AMP-activated protein kinase (AMPK) is a highly evolutionarily conserved heterotrimeric
kinase, characterised as a critical sensor of energetic stress and is therefore being investigated as a therapeutic target for cancer and metabolic diseases. AMPK is made up of a catalytic α subunit with 2 isoforms, a regulatory and scaffolding β subunit with 2 isoforms
and a regulatory γ subunit with 3 isoforms. Each of the 12 possible combinations of the AMPK complex has distinct tissue distribution and little is known about differences in regulation and function. Using novel AMPKα1 and AMPKα2 single and double knockout HEK293T cells, differences in the regulation of AMPKα isoforms in response to activation by either upstream kinase LKB1 or CAMKK2 revealed AMPKα1 responds uniquely to activation
by CAMKK2. To elucidate isoform and upstream kinase specific AMPK-dependent signaling pathways, a global quantitative phosphoproteomic screen using wild-type and AMPKα1/α2 single and double knockout HEK293T cells treated with 2-DG or Ionomycin was performed. FAM122A S37 emerged as a novel AMPK substrate and further investigation revealed that S37 is a crucial residue for regulating the switch in FAM122A binding to 14-3-3 and PP2A. A proteomics approach was used to identify the interactome of AMPKγ1, AMPKγ2 and AMPKγ3 containing complexes. 14-3-3 was identified as a unique AMPKγ2 binding partner which requires phosphorylation of AMPKγ2 at S122, a residue not conserved between AMPKγ isoforms. Increased 14-3-3 binding to AMPKγ2 following AMPKγ2 S122 phosphorylation by AMPK and PKA has no obvious effect on AMPK activity but may sequester AMPK in the cytosol. A 14-3-3 interactome screen revealed known and novel 14-3-3 binding partners regulated by AMPK. Together these findings confirm the existence of differences in the behaviour of AMPK subunit isoforms
and therefore support the investigation of AMPK isoform-specific drugs.