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Cited by 923interferon and immune responsesImmune Response and InflammationCytokine Signaling Pathways and Interactions

Virus-Dependent Phosphorylation of the IRF-3 Transcription Factor Regulates Nuclear Translocation, Transactivation Potential, and Proteasome-Mediated Degradation

Open access
Lin, Rongtuan, Heylbroeck, Christophe, Pitha, Paula M., Hiscott, John·1998·Molecular and Cellular Biology
DOI 10.1128/mcb.18.5.2986PMC PMC110678
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Abstract

The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta interferon (IFN-alpha/beta) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. In the present study, we demonstrate that following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carboxy terminus of IRF-3. A combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407; point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein, although residues Ser-402, Thr-404, and Ser-405 were also targets. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-3, DNA binding, and increased transcriptional activation. Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements. Phosphorylation also appears to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-3 was prevented by mutation of the IRF-3 Ser-Thr cluster or by proteasome inhibitors. Interestingly, virus infection resulted in the association of IRF-3 with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of residues Ser-396 and Ser-398 in IRF-3 abrogated its binding to CBP. These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.

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Written from the paper’s full text.

TL;DR

In Sendai virus-infected cells, IRF-3 is phosphorylated at C-terminal Ser/Thr residues, which drives nuclear translocation, DNA binding/transactivation, CBP association, and subsequent proteasome-mediated degradation. Phosphomimetic IRF-3(5D) acts as a constitutive strong activator of IFN-β and ISG-15 promoters.

Key findings

  • Virus-induced phosphorylation mapped to the C-terminal region -ISNSHPLSLTSDQ- between amino acids 395 and 407; Ser-396 and Ser-398 were critical, since IRF-3(2A) and IRF-3(5A) eliminated virus-induced phosphorylation, while IRF-3(3A) was weakly phosphorylated, implicating Ser-402, Thr-404, and Ser-405 as secondary sites.
  • Phosphorylation was required for cytoplasm-to-nucleus translocation: Sendai virus induced nuclear translocation in 90 to 95% of cells within 8 h, GFP–IRF-3(5A) completely abrogated this, and GFP–IRF-3(5D) localized partly to the nucleus in uninfected cells and intensely to the nucleus after infection.
  • IRF-3(5D) alone stimulated the IFN-β promoter by 90-fold and the ISG-15 promoter by 50-fold; with RelA(p65), IRF-3(5D) produced an 80-fold stimulation of IFN-β, while IRF-3(407) stimulated IFN-β about 6-fold and ISG-15 12-fold.
  • Phosphorylation signaled proteasome-mediated degradation and CBP binding: calpain inhibitor I or MG132 blocked virus-induced IRF-3 degradation, IRF-3(5A) was resistant, and IRF-3 coimmunoprecipitated with CBP only from virus-infected cells, requiring Ser-396/Ser-398 and the C-terminal CBP-3 domain.

Methods

  • Transient transfections were performed in human embryonic kidney 293 cells, murine NIH 3T3 cells, and COS-7 cells, plus a stable rtTA-IRF-3 293 line; cells were infected with Sendai virus at 80 hemagglutinating units/ml, and IRF-3 deletion and point mutants were generated by PCR mutagenesis.
  • Analyses included Western blotting for IRF-3 mobility and degradation, phosphatase treatment with PPA or CIP, CAT reporter assays for IFN-β (SVoβ CAT) and ISG-15 promoters normalized to β-galactosidase, EMSA with PRDIII or ISRE probes, GFP–IRF-3 fluorescence microscopy, and coimmunoprecipitation with anti-CBP or anti-myc antibodies.

Limitations

  • The paper states that the nature of the subnuclear changes in IRF-3(NES) localization is not explained, it is unclear whether the upper virus-induced protein-DNA complex contains other proteins such as VIC or DRAF or whether the lower complex is an IRF-3 breakdown product, the functional consequences of IRF-3 interaction with CBP/p300 remain to be elucidated, and the nuclear localization sequence is unidentified.
  • Evident from the text: many experiments rely on transfected or overexpressed IRF-3 in immortalized cell lines, with endogenous IRF-3 turnover sometimes more pronounced than that of transfected protein; no in vivo animal model is presented, and some supporting observations are cited as unpublished data.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1128/mcb.18.5.2986

Cites

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Statistics

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Claims the paper makes

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  • Findingown data
    § RESULTS

    Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements.

