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

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.

. Identification of TBK1-interacting proteins by Co-IP coupled with mass spectrometry analysis TBK1 was purified using S-protein agarose beads from HEK293T cells expressing S protein-Flag-Streptavidin binding peptide (SFB)-tagged mouse TBK1 or control vector cells, and then the protein was incubated with cell lysates from RAW 264.7 cells, followed by Co-IP assays and mass spectrometry analysis.A couple of the selected TBK1-interacting proteins identified by MS analysis were listed.(d, e) THP1 cells were infected with shRNA lentivirus targeting two different regions of PTK2B(shPTK2B-1, shPTK2B-2) or negative control(shCon), followed by infection with VSVΔM51-GFP for 6 h.qPCR assays were performed to measure mRNA levels of IFNB1 (d) and IFIT1 (e).
(f) Similar to (a), except the cells were transfected with poly (I:C).
(g) THP-1 cells stably expressing shRNA targeting PTK2B or control cells were infected with VSVΔM51-GFP for the indicated times, followed by immunoblotting.
(h, i) A549 cells were transfected with ASO-1 or ASO-2 targeting PTK2B, then mock infected or infected with HSV1-GFP (h) for 18 h and VSV-GFP (i) 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).
Data shown in (a-f, h, i) are from one representative experiment of three independent experiments (mean ± SD, n=3 independent samples), two-tailed Student's t-test.Data are one representative of two independent experiments with similar results in (g).Source data in (a-f, h, i) are provided as a Source Data file, source data in (g) are provided in Fig. S13.VSVΔM51-GFP(h) VSVΔM51-GFP(h)  (e, f) Immortalized MEFs stably expressing PTK2B or control cells were infected with HSV1-GFP (e) or VSVΔM51-GFP (f) for the indicated times, followed by immunoblotting with the indicated antibodies.
(k) Similar to (h), except that VSVΔM51-GFP was used for stimulation.
(o) Ptk2b +/+ and Ptk2b -/-Raw264.7 cells were infected with VSVΔM51-GFP for the indicated times, and then analyzed by immunoblotting with the indicated antibodies.
Data shown in (a-d, g-n) are from one representative experiment of three independent experiments (mean ± SD, n=3 independent samples).n.s.not significant, two-tailed Student's t-test.Data are one representative of two independent experiments with similar results in (e, f, o).Source data in (b-d, g-n) are provided as a Source Data file, source data in (e, f, o) are provided in Fig. S13.(d-e) Ptk2b +/+ and Ptk2b −/− BMDMs were infected with VSVΔM51-GFP for the indicated times, followed by qPCR to measure mRNA levels of Ifnb1 (d) and Ifit1 (e).
Data are one representative of two independent experiments with similar results in (f).Data shown in (a-e, g-l) are from one representative experiment of three independent experiments (mean ± SD, n=3 independent samples), two-tailed Student's t-test.Source data in (a-e, g-l) are provided as a Source Data file, source data in (f) is provided in Fig. S13.(c) Ptk2b +/+ and Ptk2b −/− mice were infected with HSV-1 via tail vein injection at 3x10 7 pfu per mouse for 4 days.Viral titers in the infected brain were measured using a plaque assay.n =5 mice for each group.
(g, h) Ptk2b +/+ and Ptk2b −/− mice were infected with VSV via tail vein injection at 1×10 8 pfu per mouse.Sera were collected at 6 and 12 h after infection to measure the levels of IFNβ (g) and IL-6 (h) by ELISA.n =8 mice for each group.
(i) Ptk2b +/+ and Ptk2b −/− mice were infected with VSV via tail vein injection at 1×10 8 pfu per mouse and the survival of mice was monitored for 15 days.n =9 mice for each group.
Data shown in (a-h) are from one representative experiment of two independent experiments (mean ± SD). n.s.not significant, two-tailed Student's t-test.The log-rank (Mantel-Cox) test was used in Data (i).Source data are provided as a Source Data file.

