ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS

Abstract

Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. However, ATM also has been implicated in metabolic regulation, and ATM deficiency is associated with elevated reactive oxygen species (ROS). ROS has a central role in many physiological and pathophysiological processes including inflammation and chronic diseases such as atherosclerosis and cancer, underscoring the importance of cellular pathways involved in redox homeostasis. We have identified a cytoplasmic function for ATM that participates in the cellular damage response to ROS. We show that in response to elevated ROS, ATM activates the TSC2 tumor suppressor via the LKB1/AMPK metabolic pathway in the cytoplasm to repress mTORC1 and induce autophagy. Importantly, elevated ROS and dysregulation of mTORC1 in ATM-deficient cells is inhibited by rapamycin, which also rescues lymphomagenesis in Atm-deficient mice. Our results identify a cytoplasmic pathway for ROS-induced ATM activation of TSC2 to regulate mTORC1 signaling and autophagy, identifying an integration node for the cellular damage response with key pathways involved in metabolism, protein synthesis, and cell survival.

Fig. 1.

mTORC1 repression by ROS in the cytoplasm (A and B) Western analysis of H₂O₂ treated MCF7 cells. (C) Western analysis of cytoplasmic and nuclear fractions of H₂O₂ treated MCF7 cells. (D) Western analysis of MCF7 cells treated with H₂O₂ in the presence or absence of 100 ng/mL leptomycin B. LDH and LAMIN are controls for cytoplasmic and nuclear fractions, respectively.

Fig. 2.

mTORC1 repression induces autophagy (A) MCF7 cells stably transfected with GFP-LC3 were treated with H₂O₂ for 1 h and analyzed by microscopy for the presence of fluorescent puncta. Cells undergoing autophagy were quantitated as a percentage of total GFP positive cells. The graph shows the total number of puncta positive cells divided by total GFP positive cells, normalized to the vehicle control [n = 2, * P < 0.001 (χ² test)]. (B and C) Western analysis of H₂O₂ treated SKOV-3 cells. (D) Electron microscopy images of H₂O₂ treated MEFs. Arrows indicate autophagosomes.

Fig. 3.

Participation of ATM in mTORC1 repression by ROS (A) Western analysis of EBV-immortalized B-lymphocytes obtained from an AT-patient (AT-B), or nonaffected individual (WT-B) were treated with H₂O₂ for 1 h. (B) MEFs derived from Atm^+/+, Atm^+/-, or Atm^-/- mice were treated with H₂O₂ for 1 h. Western analyses shown above and quantitation of the response (percent change in ratio of phosphorylated S6K/total S6K setting the NT control to 100%) of independent clonal isolates from Atm^+/+ (n = 3), Atm^+/- (n = 3), or Atm^-/- (n = 3) mice shown in the graph below. *P < 0.05 (t test) (C) Immunohistochemistry of thymi from Atm^-/- mice. The thymi of Atm^-/- mice treated with rapamycin were markedly atrophic and hypocellular, with few if any residual tumor cells apparent (Right). (D) Quantitation of Western analysis to determine the ratio of phospho-S6K/total S6K (n = 5 normal, n = 9 tumors, n = 4 tumor+rapa), and phospho-S6/total S6 (n = 7 normal, n = 12 tumors, n = 6 tumor+rapa) in thymi in response to rapamycin. *P ≤ 0.05 (Mann-Whitney test, compared to normal) (E) Kaplan-Meier survival curves for Atm^-/- mice treated with 15 mg/kg rapamycin, and control Atm^-/- mice and Atm^+/- mice. P < 0.001 (log-rank test).

Fig. 4.

LKB1 mediates AMPK activation and mTORC1 repression by H₂O₂ (A) Immunoprecipitation showing that LKB1 is phosphorylated at Thr366 by ATM in response to H₂O₂ and 20Gy IR (positive control) in HEK293 cells. (B) Western analysis of LKB1-deficient HeLa S3 cells (parental) and stable clones expressing wild-type Lkb1 (WT) (n = 4) or T363A mutant Lkb1 (MT) (n = 2) treated with H₂O₂. *P < 0.03 versus parental. **P < 0.03 versus WT, 0.05 versus MT. (t test).

Fig. 5.

ROS induces AMPK activation of Tsc2 (A) Western analyses of Tsc2-proficient and deficient MEFs treated with H₂O₂. (B) Western analysis of HEK293 functional assay showing TSC2_AMPK2A mutant is deficient in ROS-induced mTORC1 repression.

Fig. 6.

Schematic showing cytoplasmic signaling pathway from ATM to TSC2 via LKB1 and AMPK to repress mTORC1 and induce autophagy.