    “Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements.”
    • Also stated in: § RESULTS · boosted
      “Strikingly, the substitution of the Ser-Thr cluster at aa 396 to 405 in IRF-3 with the phosphomimetic Asp generated a very strong, constitutive transactivator protein that alone stimulated the IFN-β promoter by 90-fold.”
    Quote in the paperSubstantial claim
  • Findingown data
    § Abstract

    Point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein.

    “point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein”
    • Also stated in: § RESULTS · hedged
      “These experiments thus implicate Ser-396 and Ser-398 as critical sites of virus-induced phosphorylation of IRF-3”
    Quote in the paperSubstantial claim
  • Findingown data
    § Abstract

    The inducible phosphorylation sites of IRF-3 are localized to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407.

    “A combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407”
    • Also stated in: § RESULTS · plain
      “Thus, by deletion analysis, a phosphorylation domain of IRF-3 protein was localized to the region -ISNSHPLSLTSDQ- between aa 395 and 407.”
    Quote in the paperSubstantial claim
  • Findingown data
    § RESULTS

    No evidence was found for inducible phosphorylation at Ser-385 and Ser-386 of IRF-3.

    “in studies with the S385A and S386A mutation, we found no evidence for inducible phosphorylation at these sites”
    Quote in the paperSubstantial claim
  • Findingown data
    § Abstract

    Residues Ser-402, Thr-404, and Ser-405 also contribute to virus-induced phosphorylation of IRF-3.

    “although residues Ser-402, Thr-404, and Ser-405 were also targets.”
    • Also stated in: § RESULTS · plain
      “residues Ser-402, Thr-404, and Ser-405 also contribute to the observed phosphorylation”
    Quote in the paperNot a substantial claim
  • May combine two claims. 23
  • Classified as probably not a substantial claim. 21

  • May combine two claims.

    Findingown data
    § DISCUSSION

    The residues implicated in this regulatory phosphorylation event are Ser-396, Ser-398, Ser-402, Thr-404, and Ser-405, particularly Ser-396 and Ser-398.

    “The residues implicated in this regulatory phosphorylation event are Ser-396, Ser-398, Ser-402, Thr-404, and Ser-405, particularly the Ser-396 and Ser-398 residues.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    IRF-3(357) did not stimulate gene expression but rather slightly repressed ISG-15.

    “IRF-3(357) did not stimulate gene expression but rather slightly repressed ISG-15”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    IRF-3 alone weakly activated the ISG-15 promoter; IRF-3 was weaker than IRF-1, which produced a 9-fold stimulation.

    “IRF-3 alone weakly activated the ISG-15 promoter; in the context of this regulatory element, IRF-3 was weaker than IRF-1, which produced a 9-fold stimulation”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    The interaction between IRF-3 and CBP was observed clearly in cells cotransfected with the IRF-3 expression plasmid but was not seen when immunoprecipitation was performed with preimmune serum.

    “This interaction was observed clearly in cells cotransfected with the IRF-3 expression plasmid (Fig. 8B, lane 3) but was not seen when the immunoprecipitation was performed with preimmune serum”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    Immunoblot analysis of whole-cell extracts revealed that phosphorylated IRF-3, as well as forms I and II, was present in virus-infected cells, and in cells transfected with 2A, 3A, and 5A the forms I and II but not the phosphorylated form of IRF-3 were observed.

    “Immunoblot analysis of the whole-cell extracts revealed that phosphorylated IRF-3, as well as forms I and II, was present in virus-infected cells (Fig. 8B, lane 10), and in cells transfected with 2A, 3A, and 5A the forms I and II but not the phosphorylated form of IRF-3 were observed”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    In uninfected cells, GFP-IRF-3 localized exclusively to the cytoplasm; Sendai virus infection resulted in translocation of IRF-3 to the nucleus within 8 h in 90 to 95% of the cells.