Fig
Fig. S1 PTK2B associates with STING and TBK1, not RIG-I and MAVS (a) Raw 264.7 cells were pretreated with MG132 (25uΜ) for 6 h, then mock infected or infected with HSV1-GFP for the indicated times.The cell lysates were immunoprecipitated with anti-STING antibody or control IgG and analyzed by immunoblotting.(b) THP1 cells were mock infected or infected with VSVΔM51-GFP for the indicated times.The cell lysates were immunoprecipitated with anti-PTK2B antibody or control IgG and analyzed by immunoblotting.(c) Schematic diagram of STING domains (top).HEK293T cells were co-transfected with PTK2B-Myc and STING-Flag or its truncated mutants as indicated.Co-IP assays were performed with Flag M2 beads and the pulled-down proteins were analyzed by immunoblotting (bottom).(d) Schematic diagram of PTK2B domains (top).HEK293T cells were co-transfected with STING-Myc 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 are one representative of two independent experiments with similar results in (a-d).Source data are provided in Fig.S13.
Fig. S3 Alteration of PTK2B expression affects antiviral signaling in mouse MEFs and Raw264.7 cells (a) Immortalized MEFs stably expressing PTK2B or control cells were lysed for immunoblotting.(b-d) Immortalized MEFs stably expressing PTK2B or control cells were infected with VSVΔM51-GFP for 3 and 6 h.The mRNA levels of Ifnb1 (b), Ifit1 (c) and Cxcl10 (d) were measured by qPCR.
Fig. S5 Ptk2b knockout reduces antiviral signaling in vivo (a, b) Ptk2b +/+ and Ptk2b −/− mice were infected with HSV-1 via tail vein injection at 4×10 7 pfu per mouse.Sera were collected at 4 and 8 h after infection to measure the levels of IL-6 (a) and TNFα (b) by ELISA.n =5 mice for each group.
Fig. S6 Identification of phosphorylated Tyr residues of TBK1 by PTK2B(a) Flag-tagged TBK1 was co-transfected into HEK293T cells with Myc-tagged PTK2B or control vector, cell lysates were immunoprecipitated with anti-Flag M2 beads, followed by mass spectrometric analysis.Mass spectrometric analysis identified Tyr residues Y394, Y577, Y591 and Y592 of TBK1 phosphorylated by PTK2B.
Fig. S7 Phosphorylated Y591 of TBK1 by PTK2B plays an important role in regulating TBK1 activation (a) Immortalized TBK1-deficent MEF cells were co-expressed with PTK2B and wild-type TBK1 or its mutants, followed by infection with HSV1-GFP for 3 h.The cells were harvested for immunoblotting with the indicated antibodies.(b) Immortalized TBK1-deficent and control MEFs were lysed for immunoblotting with the indicated antibodies.(c, d) PTK2B and wild-type TBK1 or its mutants were co-expressed in TBK1-deficent MEF cells, and then the cells were infected with HSV1-GFP for 3 h.The cells were harvested for qPCR to measure the mRNA levels of Ifnb1 (c) or for immunoblotting with the indicated antibodies (d).Data are one representative of two independent experiments with similar results in (a, b ,d).Data shown in (c) are one representative experiment of three independent experiments (mean ± SD, n=3 independent samples), two-tailed Student's t-test.Source data in (a, b ,d) are provided in Fig. S13, source data in (c) are provided as a Source Data file.
Fig. S11 Src and PTK2 are not required for the formation of TBK1 oligomerization (a, b) Immortalized MEFs stably expressing shRNA targeting Src or negative control were infected with HSV1-GFP for 6 h, followed by qPCR (a) and immunofluorescence analysis of TBK1 (Red) and DAPI (b, left).The percentage of cells with TBK1 foci was quantified; n=105 cells from each group were analyzed (b, right).(c) HEK293T cells were co-transfected with the indicated plasmids.IP assays were performed with anti-Flag M2 beads and analyzed by immunoblotting.(d) 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.(e, f) Immortalized MEFs stably expressing shRNA targeting Ptk2 or negative control were infected with HSV1-GFP infection for 6 h, followed by qPCR (e) and immunofluorescence analysis of TBK1 (Red) and DAPI (f, left).The percentage of cells with TBK1 foci was quantified; n=105 cells from each group were analyzed (f, right).Data shown in (a, b, e, f) are from one representative experiment of three independent experiments (mean ± SD, n=3 independent samples), two-tailed Student's t-test.Data are one representative of two independent experiments with similar results in (c, d).Source data in (a, b, e, f) are provided as a Source Data file, source data in (c, d) are provided in Fig. S13.

Fig. S12
Fig. S12 Schematic representation of PTK2B-mediated antiviral signalingUpon virus infection, PTK2B forms oligomerization, and enhances the oligomerization of TBK1 and STING, subsequently positively regulates STING-TBK1 activation to ensure efficient antiviral innate immune responses.

Fig. S13
Fig. S13 Original western blots in supplementary figuresPanels corresponding to figures are indicated.