AMPK promotes TFEB transcriptional activity through dephosphorylation at both MTORC1-dependent and -independent sites

TFEB (transcription factor EB) is a critical regulator of lysosomal biogenesis, macroautophagy/autophagy and energy homeostasis through controlling expression of genes belonging to the coordinated lysosomal expression and regulation network. AMP-activated protein kinase (AMPK) has been reported to phosphorylate TFEB at three conserved C-terminal serine residues (S466, S467, S469) and these phosphorylation events were reported to be essential for transcriptional activation of TFEB. In sharp contrast to this proposition, we demonstrate that AMPK activation leads to the dephosphorylation of the C-terminal sites. We show that a synthetic peptide encompassing the C-terminal serine residues of TFEB is a poor substrate of AMPK in vitro. Treatment of cells with an AMPK activator (MK-8722), glucose deprivation or MTOR inhibitor (torin1) robustly dephosphorylated TFEB not only at the MTORC1-targeted N-terminal serine sites, but also at the C-terminal sites. Loss of function of AMPK abrogated MK-8722- but not torin1-induced dephosphorylation and induction of the TFEB target genes.<b>Abbreviations:</b> AMPK: 5'-adenosine monophosphate-activated protein kinase; ACAC/ACC: acetyl-CoA carboxylase; AICAR: 5-aminoimidazole-4-carbox-amide ribonucleotide; CLEAR: coordinated lysosomal expression and regulation; DKO: double knockout; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; DQ-BSA: self-quenched BODIPY® dye conjugates of bovine serum albumin; KI: knock-in; KO: knockout; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; RRAGC: Ras related GTP binding C; RPTOR: regulatory associated protein of MTOR complex 1; RPS6KA/RSK: ribosomal protein S6 kinase A; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; RT-qPCR: reverse transcription quantitative polymerase chain reaction; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.