    “In uninfected cells, GFP–IRF-3 localized exclusively to the cytoplasm; Sendai virus infection resulted in translocation of IRF-3 to the nucleus within 8 h in 90 to 95% of the cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    Virus-induced degradation of full-length (endogenous) forms of IRF-3 and the truncated ΔN form was detected.

    “virus-induced degradation of full-length (endogenous) forms of IRF-3 (Fig. 7A, lanes 1 and 4) and the truncated ΔN (Fig. 7B, lanes 1 and 4) was detected”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    IRF-3(2A) and IRF-3(5A) were detected in whole-cell extract immunoblot but not in the CBP immunoprecipitate.

    “IRF-3(2A) and IRF-3(5A) were detected in whole-cell extract immunoblot but not in the CBP immunoprecipitate”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    No further stabilization of IRF-3-ΔN5A occurred with calpain inhibitor I or MG132 addition, and no phosphorylated IRF-3 was detected.

    “no further stabilization of IRF-3-ΔN5A occurred with calpain inhibitor I or MG132 addition, and no phosphorylated IRF-3 was detected”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS

    IRF-3(5D) localized both to the nucleus and to the cytoplasm in uninfected cells, while virus infection resulted in an intense nuclear pattern of IRF-3(5D) fluorescence.

    “IRF-3(5D) localized both to the nucleus and to the cytoplasm in uninfected cells (Fig. 5E), while virus infection resulted in an intense nuclear pattern of IRF-3(5D) fluorescence”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given
    § DISCUSSION

    In the case of IRF-3, phosphorylation is not detected until 6 to 8 h after infection and continues in a heterogeneous manner over the following 10 to 12 h.

    “In the case of IRF-3, phosphorylation is not detected until 6 to 8 h after infection and continues in a heterogeneous manner over the following 10 to 12 h.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation
    § DISCUSSION

    IRF-3 phosphorylation appears to be induced only by virus infection and dsRNA addition; other inducers have not resulted in IRF-3 turnover.

    “whereas IRF-3 phosphorylation appears to be induced only by virus infection and dsRNA addition; other inducers have not resulted in IRF-3 turnover (40a).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites 40a

      Not checked in the free run.

  • May combine two claims.

    Findingnone given
    § DISCUSSION

    The protease inhibitor calpain inhibitor I and the more specific proteasome inhibitor MG132 block IRF-3 turnover.

    “Furthermore, the protease inhibitor calpain inhibitor I and the more specific proteasome inhibitor MG132 block IRF-3 turnover.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § DISCUSSION

    Mutation of Ser-396 and Ser-398 or the use of proteasome inhibitors prevents postinfection degradation of IRF-3.

    “since mutation of Ser-396 and Ser-398 or the use of proteasome inhibitors prevented postinfection degradation of IRF-3.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation
    § Abstract

    IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells.

    “IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells.”
    • Also stated in: § Abstract · plain
      “The IRF-3 gene encodes a 55-kDa protein which is expressed constitutively in all tissues.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Au W-C, Moore P A, Lowther W, Juang Y-T, Pitha P M. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc Natl Acad Sci USA. 1995;92:11657–11661. doi: 10.1073/pnas.92.25.11657. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    CBP/p300 possesses histone acetyltransferase activity.

    “As a critical determinant of its global transcriptional coactivator activity, CBP/p300 possesses histone acetyltransferase activity (5, 50).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [5] Bannister A J, Kouzarides T. The CBP coactivator is a histone acetyltransferase. Nature. 1996;384:641–643. doi: 10.1038/384641a0. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [50] Ogryzko V V, Schiltz R L, Russanova V, Howard B H, Nakatani Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell. 1996;87:953–959. doi: 10.1016/s0092-8674(00)82001-2. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning
    § Abstract

    Virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.

