AMP-activated protein kinase

5' AMP-activated protein kinase or AMPK or 5' adenosine monophosphate-activated protein kinase is an enzyme (EC 2.7.11.31) that plays a role in cellular energy homeostasis, largely to activate glucose and fatty acid uptake and oxidation when cellular energy is low. It belongs to a highly conserved eukaryotic protein family and its orthologues are SNF1 in yeast, and SnRK1 in plants. It consists of three proteins ( subunits) that together make a functional enzyme, conserved from yeast to humans. It is expressed in a number of tissues, including the liver, brain, and skeletal muscle. In response to binding AMP and ADP, the net effect of AMPK activation is stimulation of hepatic fatty acid oxidation, ketogenesis, stimulation of skeletal muscle fatty acid oxidation and glucose uptake, inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibition of adipocyte lipogenesis, inhibition of adipocyte lipolysis, and modulation of insulin secretion by pancreatic β-cells.

It should not be confused with cyclic AMP-activated protein kinase ( protein kinase A).

Structure

AMPK is a heterotrimeric protein complex that is formed by α, β, and γ subunits. Each of these three subunits takes on a specific role in both the stability and activity of AMPK. Specifically, the γ subunit includes four particular Cystathionine-β-synthase (CBS) domains, giving AMPK its ability to sensitively detect shifts in the AMP/ ATP ratio. AMPK is deactivated upon AMP displacement by ATP at CBS site 3, suggesting CBS3 to be the primary allosteric regulatory site. The four CBS domains create two binding sites for AMP commonly referred to as Bateman domains. Binding of one AMP to a Bateman domain cooperatively increases the binding affinity of the second AMP to the other Bateman domain. As AMP binds both Bateman domains the γ subunit undergoes a conformational change which exposes the catalytic domain found on the α subunit. It is in this catalytic domain where AMPK becomes activated when phosphorylation takes place at threonine-172 (on α1 isoform) or Thr-174 (on α2 isoform) by an upstream AMPK kinase ( AMPKK). The α, β, and γ subunits can also be found in different isoforms: the γ subunit can exist as either the γ1, γ2 or γ3 isoform; the β subunit can exist as either the β1 or β2 isoform; and the α subunit can exist as either the α1 or α2 isoform. Although the most common isoforms expressed in most cells are the α1, β1, and γ1 isoforms, it has been demonstrated that the α2, β2, γ2, and γ3 isoforms are also expressed in cardiac and skeletal muscle.

The following human genes encode AMPK subunits:
* α – '' PRKAA1'', '' PRKAA2''
* β – '' PRKAB1'', '' PRKAB2''
* γ – '' PRKAG1'', '' PRKAG2'', '' PRKAG3''

The crystal structure of mammalian AMPK regulatory core domain (α C terminal, β C terminal, γ) has been solved in complex with AMP, ADP or ATP.

Regulation

Due to the presence of isoforms of its components, there are 12 versions of AMPK in mammals, each of which can have different tissue localizations, and different functions under different conditions. AMPK is regulated allosterically and by post-translational modification, which work together.

If residue Thr-172 of AMPK's α1-subunit (or Thr-174 of AMPK's α2-subunit) is phosphorylated, AMPK is activated around 100-fold; access to that residue by phosphatases is blocked if AMP or ADP can block access for and ATP can displace AMP and ADP. That residue is phosphorylated by at least three kinases ( liver kinase B1 (LKB1), which works in a complex with STRAD and MO25, Calcium/calmodulin-dependent protein kinase kinase II-( CAMKK2), and TGFβ-activated kinase 1 (TAK1)) and is dephosphorylated by three phosphatases ( protein phosphatase 2A (PP2A); protein phosphatase 2C (PP2C) and Mg²⁺-/Mn²⁺-dependent protein phosphatase 1E ( PPM1E)).

Regulation of AMPK by CaMKK2 requires a direct interaction of these two proteins via their kinase domains. The interaction of CaMKK2 with AMPK only involves the α and β subunits of AMPK (AMPK γ is absent from the CaMKK2 complex), thus rendering regulation of AMPK in this context to changes in calcium levels but not AMP or ADP.

AMPK is regulated allosterically mostly by competitive binding to the CBS sites on its γ subunit between ATP (which allows phosphatase access to Thr-172) and AMP or ADP (each of which blocks access to phosphatases). It thus appears that AMPK is a sensor of AMP/ATP or ADP/ATP ratios and thus cell energy level. AMPK undergoes a large conformational change upon ATP binding. A region on the α subunit known as the kinase domain (KD) dissociates from its active-state conformation and loosely associates with the γ subunit ~100Å away. The KD also rotates ~180° in the conformational change. Upon KD dissociation, the active loop (AL) of the α subunit which contains the critical phosphorylated Thr residue is fully exposed to upstream phosphatases. This conformational change represents a plausible mechanism for AMPK modulation. When cellular energy states are low (high AMP/ATP or ADP/ATP levels), AMPK adopts the KD-associated conformation and AMPK is protected from dephosphorylation and remains activated. When cellular energy states are high, AMPK adopts the KD-displaced conformation, the AL is exposed to upstream phosphatases, and AMPK is deactivated.

The pharmacological compounds Merck Compound 991 and Abbott A769662 bind to the allosteric drug and metabolism site (ADaM) on the β subunit and have been shown to activate AMPK up to 10-fold. ADaM site binding may have roles in AMPK activation as well as protection against dephosphorylation.

There are other mechanisms by which AMPK is inhibited or activated by insulin, leptin, and diacylglycerol by inducing various other phosphorylations.

AMPK may be inhibited or activated by various tissue-specific ubiquitinations.

It is also regulated by several protein-protein interactions, and may either be activated or inhibited by oxidative factors; the role of oxidation in regulating AMPK was controversial as of 2016.

