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AMP kinase (PRKAA1)
  1. Sukriti Krishan,
  2. Des R Richardson,
  3. Sumit Sahni
  1. Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales, Australia
  1. Correspondence to Dr Sumit Sahni and Prof Des Richardson, Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Blackburn Building (D06), Sydney, NSW 2006, Australia; sumit.sahni{at}sydney.edu.au

Abstract

The PRKAA1 gene encodes the catalytic α-subunit of 5′ AMP-activated protein kinase (AMPK). AMPK is a cellular energy sensor that maintains energy homeostasis within the cell and is activated when the AMP/ATP ratio increases. When activated, AMPK increases catabolic processes that increase ATP synthesis and inhibit anabolic processes that require ATP. Additionally, AMPK also plays a role in activating autophagy and inhibiting energy consuming processes, such as cellular growth and proliferation. Due to its role in energy metabolism, it could act as a potential target of many therapeutic drugs that could be useful in the treatment of several diseases, for example, diabetes. Moreover, AMPK has been shown to be involved in inhibiting tumour growth and metastasis, and has also been implicated in the pathology of neurodegenerative and cardiac disorders. Hence, a better understanding of AMPK and its role in various pathological conditions could enable the development of strategies to use it as a therapeutic target.

  • Biochemistry
  • Cancer
  • Diabetes
  • Cardiovascular

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Structure and regulation of AMPK

The PRKAA1 gene constitutes a 39 kb region located on chromosome 5p12.1 The gene encodes the 574 amino acid long 5′ AMP-activated protein kinase (AMPK) α1 protein subunit. AMPK is a heterotrimeric protein composed of three subunits: a catalytic α subunit (64 kDa) and two regulatory subunits, β (40 kDa) and γ (38 kDa),2 ,3 with each of these subunits being present as two to three isoforms.3 The protein AMPK is an energy sensor and is activated by an increase of the AMP/ATP ratio4–6 (figure 1), with AMP binding to the γ subunit of AMPK and acting as an allosteric activator.2 Along with activating AMPK, AMP also protects the protein from phosphatases that act to dephosphorylate Thr172 in the activation loop of the catalytic α subunit.7

Figure 1

Energy homeostasis mechanisms mediated by AMP-activated protein kinase (AMPK). Under energy-deficient conditions, there is an increase in the AMP/ATP ratio, which results in activation of the liver kinase B1 (LKB1)—5′ AMPK pathway. AMPK is phosphorylated at Thr172 via upstream kinases, such as LKB1, calmodulin-dependent protein kinase-β (CaMKKβ1) and transforming growth factor β activated kinase 1 (TAK1). This phosphorylation activates the enzyme and leads to further downstream kinase activity, such as phosphorylation of acetyl CoA carboxylase 1 (ACC1) and hydroxymethylglutaryl CoA reductase (HMGCR). The phosphorylation of ACC1 and HMGCR inactivates the enzymes resulting in inhibition of fatty acid and cholesterol synthesis, respectively. The end product of ACC1 activity, namely, malonyl CoA, inhibits carnitine palmitoyltransferase 1 (CPT1) which is a positive regulator of β-oxidation. Hence, the inhibition of ACC1 will lead to an increase of CPT1 activity and the oxidation of fatty acids. Additionally, AMPK directly inhibits the transcription factor, sterol regulatory element-binding protein-1 (SREBP1), which is known to regulate expression of ACC1 and fatty acid synthase (FASN). FASN is an essential enzyme involved in fatty acid synthesis. Overall, activation of AMPK leads to decreased fatty acid synthesis and increased β-oxidation, thus providing an alternate source of energy under conditions of stress.

Phosphorylation of Thr172 in the AMPK α subunit can be mediated by liver kinase B1 (LKB1)8 and also calmodulin-dependent protein kinase-β (CaMKKβ)9 ,10 (figure 1). The latter occurs in response to increased cellular calcium levels that can take place during the integrated stress response.9 ,10 Moreover, studies have shown that transforming growth factor β-activated kinase 1 (TAK1) can also activate AMPK (figure 1), but the detailed mechanism remains elusive.11 Once activated, AMPK inhibits processes that require ATP consumption with the enzyme having a multitude of downstream targets. Critical molecular processes which are affected by AMPK include (1) fatty acid12 and cholesterol synthesis13 (figure 1); (2) autophagy14 (figure 2); (3) protein synthesis and cellular proliferation (via the mammalian target of rapamycin complex 1 (mTORC1); figure 2)15 and (4) cell polarity (figure 2).16 These effects are described in detail below.