    “These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    The recent crystal structure of IRF-1 bound to PRDI provides evidence for a novel helix-turn-helix motif that latches onto a GAAA core sequence via three of the five conserved tryptophan amino acids of the DNA binding domain.

    “The recent crystal structure of the related IRF-1 protein bound to PRDI provides evidence for a novel helix-turn-helix motif that latches onto a GAAA core sequence via three of the five conserved tryptophan amino acids of the DNA binding domain (20).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Escalante C R, Yie J, Thanos D, Aggarwal A K. Structure of IRF-1 with bound DNA reveals determinants of interferon regulation. Nature. 1998;391:103–106. doi: 10.1038/34224. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • May combine two claims.

    Premisecitation
    § DISCUSSION

    IRF-3 is able to bind the ISRE element of ISG-15, as well as the PRDIII-PRDI and IE regions of the IFN-β and IFN-α promoters, respectively.

    “In previous experiments, we demonstrated that IRF-3 was able to bind the ISRE element of ISG-15, as well as the PRDIII-PRDI and IE regions of the IFN-β and IFN-α promoters, respectively (2, 56).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [2] Au W-C, Moore P A, Lowther W, Juang Y-T, Pitha P M. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc Natl Acad Sci USA. 1995;92:11657–11661. doi: 10.1073/pnas.92.25.11657. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [56] Schafer S, Lin R, Moore P, Hiscott J, Pitha P M. Regulation of type 1 interferon gene expression by interferon regulatory factor 3. J Biol Chem. 1998;273:2714–2720. doi: 10.1074/jbc.273.5.2714. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Synergistic activation of the IFN-β promoter requires recruitment of CBP/p300 to the enhanceosome, via a new activating surface assembled from the activation domains of all of the transcription factors in the enhanceosome.

    “Elegant studies published during review of the manuscript demonstrated that synergistic activation of the IFN-β promoter requires recruitment of CBP/p300 to the enhanceosome, via a new activating surface assembled from the activation domains of all of the transcription factors in the enhanceosome (37, 45).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] Kim T K, Maniatis T. The mechanism of transcriptional synergy of an in vitro assembled interferon β enhanceosome. Mol Cell. 1998;1:119–129. doi: 10.1016/s1097-2765(00)80013-1. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [45] Merika M, Williams A, Chen G, Collins T, Thanos D. Recruitment of CBP/p300 by the IFNβ enhanceosome is required for synergistic activation of transcription. Mol Cell. 1998;1:277–287. doi: 10.1016/s1097-2765(00)80028-3. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning
    § DISCUSSION

    Ser-402, Ser-404, and Ser-405 may represent secondary phosphorylation sites for IRF-3 degradation.

    “although Ser-402, Ser-404, and Ser-405 may represent secondary phosphorylation sites.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Upon stimulation by many activating agents, including cytokines, viruses, and dsRNA, IκBα is rapidly phosphorylated and degraded, resulting in the release and nuclear translocation of NF-κB.

    “Upon stimulation by many activating agents, including cytokines, viruses, and dsRNA, IκBα is rapidly phosphorylated and degraded, resulting in the release and nuclear translocation of NF-κB.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Baldwin A S., Jr The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol. 1996;14:649–681. doi: 10.1146/annurev.immunol.14.1.649. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [6] Beauparlant P, Hiscott J. Biological and biochemical inhibitors of the NF-κB/Rel proteins and cytokine synthesis. Cytokine Growth Factor Rev. 1996;7:175–190. doi: 10.1016/1359-6101(96)00020-2. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § DISCUSSION

    TH3 binds DNA and represses transcription more efficiently than the full-length IRF-2 protein.

    “Although TH3 has been shown to bind DNA and to repress transcription more efficiently than the full-length IRF-2 protein (42)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Lin R, Mustafa A, Nguyen H, Hiscott J. Mutational analysis of interferon (IFN) regulatory factors 1 and 2: effects on the induction of IFN-β gene expression. J Biol Chem. 1994;269:17542–17549. [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Sendai virus-induced turnover of IκBα also occurs slowly over several hours.

