Mol. Cell 44, 878–892 (2011)

AMP-activated protein kinase (AMPK) is generally viewed as a key regulator of energy homeostasis that responds to external stimuli such as nutrient deprivation by phosphorylating protein substrates that reset cellular energy balance. Banko et al. now apply a chemical-genetic strategy to identify new AMPK substrates and show its involvement in regulating mitotic progression. Mutating the gatekeeper residue of the catalytic domain of AMPK to a glycine residue resulted in an analog-specific variant (AS-AMPKα2) that accepts a bulky N6-modified ATP analog as a substrate. AS-AMPKα2 and a thiolated bulky ATP analog were used as reagents in a thiophosphate tagging method that was applied to identify 28 potentially new AMPK targets in 293T cells. Several of the candidates were validated as bona fide AMPK substrates in vitro and in cells, and their AMPKα2 phosphorylation sites were mapped by mutational studies. Bioinformatic analysis revealed that many of the hits, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2), were proteins associated with mitotic regulation. Further characterization showed that mitotic cells have higher AMPK activities and that inhibition of AMPK activity blocks mitosis in a PPP1R12C phosphorylation–dependent manner. The analog-specific kinase engineering approach revealed a previously unknown role of AMPK in mitotic regulation and may offer a general strategy for identifying cellular substrates of kinases.