Fig. 3: LegK3 directly phosphorylates Caspase-3.
From: Phosphorylation of caspases by a bacterial kinase inhibits host programmed cell death
A HEK293T cells were transfected to express GFP-LegK3 and HA-Caspase-3, samples transfected with the indicated mutants were also established. After immunoprecipitation with anti-HA agarose, the beads-bound proteins were detected by immunoblotting using anti-HA or anti-Phospho-(Ser) antibodies. B HA-Caspase-3 obtained in (A) by elution with 3xHA peptide was analyzed by mass spectrometry. Extracted ion chromatograms of the Ser29-phosphorylated peptide (IIHGSESMDpSGISLDNSYK) and a control peptide (SGTDVDAANLR) were shown. C Phosphorylation of Caspase-3 by LegK3 in E. coli. Flag-tagged LegK3 or LegK3D/A and His6-tagged Caspase-3* were co-expressed in E. coli. His6-Caspase-3* was obtained by affinity purification. Phosphorylation of Caspase-3* was detected by phospho-protein staining, immunoblotting with anti-Phospho-(Ser) antibodies or native PAGE. Expression of LegK3 was probed by immunoblotting with the Flag antibody. Caspase-3* represents for Caspase-3C163A, a catalytic inactive mutant of Caspase-3 that can be purified as full-length protein from the E. coli expression system. D, E LegK3 targets Caspase-3 for phosphorylation during L. pneumophila infection. HEK293 cells transfected to express the FcγRII receptor and Flag-Caspase-3 were either left uninfected or challenged with the indicated L. pneumophila strains for 3 h. After immunoprecipitation with the Flag antibody, the beads-associated proteins were analyzed by immunoblotting using Flag or Phospho-(Ser)-specific antibodies, respectively (D). Post-exponential bacteria were lysed and probed for the production of LegK3 by immunoblotting using Flag antibody (E). The housekeeping protein ICDH (isocitrate dehydrogenase) was probed as a loading control (E). F Flag-Caspase-3 immuniprecipitated from wild-type and dotA- L. pneumophila infected samples (D) were eluted by 3xFlag peptide and further detected by mass spectrometry. Extracted ion chromatograms of the Ser29-phosphorylated peptide (IIHGSESMDpSGISLDNSYK) and a control peptide (KQIPCIVSMLTK) were shown. Data in A, C–E are representative from three independent experiments. Source data are provided as a Source Data file.
