Figure 4.

PRKDC phosphorylates PRKAG1. (A) In vitro PRKDC kinase assay with recombinant PRKDC (+) and PRKAG1 immunoprecipitated from MCF7 cells in stringent conditions. When indicated 100 nM NU7441 was added to the reaction. (B) Predicted PRKDC/ATM and ATM phosphorylation sites in PRKAG1. Left, crystal structure of holo-AMPK complex consisting of PRKAA1, PRKAB2 and PRKAG1 [30]. Ser192 and Thr284 are highlighted with yellow. Right, domain structure of PRKAG1 with CBS domains 1–4 and predicted phosphorylation sites highlighted. (C) In vitro PRKDC kinase assay with indicated PRKAG1 peptides as substrates. Radioactivity was analyzed by FUJI phospho-imager plate and spots were quantitated with FujiFilm MultiGauge version 3.2. (D) In vitro PRKDC kinase assay with WT PRKAG1-Ch-Flag or its S192A, T284A and S192A,T284A mutants immunopurified from U2OS cells as substrates. (E) Representative immunoblots of indicated proteins from MCF7 cells transfected with non-targeting control (NTC) or PRKDC siRNA for 60 h and starved for glucose for the last 12 h when indicated (12). (F) Representative immunoblots of indicated proteins from MCF7 cells left untreated or starved for glucose for 12 h with or without 1 h recovery. When indicated cells were retreated with 100 nM NU7441 for indicated times. (G) Representative Immunoblots of indicated proteins from U2OS cells left untreated or starved for glucose for indicated times. (H) Representative immunoblots of indicated proteins from control and PRKDC shRNA-infected U2OS cells left untreated, starved for glucose or treated with 5 µM compound C as indicated. (I) Representative immunoblots of indicated proteins from MCF7 cells and BJ fibroblasts left untreated or starved for glucose for 6 h.