Figure 2

AMP-activated protein kinase (AMPK) regulates mammalian target of rapamycin complex (mTORC) signalling, autophagy and cell polarity. AMPK inhibits the mTORC1 via activation of the tuberous sclerosis complex 1/2 (TSC1/2) which is known to inhibit the mTORC1 activator, Ras homologue enriched in brain (Rheb). The mTORC1 is responsible for phosphorylating translation factors such as 4e-binding protein (4eBP1) and ribosomal S6 kinase (S6K). Phosphorylation of 4eBP1 prevents it from binding to the translation initiation factor, eIF4E. Thus, inhibition of mTORC1 by AMPK prevents the phosphorylation of 4eBP1, allowing it to bind to eIF4E and inhibiting it. Hence, this leads to a decrease in protein translation. AMPK also inhibits S6K-mediated protein translation via inhibition of the mTORC1 pathway. Upon activation, AMPK phosphorylates unc-51 like autophagy-activating kinase 1 (ULK1) at Ser317 and Ser777, leading to initiation of the autophagy pathway; mTORC1 is also known to phosphorylate ULK1 at Ser757 which, in turn, inhibits phosphorylation of ULK1 at Ser317 and Ser777. AMPK initiates autophagy via a direct increase in phosphorylation of ULK1 at Ser317 and Ser777, as well as by inhibition of the mTORC1-mediated suppressive effect on ULK1 phosphorylation at Ser317 and Ser777. Furthermore, AMPK phosphorylates the microtubule plus end protein, cytoplasmic linker protein-170 (CLIP-170), which is involved in microtubule dynamics. Hence, by phosphorylating CLIP-170, AMPK maintains cell polarity.

Biological functions

Role of AMPK in fatty acid and cholesterol metabolism: activation of catabolism and inhibition of anabolism

AMPK is a key molecule involved in the maintenance of cellular energy homeostasis.2 During low energy conditions, it inhibits fatty acid synthesis via phosphorylation of enzymes such as acetyl-CoA carboxylase 1 (ACC1),17 which catalyses the rate-limiting step in fatty acid synthesis18 (figure 1). Importantly, ACC1 catalyses the carboxylation of acetyl CoA to malonyl CoA,18–20 with malonyl CoA subsequently inhibiting carnitine palmitoyltransferase 1 (CPT1)21 (figure 1). CPT1 enables activated long-chain fatty acids to enter the mitochondrion for metabolism via the β-oxidation pathway,21 ,22 resulting in increased ATP synthesis.21 Hence, phosphorylation of ACC1 by AMPK results in increased activity of CPT1, leading to enhanced β-oxidation and ATP synthesis23 ,24 (figure 1).

AMPK has also been shown to inhibit the activity of sterol regulatory element-binding protein-1 (SREBP1),25 a transcription factor which upregulates the expression of fatty acid synthase (FASN) and ACC112 (figure 1). These two enzymes are essential for the de novo synthesis of fatty acids, and by inhibiting SREBP1,26 AMPK downregulates the expression of FASN and ACC1 and, therefore, inhibits fatty acid synthesis which requires ATP consumption.27 Importantly, activated AMPK is also known to phosphorylate and inhibit hydroxymethylglutaryl CoA reductase (HMGCR), which is a rate-limiting enzyme in cholesterol synthesis.13 Collectively, AMPK plays an important role in energy homeostasis via increasing catabolic pathways, including β-oxidation, and inhibiting anabolic processes, such as fatty acid and cholesterol synthesis.

