Eukaryotic cells not only sense carbon nutrients directly, but also sense them indirectly by monitoring the levels of cellular adenine nucleotides produced by their catabolism. Starvation for a key carbon source may cause increases in ADP:ATP ratios that are amplified by the adenylate kinase reaction (2ADP ↔ ATP + AMP) into even larger increases in AMP:ATP ratios. The major cellular sensor of such changes in nucleotide levels is the AMP-activated protein kinase (AMPK) which, once activated, acts to restore energy balance within cells. Increases in the AMP:ATP ratio result in conformational changes in AMPK that: (1) cause allosteric activation
(2) promote phosphorylation at a critical threonine residue (Thr172) in the kinase domain of AMPK-α subunits by the upstream kinase LKB1
(3) protect against Thr172 dephosphorylation by protein phosphatases. Together, these three mechanisms of activation can cause up to a 1000-fold increase in AMPK activity, and this chapter will describe assays to monitor these different activation mechanisms in cell-free assays. Adenine nucleotides (AMP/ADP/ATP) bind to sites formed by sequence repeats termed CBS motifs in the AMPK-γ subunits. Several pathogenic mutations in these motifs have been identified that either reduce or eliminate the sensitivity of AMPK to changes in AMP and other nucleotides. We also describe the use of cells expressing such mutants to determine whether agents that activate AMPK do so by disturbing adenine nucleotide levels.