The AMPK alpha-beta-gamma heterotrimer is a crucial protein kinase that maintains cellular energy homeostasis. It forms up to 12 unique complexes composed of subunit isoform variations (alpha1/2, beta1/2, gamma1/2/3) in a 1:1:1 ratio. AMPK is modified post-translationally by phosphorylation across most subunits, while some phosphorylation sites have been mechanistically characterised the function of most remains elusive. We utilised label-free quantitative mass spectrometry to generate phosphorylation stoichiometry profiles of all 12 AMPK complexes expressed in HEK293T cells. Eighteen phosphorylation sites were detected and quantified, including the discovery of 10 novel sites. Nine quantified sites contain Ser-Pro motifs, including known mTORC1 substrates alpha1-S347/alpha2-S345 and alpha2-S377. Seven of these were sensitive to pharmacological mTORC1 inhibition by torin1, including beta1-S182/beta2-S184 which is basally near-maximally phosphorylated.
We confirmed beta1-S182/beta2-S184 is an mTORC1 substrate in vitro. Phosphorylation on beta1-S182/beta2-S184 was elevated in alpha1-containing complexes, an effect partly attributable to the non-conserved alpha-subunit serine/threonine-rich loop. Despite previous reports of this site regulating nuclear transport, prolonged dephosphorylation of beta1-S182/beta2-S184 had negligible effects on nuclear AMPK localisation. While the beta1-S182A mutation had no effect on basal and ligand-stimulated AMPK activity, beta2-S184A increased nuclear AMPK activity and enhanced cell proliferation under nutrient stress. RNAseq analysis of WT vs. beta2-S184A KI HEK293T cells revealed loss of beta2-S184 phosphorylation induces significant changes in the expression of key enzymes involved in gluconeogenesis, glycolysis, glucose uptake, hexosamine biosynthesis, and the pentose phosphate pathway (PPP), together suggesting loss of beta2-S184 phosphorylation redirects carbon flux through the PPP. Additionally, caspase-3 cleavage of the nuclear export signal at the extreme COOH-terminus of alpha1, which structurally neighbours beta2-S184, promotes dephosphorylation of beta2-S184. We conclude that mTORC1 governs the nuclear activity of AMPK through beta2-S184 phosphorylation, where relinquishing this inhibitory input allows AMPK to remodel metabolism to induce adaptive and protective responses during nutrient shortage, ultimately promoting cell survival.