John W Scott

Management of cellular energy by the AMP-activated protein kinase system

The AMP‐activated protein kinase is a sensor of cellular energy status that is found in all eukaryotic cells. It is activated by rising AMP and falling ATP by a complex mechanism that results in an ultrasensitive response. The functions of the different domains on the three subunits of the αβγ heterotrimer are slowly being unravelled, and a recent development has been the identification of a glycogen‐binding domain on the β subunit. Along with findings that high cellular glycogen represses kinase activation, this suggests that the system may be a sensor of glycogen content as well as of AMP and ATP. New insights have been obtained into the sequence and structural features by which the kinase recognises its downstream target proteins, and these are discussed. Once activated by depletion of cellular energy reserves, the kinase switches on ATP‐producing catabolic pathways and switches off ATP‐consuming processes, both via direct phosphorylation of regulatory proteins and via indirect effects on gene expression. A survey of the range of downstream targets for this important signalling pathway is presented.

AMP-activated protein kinase – development of the energy sensor concept

The LKB1→AMPK cascade is switched on by metabolic stresses that either inhibit ATP production (e.g. hypoxia, hypoglycaemia) or that accelerate ATP consumption (e.g. muscle contraction). Any decline in cellular energy status is accompanied by a rise in the cellular AMP: ATP ratio, and this activates AMPK by a complex and sensitive mechanism involving antagonistic binding of the nucleotides to two sites on the regulatory γ subunits of AMPK. Once activated by metabolic stress, AMPK activates catabolic pathways that generate ATP, while inhibiting cell growth and biosynthesis and other processes that consume ATP. While the AMPK system probably evolved in single‐celled eukaryotes to maintain energy balance at the cellular level, in multicellular organisms its role has become adapted so that it is also involved in maintaining whole body energy balance. Thus, it is regulated by hormones and cytokines, especially the adipokines leptin and adiponectin, increasing whole body energy expenditure while regulating food intake. Some hormones may activate AMPK by an LKB1‐independent mechanism involving Ca2+/calmodulin dependent protein kinase kinases. Low levels of activation of AMPK are likely to play a role in the current global rise in obesity and Type 2 diabetes, and AMPK is the target for the widely used antidiabetic drug metformin.

CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations

CBS domains are defined as sequence motifs that occur in several different proteins in all kingdoms of life. Although thought to be regulatory, their exact functions have been unknown. However, their importance was underlined by findings that mutations in conserved residues within them cause a variety of human hereditary diseases, including (with the gene mutated in parentheses): Wolff-Parkinson-White syndrome (gamma 2 subunit of AMP-activated protein kinase); retinitis pigmentosa (IMP dehydrogenase-1); congenital myotonia, idiopathic generalized epilepsy, hypercalciuric nephrolithiasis, and classic Bartter syndrome (CLC chloride channel family members); and homocystinuria (cystathionine beta-synthase). AMP-activated protein kinase is a sensor of cellular energy status that is activated by AMP and inhibited by ATP, but the location of the regulatory nucleotide-binding sites (which are prime targets for drugs to treat obesity and diabetes) was not characterized. We now show that tandem pairs of CBS domains from AMP-activated protein kinase, IMP dehydrogenase-2, the chloride channel CLC2, and cystathionine beta-synthase bind AMP, ATP, or S-adenosyl methionine,while mutations that cause hereditary diseases impair this binding. This shows that tandem pairs of CBS domains act, in most cases, as sensors of cellular energy status and, as such, represent a newly identified class of binding domain for adenosine derivatives.

