AMPK: An Energy-Sensing Pathway with Multiple Inputs and Outputs

Abstract

AMP-activated protein kinase (AMPK) is a key regulator of energy balance expressed ubiquitously in eukaryotic cells. Here we review the canonical adenine nucleotide-dependent mechanism that activates AMPK when cellular energy status is compromised, as well as other, noncanonical activation mechanisms. Once activated, AMPK acts to restore energy homeostasis by promoting catabolic pathways, resulting in ATP generation, and inhibiting anabolic pathways that consume ATP. We also review the various hypothesis-driven and unbiased approaches that have been used to identify AMPK substrates and have revealed substrates involved in both metabolic and non-metabolic processes. We particularly focus on methods for identifying the AMPK target recognition motif and how it can be used to predict new substrates.

Keywords: AMPK; allosteric activation; energy sensing; kinase recognition motif; kinase target identification; pharmacological activators.

Figure 1. Canonical mechanism of activation of AMPK by adenine nucleotides, and the Ca²⁺-dependent mechanism mediated by CaMKKβ

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⁺.

Figure 2. Crystal structure of the human α1β2γ1 heterotrimer in complex with β-cyclodextrin, staurosporine, and AMP, with Thr172 phosphorylated

Atomic coordinates are from the PDB file 4RER [8]. 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.

Figure 3. Structures of the kinase domain (α-KD) and auto-inhibitory domains (α-AID) of the α subunit in (A) inactive and (B) active conformations

Atomic co-ordinates are from the PDB files 4RED (A) and 4RER (B) [8], 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 Fig. 2. Most of the structures are rendered in “cartoon” view (PyMOL v1.7.4.2), but the side chains of the regulatory spine [18] (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 [8].

Figure 4. Selection of AMPK activating compounds grouped according to their mechanisms of action

(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 Fig. 1 in [71].

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.

Figure 6. AMPK recognizes a well-defined phosphorylation motif

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 Figure 5. The logo motif was generated by WebLogo [72], using a previously described list of 50 well-validated phosphorylation sites [54] updated with 14 additional sites from the literature. This full list of 64 well-validated AMPK targets is provided as supplementary Table 1. Human sequences were used to generate this motif.