Li Z, Cai S, Sun Y, Li L, Ding S, Wang X. gene induction. Consequently, in the absence of Syk, HSV-1 could not induce interferon, and it replicated more robustly. test. (D) Syk KD HeLa cells were treated with cGAMP; after 5 h, Ccl20 and IFN- mRNAs were measured (test. (B) HT1080 and HT1080 STING?/? cells were pretreated with the Syk inhibitor R406 followed by HSV-1 contamination, and viral titers were determined by plaque assays at 16 h postinfection (MOI?=?5). Bars show means and SD from Peiminine 3 impartial experiments. *test. (C) WT HeLa cells and Syk KD cells were infected with HSV-1 (MOI?=?5), and viral titers were determined by plaque assays at 16 h postinfection Bars show means and SD from 3 independent experiments. **test. (D) WT HT1080 cells, STING?/? HT1080 cells, and Syk?/? cells were infected with HSV-1 (MOI?=?5), and viral titers were determined by plaque assays at 16 h postinfection. Bars show means and SD from 3 impartial experiments. **test. Syk promotes STING signaling by phosphorylating its Tyr 240 residue. After confirming the need of Syk for STING signaling, we set out to investigate its mechanistic basis. In view of the fact that phosphorylation of Y245 of STING by EGFR is needed for IFN induction (16, 28), we inquired Peiminine whether another Tyr residue of STING is usually phosphorylated upon stimulation and whether Syk is required for the process. For Peiminine this purpose, STING-green fluorescent protein (GFP)-expressing cells were stimulated with cGAMP; STING-GFP was purified from the cell extract and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis after protease digestion. Two isoforms of a phosphopeptide spanning V239 to R253 were identified; the mass of one was consistent with modification at Y240 and that of the other with modification of Y245 (Fig.?3A). The degree of modification was determined by plotting chromatograms of both altered and unmodified forms of the peptide (Fig.?3B). A doubly phosphorylated form of the same peptide was identified as well (Fig.?3C). The abundance of singly or doubly phosphorylated peptides was low compared to that of the unmodified peptide (Fig.?3D), possibly because only a small portion of the highly expressed STING-GFP was engaged in signaling. For the sake of facilitating detection of Y240 or Y245 phosphorylation of STING, we raised antibodies to the two respective phosphopeptides. As expected, these antibodies did not recognize STING-GFP pulled down from extracts of untreated cells, but both reacted with it if cells were stimulated with cGAMP (Fig.?3E). These phosphospecific antibodies were extensively used for many experiments, as described below. Open in a separate windows FIG?3 Tyr240 of STING is phosphorylated upon cGAMP stimulation. (A) After 3 h of cGAMP stimulation, STING-GFP was purified from 293XL cells via GFP trap beads, fractionated on an SDS-PAGE gel, and digested with trypsin and Peiminine chymotrypsin, and the digests were analyzed by LC-MS/MS. A doubly charged peptide with a mass of 932.93?Da was identified in a targeted analysis of GFP-STING. The collision-induced dissociation (CID) spectra for this peptide Rabbit Polyclonal to DGKD contains several C-terminal y ions, and the masses of these ions are consistent with the presence of more than one phosphoisoform. The mass difference of the y13* peptide is usually consistent with modification at Y240, and the mass of the y9** peptide is usually consistent with modification at Y245. (B) The degree of modification from A was determined by plotting chromatograms for both the unmodified and altered forms of the Y240/Y245 peptides. The chromatograms for the and peptides are shown. (C) A doubly charged peptide with a mass of 972.911?Da was identified in a targeted analysis of STING-GFP. The CID spectrum for this peptide contains several C-terminal y ions, and the masses of the y8, y9, and y13 ions are consistent with phosphorylation at Y240 and Y245. (D) Chromatograms for the unmodified, pY240, pY245,.