Analysis of the LKB1-STRAD-MO25 complex

Mutations in the LKB1 tumour suppressor threonine kinase cause the inherited Peutz-Jeghers cancer syndrome and are also observed in some sporadic cancers. Recent work indicates that LKB1 exerts effects on metabolism, polarity and proliferation by phosphorylating and activating protein kinases belonging to the AMPK subfamily. In vivo, LKB1 forms a complex with STRAD, an inactive pseudokinase, and MO25, an armadillo repeat scaffolding-like protein. Binding of LKB1 to STRAD-MO25 activates LKB1 and re-localises it from the nucleus to the cytoplasm. To learn more about the inherent properties of the LKB1-STRAD-MO25 complex, we first investigated the activity of 34 point mutants of LKB1 found in human cancers and their ability to interact with STRAD and MO25. Interestingly, 12 of these mutants failed to interact with STRAD-MO25. Performing mutagenesis analysis, we defined two binding sites located on opposite surfaces of MO25α, which are required for the assembly of MO25α into a complex with STRADα and LKB1. In addition, we demonstrate that LKB1 does not require phosphorylation of its own T-loop to be activated by STRADα-MO25α, and discuss the possibility that this unusual mechanism of regulation arises from LKB1 functioning as an upstream kinase. Finally, we establish that STRADα, despite being catalytically inactive, is still capable of binding ATP with high affinity, but that this is not required for activation of LKB1. Taken together, our findings reinforce the functional importance of the binding of LKB1 to STRAD, and provide a greater understanding of the mechanism by which LKB1 is regulated and activated through its interaction with STRAD and MO25.

Fatal Congenital Heart Glycogenosis Caused by a Recurrent Activating R531Q Mutation in the γ2-Subunit of AMP-Activated Protein Kinase (PRKAG2), Not by Phosphorylase Kinase Deficiency

Fatal congenital nonlysosomal cardiac glycogenosis has been attributed to a subtype of phosphorylase kinase deficiency, but the underlying genes and mutations have not been identified. Analyzing four sporadic, unrelated patients, we found no mutations either in the eight genes encoding phosphorylase kinase subunits or in the two genes encoding the muscle and brain isoforms of glycogen phosphorylase. However, in three of five patients, we identified identical heterozygous R531Q missense mutations of the PRKAG2 gene, which encodes the gamma 2-subunit of AMP-activated protein kinase, a key regulator of energy balance. Biochemical characterization of the recombinant R531Q mutant protein showed >100-fold reduction of binding affinities for the regulatory nucleotides AMP and ATP but an enhanced basal activity and increased phosphorylation of the alpha -subunit. Other PRKAG2 missense mutations were previously identified in patients with autosomal dominant hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome, characterized by juvenile-to-adult clinical onset, moderate cardiac glycogenosis, disturbed excitation conduction, risk of sudden cardiac death in midlife, and molecular perturbations that are similar to--but less severe than--those observed for the R531Q mutation. Thus, recurrent heterozygous R531Q missense mutations in PRKAG2 give rise to a massive nonlysosomal cardiac glycogenosis of fetal symptomatic onset and rapidly fatal course, constituting a genotypically and clinically distinct variant of hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome. R531Q and other PRKAG2 mutations enhance the basal activity and alpha -subunit phosphorylation of AMP-activated protein kinase, explaining the dominant nature of PRKAG2 disease mutations. Since not all cases displayed PRKAG2 mutations, fatal congenital nonlysosomal cardiac glycogenosis seems to be genetically heterogeneous. However, the existence of a heart-specific primary phosphorylase kinase deficiency is questionable, because no phosphorylase kinase mutations were found.

Phenformin and 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) activation of AMP‐activated protein kinase inhibits transepithelial Na+ transport across H441 lung cells

