The signaling adaptor TRAF3 is a highly versatile regulator of both innate immunity and adaptive immunity, but how its phosphorylation is regulated is still unknown. Here we report that deficiency in or inhibition of the conserved serine-threonine kinase CK1ɛ suppressed the production of type I interferon in response to viral infection. CK1ɛ interacted with and phosphorylated TRAF3 at Ser349, which thereby promoted the Lys63 (K63)-linked ubiquitination of TRAF3 and subsequent recruitment of the kinase TBK1 to TRAF3. Consequently, CK1ɛ-deficient mice were more susceptible to viral infection. Our findings establish CK1ɛ as a regulator of antiviral innate immune responses and indicate a novel mechanism of immunoregulation that involves CK1ɛ-mediated phosphorylation of TRAF3.
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We thank B. Zhao (Zhejiang University) for CK1ɛ and CK1ɛ(K38R) constructs, J. Yang (Tongji University) for HA-Ub(K63) and HA-Ub(K48) constructs, X. Cao (Zhejiang University) for HSV-1 and VSV; F. Shao (National Institute of Biological Sciences, Beijing) for immortalized BMDMs; G. Chen (University of California) for Traf3−/− MEFs; X. Wang (National Institute of Biological Sciences, Beijing) for mit-RFP; and Abclonal Biotech for assistance with the antibody to phosphorylated TRAF3. Supported by the National Basic Research Program of China (973 Programs 2012CB578100 and 2011CB505000) and the National Natural Science Foundation of China (projects 81330069 and 31030028).
The authors declare no competing financial interests.
Integrated supplementary information
(a) HEK293T cells were cultured in 384 wells and transiently transfected with 50 ng of the IFN-β promoter-Luc construct along with 5 ng TK-Luc construct for 12 hours. Various compounds were then added to culture media for 12 hours and luciferase activity was detected. Sample with a low TK activity (smaller than half of control) was considered to be cytotoxic and was ruled out. (b-i) Q-PCR analysis of DMSO- or IC261 (10 μM)-treated primary peritoneal macrophages stimulated by VSV (12h) and poly(I:C) (3h). (j-l) Q-PCR analysis of DMSO- or IC261 (10 μM)-treated primary peritoneal macrophages stimulated by VSV. (m) IB analysis of p52 in lysates of DMSO- or IC261-treated primary peritoneal macrophages infected with VSV. (n) Primary peritoneal macrophages cells were pretreated with DMSO or IC261 (10 μM) for 60 minutes, and then infected with VSV (MOI=0.01). The mRNA level of VSV was examined by Q-PCR analysis. (o) HeLa cells were pretreated with DMSO or IC261 (10 μM) for 60 minutes, and then infected with HSV (MOI=0.001). The mRNA level of HSV was examined by Q-PCR analysis. NS, not significant (P > 0.05); *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired t-test (a-h)). Data are from three independent experiments with biological duplicates in each (b-o; mean and s.e.m. of n = 3). Data are representative of three independent experiments (m).
(a-h) Q-PCR analysis of WT and Csnk1e−/− primary peritoneal macrophages infected by VSV (12h) and poly(I:C) (3h). (i-k) Q-PCR analysis of WT and Csnk1e−/− primary peritoneal macrophages infected by VSV. (l) IB analysis of IRF3 phosphorylation and p52 abundance in lysates of WT and Csnk1e−/− primary peritoneal macrophages infected with VSV. (m, n) WT and Csnk1e−/− primary peritoneal macrophages cells were stimulated with poly(I:C) or LPS, and then IRF3 phosphorylation was examined by western blot. (o-q) Densitometry quantification of IRF3 phosphorylation in supplementary Fig 2l-m. (r-t) Primary peritoneal macrophages were infected with WNV (MOI=1) (r, t) or VSV (MOI=0.01) (s). The WNV-E protein level (r) or mRNA level of VSV or WNV (s, t) and the WNV-E protein level (r) were examined. (u) Q-PCR analysis of VSV-infected Csnk1e+/+ and Csnk1e−/− macrophages treated as indicated. NS, not significant (P > 0.05); *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired t-test (a-k, s-u)). Data are from three independent experiments with biological duplicates in each (a-k, s-u; mean and s.e.m. of n = 3). Data are representative of three independent experiments (l-n, r).
(a) IP and IB of cell lysates from HEK293T cells expressing HA-CK1ɛ and Flag-TRAF1-6 with antibodies against HA or Flag. (b) IP and IB of cell lysates from HEK293T cells expressing indicated constructs. (c) In vitro GST precipitation assay using different purified histidine (His)-tagged CK1ɛ deletion constructs combined with GST-TRAF3. (d) In vitro GST precipitation assay using purified histidine-tagged CK1ɛ combined with GST-MAVS or GST-TRAF3. (e) IB of mitochondria or whole cell lysate (WCL) from BMDM infected with VSV. (f-h) Confocal microscopy of MEF cells (f, g) or iBMDM (h) infected with VSV for indicated hours. Data are representative of three independent experiments (a-h).
Dot blot analysis of anti-pSer349, TS349-p: Ser349 phosphorylated peptide, TS349-c: Ser349 non-phosphorylated peptide. Data are one representative from two experiments.
Traf3 −/− MEFs were transfected with indicated plasmids, and then infected with VSV for indicated times. Cell lysates were subjected to IB analysis. Data is a representative of the same experiment sample as shown in Fig. 5g, 5h.
(a) HEK293T cells transfected with indicated plasmids were treated with IC261, and lysates and anti-TRAF3 IP were subjected to IB analysis as indicated. (b) IB of lysates and anti-Flag IP from HEK293T cells transfected with indicated plasmids. (c) Traf3 −/− MEFs were transfected with indicated plasmids, and then infected with VSV for indicated times. Cell lysates or anti-TRAF3 IP were subjected to IB analysis. (d, e) IB of cell lysates and anti-TRAF3 IP of Csnk1e+/+ or Csnk1e−/− primary peritoneal macrophages infected with VSV (d) or stimulated with LPS (e) for the indicated times. Data are representative of three independent experiments (a-e).
(a-c) IBA1 staining for macrophage (a), MPO staining for neutrophil (b) and CD3 staining for lymphocyte (c) in the central nervous system (CNS) of WT or Csnk1e−/− mice infected with WNV for 7 days. Data are representative of three independent experiments (a-c).
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Zhou, Y., He, C., Yan, D. et al. The kinase CK1ɛ controls the antiviral immune response by phosphorylating the signaling adaptor TRAF3. Nat Immunol 17, 397–405 (2016). https://doi.org/10.1038/ni.3395
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