    “Previous experiments, however, have demonstrated that Sendai virus-induced turnover of IκBα also occurs slowly over several hours (24).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [24] Garoufalis E, Kwan I, Lin R, Mustafa A, Pepin N, Roulston A, Lacoste J, Hiscott J. Viral induction of the human interferon beta promoter: modulation of transcription by NF-κB/rel proteins and interferon regulatory factors. J Virol. 1994;68:4707–4715. doi: 10.1128/jvi.68.8.4707-4715.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § DISCUSSION

    CBP/p300 acetylates p53 and stimulates its transactivation potential.

    “interestingly, CBP/p300 was shown recently to acetylate p53 and to stimulate its transactivation potential (28).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Gu W, Roeder R G. Activation of p53 sequence-specific DNA binding by acetylation of p53 C-terminal domain. Cell. 1997;90:595–606. doi: 10.1016/s0092-8674(00)80521-8. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    In response to dsRNA or viral induction, the 50-kDa IRF-2 protein is proteolytically processed into a smaller, 24- to 27-kDa protein comprising the 160-aa DNA-binding domain of IRF-2, termed TH3 or In4.

    “In response to double-stranded RNA (dsRNA) or viral induction, the 50-kDa IRF-2 protein is proteolytically processed into a smaller, 24- to 27-kDa protein (51) comprising the 160-aa DNA-binding domain (DBD) of IRF-2, which was termed TH3 (14) or In4 (65).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [51] Palombella V J, Maniatis T. Inducible processing of interferon regulatory factor-2. Mol Cell Biol. 1992;12:3325–3336. doi: 10.1128/mcb.12.8.3325-3336.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [14] Cohen L, Hiscott J. Characterization of TH3, an induction specific protein interacting with the interferon-β promoter. Virology. 1992;191:589–599. doi: 10.1016/0042-6822(92)90234-g. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [65] Whiteside S T, King P, Goodbourn S. A truncated form of the IRF-2 transcription factor has the properties of a postinduction repressor of interferon-β gene expression. J Biol Chem. 1994;269:27059–27065. [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § DISCUSSION

    The amino terminus of IκBα is necessary for signal-induced phosphorylation and ubiquitination, but degradation requires the C-terminal PEST domain.

    “The amino terminus of IκBα is necessary for signal-induced phosphorylation and ubiquitination, but for degradation to occur, there is an absolute requirement for the C-terminal PEST domain (reviewed in references 4 and 6).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Baldwin A S., Jr The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol. 1996;14:649–681. doi: 10.1146/annurev.immunol.14.1.649. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [6] Beauparlant P, Hiscott J. Biological and biochemical inhibitors of the NF-κB/Rel proteins and cytokine synthesis. Cytokine Growth Factor Rev. 1996;7:175–190. doi: 10.1016/1359-6101(96)00020-2. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § DISCUSSION

    Substitution of alanine for either Ser-32 or Ser-36 completely abolishes the signal-induced phosphorylation and degradation of IκBα and blocks activation of NF-κB.

    “substitution of alanine for either Ser-32 or Ser-36 completely abolished the signal-induced phosphorylation and degradation of IκBα and blocked activation of NF-κB.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Baldwin A S., Jr The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol. 1996;14:649–681. doi: 10.1146/annurev.immunol.14.1.649. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [6] Beauparlant P, Hiscott J. Biological and biochemical inhibitors of the NF-κB/Rel proteins and cytokine synthesis. Cytokine Growth Factor Rev. 1996;7:175–190. doi: 10.1016/1359-6101(96)00020-2. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    In unstimulated cells, NF-κB heterodimers are retained in the cytoplasm by inhibitory IκB proteins.

    “In unstimulated cells, NF-κB heterodimers are retained in the cytoplasm by inhibitory IκB proteins.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Baldwin A S., Jr The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol. 1996;14:649–681. doi: 10.1146/annurev.immunol.14.1.649. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [6] Beauparlant P, Hiscott J. Biological and biochemical inhibitors of the NF-κB/Rel proteins and cytokine synthesis. Cytokine Growth Factor Rev. 1996;7:175–190. doi: 10.1016/1359-6101(96)00020-2. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    CBP/p300 is implicated as a negative regulator of cell growth based on its interactions with adenovirus E1a, simian virus 40 large T antigen, and the tumor suppressor p53 among others.