Active re‐absorption of Na+ across the alveolar epithelium is essential to maintain lung fluid balance. Na+ entry at the luminal membrane is predominantly via the amiloride‐sensitive Na+ channel (ENaC) down its electrochemical gradient. This gradient is generated and maintained by basolateral Na+ extrusion via Na+,K+‐ATPase an energy‐dependent process. Several kinases and factors that activate them are known to regulate these processes; however, the role of AMP‐activated protein kinase (AMPK) in the lung is unknown. AMPK is an ultra‐sensitive cellular energy sensor that monitors energy consumption and down‐regulates ATP‐consuming processes when activated. The biguanide phenformin has been shown to independently decrease ion transport processes, influence cellular metabolism and activate AMPK. The AMP mimetic drug 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside (AICAR) also activates AMPK in intact cells. Western blotting revealed that both the α1 and α2 catalytic subunits of AMPK are present in Na+ transporting H441 human lung epithelial cells. Phenformin and AICAR increased AMPK activity in H441 cells in a dose‐dependent fashion, stimulating the kinase maximally at 5–10 mm (P= 0.001, n= 3) and 2 mm (P < 0.005, n= 3), respectively. Both agents significantly decreased basal ion transport (measured as short circuit current) across H441 monolayers by approximately 50% compared with that of controls (P < 0.05, n= 4). Neither treatment altered the resistance of the monolayers. Phenformin and AICAR significantly reduced amiloride‐sensitive transepithelial Na+ transport compared with controls (P < 0.05, n= 4). This was a result of both decreased Na+,K+‐ATPase activity and amiloride‐sensitive apical Na+ conductance. Transepithelial Na+ transport decreased with increasing concentrations of phenformin (0.1–10 mm) and showed a significant correlation with AMPK activity. Taken together, these results show that phenformin and AICAR suppress amiloride‐sensitive Na+ transport across H441 cells via a pathway that includes activation of AMPK and inhibition of both apical Na+ entry through ENaC and basolateral Na+ extrusion via the Na+,K+‐ATPase. These are the first studies to provide a cellular signalling mechanism for the action of phenformin on ion transport processes, and also the first studies showing AMPK as a regulator of Na+ absorption in the lung.

Regulation of AMP‐activated protein kinase by a pseudosubstrate sequence on the γ subunit

The AMP‐activated protein kinase (AMPK) system monitors cellular energy status by sensing AMP and ATP, and is a key regulator of energy balance at the cellular and whole‐body levels. AMPK exists as heterotrimeric αβγ complexes, and the γ subunits contain two tandem domains that bind the regulatory nucleotides. There is a sequence in the first of these domains that is conserved in γ subunit homologues in all eukaryotes, and which resembles the sequence around sites phosphorylated on target proteins of AMPK, except that it has a non‐phosphorylatable residue in place of serine. We propose that in the absence of AMP this pseudosubstrate sequence binds to the active site groove on the α subunit, preventing phosphorylation by the upstream kinase, LKB1, and access to downstream targets. Binding of AMP causes a conformational change that prevents this interaction and relieves the inhibition. We present several lines of evidence supporting this hypothesis.

Fatal infantile cardiac glycogenosis with phosphorylase kinase deficiency and a mutation in the γ2-subunit of AMP-activated protein kinase

A 10-wk-old infant girl with severe hypertrophy of the septal and atrial walls by cardiac ultrasound, developed progressive ventricular wall thickening and died of aspiration pneumonia at 5 mo of age. Postmortem examination revealed ventricular hypertrophy and massive atrial wall thickening due to glycogen accumulation. A skeletal muscle biopsy showed increased free glycogen and decreased activity of phosphorylase b kinase (PHK). The report of a pathogenic mutation (R531Q) in the gene (PRKAG2) encoding the γ2 subunit of AMP-activated protein kinase (AMPK) in three infants with congenital hypertrophic cardiomyopathy, glycogen storage, and “pseudo PHK deficiency” prompted us to screen this gene in our patient. We found a novel (R384T) heterozygous mutation in PRKAG2, affecting an arginine residue in the N-terminal AMP-binding domain. Like R531Q, this mutation reduces the binding of AMP and ATP to the isolated nucleotide-binding domains, and prevents activation of the heterotrimer by metabolic stress in intact cells. The mutation was not found in DNA from the patients father, the only available parent, and is likely to have arisen de novo. Our studies confirm that mutations in PRKAG2 can cause fatal infantile cardiomyopathy, often associated with apparent PHK deficiency.