Role of AMPK in initiating autophagy

Autophagy is a cellular catabolic degradation process that is induced usually as a response to starvation or stress.28 ,29 Significantly, AMPK plays an important role in the initiation of autophagy.14 Once activated by LKB1, AMPK phosphorylates unc-51-like autophagy-activating kinase 1 (ULK1) at Ser317 and Ser77714 ,30 (figure 2). These phosphorylation sites are important for ULK1 in terms of it acting as an initiator for autophagy.14

ULK1 is also phosphorylated by mTORC1 at Ser757,14 which then has an inhibitory effect on the phosphorylation of ULK1 at Ser317 and Ser777, resulting in suppression of the autophagic pathway14 (figure 2). When AMPK is activated, it results in a suppression of mTORC1 activity, leading to decreased phosphorylation of ULK1 at Ser757.14 This allows ULK1 to be phosphorylated at Ser317 and Ser777 by AMPK, which results in the initiation of autophagy (figure 2).14 Thus, AMPK initiates autophagy via a direct increase in phosphorylation of ULK1 at Ser317 and Ser777, but also by inhibition of mTORC1-mediated suppressive effect on ULK1 phosphorylation at Ser317 and Ser777.

Effect of AMPK on inhibiting protein synthesis and cell proliferation via the mTORC1 signalling pathway

The mTORC1 is a known downstream target for AMPK15 (figure 2). Significantly, mTORC1 plays a critical role as a nutrient/energy/redox sensor and controls protein synthesis.31 Notably, mTORC1 consist of mTOR1 and the regulatory-associated protein of mTOR (raptor), which phosphorylates eukaryotic translation initiation factor 4e-binding protein (4eBP1) and ribosomal S6 kinase.32 Phosphorylation of 4eBP1 results in its inability to bind and inhibit the translation initiation factor eIF4E.33 This leads to an accumulation of free eIF4E and increased protein translation.34 Additionally, phosphorylation of ribosomal S6 kinase (S6K) by mTORC1 upregulates the translation of proteins such as HIF-1α, MYC and cyclin D1.35 Thus, mTORC1 integrates nutrient and growth factor input into phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signalling, and controls cell growth in all eukaryotes.35

The mechanism of how AMPK regulates AKT signalling involves the ability of AKT to inhibit the tuberous sclerosis 1/2 (TSC1/2) complex36 (figure 2). This occurs via the ability of AKT to phosphorylate TSC2 serine residues,37 resulting in recruitment of the cytosolic anchoring protein 14-3-3 to TSC2, disrupting the TSC1/TSC2 dimer.37 When TSC2 is not associated with TSC1, TSC2 loses its GTPase activating protein activity and can no longer hydrolyse GTP bound to the Ras homologue enriched in brain (Rheb).38 The active Rheb-GTP then stimulates mTORC1 through unknown pathways38 (figure 2). This effect results in activation of mTORC1, allowing protein synthesis through insulin signalling.39

Suppression of mTORC1 by AMPK is mediated via phosphorylation of at least two proteins to rapidly induce inhibition. Indeed, AMPK has been shown to phosphorylate TSC2 at distinct serine sites from those targeted by other kinases (such as Ser1387) to activate the TSC1/2 complex, thus inhibiting Rheb (figure 2).40 ,41 This results in inhibition of mTORC1 signalling (figure 2).42 Moreover, AMPK also directly phosphorylates the critical mTORC1 binding subunit raptor in mTORC1 complex and inhibits it directly.43 Thus, AMPK acts via a dual mechanism to suppress the mTORC1 signalling pathway which is involved in increasing protein synthesis (figure 2).31 Taken together, these studies suggest that by regulating the mTORC1 signalling pathway, AMPK inhibits protein synthesis (an ATP consuming process) and activates autophagy in the cell, leading to prolonged survival.

AMPK and cell polarity

Additionally, AMPK phosphorylates the microtubule plus end protein known as cytoplasmic linker protein-170 (CLIP-170), thus regulating cell polarity via microtubule dynamics (figure 2).44 CLIP-170 directly binds freshly polymerised distal ends of growing microtubules and rapidly dissociates from the older microtubule lattice.45 Microtubules induce cell polarity by transporting protein complexes to the new end of the cell.46 Inhibition of AMPK leads to an accumulation of non-phosphorylated CLIP-170 and disturbed cell polarity,44 suggesting a role of AMPK in maintaining polarity. Collectively, AMPK plays an important role in physiological functioning of the cell by executing cellular processes critical for cell survival.

