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The tumor suppressor PTEN has a critical role in antiviral innate immunity

Nature Immunology volume 17, pages 241–249 (2016)Cite this article

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Abstract

The gene encoding PTEN is one of the most frequently mutated tumor suppressor–encoding genes in human cancer. While PTEN's function in tumor suppression is well established, its relationship to anti-microbial immunity remains unknown. Here we found a pivotal role for PTEN in the induction of type I interferon, the hallmark of antiviral innate immunity, that was independent of the pathway of the kinases PI(3)K and Akt. PTEN controlled the import of IRF3, a master transcription factor responsible for IFN-β production, into the nucleus. We further identified a PTEN-controlled negative phosphorylation site at Ser97 of IRF3 and found that release from this negative regulation via the phosphatase activity of PTEN was essential for the activation of IRF3 and its import into the nucleus. Our study identifies crosstalk between PTEN and IRF3 in tumor suppression and innate immunity.

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Figure 1: PTEN is involved in virus-induced production of type I interferon.
Figure 2: Pivotal role for PTEN in the induction of type I interferon and antiviral response in vivo.
Figure 3: PTEN promotes the induction of IFNB1 in manner dependent on the phosphatase activity of PTEN and independent of the Akt pathway.
Figure 4: PTEN regulates the IRF3-mediated induction of IFNB1.
Figure 5: PTEN controls the import of IRF3 into the nucleus.
Figure 6: PTEN releases the negative regulatory phosphorylation of IRF3 at Ser97.
Figure 7: Virus-induced dynamic interaction of PTEN and IRF3 in the cytoplasm.

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Acknowledgements

We thank P. Feng (University of Southern California) for Irf3−/−Irf7−/− MEFs; H. Wu (University of California, Los Angeles) for primary Pten+/+ and Pten−/− MEFs; H. Li (Wuhan University) for PtenLoxP/LoxP mice; Y. Zhu and S. Liu (Wuhan University) for plasmid encoding the IL-6 or IL-8 firefly luciferase reporter; X. Chen for help with mass spectrometry; X. Zou and S. Jin for help with statistical analysis; K. Meng and H. Zhang for technical assistance; and X. Fu and Z. Jiang for critical reading and comment on the manuscripts. Supported by the National Basic Research Program of China (2010CB911803), the National Science Foundation of China (81130083 and 31221061) and Hubei Province's Outstanding Medical Academic Leader Program (D.G.).

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Authors and Affiliations

  1. State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China

    Shun Li, Mingzhu Zhu, Ruangang Pan, Ting Fang, Yuan-Yuan Cao, Xiaolu Zhao, Cao-Qi Lei, Lin Guo, Yu Chen, Hong-Bing Shu & Deyin Guo

  2. Medical Research Institute, School of Basic Medical Sciences, Wuhan University, Wuhan, China

    Shuliang Chen & Deyin Guo

  3. Institute of Biotechnology, University of Helsinki, Helsinki, Finland

    Chun-Mei Li & Eija Jokitalo

  4. Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China

    Yuxin Yin

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Contributions

S.L. and D.G. designed the experiments and wrote the manuscript.; S.L., M.Z., R.P., T.F., Y.-Y.C. and C.-M.L. performed experimental work; S.L., M.Z., S.C., X.Z., L.G., Y.C., E.J. and D.G. analyzed the data; C.-Q.L., Y.Y. and H.-B.S. provided experimental material; and D.G. conceived of and supervised the study.

Corresponding author

Correspondence to Deyin Guo.

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Competing interests

D.G. and S.L. are applying for a patent related to PTEN's function in antiviral immunity.

Integrated supplementary information

Supplementary Figure 1 PTEN promotes virus-induced expression of IFNB1 and its downstream genes.

