Fig 2: Syk kinase activity is required for gene induction by cGAMP-activated STING. (A) Different protein tyrosine kinase inhibitors were tested for their ability to inhibit STING-mediated gene induction; cells were pretreated with the inhibitor before cGAMP stimulation. The inhibitors were PP2 (Src inhibitor; 20 µM), ibrutinib (BTK inhibitor; 10 µM), PF-562271 (FAK inhibitor; 20 µM), saracatinib (Fyn inhibitor; 10 µM), amuvatinib (c-Kit inhibitor; 10 µM), ruxolitinib (Jak1/2 inhibitor; 10 µM), alectinib (ALK inhibitor; 10 µM), gefitinib (EGFR inhibitor; 10 µM), and R406 (Syk inhibitor; 10 µM). (B and C) The Syk inhibitor R406 inhibited STING-mediated induction of IFN-ß and Ccl20 mRNAs in (B) human HT 1080 and (C) mouse L929 cells. Cells were pretreated with dimethyl sulfoxide (DMSO), gefitinib, or the Syk inhibitor R406 (10 µM) before cGAMP transfection; after 5 h, mRNAs were measured (n = 3). Bars show means and standard deviations (SD) for 3 independent experiments. ***, P < 0.001 compared to the R406+cGAMP-treated group by two-tailed Student’s t test. (D) Syk KD HeLa cells were treated with cGAMP; after 5 h, Ccl20 and IFN-ß mRNAs were measured (n = 3). Bars show means and SD for 3 independent experiments. ***, P < 0.001 compared to the Syk-KD+cGAMP group. (E) The Syk inhibitor fostamatinib (10 µM) inhibited STING-mediated induction of IFN-ß and Ccl20 mRNAs in L929 cells. (F) Syk KO HT1080 cells were treated with cGAMP; after 5 h, Ccl20 and IFN-ß mRNAs were measured (n = 3). The Syk KO effect were verified through WB using Syk antibody; two clonal lines were tested (inset). Bars show means and SD for 3 independent experiments. ***, P < 0.001 compared to the Syk KO+cGAMP group.

Fig 3: STING, EGFR, and Syk physically interact in the signaling complex. (A) EGFR interacts with Syk and STING upon cGAMP treatment, and this association does not depend on Syk activity. HT1080 cells were pretreated with R406 for 1 h and treated with cGAMP for 1 h, and the cell lysates were subjected to IP with EGFR antibody and analyzed by WB with Syk, STING, and EGFR antibodies. (B) Syk interacts with EGFR upon cGAMP treatment but not STING. HT1080 cells were pretreated with R406 for 1 h and treated with cGAMP for 1 h, and the cell lysates were subjected to IP with anti-Syk and analyzed via WB with the indicated antibodies. (C) STING binds to EGFR, but not Syk, upon cGAMP treatment. HT1080 cells were pretreated with R406 for 1 h and treated with cGAMP for 1 h, and the cell lysates were subjected to IP with anti-STING and analyzed via WB with the indicated antibodies. (D) Confocal experiment showed that STING and Syk colocalized on ER. HeLa cells were transfected with mGST-Syk and STING-GFP plasmids, treated with cGAMP for 1 h, fixed, and stained with calnexin (red) and GST antibody (purple). The white dots show STING-Syk colocalization on ER as revealed by Fuji analysis. (E) PLA technology showed that STING and Syk interacted in stimulated cells. The red dots show that STING and Syk were in close proximity. HeLa cells or HeLa EGFR KO cells were transfected with STING-GFP for 24 h, treated with cGAMP for 1 h, fixed, and stained with GFP and Syk antibody followed by PLA. (F) Quantification of PLA dots per cell from panel E (means and SD). P < 0.001. At least 30 cells were quantified from each independent experiment, which was repeated three times with similar results.

Fig 4: Both Syk and EGFR are needed for phosphorylation of Tyr240 and Tyr245. (A) Syk activity is required for Y245 phosphorylation. HT1080 cells were pretreated with R406 for 1 h and treated with cGAMP for 3 h, and the cell lysates were subjected to WB with the pY245 antibody of STING. (B) Gefitinib and EGFR KO inhibited both Y245 and Y240 phosphorylations. (C) Gefitinib inhibited the phosphorylation of Y240 and Y245, as revealed by LC-MS/MS analysis. Bars show means and SD from 3 independent LC-MS/MS experiments. ***, P < 0.001 compared to the gefitinib‐pretreated group by two‐tailed Student’s t test. (D) The absence of EGFR inhibited the phosphorylation of both Y240 and Y245. STING phosphorylation was analyzed by LC‐MS/MS as described above. Bars show means and SD from 3 independent LC-MS/MS experiments. ***, P < 0.001 compared to the EGFR KD group by two‐tailed Student’s t test.

Fig 5: Steps of STING signaling in the ER. cGAMP binds to STING, causes its conformation change, and stabilizes STING dimers and oligomers. STING interacts with ER membrane-bound EGFR; EGFR then autophosphorylates, and Syk docks on p-EGFR and is activated. Syk phosphorylates STING on Y240; EGFR phosphorylates STING on Y245, and STING translocates to the ERGIC for signaling transduction.