Fig. 4: AMPK targets GSDMD-Ser46.

A Nonradioactive in vitro AMPK kinase assay. HEK293 cells were transfected with HA-AMPKα1 or AMPKα2 and subjected to anti-HA IP assay. The precipitants were incubated with purified GST-GD-NT in a kinase reaction containing 1 mM [γS] ATP. The generated thiophosphorylation sites were alkylated with pNitrobenzyl mesylate. The products were immunoblotted with the antibody against thiophosphate ester (Anti-αThioP), HA-tag, or GST-tag. B Ser46 of GSDMD is an evolutionarily conserved site and matches the well-defined AMPK motif found in most substrates. Examples of classical motifs in well-known AMPK substrates are also shown. C HEK293 cells were co-transfected with Flag-GD-NT mutants and HA-AMPKα1 or AMPKα2. 16 hours later, cells were harvested for anti-Flag IP assay. The precipitants and WCL were immunoblotted with indicated antibodies. GD-NT-Δ39-53 is the mutant lacking the AMPK substrate motif. D Specificity of the generated antibody pS46-GD. HEK293 cells were transfected with Flag-GD-NT wildtype or its mutant Δ39-53 or S46A. 16 hours later, cells were harvested for anti-Flag IP assay. The precipitants were immunoblotted with indicated antibodies. E Similar to (A), except that the kinase reaction contained 200 μM regular ATP, and GD-NT phosphorylation was detected using the antibody pS46-GD. F Similar to (E), except that GST-CA-AMPKα1 or AMPKα2 were used as the phosphorylation kinase in the kinase reaction. G CRISPR/Cas9 technology was used to genetically disrupt the expression of AMPKα. HEK293 cells were primarily transfected with the vector expressing sgPRKAA1 and/or sgPRKAA2. After 2 days of puromycin selection, cells were further transfected with Flag-GD-NT and subjected to anti-Flag IP assay. Shown is IB analysis of anti-Flag IP and WCL with indicated antibodies. H, HEK293 cells were co-transfected with Flag-GD-NT and GST-CA-AMPKα1 or AMPKα2. 16 hours later, cells were harvested for anti-Flag IP assay. The precipitants and WCL were immunoblotted with indicated antibodies. I, HeLa cells expressing Dox-inducible Flag-GD-NT were treated with Dox (2 µg/ml) and AMPK agonist metformin (1 mM), AICAR (0.2 mM), 2-Deoxy-D-glucose (2DG, 3 mM) or AMPK antagonist Compound C (5-10 µM). 16 hours later, cells were harvested for anti-Flag IP assay. The precipitants and WCL were immunoblotted with indicated antibodies. J Similar to (I), except that the experiments were performed in E0771 cells. Data are representative of at least two independent experiments.