(a) Quantitative RT-PCR analysis of IFNB1 mRNA (left) and ELISA of IFN-β (right) in HEK 293 cells (2 × 105) transfected for 24 h with empty vector (EV) or plasmid for the expression of PTEN, and then untreated (UT) or transfected for another 8 h with poly(I:C) (1 μg/ml). (b, c) Luciferase assay (as in Fig. 1b) (top) and immunoblot analysis of Flag-tagged PTEN and β-actin (below) in HEK293 cells transfected for 24 h with luciferase reporter plasmid for IFN-β (IFN-β-Luc; b) or ISRE (ISRE-Luc; c), plus empty vector or increasing amounts (Wedge: 0, 0.01, 0.05, 0.2 and 1 μg) of plasmid encoding PTEN (below lanes), then left uninfected (UI; left) or infected (right) for 8 h with SeV. (d) Quantitative RT-PCR analysis of IFIT1, ISG15, CXCL10 or RIG-I mRNA in HEK 293 cells (2 × 105) transfected for 24 h with empty vector (EV) or plasmid for the expression of PTEN, and then untreated or transfected for another 8 h with SeV; results were normalized to those of the control gene GAPDH and are presented relative to those of untreated cells transfected with empty vector. (e) Luciferase assay (as in Fig. 1b) in HEK293 cells transfected for 24 h with luciferase reporter plasmid for IL-6 (IL-6-Luc; top) or IL-8 (IL-8-Luc; bottom), plus empty vector (EV) or plasmid encoding PTEN, then left untreated (UT) or infected with SeV or treated with IL-1β (0.02 μg/ml) for 10 h. *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired t-test (a, d, e) or one-way analysis of variant (ANOVA) with post hoc Bonferroni t-test (b, c)). Data are from three independent experiments (a; b, c, top; d, e; mean and s.d. of three independent biological replicates per group in each) or are representative of three independent experiments (b, c, bottom).

Supplementary Figure 2 PTEN deficiency impairs cellular antiviral responses.

(a) Microscopy analyzing VSV replication (left) and immunoblot analysis (right) of VSV-GFP, PTEN and β-actin with the indicated antibodies in HEK293 cells (2 × 105) transfected for 36 h with plasmid encoding non-targeting control siRNA (siControl) or siRNA targeting PTEN (siPTEN1 or siPTEN2), then infected for another 24 h with VSV-GFP (MOI = 0.001). (b) Plaque assay of VSV in HEK293 cells (2 × 105) transfected for 24 h with siRNA-encoding plasmid as in a and left untreated or transfected for 12 h with poly(I:C), then infected or another 24 h with VSV-GFP (MOI = 0.001). (c) Plaque assay of VSV (left) and quantitative RT-PCR analysis of IFNB1 mRNA (right) in 786-O cells stably expressing empty vector (EV) or plasmid encoding wild-type PTEN or PTEN(C124S), and left uninfected or infected for 24 h with VSV-GFP (MOI = 0.01). (d) Cell cytotoxicity assays of PC-3 cells stably expressing empty vector (EV) or plasmid encoding wild-type PTEN or PTEN(C124S), then infected for 24 h with increments of VSV-GFP titers. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA (c) or two-way ANOVA with post hoc Bonferroni t-test (b, d)). Data are from three independent experiments (b, c, d; mean and s.d. of three independent biological replicates per group in each) or are representative of three independent experiments (a). BF, bright field.

Supplementary Figure 3 PTEN promotes the induction of IFNB1 independently of Akt pathway.

(a) Breeding scheme for the generation of inducible PTEN-mutant mice and PTEN-wild-type mice. (b) Schematic presentation (left) and PCR analysis (right) of the genotype of mice with respect to the CreER transgene and the target Pten gene by using mice tails as PCR templates. P1, P2, P3 and P4 indicated the primers specific for PtenloxP and Cre. (c) Immunoblotting analysis of PTEN expression in mice spleen from PTEN-wild-type mice and PTEN-mutant mice (line 1, 2 and 3). (d) Luciferase assay (as in Fig. 1b) (top) and immunoblot analysis (below lanes) in HEK 293 cells transfected for 24 h with empty vector (EV) or plasmid encoding PTEN, VHR, PTP1B, Cdc25A, SHP1 or SHIP1, and then left uninfected (UI; left) or infected (right) for 10 h with SeV. (e) Luciferase assay (as in Fig. 1b) in HEK 293 cells transfected for 24 h with empty vector (EV) or plasmid encoding PTEN, and untreated (left half) or treated for 2 h with LY294002 (5 μM each), then left uninfected (UI) or infected for 10 h with SeV. (f) Luciferase assay (as in Fig. 1b) in HEK 293 cells transfected for 24 h with empty vector (EV) or plasmid encoding PTEN, plus empty vector (left half) or plasmid encoding Akt-AA (right top) or Myr-Akt (right bottom), and then left uninfected or infected for 10 h with SeV. Akt-AA, the dominant-negative form of Akt, whose phosphorylation sites (Thr308 and Ser473) are replaced by alanine; Myr-Akt, constitutively active Akt with a myristoylation moiety that results in enhanced plasma membrane binding. (g) Luciferase assay (as in Fig. 1b) in MCF-7 cells (left), HeLa cells (middle) and PC-3 cells (right) transfected for 24 h with empty vector (EV) or plasmid encoding PTEN, and untreated (left half) or treated for 2 h with wortmannin (1 μM each), then left uninfected (UI) or infected for 10 h with SeV. (h) Quantitative RT-PCR analysis (left) of IFNB1 and VSV nucleocapsid (VSV-N) mRNA and immunoblot analysis (right) of Flag-tagged Myr-Akt, Akt-AA and total GAPDH in the livers of wild-type mice (n = 3) with empty vector (EV), plasmid encoding Myr-Akt or Akt-AA delivered to the livers for 9 h by hydrodynamic tail vein injection, infected intrahepaticly with VSV (1 x 107 PFU per mouse), and assessed 6 h after infection. (i) Quantitative RT-PCR analysis (left) of IFNB1 and VSV-N mRNA and immunoblot analysis (right) of total Akt, Akt phosphorylation at Ser473 (p-Akt(S473)) and total GAPDH in the livers of wild-type mice (n = 3) left untreated (UT) or treated intravenously with wortmannin (0.7 mg/kg) or LY294002 (75 mg/kg) for 0.5 h before intrahepatic infection with VSV (1 x 107 PFU per mouse), assessed 6 h after infection. *p < 0.05; **p < 0.01 (one-way ANOVA (d, h, i) or two-way ANOVA with post hoc Bonferroni t-test (e, f, g)). Data are from three independent experiments (d, top; e-g; h, i, left; mean and s.d. of three independent biological replicates per group in each) or are representative of three independent experiments (b, right; c; d, bottom; h, i, right).

