AMPK (AMP-activated protein kinase)
Protein Phosphorylation

Author: Gianpiero Pescarmona
Date: 18/06/2009

Description

DEFINITION

AMP-activated protein kinase or AMPK is an enzyme that plays a role in cellular energy homeostasis.

The net effect of AMPK activation is:

  • stimulation of hepatic fatty acid oxidation and ketogenesis
  • inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis
  • inhibition of adipocyte lipolysis and lipogenesis
  • stimulation of skeletal muscle fatty acid oxidation and muscle glucose uptake
  • modulation of insulin secretion by pancreatic beta-cells
Database"URL":
WikigenesURL
GeneCards"URL":
iHOP"URL":
OMIM"URL":

CHEMICAL STRUCTURE AND IMAGES

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 beta synthase (CBS) domains giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio. 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 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.

When relevant for the function

  • Primary structure
  • Secondary structure
  • Tertiary structure
  • Quaternary structure

Protein Aminoacids Percentage (Width 700 px)

From the evolutionary point of view the sequence is from the oldest to the newest:

mTOR --> AAPK1 --> AAKB1 --> AAKG1

mTOR regulation of protein synthesis was probably strictly regulated by AA availability, mainly by methionine and leucine
Later on the control of the activity was dependent on AAPK1, on AAPK1+AAPKB1 and finally in recent times by AAPK1+AAPKB1+AAKG1

SYNTHESIS AND TURNOVER

mRNA synthesis
protein synthesis
post-translational modifications
degradation

CELLULAR FUNCTIONS

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.

cellular localization,

Nuclear/cytoplasmic localization

Localization of AMP kinase is regulated by stress, cell density, and signaling through the MEK→ERK1/2 pathway, 2007

Environmental stress regulates the intracellular localization of AMPK, and upon recovery from heat shock or oxidant exposure AMPK accumulates in the nuclei. We show that under normal growth conditions AMPK shuttles between the nucleus and the cytoplasm, a process that depends on the nuclear exporter Crm1. However, nucleocytoplasmic shuttling does not take place in high-density cell cultures, for which AMPK is confined to the cytoplasm. Furthermore, we demonstrate that signaling through the mitogen-activated protein kinase kinase (MEK)→extracellular signal-regulated kinase 1/2 (ERK1/2) cascade plays a crucial role in controlling the proper localization of AMPK.

biological function

The core mechanism of the mammalian circadian clock and its link to energy metabolism. (A) High NADH levels promote CLOCK:BMAL1 binding to E-box sequences leading to the acetylation of BMAL1 and expression of Pers, Crys, and other clock-controlled genes. The negative feedback loop, PERs:CRYs, binds to CLOCK:BMAL1 and consequently PERs are acetylated. Activated AMPK leads to a rise in NAD+ levels, phosphorylation of CRYs, and phosphorylation of CKI?, which then phosphorylates the PERs. As a result of increased NAD+ levels, SIRT1 deacetylates PERs and BMAL1. This and the destabilization of phosphorylated PERs and CRYs relieves PERs:CRYs repression and another cycle starts. (B) Expression of Bmal1 and Rev-erbα genes are controlled by PPARα and binding of RORs to RORE sequences. RORs need a co-activator, PGC-1α, which is phosphorylated by activated AMPK. In parallel, AMPK activation leads to an increase in NAD+ levels, which, in turn activate SIRT1. SIRT1 activation leads to PGC-1α deacetylation and activation. Acetyl adenosine diphosphate ribose (Ac-ADP-r) and nicotinamide (NAM) are released after deacetylation by SIRT1.
* Enzymes
* Cell signaling and Ligand transport
* Structural proteins

REGULATION

DOWNSTREAM EFFECTS

Fatty Acid metabolism

Acta Physiol (Oxf). 2009 May;196(1):27-35. Epub 2009 Feb 19.
Regulation of glucose transporter 4 traffic by energy deprivation from mitochondrial compromise. 2009

Klip A, Schertzer JD, Bilan PJ, Thong F, Antonescu C.