Function

When AMPK phosphorylates acetyl-CoA carboxylase 1 (ACC1) or sterol regulatory element-binding protein 1c (SREBP1c), it inhibits synthesis of fatty acids, cholesterol, and triglycerides, and activates fatty acid uptake and β-oxidation.

AMPK stimulates glucose uptake in skeletal muscle by phosphorylating Rab-GTPase-activating protein TBC1D1, which ultimately induces fusion of GLUT4 vesicles with the plasma membrane. AMPK stimulates glycolysis by activating phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2/3 and activating phosphorylation of glycogen phosphorylase, and it inhibits glycogen synthesis through inhibitory phosphorylation of glycogen synthase. In the liver, AMPK inhibits gluconeogenesis by inhibiting transcription factors including hepatocyte nuclear factor 4 (HNF4) and CREB regulated transcription coactivator 2 (CRTC2).

AMPK inhibits the energy-intensive protein biosynthesis process and can also force a switch from cap-dependent translation to cap-independent translation, which requires less energy, by phosphorylation of TSC2, RPTOR, transcription initiation factor 1A.66, and eEF2K. When TSC2 is activated it inhibits mTORC1. As a result of inhibition of mTORC1 by AMPK, protein synthesis comes to a halt. Activation of AMPK signifies low energy within the cell, so all of the energy consuming pathways like protein synthesis are inhibited, and pathways that generate energy are activated to restore appropriate energy levels in the cell.

AMPK activates autophagy by directly and indirectly activating ULK1. AMPK also appears to stimulate mitochondrial biogenesis by regulating PGC-1α which in turn promotes gene transcription in mitochondria. AMPK also activates anti-oxidant defenses.

Clinical significance

Exercise/training

Many biochemical adaptations of skeletal muscle that take place during a single bout of exercise or an extended duration of training, such as increased mitochondrial biogenesis and capacity, increased muscle glycogen, and an increase in enzymes which specialize in glucose uptake in cells such as GLUT4 and hexokinase II are thought to be mediated in part by AMPK when it is activated. Additionally, recent discoveries can conceivably suggest a direct AMPK role in increasing blood supply to exercised/trained muscle cells by stimulating and stabilizing both vasculogenesis and angiogenesis. Taken together, these adaptations most likely transpire as a result of both temporary and maintained increases in AMPK activity brought about by increases in the AMP:ATP ratio during single bouts of exercise and long-term training.

During a single acute exercise bout, AMPK allows the contracting muscle cells to adapt to the energy challenges by increasing expression of hexokinase II, translocation of GLUT4 to the plasma membrane, for glucose uptake, and by stimulating glycolysis. If bouts of exercise continue through a long-term training regimen, AMPK and other signals will facilitate contracting muscle adaptations by escorting muscle cell activity to a metabolic transition resulting in a fatty-acid oxidation approach to ATP generation as opposed to a glycolytic approach. AMPK accomplishes this transition to the oxidative mode of metabolism by upregulating and activating oxidative enzymes such as hexokinase II, PPAR-α, PPAR-δ, PGC-1, UCP-3, cytochrome C and TFAM.

Mutations in the skeletal muscle calcium release channel ( RYR1) underlies a life- threatening response to heat in patients with malignant hyperthermia susceptibility (MHS). Upon acute exposure to heat, these mutations cause uncontrolled Ca²⁺ release from the sarcoplasmic reticulum, leading to sustained muscle contractures, severe hyperthermia, and sudden death. At basal conditions, the temperature-dependent Ca²⁺ leak also leads to increased energy demand and activation of energy sensing AMP kinase (AMPK) in skeletal muscle. The activated AMPK increases muscle metabolic activity, including glycolysis, which leads to marked elevation of circulating lactate.

AMPK activity increases with exercise and the LKB1/MO25/STRAD complex is considered to be the major upstream AMPKK of the 5’-AMP-activated protein kinase phosphorylating the α subunit of AMPK at Thr-172. This fact is puzzling considering that although AMPK protein abundance has been shown to increase in skeletal tissue with endurance training, its level of activity has been shown to decrease with endurance training in both trained and untrained tissue. Currently, the activity of AMPK immediately following a 2 hour bout of exercise of an endurance trained rat is unclear. It is possible that a direct link exists between the observed decrease in AMPK activity in endurance trained skeletal muscle and the apparent decrease in the AMPK response to exercise with endurance training.

Although AMPKα2 activation has been thought to be important for mitochondrial adaptations to exercise training, a recent study investigating the response to exercise training in AMPKα2 knockout mice opposes this idea. Their study compared the response to exercise training of several proteins and enzymes in wild type and AMPKα2 knockout mice. And even though the knockout mice had lower basal markers of mitochondrial density (COX-1, CS, and HAD), these markers increased similarly to the wild type mice after exercise training. These findings are supported by another study also showing no difference in mitochondrial adaptations to exercise training between wild type and knockout mice.

Maximum life span

The ''C. elegans'' homologue of AMPK, aak-2, has been shown by Michael Ristow and colleagues to be required for extension of life span in states of glucose restriction mediating a process named mitohormesis.

Lipid metabolism

One of the effects of exercise is an increase in fatty acid metabolism, which provides more energy for the cell. One of the key pathways in AMPK's regulation of fatty acid oxidation is the phosphorylation and inactivation of acetyl-CoA carboxylase. Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 ( CPT-1). CPT-1 transports fatty acids into the mitochondria for oxidation. Inactivation of ACC, therefore, results in increased fatty acid transport and subsequent oxidation. It is also thought that the decrease in malonyl-CoA occurs as a result of malonyl-CoA decarboxylase (MCD), which may be regulated by AMPK. MCD is an antagonist

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