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

In the paper

“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.”

In the paper

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

In the paper

“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”

In the paper

“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”

In the paper

“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”

In the paper

“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 the paper

“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”

In the paper

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

In the paper

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

In the paper

“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”

In the paper

“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 paper

“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).”

What the sources say

• Not checked yetcites 40a

In the paper

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

In the paper

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

In the paper

“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.”

What the sources say

• Not checked yetcites [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]

In the paper

“As a critical determinant of its global transcriptional coactivator activity, CBP/p300 possesses histone acetyltransferase activity (5, 50).”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“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.”

In the paper

“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).”

What the sources say

• Not checked yetcites [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]

In the paper

“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).”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“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).”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“although Ser-402, Ser-404, and Ser-405 may represent secondary phosphorylation sites.”

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“Although TH3 has been shown to bind DNA and to repress transcription more efficiently than the full-length IRF-2 protein (42)”

What the sources say

• Not checked yetcites [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]

In the paper

“Previous experiments, however, have demonstrated that Sendai virus-induced turnover of IκBα also occurs slowly over several hours (24).”

What the sources say

• Not checked yetcites [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]

In the paper

“interestingly, CBP/p300 was shown recently to acetylate p53 and to stimulate its transactivation potential (28).”

What the sources say

• Not checked yetcites [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]

In the paper

“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).”

What the sources say

• Not checked yetcites [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 yetcites [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 yetcites [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]

In the paper

“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).”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

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

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“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.”

In the paper

“Likewise, in the case of IκBα, phosphorylation of Ser-32 and Ser-36 are required for inducer-mediated degradation.”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“Alterations in any of the activation domains decreased both CBP recruitment and transcriptional synergy.”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“functional interaction between these two important growth regulatory proteins accounts for several of the known activities of p53 (3, 29, 40)”

What the sources say

• Not checked yetcites [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 yetcites [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 yetcites [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]

In the paper

“By analogy, recruitment of CBP/p300 to DNA-bound IRF-3 is likely required for maximal transcriptional activation.”

In the paper

“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).”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“These mutations also blocked in vitro ubiquitination of the IκBα protein.”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“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.”

What the sources say

• Not checked yetcites [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 yetcites [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]

In the paper

“a region previously shown to associate with the p53 tumor suppressor, whereas STAT1 and STAT2 associate with different regions of CBP (7, 68).”

What the sources say

• Not checked yetcites [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 yetcites [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]