1. AMPK directly increases mTORC2 catalytic activity.
AMPK directly increases mTORC2 catalytic activity. (A) AMPKα1/α2 DKO MEFs were serum-starved (20 hours), pretreated with torin1 (100 nM; 30 min), and treated without (−) or with (+) AICAR (2.5 mM; 2 hours) or insulin (INS; 100 nM; 30 min). Immunoprecipitations with anti-rictor antibodies or beads only (b/o) were incubated with recombinant His-Akt1 substrate (100 ng; 30 min) at 30°C. IVK reactions, immunoprecipitates, and whole-cell lysates were immunoblotted as indicated. Graph represents the mean ratio ± SD of AktS473 phosphorylation over total Akt levels. n = 3 samples from three independent experiments. *P < 0.05 by unpaired t test. (B) AMPKα1/α2 DKO MEFs were serum-starved (20 hours) and stimulated without (−) (lanes 1 to 7) or with (+) (lanes 8 and 9) insulin (100 nM; 30 min). Immunoprecipitations with anti-rictor antibodies (+) or beads only (−) were preincubated without or with compound C (CC; 0.5 mM) or torin1 (5 μM; 30 min) on ice, incubated without (−) or with (+) recombinant GST-AMPKα1β1γ1 (100 ng; 30 min) at 30°C (stage 1), washed twice, and incubated without (−) or with (+) His-Akt1 (100 ng; 30 min) at 30°C (stage 2). IVK reactions were immunoblotted as indicated. Graph represents the mean ratio ± SD of AktS473 phosphorylation over total Akt levels. n = 4 samples from four independent experiments. **P < 0.01 by unpaired t test. (C) Rictor (+) or beads-only (−) immunoprecipitates were incubated without (−) or with (+) recombinant active GST-AMPKα1/β1/γ1 (100 ng) and without (−) or with (+) compound C (25 μM; 30 min) at 30°C. Blots are representative of four independent experiments.
Dubek Kazyken et al., Sci. Signal. 2019;12:eaav3249
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