Trends in Cell Biology
Volume 26, Issue 3, March 2016, Pages 190-201
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Review
AMPK: An Energy-Sensing Pathway with Multiple Inputs and Outputs

https://doi.org/10.1016/j.tcb.2015.10.013Get rights and content

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AMPK is an energy-sensing protein kinase activated by phosphorylation of Thr172 within its catalytic α subunit. It binds AMP and/or ADP, both signals of energy stress, via its regulatory γ subunit. This activates the kinase by promoting Thr172 phosphorylation, inhibiting Thr172 dephosphorylation and allosteric activation.

AMP binding to the γ subunit causes its interaction with the α linker region of the α subunit. This pulls the autoinhibitory domain on the α subunit away from the kinase domain, triggering activation.

AMPK is also activated by the binding of synthetic and naturally occurring drug-like molecules that bind in the allosteric drug and metabolite (ADaM) site between the α and β subunits.

AMPK has a well-defined recognition motif that has been established both by hypothesis-driven approaches and by various unbiased screens. It now has over 60 well-validated substrates.

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.

Section snippets

AMPK: Subunit Structure and Regulation

AMPK is a key sensor of cellular energy status present in essentially all eukaryotic cells, where it occurs as heterotrimers comprising catalytic α subunits and regulatory β and γ subunits 1, 2, 3. Genes encoding at least one of these subunits are found in the genomes of essentially all eukaryotes, while mammals have genes encoding multiple isoforms (α1, α2; β1, β2; γ1, γ2, γ3). AMPK heterotrimers are normally significantly active only after phosphorylation of a conserved threonine residue

Canonical Inputs: Adenine Nucleotide Binding to the AMPK γ Subunit

AMPK senses changes in AMP through its direct binding to the γ subunit. AMPK γ subunits in all species contain four tandem repeats of sequence motifs known as cystathionine-β-synthase (CBS) repeats (see Glossary). These also occur in a small number of other proteins in the human genome, although usually as just two tandem repeats. In many cases, each tandem pair of repeats binds a regulatory adenosine-containing ligand, such as ATP or S-adenosyl methionine, in the cleft between the repeats [9].

Outputs: Identification of Downstream Targets

Once AMPK is activated, it acts to restore energy homeostasis by activating catabolic pathways that generate ATP and inhibiting anabolic pathways that consume ATP. However, since the great majority of cellular processes consume energy and most are coupled to ATP hydrolysis, there is no reason why processes switched off by AMPK should be restricted to metabolic roles. There has therefore been a need to understand how AMPK recognizes its target phosphorylation sites and to develop methods to

Prediction of Novel Targets Using the AMPK Recognition Motif

AMPK now has a recognition motif (Figure 6) that is among the best defined of any protein kinase, making it potentially useful for predicting novel targets. It is important to note, however, that there are likely to be many sites that conform to this motif that are not targets for AMPK, either because they are not accessible to the kinase due to some aspect of their structure or protein–protein interaction or because of their subcellular location. Since important phosphorylation sites are

Concluding Remarks

Some important unresolved issues regarding the inputs and outputs impinging on AMPK are outlined in the Outstanding Questions. AMPK is now known to be activated through various upstream inputs, including the canonical mechanism involving sensing of adenine nucleotide ratios, noncanonical mechanisms such as those used by compounds that bind at the ADaM site, and the Ca2+-dependent pathway involving CaMKKβ.

The number of outputs (i.e., identified downstream targets) is also expanding rapidly. The

Note Added to Proof

A manuscript [73] describing a phosphoproteomic approach to identify AMPK targets during exercise in human muscle has just been published.

Acknowledgments

Research in the D.G.H. laboratory is funded by the Wellcome Trust (097726) and a Programme Grant (C37030/A15101) from Cancer Research UK. Research in the A.B. laboratory is supported by grants CIRM RB4-06087 and NIH R01 AG031198 (A.B.), NSF GRFP (B.E.S.), the Robert M. and Anne T. Bass Stanford Graduate Fellowship (B.E.S.), and NIH T32 CA09302 (B.E.S.). The authors thank all of those researchers who have contributed to the development of the AMPK story whose work they could not cite due to

Glossary

α linker
region of the AMPK α subunit that connects the α-AID to the C-terminal domain, important in the mechanism of regulation by AMP.
α regulatory subunit interacting motif (α-RIM1/α-RIM2)
conserved sequences within the α linker that interact with the AMPK γ subunit when AMP is bound at site 3.
α subunit autoinhibitory domain (α-AID)
the domain that follows the kinase domain in AMPK α subunits and that inhibits the kinase domain in the absence of AMP.
α subunit kinase domain (α-KD)
kinase domains

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