    “A growing body of biochemical and genetic evidence also implicates CBP/p300 as a negative regulator of cell growth, based on its interactions with adenovirus E1a, simian virus 40 large T antigen, and the tumor suppressor p53 among others.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    In IκBα, phosphorylation of Ser-32 and Ser-36 is required for inducer-mediated degradation.

    “Likewise, in the case of IκBα, phosphorylation of Ser-32 and Ser-36 are required for inducer-mediated degradation.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Baldwin A S., Jr The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol. 1996;14:649–681. doi: 10.1146/annurev.immunol.14.1.649. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [6] Beauparlant P, Hiscott J. Biological and biochemical inhibitors of the NF-κB/Rel proteins and cytokine synthesis. Cytokine Growth Factor Rev. 1996;7:175–190. doi: 10.1016/1359-6101(96)00020-2. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Alterations in any of the activation domains decrease both CBP recruitment and transcriptional synergy.

    “Alterations in any of the activation domains decreased both CBP recruitment and transcriptional synergy.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] Kim T K, Maniatis T. The mechanism of transcriptional synergy of an in vitro assembled interferon β enhanceosome. Mol Cell. 1998;1:119–129. doi: 10.1016/s1097-2765(00)80013-1. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [45] Merika M, Williams A, Chen G, Collins T, Thanos D. Recruitment of CBP/p300 by the IFNβ enhanceosome is required for synergistic activation of transcription. Mol Cell. 1998;1:277–287. doi: 10.1016/s1097-2765(00)80028-3. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    Functional interaction between p53 and CBP/p300 accounts for several of the known activities of p53.

    “functional interaction between these two important growth regulatory proteins accounts for several of the known activities of p53 (3, 29, 40)”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [3] Avantaggiati M L, Ogryzko V, Gardner K, Giordano A, Levine A S, Kelly K. Recruitment of p300/CBP in p53-dependent signal pathways. Cell. 1997;89:1175–1184. doi: 10.1016/s0092-8674(00)80304-9. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [29] Gu W, Shi X L, Roeder R G. Synergistic activation of transcription by CBP and p53. Nature. 1997;387:819–823. doi: 10.1038/42972. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [40] Lill N L, Grossman S R, Ginsberg D, DeCaprio J, Livingston D M. Binding and modulation of p53 by p300/CBP coactivators. Nature. 1997;387:823–827. doi: 10.1038/42981. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning
    § DISCUSSION

    Recruitment of CBP/p300 to DNA-bound IRF-3 is likely required for maximal transcriptional activation.

    “By analogy, recruitment of CBP/p300 to DNA-bound IRF-3 is likely required for maximal transcriptional activation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    CBP/p300 participates in the transcriptional process by providing a scaffold for different classes of transcriptional regulators on specific chromatin domains.

    “Several studies have now demonstrated that CBP/p300 participates in the transcriptional process by providing a scaffold for different classes of transcriptional regulators on specific chromatin domains (12, 50).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [12] Chen H, Lin R J, Schiltz R L, Chakravarti D, Nash A, Nagy L, Privalsky M L, Nakatani Y, Evans R M. Nuclear receptor coactivator ACTR is a novel histone acetyltransferases and forms a multimeric activation complex with P/CAF and CBP/p300. Cell. 1997;90:569–580. doi: 10.1016/s0092-8674(00)80516-4. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [50] Ogryzko V V, Schiltz R L, Russanova V, Howard B H, Nakatani Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell. 1996;87:953–959. doi: 10.1016/s0092-8674(00)82001-2. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § DISCUSSION

    These mutations also block in vitro ubiquitination of the IκBα protein.