Supplementary Figure 4 PTEN promotes IRF3 but not NF-κB-dependent gene transcription.

(a) Quantitative RT-PCR analysis of Isg15, Ifit1, Cxcl10, Ccl5, Cxcl1, Il6, Nfkbia or Nfkb1 mRNA in Pten+/+ MEFs and Pten−/− MEFs left uninfected (UI) or infected for 8 h with SeV. (b) Luciferase assay (as in Fig. 1b) in 293-TLR3 (left), 293 (middle) or 293-TLR4+MD2+CD14+ cells (right) transfected for 36 h with a luciferase reporter plasmid for NF-κB promoter (NF-κB-Luc), plus plasmid encoding non-targeting control siRNA (siControl) or siRNA targeting PTEN, left untreated (UT) or treated with poly(I:C) (10 μg/ml) or LPS (10 μg/ml), or transfected with poly(I:C) (1 μg/ml) for 10 h. *p < 0.05; **p < 0.01 (unpaired t-test (a, b)). Data are from three independent experiments (a, b; mean and s.d. of three independent biological replicates per group in each).

Supplementary Figure 5 Functional screen for the negative phosphorylation site on IRF3 targeted by PTEN.

(a) Luciferase assay (as in Fig. 1b) (top) and immunoblot analysis (below lanes) of IRF3 variants in Irf3−/−Irf7−/− MEFs (1 × 105) transfected with a luciferase reporter plasmid for IFNB1 promoter (IFN-β-Luc), plus empty vector (EV), or plasmid encoding wild-type IRF3 (WT), IRF3 phospho-mimetic or phospho-defective site mutants, left uninfected (UI) or infected for 10 h with SeV. (b) Immunofluorescence microscopy of Irf3−/−Irf7−/− MEFs transfected for 20 h with plasmid encoding wild-type IRF3 (WT) or mutant IRF3 with S97D substitution, left uninfected (UI) or infected for 12 h with SeV, stained with antibody to IRF3 (green) and DAPI (blue). Scale bars, 10 μm. (c) Immunoblot analysis (as in Fig. 5d) of HEK293 cells transfected for 24 h with wild-type IRF3 (WT) or mutant IRF3 with S97A substitution, then left uninfected (–) or infected (+) for 8 h with SeV. (d) Immunoblot analysis of HEK293 cells transfected and infected as in c, followed by nucleus-cytoplasm extraction (and SDS-PAGE of the extracts). (e, f) Luciferase assay (as in Fig. 1b) (top) and immunoblot analysis (below) in Irf3−/−Irf7−/− MEFs transfected for 24 h with luciferase reporter plasmid for IFNB1 (IFN-β-Luc; e) or IFNA4 (IFN-α4; f) promoter, plus plasmid encoding wild-type IRF7 (WT) or mutant IRF7 with the S112A or S112D substitution (top), then left uninfected (UI) or infected for 8 h with SeV. (g) Luciferase assay (as in Fig. 1b) (top) and immunoblot analysis (below) in Pten+/+ MEFs and Pten−/− MEFs transfected for 24 h with luciferase reporter plasmid for IFNB1 (IFN-β-Luc), plus plasmid encoding wild-type IRF7 (WT) or mutant IRF7 with the S112A or S112D substitution. **p < 0.01 (two-way ANOVA with post hoc Bonferroni t-test (g)). Data are from three independent experiments (a (top), e-g (top); mean and s.d. of three independent biological replicates per group in each) or are representative of three independent experiments (a (below lanes), c, d, e-g (bottom)).

