Fig. 2: The kinase activity of LegK3 is required for the inhibition of host apoptosis.
From: Phosphorylation of caspases by a bacterial kinase inhibits host programmed cell death
A Predicted domain structure of LegK3 and alignment of its catalytic loop with several known bacterial S/T kinases. The conserved residues are highlighted in red. B, C HeLa cells expressing GFP-LegK3 or GFP-LegK3D/A were stimulated with STS for 4 h, the cell lysates were subjected to immunoblotting with anti-Caspase-3, anti-Caspase-7, and anti-PARP antibodies (B) or measuring Caspase-3/-7 activity using the substrate Ac-DEVD-pNA (C). D, E Flow cytometry analysis of apoptotic cell death of cells expressing LegK3 or LegK3D/A that had been treated with STS. HeLa cells were transfected to produce BFP, BFP-LegK3, or BFP-LegK3D/A for 24 h, cells were either left untreated or treated with 1 μM STS for 4 h. After staining the cells with PI and DEVD-GreenNucTM, samples were analyzed by a flow cytometry under dark. Representative images and quantification of apoptotic rates were shown in (D) and (E), respectively. F–I LegK3 inhibits Caspase-3/-7 activation in L. pneumophila-infected cells. FcγRII-expressing HeLa cells were infected with the indicated L. pneumophila strains for 3 h. Infected cells were either left untreated (F, G) or incubated with 1 μM of STS for 4 h (H–I). The amounts of mature Caspase-3/-7 were assessed by immunoblotting (F, H) or by the cleavage of the substrate Ac-DEVD-pNA (G, I). Data in B, F, and H are representative from three independent experiments. For C, E, G, and I, data shown are mean ± SD from three replicates. Unpaired two-tailed Student’s t tests were performed. Source data are provided as a Source Data file.
