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. 2023 May 24;14:2994. doi: 10.1038/s41467-023-38401-z

Fig. 1. AMPK inhibits ULK1 signaling to the autophagy initiation machinery.

Fig. 1

a mTORC1 inhibition suppresses ULK1 Ser556 phosphorylation. HCT116 cells were starved of amino acids or glucose for 2 h or treated with Torin1 (250 nM) or rapamycin (100 nM) for 1 h. b mTORC1 inhibition disrupts the AMPK-ULK1 interaction. ULK1 KO HEK293T cells transiently transduced with myc-ULK1 were treated as in a. Anti-myc immunoprecipitates were analyzed for endogenous AMPKα. c AMPK activation suppresses ULK1 activity. HCT116 cells were starved of amino acids in the presence or absence of A769662 (100 µM). d, e A769662 prevents the ability of amino acid starvation or Torin1 to increase ULK1 activity. MEFs were pre-treated with vehicle (−) or A769662 (+) for 30 min, then incubated with either full or amino acid-deprived medium or treated with Torin1 for 1 h. f, g AMPK activators suppress ULK1 activity. HCT116 cells were treated with A769662, 991 (10 µM) or MK8722 (10 µM) for 30 min then starved of amino acids for 2 h. h, i AMPKα depletion enhances ULK1 activity. AMPKα WT and DKO HCT116 cells were starved of amino acids during the indicated periods. j, k AMPKα depletion blunts the suppressive effect of A769662 on ULK1 activity. HCT116 cells were treated with A769662 (100 µM) and/or Torin1 (250 nM) for 1 h. l AMPK kinase activity is necessary to suppress ULK1 activity. AMPKα DKO HEK293T (left) and HCT116 cells (right) stably reconstituted with WT or KD AMPKα were treated as described in j. m, n AMPK activation suppresses Torin1-induced ULK1 activation in the liver and skeletal muscle (tibialis anterior). C57BL6 mouse was intraperitoneally injected with Torin1 alone or together with MK8722 for 1 h. o Model for AMPK role in autophagy. The dotted line indicates that the AMPK-mTORC1 axis plays a lesser role in autophagy. Data in the graphs of this figure are shown as mean ± standard error of the mean (SEM). Data were analyzed by two-tailed Student’s t-test. The number of independent experiments is denoted by the ‘n’ value on each graph. Source data are provided as a Source Data file.