Supplementary Figure 6 Identification of phosphorylation of IRF3 at Ser97 in vivo.

(a) Schematic presentation of IRF3 domain and amino acid sequence with phosphorylation sites identified by mass spectrometry highlighted in red. (b) Tandem mass spectrometry analysis of Ser97-phosphorylated peptide EGLRLAEDRpSK. The observation of fragment ions in the CID product ion spectra was used to localize the phosphorylation site. The presence of the intensive signal at m/z (mass to charge ratio) 628.34 (doubly charged) corresponding to the neutral loss of phosphoric acid (98 Th, doubly charged) due to gas-phase β-elimination from the doubly charged parent ion at m/z 677.34 indicated that the peptide was serine (or threonine)-phosphorylated. The difference of 167 Th between b92+ and b102+ at m/z 520.78 and 604.28 respectively, which corresponded to a phosphoserine residue, was observed in the spectrum. This detection established the phosphorylation site as Ser97 in the sequence. (c, d) The phosphorylation sites stoichiometry (phosphorylation intensity versus total intensity) (c) and Commassie blue staining (d) of recombinant IRF3 (fused with two tags SBP (streptavidin-binding peptide) and CBP (calmodulin-binding peptide)) expressed in 293 cells, purified and treated with GST or GST-PTEN, and then separated by SDS-PAGE and mass spectrometry analysis. (e) ELISA of rabbit immune serum (1), antibody flow-through (2), anti-phospho-Ser97 antibody (3) or anti-nonphospho-Ser97 antibody (4), diluted from 1:500 to 1:106 (right margin), with Ser97 phospho-peptide (left half) and its relative non-phospho peptide (right half) coated as an ELISA plate. (f) Immunoblot analysis of IRF3 with anti-phospho-Ser97 antibody, plus Ser97-phosphorylated peptide (first row) or its relative non-phosphorylation peptide (second row) in Irf3−/−Irf7−/− MEFs transfected with empty vector (EV), wild-type IRF3 (WT) and mutant IRF3 with the S97A substitution (top); below (input), immunoblot analysis with antibody to IRF3 and β-actin. (g) Immunoblot analysis of PC-3 cells infected for 0-8 h with SeV, followed by nucleus-cytoplasm extraction (5% of cytoplasmic extracts and 10% of nuclear extracts separated by SDS-PAGE). Data are representative of three independent experiments (a-g).

Supplementary Figure 7 In vitro and in vivo phosphatase activity of PTEN.

(a) Commassie blue staining of recombinant GST-tagged wild-type PTEN, VHR, PTP1B, Cdc25A and mutant PTEN with the C124S, G129E or G129R substitution expressed in E. coli BL21 (DE3) cells and purified. (b, c) Lipid phosphatase activity assay and serine or threonine phosphatase activity assay of the recombinant proteins as in a, incubated for 2 h with PtdIns-(3,4,5)-P3 (b) or Ser or Thr-phosphorylated peptide (c). (d) In vitro phosphatase assay of GST, recombinant wild-type PTEN, mutant PTEN with the C124S substitution, CIP (Calf intestinal alkaline phosphatase) and λ-PPase (Lambda protein phosphatase) (positive control), incubated for 1 h with recombinant Flag-tagged IRF3 (expressed in HEK293 cells and immunoprecipitated with antibody to Flag). Numbers under lanes indicate IRF3 Ser97 phosphorylation band intensity, normalized to that of total IRF3. (e, f) In vitro phosphatase assay of GST, recombinant wild-type PTEN and CIP, incubated for 1 h with the cell lysates of PC-3 cells left uninfected (–) or infected for 6 h with SeV (+). (g, h) Immunoblot analysis of IRF3 phosphorylated at Ser97 (p-IRF3(S97)) and total IRF3, PTEN and GAPDH (g) and ELISA of IFN-β (h) in PC-3 cells stably expressing empty vector (EV) or plasmid encoding wild-type PTEN and infected for 0-12 h with SeV. Data are from three independent experiments (b, c, h; mean and s.d. of three independent biological replicates per group in each) or are representative of three independent experiments (a, d-g).

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Li, S., Zhu, M., Pan, R. et al. The tumor suppressor PTEN has a critical role in antiviral innate immunity. Nat Immunol 17, 241–249 (2016). https://doi.org/10.1038/ni.3311

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