Figure 5. Approaches used to characterize the AMPK recognition motif.

(A) Hypothesis-driven approach using mutations Constructs containing 34 residues around Ser79 on ACC1, with and without the indicated mutations, were expressed in bacteria and phosphorylated by AMPK in cell-free assays. Changes in kinetic parameters (k_cat/K_m) for each mutant relative to the wild type are represented by the lengths of the bars above (increases) or below (decreases) the indicated amino acid [46]. (B) Positional scanning peptide library. Phosphorylation by AMPK in cell-free assays of peptide mixtures (10-mers) containing serine and threonine at position 6, a fixed amino acid at one position (e.g. P-5, illustrated), and random mixtures at all others, revealed preferences for specific amino acids at each position [48]; “x”, any amino acid. (C) Direct thiophosphorylation with gatekeeper mutation. An AMPK mutant uses a bulky derivative of ATP-γ–S (A*TPγS) to thiophosphorylate direct targets in permeabilized cells [50]. Many direct AMPK phosphorylation sites were identified through isolation and identification by tandem mass spectrometry of the thiophosphorylated peptides [54]. Hydrophobic amino acids are in green, neutral polar in blue, acidic in orange, and basic in red.