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. 2021 Jan 21;24(2):102078.
doi: 10.1016/j.isci.2021.102078. eCollection 2021 Feb 19.

The tyrosine kinase c-Abl potentiates interferon-mediated antiviral immunity by STAT1 phosphorylation

Hainan Liu  1 , Yan Cui  1   2 , Yu Bai  3 , Yi Fang  4 , Ting Gao  1 , Guangfei Wang  1 , Lin Zhu  1 , Qincai Dong  1 , Shuwei Zhang  1 , Yi Yao  1 , Caiwei Song  1 , Xiayang Niu  3 , Yanwen Jin  1 , Ping Li  1 , Cheng Cao  1 , Xuan Liu  1
Affiliations

The tyrosine kinase c-Abl potentiates interferon-mediated antiviral immunity by STAT1 phosphorylation

Hainan Liu et al. iScience. .

Abstract

Interferon (IFN)-induced activation of the signal transducer and activator of transcription (STAT) family is an important event in antiviral immunity. Here, we show that the nonreceptor kinases c-Abl and Arg directly interact with STAT1 and potentiate the phosphorylation of STAT1 on Y701. c-Abl/Arg could mediate STAT1 phosphorylation independent of Janus kinases in the absence of IFNγ and potentiate IFNγ-mediated STAT1 phosphorylation. Moreover, STAT1 dimerization, nuclear translocation, and downstream gene transcription are regulated by c-Abl/Arg. c-Abl/Arg (abl1/abl2) deficiency significantly suppresses antiviral responses in vesicular stomatitis virus-infected cells. Compared to vehicle, administration of the c-Abl/Arg selective inhibitor AMN107 resulted in significantly increased mortality in mice infected with human influenza virus. Our study demonstrates that c-Abl plays an essential role in the STAT1 activation signaling pathway and provides an important approach for antiviral immunity regulation.