Role of AMPK in pathological conditions

AMPK is shown to be involved in the pathophysiology of a number of disorders, such as diabetes and cancer.41 ,47 This is mainly attributed to its ability to regulate important cellular processes, such as energy homeostasis, autophagy and protein synthesis.

Role of AMPK in diabetes

Since AMPK plays an important role in energy metabolism (figure 1), it has been shown to be involved in type 2 diabetes mellitus (T2DM).48 In fact, since AMPK is involved in fatty acid oxidation, it is a potential target for therapeutic intervention. Studies have shown that chronic elevation of plasma free fatty acids is commonly associated with impaired insulin-mediated glucose uptake,49 and often coexists with obesity and T2DM.49 Insulin-mediated glucose transport requires translocation of the glucose transporters (GLUT) to the plasma membrane, upregulating glucose transport into the cell.50 Considering this, it has been demonstrated that AMPK enhances GLUT4 translocation to the membrane, and this is responsible for increased glucose uptake by the cell.13 ,51 This effect is consistent with AMPK's role as an energy-conserving and ATP-generating mechanism in the cell.13 ,51

Further investigations in rodent models of the metabolic syndrome have shown that treatment with the AMPK activator, aminoimidazole carboxamide ribonucleotide (AICAR), improved insulin resistance and glucose uptake.13 ,52–54 Additionally, AICAR also prevented the development of diabetes as well as ectopic lipid deposition and degranulation of the pancreatic islet β cells.55 Moreover, AMPK activators, such as thiazolidinediones (TZD)56 and metformin57 have shown similar results in preventing the metabolic syndrome in rodents, suggesting a role of AMPK in preventing diabetes. Overall, these studies suggest AMPK plays an important role in T2DM and acts as a novel therapeutic target.

Effects of AMPK in cancer

The findings that the tumour suppressor LKB1 was the major upstream kinase required for AMPK activation8 ,58 ,59 (figure 1), introduced a link between AMPK and cancer. Activation of AMPK results in cell cycle arrest mediated through p53,60 ,61 and also regulates protein synthesis through inhibition of mTORC13 (figure 2). Moreover, the AMPK pathway includes a number of tumour suppressors, such as LKB1,8 ,58 ,59 TSC1/237 and p53,60 ,61 and overcomes growth factor signalling from PI3K, extracellular signal-regulated kinases (ERK) and AKT.35 Overall, the AMPK pathway acts to suppress tumour growth.62

Several studies have shown that AMPK also plays a role in metastasis through the regulation of cell polarity.16 ,63 ,64 Metastasis plays a critical role in the progression of tumours and is the main cause of all cancer-related deaths.65 It involves the migration and spread of tumour cells from their primary site via blood vessels and the lymphatic system to a distal location.65 Multiple molecules are responsible for this process and AMPK is believed to play a role through its effects on cell migration and polarity.16 ,63 ,64 In fact, it has been demonstrated that AMPK stimulates cell motility via microtubule polymerisation, and silencing AMPK results in disrupted front-rear polarity, as well as directional migration defects.44 Furthermore, LKB1 which phosphorylates AMPK, plays a role in cell polarisation, which is involved in the directional migration of tumour cells.16 Epithelial cells possess apical to basal cell polarity, and adhere to each other via tight junctions, desmosomes and adherens junctions.66 Loss of polarity may be responsible for the epithelial to mesenchymal transition (EMT) and subsequent tumour invasion.67

Berberine is a pharmacological agent derived from plants of the species Berberis.68 ,69 Berberine has been shown to suppress metastasis through the activation of AMPK70 which reduces metastasis and invasion by downregulating the levels of integrin β1.71 Another study has shown that berberine-induced AMPK activation inhibits the metastatic potential of melanoma cells via reduction of ERK activity and COX-2 protein expression.72 The ERK/COX-2 pathway is involved in the regulation of cell proliferation, angiogenesis and metastasis in many cancers.73 Collectively, these studies reveal that AMPK can be targeted for development of new anticancer drugs, in particular those targeting metastasis.

