PTK2B promotes TBK1 and STING oligomerization and enhances the STING-TBK1 signaling

Abstract

TANK-binding kinase 1 (TBK1) is a key kinase in regulating antiviral innate immune responses. While the oligomerization of TBK1 is critical for its full activation, the molecular mechanism of how TBK1 forms oligomers remains unclear. Here, we show that protein tyrosine kinase 2 beta (PTK2B) acts as a TBK1-interacting protein and regulates TBK1 oligomerization. Functional assays reveal that PTK2B depletion reduces antiviral signaling in mouse embryonic fibroblasts, macrophages and dendritic cells, and genetic experiments show that Ptk2b-deficient mice are more susceptible to viral infection than control mice. Mechanistically, we demonstrate that PTK2B directly phosphorylates residue Tyr591 of TBK1, which increases TBK1 oligomerization and activation. In addition, we find that PTK2B also interacts with the stimulator of interferon genes (STING) and can promote its oligomerization in a kinase-independent manner. Collectively, PTK2B enhances the oligomerization of TBK1 and STING via different mechanisms, subsequently regulating STING-TBK1 activation to ensure efficient antiviral innate immune responses.

Fig. 1. PTK2B interacts with TBK1 and STING.

a HEK293T cells were transfected with Myc-tagged PTK2B and Flag-tagged TBK1 or empty vector and lysed 24 h after transfection for co-immunoprecipitation (Co-IP) with anti-Flag M2 beads, and then the pulled-down proteins were analyzed by immunoblotting. b HEK293T cells were transfected with the indicated expression plasmids. Co-IP assays were performed with anti-Flag M2 beads and analyzed by immunoblotting. c RAW 264.7 cells were mock infected or infected with HSV1-GFP for the indicated times. The cell lysates were immunoprecipitated with anti-PTK2B antibody or control IgG and analyzed by immunoblotting. d Immortalized mouse embryonic fibroblasts (MEFs) were mock infected or infected with HSV-1 for 6 h. Cells were then fixed, stained with PTK2B (green) and TBK1 (red) antibodies, then imaged by confocal microscopy (left). Scale bars, 10 μm. Relative co-localization of PTK2B and TBK1 was quantified with Pearson’s correlation coefficient by using NIKON NIS-Elements Analysis software (right), the cells from mock infected (n = 104) or infected (n = 110) group were analyzed. e A mixture of purified GST-PTK2B and His-TBK1 (residues 1–657) or His-GFP, expressed in E. coli, was pulled down with Ni-Sepharose beads, and then analyzed by immunoblotting. f A mixture of purified His-STING (residues 153–379) and GST-PTK2B or GST-GFP, expressed in E. coli, was pulled down with Glutathione-Sepharose beads, and then analyzed by immunoblotting. g Schematic diagram of TBK1 domains (top). HEK293T cells were co-transfected with PTK2B-Myc and TBK1-Flag or its truncated mutants as indicated. Co-IP assays were performed with anti-Flag M2 beads and the pulled-down proteins were analyzed by immunoblotting (bottom). h Schematic diagram of PTK2B domains (top). HEK293T cells were co-transfected with TBK1-HA and PTK2B-Flag or its truncated mutants as indicated. Co-IP assays were performed with anti-Flag M2 beads and the pulled-down proteins were analyzed by immunoblotting (bottom). Data shown in (a–c, e–h) are from one representative of two independent experiments with similar results. Data shown in (d) are from one representative experiment of three independent experiments (mean ± SD), two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 2. PTK2B plays a positive role in regulating antiviral signaling.

a–d THP-1 cells were infected with short hairpin RNA (shRNA) lentivirus targeting two different regions of PTK2B (shPTK2B-1, shPTK2B-2) or negative control (shCon), followed by infection with HSV1-GFP for 6 h. Quantitative PCR (qPCR) assays were performed to measure the mRNA levels of IFNB1 (a), IFIT1 (b), CXCL10 (c) and PTK2B (d). e THP-1 cells stably expressing shRNA targeting PTK2B or control cells were infected with HSV1-GFP for the indicated times, followed by immunoblotting. f–h Immortalized mouse embryonic fibroblasts (MEFs) stably expressing PTK2B or control cells were infected with HSV1-GFP for 3 and 6 h. Ifnb1 (f), Ifit1 (g) and Cxcl10 (h) mRNA levels were measured by qPCR. i–k Ptk2b^+/+ and Ptk2b^−/− RAW 264.7 cells were infected with HSV1-GFP for 3 and 6 h and then analyzed by qPCR to quantify Ifnb1 (i), Ifit1 (j) and Cxcl10 (k) mRNA levels. l Ptk2b^+/+ and Ptk2b^−/− RAW 264.7 cells were infected with HSV1-GFP for the indicated times and then analyzed by immunoblotting with the indicated antibodies. m Ptk2b^+/+ and Ptk2b^−/− RAW 264.7 cells were infected with HSV1-GFP for 24 h, or VSVΔM51-GFP for 12 h. The cells were imaged by fluorescence microscopy (left, Scale bars, 200 μm.), or the culture supernatants were harvested to quantify the viral titer using a plaque assay (right). Data shown in (a–d, f–k, m) are from one representative experiment of three independent experiments (mean ± SD, n = 3 independent samples), two-tailed Student’s t-test. Data shown in (e, l) are one representative of two independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 3. PTK2B deficiency reduces innate immune responses to virus infection in vivo.

