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. 2021 Sep 27;24(11):103177. doi: 10.1016/j.isci.2021.103177

Figure 5.

Figure 5

IRF3 S386 phosphorylation by mTOR is for antiviral genes expression

(A) The mTOR stable HeLa cell line and control cell line were treated with or without 5 nM Rapamycin for 24 h, then the cells infected with VSV, the infected cells were analyzed by flow cytometry. Left, representative image; right, statistical results of 3 independent experiments. Data are represented as mean ± SD; ∗∗, p < 0.01, two-tailed t test.

(B) HEK293T cells were transfected with or without IRF3 as indicated followed by AA treatment or FBS treatment. RT-PCR was performed to analyze IFNα mRNA expression level. Data are represented as mean ± SD; ∗∗∗∗, p < 0.0001, one-way ANOVA.

(C) IRF3 protein S386 phosphorylation identified with mass spectrometry. IRF3 was purified from HEK293T cells transfected with IRF3 along with mTOR.

(D) IRF3 S385S386 motif was aligned with T36T37 and T45T46 motifs of 4EBP.

(E) WCLs were prepared from HeLa cells receiving AA treatment for indicated times. IRF3 phosphorylation was then analyzed in Western blot with anti-IRF3-pS386.

(F) WCLs were prepared from mouse fibroblasts receiving FBS treatment for indicated times. Anti-IRF3-pS386, anti-4EBP1-pS65, and anti-S6K1-pT389 were applied in Western blot.

(G) Modi-TOF analysis of S386 peptide (ARVGGASSLENTVDLHI) phosphorylation by mTOR in vitro.

(H) Western blot analysis revealed that while Rheb elevated mTOR in IRF3 phosphorylation induction, FKBP12 attenuated its activity in HEK293T cells. VSV infection induced IRF3 phosphorylation on S386 in fibroblasts.

(I) GFP-IRF3 was cotransfected with EV, mTOR, or mTOR + FKBP12 in Hela cells. GFP and DAPI staining were visualized with confocal fluorescent microscope. mTOR overexpression caused IRF3 nuclear translocation whereas FKBP12 cotransfecteion with mTOR blocked IRF3 nuclear translocation. Scale bar: 10 μm.

(J) In Hela cells, EV, Flag-mTOR, or Flag-Rheb was transiently transfected followed by rapamycin treatment, Upper panel was immune-stained with anti-IRF3-pS386. The lower panel was an overlay of DAPI staining and anti-Flag immune-staining. Scale bar: 20 μm.