AMPK and cardiac hypertrophy

There is a metabolic switch observed in cardiac hypertrophy which results in decreased fatty acid usage leading to an increase in AMPK activation.74 ,75 This, in turn, leads to increased glycolytic flux via the ability of AMPK to increase translocation of the GLUT4 glucose transporter to the plasma membrane.74–76 It has been demonstrated using a pressure-overload hypertrophy model in rats that there was increased AMPK activity and glucose uptake which resulted in cardiac hypertrophy.75

Additionally, it is known that cardiac hypertrophy requires functional protein synthesis and expansion of the cellular cytoskeleton.77 Knockout of the AMPK α2 catalytic subunit in a mouse model exacerbated cardiac hypertrophy and heart failure in response to pressure overload, and this was associated with upregulated mTORC1 signalling.78 This response in the AMPK knockout mouse was probably due to the respite from the AMPK-mediated inhibition of mTORC1,43 which subsequently increased expression of proteins associated with cardiac myocyte enlargement. It has also been shown that inhibition of protein synthesis by pharmacological activation of AMPK may be a key regulatory mechanism for controlling hypertrophic cardiac growth.79

Furthermore, a recent study has demonstrated that the microtubule cytoskeleton of cardiomyocytes is a sensitive target of AMPK.77 In fact, AMPK activation in neonatal cardiomyocytes markedly reduced microtubule stability.77 More importantly, AMPK α2 knockout mice exposed to pressure overload demonstrated increased microtubule accumulation that correlated with the severity of contractile dysfunction.77 Taken together, the studies described above indicate an essential role of AMPK in regulating cardiac hypertrophy, mainly due to its ability to attenuate microtubule proliferation and inhibit protein synthesis.

AMPK in neurodegeneration

Neurons tend to have a high-energy demand, but they themselves are poor nutrient stores.80 Hence, this gap between energetic needs and capacity to store and generate energy suggests that AMPK plays an important role in maintenance and survival of neurons. Studies in Drosophila have shown that mutations in the γ subunit of AMPK lead to neurodegeneration.81 This can be explained, as the γ subunit of AMPK is an allosteric activator of this enzyme.2 Furthermore, examination of the AMPK-β knockout (alc) in the eyeless expression domain in Drosophila resulted in neurodegeneration in the retina, the optic lobe and the antennae.82

Additionally, rearing alc mutant flies in the dark resulted in reduced retinal degeneration, suggesting that neural excitation, and therefore, energy depletion caused the neurodegeneration.82 Additionally, rats subjected to ischaemic brain injury or cultured hippocampal neurons after an anoxic insult showed increased AMPK activity, which results in enhanced phosphorylation of the GABAB receptor.83 This response inhibits neurotransmitter release84 that prevents energy depletion and reduces excitotoxicity of neurons leading to their prolonged survival.83 These studies suggest that AMPK is associated with neuronal survival via prevention of energy depletion.

Collectively, these studies show an important role of AMPK in a variety of disorders and further research is required in order to explore the intricate mechanisms via which it regulates these diseases.

Summary

AMPK is an important protein involved in energy homeostasis. It leads to activation of catabolic pathways, such as β-oxidation and autophagy, and suppression of anabolic pathways (such as fatty acid synthesis) in order to provide an alternate source of energy to the cell under stressful conditions. It is also shown to be implicated in other physiological functions such as maintenance of cellular polarity. Due to the role of AMPK in energy homeostasis, it is implicated in a number of disorders, such as diabetes and cardiac hypertrophy. In conclusion, AMPK is one of the critical cellular regulators involved in physiological and pathological conditions.

Take home messages

  • PRKAA1 gene encodes AMP-activated protein kinase (AMPK)-α1 which is a catalytic subunit of AMPK.

  • AMPK is an important regulator of energy homeostasis.

  • AMPK activates catabolic processes (such as β-oxidation) and suppresses anabolic processes (such as fatty acid synthesis).

  • AMPK initiates autophagy via a dual mechanism: (1) direct activation of ULK1 at Ser317 and Ser777 and (2) inhibition of mammalian target of rapamycin complex 1 (mTORC1).

  • AMPK is implicated in pathophysiology of variety of disorders, such as diabetes, cardiac hypertrophy, cancer and so on.

References

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Footnotes

  • Contributors SK, DRR and SS were equally involved in design and writing of the article.

  • Competing interests DRR is the recipient of a National Health and Medical Research Council Senior Principal Research Fellowship and Project Grants.

  • Provenance and peer review Not commissioned; internally peer reviewed.

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