Central to cellular metabolism and cell proliferation are highly conserved signalling pathways regulated by the enzymes AMP-activated protein kinase (AMPK) and the mechanistic target of rapamycin (mTOR). Energetic stress triggers AMPK phosphorylation of rate-limiting metabolic enzymes implicated in most branches of nutrient metabolism to down-regulate anabolic (ATP-consuming), and up-regulate catabolic (ATP-synthesising), pathways. AMPK exists as a heterotrimer comprised of catalytic α-subunits, and regulatory β- and γ-subunits, for which multiple isoforms exist (α1/2, β1/2 and γ1/2/3) creating 12 possible configurations. mTOR nucleates two functionally and structurally distinct complexes, in which the rapamycin-sensitive mTORC1 controls anabolic metabolism such as lipid and protein synthesis, whereas the rapamycin-insensitive mTORC2 mitigates growth factor signalling.
We demonstrate in vitro that mTORC1 phosphorylates AMPK on Ser182/4 of either isoform of the β-subunit (which serves as the scaffold for AMPK), provided it is complexed with α1 and not α2. To determine which region of the α-subunit is responsible for facilitating mTORC1 phosphorylation of the β-subunit, we generated several bacterial-expressed chimera constructs and treated them with mTORC1. The greatest sequence divergence between the two α-isoforms is situated in a C-terminal region termed the 'ST-loop' (due to its high proportion of Ser and Thr residues). Indeed, an α2-α1 ST-loop 'swap' recovered in vitro β1-S182 phosphorylation by mTORC1.
In cells, acute treatment of AMPK expressing each α/β-isoform combination with mTOR inhibitors failed to elicit any effect on β-S182/4 phosphorylation. In contrast, chronic mTOR inhibition resulted in a reduction of endogenous β-S182/4 phosphorylation, predominantly for β2. This treatment also reduced AMPK expression, suggesting that β-S182/4 phosphorylation is a co-translational modification. Since mTORC2 has been shown to associate with ribosomes and co-translationally phosphorylate several newly-synthesised substrates, these data could indicate that mTORC2 is the physiological β-S182/4 kinase in vivo. Furthermore, while S182/4A mutants have comparable basal and ligand-stimulated activities versus their wild-type counterparts in whole-cell lysates, our preliminary findings reveal that these mutants have markedly elevated activity in the nucleus. Therefore, this phospho-site may govern the nuclear localisation and activity of AMPK where it has been shown to regulate transcription factors involved in metabolism, and signifies a further point of interrogation.