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. 2003 Apr;4(4):368-73.
doi: 10.1038/sj.embor.embor802. Epub 2003 Mar 21.

Phosphorylation of the Stat1 transactivating domain is required for the response to type I interferons

Andreas Pilz  1 , Katrin Ramsauer, Hamid Heidari, Michael Leitges, Pavel Kovarik, Thomas Decker
Affiliations

Phosphorylation of the Stat1 transactivating domain is required for the response to type I interferons

Andreas Pilz et al. EMBO Rep. 2003 Apr.

Abstract

Stat1 (signal transducer and activator of transcription 1) regulates transcription in response to the type I interferons IFN-alpha and IFN-beta, either in its dimerized form or as a subunit of the interferon-stimulated gene factor 3 (Isgf3) complex (consisting of Stat1, Stat2 and interferon-regulating factor 9). Full-length Stat1-alpha and the splice variant Stat1-beta, which lacks the carboxyl terminus and the Ser727 phosphorylation site, are found in all cell types. IFN-induced phosphorylation of Stat1-alpha on Ser727 occurs in the absence of the candidate kinase, protein kinase C-delta. When expressed in Stat1-deficient cells, Stat1-beta and a Stat1-S727A mutant both restored the formation of Stat1 dimers and of the Isgf3 complex on treatment with IFN-beta. By contrast, only Stat1-alpha restored the ability of IFN-beta to induce high levels of transcription from target genes of Stat1 dimers and Isgf3 and to induce an antiviral state. Our data suggest an important contribution of the Stat1 C terminus and its phosphorylation at Ser727 to the transcriptional activities of the Stat1 dimer and the Isgf3 complex.

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Figures

Figure 1
Figure 1
Signal tranducer and activator of transcription 1 phosphorylation at Ser727 in the absence of protein kinase C-δ. Macrophages derived from wild-type or protein kinase C-δ (Pkc-δ)-deficient mice were treated with interferon (IFN)-β, IFN-γ or lipopolysaccharide (Lps) for 60 min. Stat1 phosphorylation was analysed by western blotting with an antiserum that specifically recognizes the Ser727-phosphorylated molecule (Stat1-pSer727). For normalization, the blot was reprobed with a polyclonal antiserum to the Stat1 carboxyl terminus (Stat1C).
Figure 2
Figure 2
Expression and activation of mutant signal tranducer and activator of transcription 1. (A) Isolation of fibroblast clones expressing signal tranducer and activator of transcription 1-β (Stat1-β). Stat1-deficient fibroblasts were transfected with a plasmid driving the expression of Stat1-β, selection was carried out, and cells were cloned by a limiting dilution procedure. Levels of Stat1-β were analysed by western blotting using a monoclonal antibody that recognizes the amino terminus (Stat1N). For normalization, the blot was reprobed with an antibody (pan-ERK) against extracellular signal-regulated protein kinases 1 and 2 (ERKs 1 and 2). The results from two clones that expressed Stat1-β (clones β1 and β8) and, for comparison, clones that expressed Stat1-α or the Stat1-S727A mutant are shown. (B) Stat1 tyrosine phosphorylation in response to interferon-β (IFN-β). Fibroblasts that expressed Stat1-α, Stat1-β or Stat1-S727A were stimulated with IFN-β for the durations indicated, and Stat1 phosphorylation at Tyr701 (pY701) was analysed by western blotting using a phospho-specific antiserum. For normalization, the blots were reprobed with a monoclonal antibodies that recognize either the N terminus or the carboxyl terminus of Stat1. (C) Formation of the interferon-stimulated gene factor 3 (Isgf3) complex. Fibroblasts expressing Stat1-α, Stat1-β or Stat1-S727A were treated for the durations indicated with IFN-β. The presence of Isgf3 in cell extracts was analysed by an electrophoretic mobility-shift assay using DNA containing an interferon-stimulated response-element sequence as a probe.
Figure 3
Figure 3
Requirement of the signal tranducer and activator of transcription 1 transactivating domain and its phosphorylation at Ser727 for the expression of interferon-β-induced genes. Stat1-deficient fibroblasts, and cells that express Stat1-α, Stat1-β or Stat1-S727A were treated with interferon-β (IFN-β) for 4 h or 6 h. Expression of the Irf1, Gbp1 and Mx1 genes was determined by quantitative real-time PCR of reverse-transcribed messenger RNA. For normalization to input quantities, the amount of Hprt mRNA was determined. (A) Inducibility in response to treatment with IFN-β (that is, the ratio of expression in IFN-β-treated and untreated cells) after 4 h and 6 h in one of three independent experiments. (B) The results of three independent experiments are shown, expressed as the percentage induction ± s.d. in cells that express Stat1-β, Stat1-S727A or that express no Stat1, compared with the values measured in cells that express Stat1-α (100%).
Figure 4
Figure 4
Effect of the signal tranducer and activator of transcription 1-S727A mutation on the expression of the endogenous Mx1 and Gbp1 genes during transient transfection. Stat1-deficient fibroblasts were co-transfected with Stat1-α or Stat1-S727A and a plasmid that drives expression of the β-galactosidase gene (β-Gal). Some of the transfected cells were used for measuring β-Gal activity in cell extracts to ensure comparable transfection efficiencies. The remaining cells were either left untreated or were treated with interferon-β (IFN-β) for 4 h, after which total RNA was isolated and processed for real-time quantitative PCR analysis of the expression of the Mx1 and Gbp1 genes. Inducibility is calculated as the ratio of expression measured in IFN-β-treated and untreated cells.
Figure 5
Figure 5
Antiviral activity. Fibroblasts that express Stat1-α, Stat1-β, Stat1-S727A or no form of Stat1 were treated with serial dilutions of interferon-β (IFN-β) for 14 h. The cells were then infected with vesicular stomatitis virus (VSV) at an MOI (multiplicity of infection) of 0.1. After 24 h, remaining viable cells were stained with crystal violet and staining intensity was determined by absorbance measurements at 620 nm.
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References

    1. Briken V., Ruffner H., Schultz U., Schwarz A., Reis L.F., Strehlow I., Decker T. & Staeheli P. (1995) Interferon regulatory factor 1 is required for mouse Gbp gene activation by γ interferon. Mol. Cell. Biol., 15, 975–982. - PMC - PubMed
    1. Bromberg J.F., Horvath C.M., Wen Z., Schreiber R.D. & Darnell J.E. (1996) Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon-α and interferon-γ. Proc. Natl Acad. Sci. USA, 93, 7673–7678. - PMC - PubMed
    1. Darnell J.E., Kerr I.M. & Stark G.R. (1994) Jakstat pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science, 264, 1415–1421. - PubMed
    1. Decker T. & Kovarik P. (2000) Serine phosphorylation of Stats. Oncogene, 19, 2628–2637. - PubMed
    1. Durbin J.E., Hackenmiller R., Simon M.C. & Levy D.E. (1996) Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell, 84, 443–450. - PubMed

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