Fig 7. Autophosphorylation of BLK at Y309 is critical for its regulation of IRF3-dependent antiviral signaling.

(A) BLK undergoes tyrosine phosphorylation upon SeV infection. U87MG cells stably expressing BLK-Flag (2 × 10⁷) were uninfected or infected with SeV (MOI, 1) for the indicated times. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (B) BLK catalyzes autophosphorylation in a kinase activity-dependent manner. Purified Flag-tagged BLK and BLK(K269A) coupled with Protein G sepharose beads were subjected to in vitro BLK autophosphorylation assays. The detailed procedures are shown in the Materials and Methods. “Vector” here means the control Protein G sepharose beads. (C) Identification of potential autophosphorylation sites of BLK by mass spectrometry. The in vitro phosphorylated BLK in Fig 7B was subjected to mass spectrometry. The list shows the phosphorylated peptide sequences and phosphorylation (STY) probabilities of BLK analyzed by mass spectrometry. (D) BLK catalyzes autophosphorylation mainly at Y309. Purified Flag-tagged BLK and its mutants coupled with Protein G sepharose beads were subjected to in vitro BLK autophosphorylation assays. (E) BLK(Y309F) fails to phosphorylate IRF3. HEK293 cells (2 × 10⁶) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. (F) Effects of BLK or its mutant recovery on SeV-induced transcription of downstream antiviral genes. BLK-deficient and control U87MG cells (2 × 10⁵) were transfected with the same amount of BLK or its mutants for 24 h. Cells were then uninfected or infected with SeV (MOI, 1) for 8 h before qPCR analysis. The lower blot shows the expression levels of BLK and its mutants as detected by anti-Flag antibody. (G) Sequence alignment of BLK from the indicated species. The sequences correspond to aa293-312 of human BLK. The conserved tyrosine residues are highlighted in black. (H) Effects of Blk or Blk(Y303F) recovery on VSV-induced transcription of downstream antiviral genes. Wild-type and Blk-deficient A20 cells were transduced with empty vector (EV), Blk or Blk(Y303F) plasmids by lentivirus-mediated gene transfer to establish the stable cell lines with blasticidin S (10 μg/mL) selection. The indicated cell lines (2 × 10⁵) were then uninfected or infected with VSV (MOI, 1) for 8 h before qPCR analysis. The lower blot shows the expression levels of Blk and Blk(Y303F) in the indicated cell lines as detected by anti-Flag antibody. Graphs show mean ± SD (n = 2 technical replicates in F and H) from one representative experiment. Data are representative of at least three independent experiments with similar results. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (unpaired, two-tailed Student’s t-test).