    “These mutations also blocked in vitro ubiquitination of the IκBα protein.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Baldwin A S., Jr The NF-κB and IκB proteins: new discoveries and insights. Annu Rev Immunol. 1996;14:649–681. doi: 10.1146/annurev.immunol.14.1.649. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [6] Beauparlant P, Hiscott J. Biological and biochemical inhibitors of the NF-κB/Rel proteins and cytokine synthesis. Cytokine Growth Factor Rev. 1996;7:175–190. doi: 10.1016/1359-6101(96)00020-2. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § DISCUSSION

    CBP/p300 functionally interacts with STAT1 and STAT2 and may contribute to IFN-α and IFN-γ nuclear signalling.

    “although recent studies demonstrated that CBP/p300 also functionally interacts with STAT1 (68) and STAT2 (7) and may contribute to IFN-α and IFN-γ nuclear signalling.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [68] Zhang J J, Vinkemeier U, Gu W, Chakravarti D, Horvath C M, Darnell J E. Two contact regions between STAT1 and CBP/p300 in interferon gamma signalling. Proc Natl Acad Sci USA. 1996;93:15092–15096. doi: 10.1073/pnas.93.26.15092. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [7] Bhattacharya S, Eckner R, Grossman S, Oldread E, Arany Z, D’Andrea A, Livingston D M. Cooperation of Stat2 and p300/CBP by interferon-α. Nature. 1996;383:344–347. doi: 10.1038/383344a0. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § DISCUSSION

    The C-terminal interaction domain of CBP is a region previously shown to associate with the p53 tumor suppressor, whereas STAT1 and STAT2 associate with different regions of CBP.

    “a region previously shown to associate with the p53 tumor suppressor, whereas STAT1 and STAT2 associate with different regions of CBP (7, 68).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [7] Bhattacharya S, Eckner R, Grossman S, Oldread E, Arany Z, D’Andrea A, Livingston D M. Cooperation of Stat2 and p300/CBP by interferon-α. Nature. 1996;383:344–347. doi: 10.1038/383344a0. [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [68] Zhang J J, Vinkemeier U, Gu W, Chakravarti D, Horvath C M, Darnell J E. Two contact regions between STAT1 and CBP/p300 in interferon gamma signalling. Proc Natl Acad Sci USA. 1996;93:15092–15096. doi: 10.1073/pnas.93.26.15092. [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

38 of 38

  • The paper relies on this without support.

    Phosphomimetic Asp at the sites of C-terminal phosphorylation functionally mimics phosphorylation.

    “IRF-3 containing the phosphomimetic Asp at the sites of C-terminal phosphorylation”

  • The authors state this as an assumption.

    IRF-3 exists in a latent state in the cytoplasm of uninfected cells.

    “IRF-3 exists in a latent state in the cytoplasm of uninfected cells”

  • The authors state this as an assumption.

    The C terminus of IRF-3 may physically interact with the DNA binding domain to obscure both the DBD and IAD regions.

    “the C terminus may physically interact with the DNA binding domain in such a way as to obscure both the DBD and the IAD regions of the protein”

  • The authors state this as an assumption.

    The C-terminal 20 aa (407 to 427) contains an autoinhibitory domain.

    “the presence of an autoinhibitory domain within the C-terminal 20 aa (407 to 427)”

  • The authors state this as an assumption.

    Translocation of IRF-3 to the nucleus occurs via an unidentified nuclear localization sequence or in conjunction with a transcriptional partner associating through the IAD region.

    “Translocation to the nucleus, occurring via an unidentified nuclear localization sequence or in conjunction with a transcriptional partner associating through the IAD region”

  • The authors state this as an assumption.

    The presence of a NES element ultimately shuttles IRF-3 from the nucleus and terminates the initial activation of IFN-responsive promoters.

    “The presence of a NES element ultimately shuttles IRF-3 from the nucleus and terminates the initial activation of IFN-responsive promoters.”

  • The authors state this as an assumption.

    The phosphorylated form of IRF-3 exported from the nucleus may then be susceptible to proteasome-mediated degradation.