Cell Biology Program, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada. amira@sickkids.ca
Abstract

Skeletal muscle is the major store and consumer of fatty acids and glucose. Glucose enters muscle through glucose transporter 4 (GLUT4). Upon insufficient oxygen availability or energy compromise, aerobic metabolism of glucose and fatty aids cannot proceed, and muscle cells rely on anaerobic metabolism of glucose to restore cellular energy status. An increase in glucose uptake into muscle is a key response to stimuli requiring rapid energy supply. This chapter analyses the mechanisms of the adaptive regulation of glucose transport that rescue muscle cells from mitochondrial uncoupling. Under these conditions, the initial drop in ATP recovers rapidly, through a compensatory increase in glucose uptake. This adaptive response involves AMPK activation by the initial ATP drop, which elevates cell surface GLUT4 and glucose uptake. The gain in surface GLUT4 involves different signals and routes of intracellular traffic compared with those engaged by insulin. The hormone increases GLUT4 exocytosis through phosphatidylinositol 3-kinase and Akt, whereas energy stress retards GLUT4 endocytosis through AMPK and calcium inputs. Given that energy stress is a component of muscle contraction, and that contraction activates AMPK and raises cytosolic calcium, we hypothesize that the increase in glucose uptake during contraction may also involve a reduction in GLUT4 endocytosis.

AMPK Activation via Modulation of De Novo Purine Biosynthesis with an Inhibitor of ATIC Homodimerization, 2015(15)00234-3

DIAGNOSTIC USE


Coenzyme Q10 increases the fatty acid oxidation through AMPK-mediated PPARα induction in 3T3-L1 preadipocytes. 2012

The promoter activity of PPARα was increased by CoQ10 in an AMPK-dependent fashion

Comments
2009-09-28T15:08:46 - edoardo giai via

Edoardo Giai Via
Eleonora Orsucci

DEFINITION

AMP-activated protein kinase or AMPK is an enzyme that plays a role in cellular energy homeostasis.
The net effect of AMPK activation is:

  • stimulation of hepatic fatty acid oxidation and ketogenesis
  • inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis
  • inhibition of adipocyte lipolysis and lipogenesis
  • stimulation of skeletal muscle fatty acid oxidation and muscle glucose uptake
  • modulation of insulin secretion by pancreatic beta-cells
Database"URL":
WikigenesURL
GeneCards"URL":
iHOP"URL":
OMIM"URL":

CHEMICAL STRUCTURE AND IMAGES

The heterotrimeric protein AMPK 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 beta synthase (CBS) domains giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio.
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 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.
The α2 isoform is the subunit of AMPK found predominantly within skeletal and cardiac muscle, whereas, approximately equal distribution of both the α1 and α2 isoforms are present in hepatic AMPK.
the β subunits of AMPK have a glycogen-binding domain, GBD
Within pancreatic islet β-cells the α1 isoform predominates.
.
The N-terminal half of the α subunits contains a typical serine/threonine kinase catalytic domain. Interaction with the β and γ subunits occurs via the C-terminal half of the α subunits.
The yeast AMPK β subunits are lipid modified with myristic acid. Myristoylation may account for the membrane association of mammalian AMPK. The core of the β subunits have a glycogen-binding domain (GBD). This domain is closely related to the isoamylase N domain subfamily and weakly related to domains in the glycogen-targeting phosphatase subunits and several starch-binding proteins. The close proximity of AMPK to cellular glycogen stores allows it to rapidly effect changes in glycogen metabolism in response to changes in metabolic demands.
The γ subunits of AMPK have been shown to contain nucleotide binding sites with similarity to cystathionine β-synthase (CBS) domains. Indeed, direct AMP-binding studies have shown that AMP is bound to the γ subunits by a pair of CBS domains.

h3. CELLULAR FUNCTIONS

It is expressed in a number of tissues, including the liver, brain, and skeletal muscle.
The signaling cascades initiated by the activation of AMPK exert effects on glucose and lipid metabolism, gene expression and protein synthesis. These effects are most important for regulating metabolic events in the liver, skeletal muscle, heart, adipose tissue, and pancreas.

