PKA‐mediated phosphorylation of AMPKα1 prevents its phosphorylation by LKB1 at Thr‐172 in vitro and phosphorylation of AMPK at Thr172 and Ser‐173 is not.

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PKA‐mediated phosphorylation of AMPKα1 prevents its phosphorylation by LKB1 at Thr‐172 in vitro and phosphorylation of AMPK at Thr172 and Ser‐173 is not mutually exclusive. PKA‐mediated phosphorylation of AMPKα1 prevents its phosphorylation by LKB1 at Thr‐172 in vitro and phosphorylation of AMPK at Thr172 and Ser‐173 is not mutually exclusive. (A) LKB1 was allowed to phosphorylate AMPKα1(D157S485A/S497A) in the presence of ATP before addition of PKA and [γ‐32P]ATP. Signals obtained by autoradiography indicate phosphorylation at Ser‐173 (lanes 3 and 4). Phosphorylation of Thr‐172 and Ser‐173 is coexistent, if PKA is allowed to phosphorylate AMPK after preincubation with LKB1 (lane 4). In this double‐phosphorylated form, phosphorylation of Thr‐172 but not Ser‐173 was recognized by the corresponding antibodies. PKi, PKA inhibitor. Samples were processed for immunoblotting with the specified antibodies. (B) PKA phosphorylation of AMPKα1 prevents LKB1‐mediated Thr‐172 phosphorylation in vitro. AMPKα1(D157A) was preincubated with/without PKA and non‐radioactive ATP as indicated, followed by LKB1 assays in the presence of [γ‐32P]ATP. LKB1 was unable to phosphorylate AMPK after PKA phosphorylation. Samples were processed for immunoblotting with the specified antibodies. (C) PKA‐mediated AMPKα1 phosphorylation inhibits AMPK activity. AMPK was preincubated with different amounts of PKA before in vitro kinase reactions with LKB1. AMPK activity assessed as the amount of phosphate incorporated into the synthetic peptide substrate SAMS. (D) Immunoblot corresponding to (B) probed with the indicated antibodies. (E) PKA‐phosphorylated AMPK was either left untreated or dephosphorylated with λ‐PPase using the indicated concentrations, then processed for in vitro kinase assays with or without LKB1. AMPK activity quantified using the SAMS assay. (F) Immunoblot corresponding to (E) probed with the indicated antibodies. All activity measurements were done in triplicates. Error bars represent±s.e.m. Immunoblots are representative of at least three independent experiments. Nabil Djouder et al. EMBO J. 2010;29:469-481 © as stated in the article, figure or figure legend