    “The phosphorylated form of IRF-3 exported from the nucleus may then be susceptible to proteasome-mediated degradation.”
7 of 7

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • IRF-3 has a C-terminal autoinhibitory region, and regulated phosphorylation upon viral infection stimulates DNA binding.

    “IRF-3 possesses a C-terminal autoinhibitory region.Regulated phosphorylation of this region upon viral infection stimulates DNA binding”

    From Assembly requirements of PU.1–Pip (IRF‐4) activator complexes: inhibiting function in vivo using fused dimers (1999) · cites this paper as Lin et al., 1998

    The Discussion proposes a C-terminal autoinhibitory domain and a model in which virus-induced phosphorylation relieves autoinhibition and leads to DNA binding; the Abstract also reports stimulated DNA binding. The claim states the proposed domain as established and implies that the autoinhibitory domain itself is phosphorylated. The Discussion places that domain at amino acids 407–427, but the phosphorylation sites at 396–405. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 2B lane 6 cited for both presence and disappearance of phosphorylated IRF-3

    Minor

    Lane 6 is cited as showing the additional IRF-3 form in infected cells and also as showing its complete disappearance after phosphatase treatment.

  • Residue 404 identified as both threonine and serine

    Minor

    The mapped phosphorylation cluster identifies residue 404 as threonine, whereas the discussion calls it serine.

  • Different residues identified for the NES mutation

    Minor

    The results identify the nuclear export signal mutation as L145A-L146A, but the discussion identifies it as L144A and L145A.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 3

  1. Crepieux P., 1994, Crit. Rev. Oncog., 5, 615
  2. Evan G. I., 1984, Mol. Cell. Biol., 4, 2843
  3. Nguyen, H., J. Hiscott, and P. M. Pitha. The growing family of IRF transcription factors. Cytokine Growth Factor Rev., in press.

Cited by

Sorted by citations

Related

Authors
Lin, R., Heylbroeck, C., Pitha, P. M., & Hiscott, J.
Affiliations
  • Lady Davis Institute for Medical Research (CA)
  • Johns Hopkins University (US)
Corresponding author
Lin, Rongtuan
ORCID
Venue
Molecular and Cellular Biology
Publisher
Informa UK Limited
Published
1998-05-01
Volume
18
Issue
5
Pages
2986-2996
ISSN
  • 0270-7306 (ISSN-L)
  • 1098-5549
DOI
10.1128/mcb.18.5.2986
PMC
PMC110678
OpenAlex
W2130576390
Semantic Scholar
d9395b576923c5fe3c0e57a18df21bd0fd11d519
Cited by
915
References
69
Citations by year
2026: 11 · 2025: 20 · 2024: 24 · 2023: 26 · 2022: 33 · 2021: 24 · 2020: 30 · 2019: 26 · 2018: 27 · 2017: 28 · 2016: 33 · 2015: 26 · 2014: 38 · 2013: 42 · 2012: 30
FWCI
10.65
Type
journal article
Language
en
Open access
Licence
journals.asm.org/non-commercial-tdm-license (tdm)
Locations
  • Molecular and Cellular Biology (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Health Sciences › Medicine › Oncology › Cytokine Signaling Pathways and Interactions
Keywords
IRF3, interferon regulatory factors, protein phosphorylation, Sendai virus, nuclear translocation, transcriptional activation, interferon-stimulated response element, proteasomal degradation, CREB-binding protein, transcriptional coactivator, post-translational modification, interferon-stimulated genes, phosphomimetic mutations
MeSH terms
Amino Acid Sequence, Animals, Biological Transport, Cell Compartmentation, Cell Nucleus, Cysteine Endopeptidases, Cytoplasm, DNA Mutational Analysis, DNA-Binding Proteins, Humans, Molecular Sequence Data, Multienzyme Complexes, Peptide Mapping, Phosphorylation, Protein Binding, Protein Processing, Post-Translational, Transcription Factors, Transcriptional Activation, Trans-Activators, Respirovirus, Proteasome Endopeptidase Complex, Interferon Regulatory Factor-3, Mice, Histone Acetyltransferases, Nuclear Receptor Coactivator 3

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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