Fig. 1: CAND1 is a kinase substrate of OspG uncovered by large-scale host phosphoproteomics. | Nature Communications

Fig. 1: CAND1 is a kinase substrate of OspG uncovered by large-scale host phosphoproteomics.

From: The Shigella kinase effector OspG modulates host ubiquitin signaling to escape septin-cage entrapment

Fig. 1

a A phosphoproteomic screen to identify the kinase substrates of the S. flexneri effector OspG. RP-LC, reversed-phase liquid chromatography. LC-MS, liquid chromatography-mass spectrometry. b A volcano plot depicting fold changes of detected phosphopeptides in cells expressing OspG versus OspG K53A. Candidate substrates (i.e., CAND1) of OspG appear to the far right. Proteins assigned from phosphopeptides with log2(fold change of OspG/OspG K53A) >2.5 and summed spectral counts >20 are labeled with their names in the graph. Colors indicate the extent of fold changes (FC). c Collision-induced dissociation (CID)-tandem mass spectrum of CAND1 peptide bearing phosphorylation at Ser558. d Extracted ion chromatograms of the Ser558-phosphorylated peptide (VIRPLDQPSSFDATPYIK) and a control peptide (LTLIDPETLLPR) from indicated CAND1 samples. 293T cells were co-transfected with Flag-CAND1 and indicated OspG mutants, and immunoprecipitated CAND1 samples were resolved by SDS-PAGE before LC-MS analysis. e GST pull-down of Flag-CAND1 by purified GST-OspG. Glutathione resins coated with GST-OspG or GST alone were incubated with lysates prepared from Flag-CAND1-expressing 293T cells. Eluted samples were blotted with anti-Flag and anti-GST antibodies. Images are representative of n  =  3 independent experiments. f Purified GST-OspG incubated with GST-CAND1 with or without ubiquitin in vitro, and the reaction mixtures were resolved by SDS-PAGE. Potential phosphorylation signals of CAND1 were measured by SRM (selected reaction monitoring) analysis. Extracted ion chromatograms of the Ser558 phosphorylated peptide and a control peptide (ITSEALLVTQQLVK) of CAND1 were shown. g SRM analysis of CAND1 Ser558 phosphorylation during S. flexneri infection. 293T cells transfected with Flag-CAND1 were infected with indicated S. flexneri strains, and immunoprecipitated CAND1 samples were resolved by SDS-PAGE and analyzed by SRM. h Extracted ion chromatograms of the Ser558-phosphorylated peptide (VIRPLDQPSSFDATPYIK) and a control peptide (LTLIDPETLLPR) from indicated CAND1 samples. 293T cells were co-transfected with Flag-CAND1 and OspG or NleH1, and immunoprecipitated CAND1 samples were resolved by SDS-PAGE before LC-MS analysis.

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