a–c Primary Ptk2b^+/+ and Ptk2b^−/− MEFs were infected with HSV1-GFP for the indicated times and then lysed for the quantification of Ifnb1 (a), Ifit1 (b) and Cxcl10 (c) mRNA levels by qPCR. d Primary Ptk2b^+/+ and Ptk2b^−/− MEFs were infected with HSV1-GFP for the indicated times and then analyzed by immunoblotting. e Primary Ptk2b^+/+ and Ptk2b^−/− MEFs were infected with lentivirus expressing wild-type PTK2B, PTK2B(K457R) or empty vector, and then infected with HSV1-GFP for 6 h. The cells were harvested for qPCR to measure the mRNA levels of Ifnb1 (top) or for immunoblotting with the indicated antibodies (bottom). f–h Ptk2b^+/+ and Ptk2b^−/− bone marrow-derived macrophages (BMDMs) were infected with HSV1-GFP for the indicated times, followed by qPCR to measure the mRNA levels of Ifnb1 (f), Ifit1 (g) and Cxcl10 (h). i 12-week-old Ptk2b^+/+ and Ptk2b^−/− mice were infected with HSV-1 via tail vein injection at 4 × 10⁷ pfu per mouse. Sera were collected at 4 and 8 h of infection to measure IFNβ levels by ELISA. n = 5 mice for each group. j 12-week-old Ptk2b^+/+ and Ptk2b^−/− mice were infected with HSV-1 via tail vein injection at 4 × 10⁷ pfu per mouse. Sera were collected at 24 h of infection to measure viral titers using a plaque assay. n = 6 mice for each group. k 12-week-old Ptk2b^+/+ (n = 10) and Ptk2b^−/− (n = 7) mice were infected with HSV-1 via tail vein injection at 4 × 10⁷ pfu per mouse and the survival of mice was monitored for 15 days. l 12-week-old Ptk2b^+/+ and Ptk2b^−/− mice were infected with HSV-1 via tail vein injection at 3 × 10⁷ pfu per mouse for 4 days. Sections of lung from infected mice were analyzed using hematoxylin and eosin staining. Scale bars, 100 μm. Data shown in (a–c, e–h) are from one representative experiment of three independent experiments (mean ± SD, n = 3 independent samples), two-tailed Student’s t-test. Data shown in (d, i, j, l) are one representative of two independent experiments with similar results. The log-rank (Mantel–Cox) test was used in Data (k). Source data are provided as a Source Data file.

Fig. 4. PTK2B augments TBK1 activation by mediating tyrosine phosphorylation.

a HEK293T cells were transfected with the indicated plasmids and lysed 24 h after transfection for immunoblotting. b HEK293T cells were co-transfected with Flag-tagged TBK1 and Myc-tagged PTK2B or PTK2B-K457R (a kinase-inactive variant). IP assays were performed with anti-Flag M2 beads and analyzed by immunoblotting. c Purified His-TBK1 protein (residues 1–657) was incubated with GST-PTK2B-KD or GST-PTK2B-KD (K457R) or GST-GFP for 30 min in kinase buffer, and then analyzed by immunoblotting. d RAW 264.7 cells were mock infected or infected with HSV1-GFP for the indicated times, followed by immunoblotting. e Ptk2b^+/+ and Ptk2b^−/− RAW 264.7 cells were mock infected or infected with HSV1-GFP for the indicated times. The cell lysates were immunoprecipitated with anti-TBK1 antibody and analyzed by immunoblotting. f HEK293T cells were co-transfected with Myc-tagged PTK2B and Flag-tagged TBK1 or its point mutants. IP assays were performed with anti-Flag M2 beads and pulled-down proteins were analyzed by immunoblotting. g HEK293T cells were co-transfected with Myc-tagged PTK2B and Flag-tagged TBK1 or TBK1-Y591F variant. IP assays were performed with anti-Flag M2 beads and pulled-down proteins were analyzed by immunoblotting. h Ptk2b^+/+ and Ptk2b^−/− RAW 264.7 cells were mock infected or infected with HSV1-GFP for 6 and 9 h. The cell lysates were immunoprecipitated with anti-TBK1 antibody and analyzed by immunoblotting. i, j HEK293T cells were co-transfected with the indicated plasmids and lysed 24 h after transfection for immunoblotting. Data shown in (a–j) are one representative of two independent experiments with similar results. Source data are provided as a Source Data file.

