Novel torin1-sensitive phosphorylation sites on the metabolic regulator AMPK revealed by label-free mass spectrometry

Abstract

AbstractAMPK and mTORC1 are nutrient-sensitive protein kinases that form a fundamental negative feedback loop that governs cell growth and proliferation. AMPK is an αβγ heterotrimer that is directly phosphorylated by mTORC1 on α2S345 to suppress AMPK activity and promote cell proliferation under nutrient stress conditions. Using mass spectrometry, we generated precise phosphorylation profiles of all 12 AMPK complexes expressed in proliferating human cells. Of the 18 phosphorylation sites detected, seven were sensitive to pharmacological mTORC1 inhibition, including four in the AMPK γ2 isoform NH2-terminal domain and α2S377 which is located in the nucleotide-sensing motif. In particular, β1S182 and β2S184 were found to be mTORC1 substrates in vitro and near-maximally or substantially phosphorylated under cellular growth conditions. βS182 phosphorylation was elevated in α1-containing complexes, relative to α2, an effect partly attributable to the non-conserved α-subunit serine/threonine-rich loop. While mutation of β1S182 to a non-phosphorylatable Ala had no effect on basal and ligand-stimulated AMPK activity, β2-S184A mutation increased nuclear AMPK activity and enhanced cell proliferation under nutrient stress. We conclude that mTORC1 governs the nuclear activity of AMPK to regulate transcription factors involved in metabolism and cell survival during nutrient shortage.