Keywords: Biological Sciences; Viral Microbiology; Virology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
c-Abl regulates the promoters of IFN-responsive genes via GAS elements (A) The differentially expressed genes were identified by RNA sequencing in c-abl/arg double-knockout (DKO) and WT MEFs. Left, the volcano plot of the c-Abl-regulated differentially expressed genes with >4-fold change. Right, the downregulation of IFN-related genes in DKO MEFs compared with that in WT MEFs. c-abl/arg double-knockout efficiency was detected in Figure S3A. (B) The transcription levels of the indicated genes were determined via quantitative RT-PCR. The data were normalized to the value of psma5 to exclude nonspecific regulation due to adenoviral infection. (C) A schematic diagram shows that the psmb9 and tap1 genes are transcribed from a shared bidirectional promoter and regulated by IFNγ. (D) The transcription of tap1 and psmb9 genes was detected by luciferase reporter constructs in RAW264.7 cells with or without AMN107 treatment. All quantitative data are shown as the mean ± SD of three independent experiments (unpaired Student's t-test). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Figure 2
Figure 2
c-Abl interacts with STAT1 (A) Lysates from MCF-7 cells were subjected to immunoprecipitation with anti-c-Abl or normal rabbit IgG as a control and subsequently analyzed by immunoblotting with the indicated antibodies. (B) 293T cells were transfected with the indicated plasmids. Anti-Flag or IgG immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. (C) Lysates from 293T cells transfected with the Flag-c-Abl expression plasmid were incubated with GST or GST-STAT1 fusion protein-conjugated agarose beads. The adsorbates were analyzed by immunoblotting and Coomassie blue staining. (D) Anti-Flag or IgG immunoprecipitates prepared from 293T cells transfected with the indicated plasmids were subjected to SDS-PAGE and transferred onto a PVDF membrane. The PVDF membrane was incubated with soluble GST-STAT1 or GST only and then detected with an anti-GST antibody. (E) Upper panel, schematic diagram of the STAT1 domain. Lower panel, 293T cells expressing Flag-c-Abl were incubated with the indicated GST-STAT1 truncated mutants or GST-conjugated agarose beads. The adsorbates were analyzed by anti-Flag immunoblotting. (F) The interaction between Flag-STAT1 and GST-Abl SH3, GST-Abl SH2, and GST only was detected by far-Western blotting as described in (D). (G) MEFs were stimulated with IFNγ at different concentrations for 3 hr. The complexes of endogenous c-Abl and STAT1 were detected by in situ PLA using anti-c-Abl and anti-STAT1 antibodies. PLA signals were shown in red, and nuclei were stained with DAPI (blue). Scale bars, 10 μm.
Figure 3
Figure 3
STAT1 is phosphorylated by c-Abl (A) The immunoprecipitates prepared from 293T cells expressing His-STAT1 or His only were incubated with 10 ng of human Abl protein (Merck, 14–529) in the presence of 0.2 mM ATP. The reaction products were analyzed with the indicated antibodies. (B and C) Anti-Flag or IgG immunoprecipitates prepared from 293T cells transfected with the indicated plasmids were analyzed by immunoblotting. (D and E) Lysates of MCF-7 cells subjected to the indicated treatment were analyzed by immunoblotting. (F) Lysates from JAK2-deficient or wild-type MCF-7 cells expressing the indicated plasmids were analyzed by immunoblotting. (G) Wild-type or c-abl/arg-knockout MEFs were stimulated with IFNγ at 40 ng/mL for the indicated time (left) or with IFNγ at the indicated concentrations for 120 min (right). Lysates were analyzed by immunoblotting.
Figure 4
Figure 4
c-Abl promotes STAT1 dimer formation and nuclear import (A) Anti-Flag or IgG immunoprecipitates prepared from 293T cells transfected with the indicated plasmids were analyzed by immunoblotting. (B) In situ cellular localization of endogenous STAT1 (green) in wild-type and c-abl/arg-knockout MCF-7 cells was detected by anti-STAT1 immunofluorescence microscopy. Nuclei were stained with DAPI (blue). c-abl/arg double-knockout efficiency was detected in Figure S3B. (C) The nuclear level of STAT1 in Figure 4B was calculated by the ratio of fluorescence intensity in the nucleus to that in the cytoplasm in the same cell. At least 15 cells were calculated, and the results are expressed as the mean ± SD. (D) Nuclear extracts isolated from 293T cells transfected with the indicated plasmids were incubated with a biotin-tagged nucleic acid probe containing the IRF1 promoter sequence, resolved via native PAGE, and analyzed with ECL. A 1000-fold molar excess of unlabeled oligonucleotide was used as a DNA-binding competitor. The efficiency of nuclear extract isolation was shown in Figure S5B. All quantitative data are shown as the mean ± SD of three independent experiments (unpaired Student's t-test). ∗∗p < 0.01.
Figure 5
Figure 5
c-Abl regulates IFN-related gene expression The mRNA transcript level of IFN-induced genes was analyzed by RT-PCR. IFN-induced gene expression was measured by the ratio of specific gene transcription with IFN treatment to that without IFN treatment. (A) Wild-type or c-abl/arg-knockout MCF-7 cells were treated with 40 ng/mL IFNγ for 6 hr (B and C) MCF-7 cells were treated with or without 5 μM AMN107 for 24 hr and then subjected to 40 ng/mL IFNγ (B) or 1000 U/ml IFNα (C) stimulation for 6 hr. All quantitative data are shown as the mean ± SD of three technical experiments (unpaired Student's t-test). ∗∗p < 0.01.
Figure 6
Figure 6
c-Abl promotes IFN-dependent antiviral effects (A) Antigen presentation by JAWS II dendritic cells was analyzed by B3Z-secreted IL2 levels. (B) Wild-type and c-abl/arg double-knockout MEFs infected with Ad-Vector or Ad-Abl or treated with or without 5 μM AMN107 were infected with VSV (MOI = 10) for 48 hr. Cell survival was then analyzed by MTS assay. (C) A549 cells were treated with 5 μM AMN107 for 18 hr or transfected with Flag-c-Abl for 24 hr and samples treated with DMSO or empty vector served as controls. Then, the cells were infected with NDV-GFP (MOI = 1) for 24 hr, and GFP fluorescence was quantified by flow cytometry analysis (left) and is shown as the geometric mean ± SD of three independent measurements (right). (D) AMN107 or ruxolitinib-treated A549 cells were stimulated with IFNγ for 24 hr. Then, the cells were infected with NDV-GFP for 24 hr, and GFP fluorescence was analyzed by FACS. (E) Survival analysis of mice following H1N1 infection. 8- to 10-week-old WT mice and c-abl-cKO mice (10 in each group) intravenously treated with AMN107 or saline for 12 days were intranasally infected with H1N1 at 0.1 LD50, and mortality was monitored. All quantitative data are shown as the mean ± SD of three independent experiments (unpaired Student's t-test or log rank test). ∗∗p < 0.01.
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