Fig. 5. PTK2B increases TBK1 oligomerization in a kinase-dependent manner.

a HEK293T cells were co-transfected with the indicated plasmids. Cell lysates were treated with or without 50 mM dithiothreitol (DTT) and then resolved by semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting. b Purified His-TBK1 protein (residues 1–657) was incubated with GST-PTK2B-KD or GST-PTK2B-KD (K457R) or GST-GFP for 30 min, and then resolved by SDD-AGE or SDS-PAGE, followed by immunoblotting. c Ptk2b^+/+ and Ptk2b^−/− RAW 264.7 cells were mock infected or infected with HSV1-GFP for 6 h. The cell lysates were resolved by Native-PAGE or SDS-PAGE, followed by immunoblotting. d, e Primary Ptk2b^+/+ and Ptk2b^−/− MEFs were stimulated by HSV1-GFP infection for 6 h (d) or poly(I:C) transfection for 2 h (e), stained with TBK1 antibody and 4′,6-diamidino-2-phenylindole (DAPI), and then imaged by confocal microscopy (left). Scale bars in (d) 20 μm and in (e) 10 μm. The percentage of cells with TBK1 foci was quantified (right, n = 108 cells in (d) and n = 103 cells in (e) for each group). f HEK293T cells were co-transfected with the indicated plasmids. Cell lysates were resolved by SDD-AGE and SDS-PAGE, followed by immunoblotting with the indicated antibodies. g Similar to (f), except that the indicated expression plasmids were transfected. h Immortalized MEFs were co-expressed with PTK2B-GA and PTK2B-GB, followed by stimulation with or without HSV-1 for 6 h. The cells were fixed and stained with DAPI (blue), antibody to TBK1 (red) and GM130 (Golgi marker, purple), then imaged by confocal microscopy. Scale bars, 10 μm. Data shown in (a–c, f–h) are one representative of two independent experiments with similar results. Data shown in (d, e) are from one representative experiment of three independent experiments (mean ± SD), two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 6. PTK2B can enhance STING oligomerization in a kinase-independent manner.

a HEK293T cells were co-transfected with Flag-STING and Myc-PTK2B or empty vector. Cell lysates were incubated in the presence or absence of 50 mM DTT for 30 min, and then resolved by SDD-AGE and SDS-PAGE, followed by immunoblotting. b Purified His-STING protein (residues 153–379) was incubated with GST-PTK2B or GST-GFP in the presence or absence of 50 mM DTT for 30 min, and then resolved by SDD-AGE or SDS-PAGE, followed by immunoblotting. c PTK2B^+/+ and PTK2B^−/− THP1 cells were infected with HSV1-GFP for 10 h. Cell lysates were resolved by SDD-AGE and SDS-PAGE, followed by immunoblotting. d PTK2B^+/+ and PTK2B^−/− THP1 cells stably expressing GFP-STING were infected with HSV-1 infection for 10 h and then imaged by confocal microscopy (left). Scale bars, 5 μm. The percentage of cells with STING foci was quantified (right, n = 102 cells for each group). e PTK2B^+/+ and PTK2B^−/− THP1 cells were stimulated with cGAMP (1 μM) for 1.5 h, then imaged by confocal microscopy (left). Scale bars, 5 μm. The percentage of cells with STING foci was quantified (right, n = 105 cells for each group). f HEK293T cells were co-transfected with Flag-tagged STING and Myc-tagged PTK2B, PTK2B-K457R variant, or empty vector. Cell lysates were immunoprecipitated with anti-Flag M2 beads, and then the pulled-down proteins were analyzed by immunoblotting. g Similar to (a), except that PTK2B-K457R expression plasmid was included. h HeLa cells were co-transfected with Flag-tagged STING and Myc-tagged PTK2B, PTK2B-K457R, or empty vector. Twenty-four hours after transfection, the cells were stained with Flag and Myc antibodies and then imaged by confocal microscopy (left). Scale bars, 10 μm. The percentage of cells with STING foci was quantified (right, n = 103 cells for each group). Data shown in (a–c, f, g) are one representative of two independent experiments with similar results. Data shown in (d, e, h) are from one representative experiment of three independent experiments (mean ± SD), n.s. not significant, two-tailed Student’s t-test. Source data are provided as a Source Data file.