The AMPK αβγ heterotrimer is a highly conserved serine/threonine protein kinase that plays a crucial role in maintaining cellular energy homeostasis by sensing metabolic fuel levels. It forms complexes composed of unique subunit isoform variations (α1, α2, β1, β2, γ1, γ2, γ3) in a 1:1:1 ratio, each exhibiting distinct tissue-expression patterns. AMPK is modified post-translationally by phosphorylation across most of its subunits, while some of these phosphorylation sites have been mechanistically characterised the function of most sites remains elusive. To investigate the function of these sites, it is important to consider the degree of phosphorylation at each site, also known as the absolute stoichiometry of phosphorylation. However, no previous studies have examined this aspect for AMPK phosphorylation sites.
We utilised mass spectrometry to generate precise phosphorylation profiles of all 12 AMPK complexes expressed in HEK293T cells. A total of 18 phosphorylation sites were detected and quantified, including the discovery of 10 novel sites, several of which are located within the understudied γ2 and γ3 subunits. We noticed nine of the quantified sites contain Ser-Pro motifs, including known mTORC1 substrates α1-S347/α2-S345, so we speculated that some of the other Ser-Pro sites might also be substrates of mTORC1. Seven sites were sensitive to pharmacological mTORC1 inhibition, including four on the NH2-terminal extension of γ2 isoform and β1-S182/β2-S184 which is near-maximally phosphorylated. We confirmed that β1-S182/β2-S184 are mTORC1 substrates in vitro. Phosphorylation on this site was elevated in α1-containing complexes, an effect partly attributable to the non-conserved α-subunit serine/threonine-rich loop.