Fig. 1

Antagonistic regulation of mTORC1 and AMPK. Nutrient depletion (i.e., in DB) or rapamycin treatment of cells in full growth media inactivates mTORC1 but activates AMPKα. a–c Nutrient depletion induced de-phosphorylation (i.e., de-activation of mTORC1) of S6K and 4EBP1, but phosphorylation/activation of AMPK. Dictyostelium cells were grown to log-phase and transferred to DB in shaking culture. Cell lysates were prepared at times indicated and analyzed by immunoblotting. Similar results were obtained in three independent experiments. d Rapamycin treatment of cells in full growth media induces de-phosphorylation of S6K and 4EBP1, but phosphorylation/activation of AMPKα. Cells were grown to log-phase and transferred to a full growth media containing 500 nM of rapamycin. Cell lysates were prepared at times indicated and analyzed by immunoblotting. Similar results were obtained in three independent experiments. e Quantification of relative AMP/ATP ratios upon nutrient withdrawal (DB) or rapamycin treatment in full growth media (Med+Rap) in shaking culture. At times indicated, 1 × 10⁷ cells were pelleted and lysed by freeze-thaw. The AMP and ATP levels were measured separately and values represent ratio changes as mean ± standard error. Results are from three independent experiments, with triplicates used for each independent set of experiments. Activation of AMPKα by pAICAR or 2-DG de-activated mTORC1. Log-phase cells in full growth media were treated with 1 mM pAICAR (f) or 80 mM 2-DG (g) in shaking culture. Cell lysates were prepared at times indicated and analyzed by immunoblotting. Immunoblots are representative of three independent experiments. h AMPKα inhibition using dorsomorphin activated mTORC1. AX3 cells were transferred to DB in the presence or absence of 40 μM dorsomorphin in shaking culture. Cell lysates were prepared at times indicated and analyzed by immunoblotting. Immunoblots are representative of three independent experiments