated from 0.1 mg of cell lysate protein with an HA antibody. D–F, same as A–C, except that HA-DEAF1 was transfected with either wild-type (WT) or the F397A mutant (F/A) of Pellino1. G and H, same as D, E except that the cells were also transfected with FLAG-tagged wild-type (WT) IRAK1 or the catalytically inactive IRAK1[D358A] mutant (D/A). I, same as A, except that the cells were transfected with HA-DEAF1 and either wild-type FLAG-IKKϵ (WT) or the catalytically inactive FLAG-IKKϵ[D157A] mutant (D/A). The cell extracts were immunoblotted with anti-HA and anti-FLAG.
Mentions: Another five positive clones identified in the screen encoded the C-terminal region of the transcription factor DEAF1, the smallest comprising residues 302–565. This finding was intriguing because Pellino1 and the Drosophila orthologue of DEAF1 are both required for the production of the anti-bacterial peptides Drosomycin and Metchnikowin in the fruit fly D. melanogaster (see Introduction), but were not considered to interact, since Pellino was reported to interact at the plasma membrane with Pelle, the Drosophila orthologue of IRAK, while DEAF1 functions in the nucleus. To investigate further whether Pellino1 and DEAF1 bind to one another, we co-expressed DNA vectors encoding hemagglutinin (HA)-tagged DEAF1 and either GFP-tagged Pellino1 (Fig. 1A) or FLAG-tagged Pellino1 (supplemental Fig. S1A) in HEK293FT cells. We found that the expressed HA-DEAF1 could be co-immunoprecipitated with GFP-Pellino1 using anti-GFP (Fig. 1B) or with FLAG-Pellino1 using anti-FLAG (supplemental Fig. S1B). Conversely, GFP-Pellino1 (Fig. 1C) or FLAG-Pellino1 (supplemental Fig. S1C) could both be immunoprecipitated with HA-DEAF1, using anti-HA. The results confirmed that these two proteins do indeed interact with one another.

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