AMP binding activates AMPK by three effects, i.e. promotion of Thr172 phosphorylation by LKB1 (effect #1), inhibition of Thr172 dephosphorylation by protein phosphatases (PP) (effect #2), and allosteric activation (effect #3). All three effects are opposed by binding of ATP, while binding of ADP mimics effect #2 and #1, but not #3. CaMKKβ phosphorylates the same site as LKB1 (Thr172) in response to increases in cellular Ca²⁺.

Atomic coordinates are from the PDB file 4RER []. The model was rendered in PyMOL v1.7.4.2 with the majority of the polypeptide in “cartoon” view and the α-linker in “sphere” view. The domains referred to in the text are color coded and labeled. The kinase inhibitor staurosporine in the active site, and the side chain of phospho-Thr172, are in “sphere” view, and β-cyclodextrin in the glycogen-binding site of the β-CBM in “stick” view, all with C atoms in green, O red, and N blue (H omitted). The curved dotted line in the center shows the approximate boundary between the “catalytic module” (containing the α-KD and β-CBM) and the “nucleotide-binding module” (containing the γ subunit and the C-terminal domains of α and β); the α-AID and α-linker form one of the flexible connectors linking these two modules. Note how the α-RIM2 section of the α-linker (in magenta) contacts site 3 of the γ subunit with its bound AMP.

Atomic co-ordinates are from the PDB files 4RED (A) and 4RER (B) [], with only the α-KD, α-AID and the start of the α–linker being displayed in (B). The color-coding of domains is as in . Most of the structures are rendered in “cartoon” view (PyMOL v1.7.4.2), but the side chains of the regulatory spine [] (Leu81, white; Leu70, red; Phe160, magenta; His139, blue), and phosphorylated Thr172 in (B), are in “sphere” view. Note how the residues of the regulatory spine are stacked in alignment in (B) but not in (A). In (A), the α-AID shown is that attached to the other molecule of α-KD:α-AID within the crystal dimer, but in solution the α-AID from the same molecule is thought to adopt this position [].

(A) pro-drugs converted into AMP analogs by cellular enzymes; (B) compounds that bind at the ADaM site; (C) compounds that act by inhibiting mitochondrial ATP synthesis and thus increase cellular AMP and ADP; (D) antifolate drugs that activate AMPK by inhibiting AICAR transformylase in the pathway of purine nucleotide biosynthesis (shown), thus increasing cellular ZMP. Figure modified from in [].

(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 []. (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 []; “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 []. Many direct AMPK phosphorylation sites were identified through isolation and identification by tandem mass spectrometry of the thiophosphorylated peptides []. Hydrophobic amino acids are in green, neutral polar in blue, acidic in orange, and basic in red.

The logo motif of 64 known AMPK phosphorylation sites from P−7 to P+7 is presented, and points of interest in the AMPK recognition motif are noted. Color scheme as in . The logo motif was generated by WebLogo [], using a previously described list of 50 well-validated phosphorylation sites [] updated with 14 additional sites from the literature. This full list of 64 well-validated AMPK targets is provided as . Human sequences were used to generate this motif.