REGULATION

In the presence of AMP the activity of AMPK is increased approximately 5-fold. However, more importantly is the role of AMP in regulating the level of phosphorylation of AMPK. An increased AMP to ATP ratio leads to a conformational change in the γ-subunit leading to increased phosphorylation and decreased dephosphorylation of AMPK. The phosphorylation of AMPK results in activation by at least 100-fold. AMPK is phosphorylated by at least three different upstream AMPK kinases (AMPKKs). Phosphorylation of AMPK occurs in the α subunit at threonine 172 (T172) which lies in the activation loop.
As the name implies, AMPK is also regulated by AMP. The effects of AMP are two-fold: a direct allosteric activation and making AMPK a poorer substrate for dephosphorylation. Because AMP affects both the rate of AMPK phoshorylation in the positive direction and dephosphorylation in the negative direction, the cascade is ultrasensitive. This means that a very small rise in AMP levels can induce a dramatic increase in the activity of AMPK. The activity of adenylate kinase, catalyzing the reaction shown below, ensures that AMPK is highly sensitive to small changes in the intracellular [ATP]/[ADP] ratio. 2 ADP ——> ATP + AMP

One kinase activator of AMPK is Ca2+-calmodulin-dependent kinase kinase β (CaMKKβ) which phosphorylates and activates AMPK in response to increased calcium. The distribution of CaMKKβ expression is primarily in the brain with detectable levels also found in the testes, thymus, and T cells. As described for the Ca2+-mediated regulation of glycogen metabolism, increased release of intracellular stores of Ca2+ create a subsequent demand for ATP. Activation of AMPK in response to Ca fluxes provides a mechanism for cells to anticipate the increased demand for ATP.

Evidence has also demonstrated that the serine-threonine kinase, LKB1(also called serine-threonine kinase 11, STK11) which is encoded by the Peutz-Jeghers syndrome tumor suppressor gene, is required for activation of AMPK in response to stress.
Unlike the limited distribution of CaMKKβ, LKB1 is widely expressed, thus making it the primary AMPK-regulating kinase. Loss of LKB1 activity in adult mouse liver leads to near complete loss of AMPK activity and is associated with hyperglycemia (The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin, 2005). The hyperglycemia is, in part, due to an increase in the transcription of gluconeogenic genes. Of particular significance is the increased expression of the peroxisome proliferator-activated receptor-γ (PPAR-γ) coactivator 1α (PGC-1α) which drives gluconeogenesis. Reduction in PGC-1α activity results in normalized blood glucose levels in LKB1-deficient mice.
The third AMPK phosphorylating kinase is transforming growth factor-β-activated kinase 1 (TAK1). However, the normal physiological conditions under which TAK1 phosphorylates AMPK are currently unclear.

Negative allosteric regulation of AMPK also occurs and this effect is exerted by phosphocreatine. As indicated above, the β subunits of AMPK have a glycogen-binding domain, GBD.
In muscle, a high glycogen content represses AMPK activity and this is likely the result of interaction between the GBD and glycogen, although this has not been shown directly. As suggested above, the GBD of AMPK allows association of the enzyme with the regulation of glycogen metabolism by placing AMPK in close proximity to one of its substrates glycogen synthase.
AMPK has also been shown to be activated by receptors that are coupled to phospholipase C-γ (PLC-γ) and by hormones secreted by adipose tissue (termed adipokines) such as leptin and adiponectin

Whereas, stress and exercise are powerful inducers of AMPK activity in skeletal muscle, additional regulators of its activity have been identified.
Insulin-sensitizing drugs of the thiazolidinedione family (activators of PPAR-γ, see below) as well as the hypoglycemia drug metformin exert a portion of their effects through regulation of the activity of AMPK. As indicated above, the activity of the AMPK activating kinase, LKB1, is critical for regulation of gluconeogenic flux and consequent glucose homeostasis. The action of metformin in reducing blood glucose levels requires the activity of LKB1 in the liver for this function. Also, several adipokines (hormones secreted by adipocytes) either stimulate or inhibit AMPK activation: leptin and adiponectin have been shown to stimulate AMPK activation, whereas, resistin inhibits AMPK activation.

EFFECTS

  • AMPK increases eNOS activity, phosphorylating it in serin 1177. Cardiac effects exerted by activation of AMPK also include phosphorylation of endothelial nitric oxide synthase, eNOS in cardiac endothelium. AMPK-mediated phosphorylation of eNOS leads to increased activity and consequent NO production and provides a link between metabolic stresses and cardiac function. In platelets, insulin action leads to an increase in eNOS activity that is due to its phosphorylation by AMPK. Activation of NO production in platelets leads to a decrease in thrombin-induced aggregation, thereby, limiting the pro-coagulant effects of platelet activation. The response of platelets to insulin function clearly indicates why disruption in insulin action is a major contributing factor in the development of the metabolic syndrome Endothelial nitric oxide synthase phosphorylation in treadmill-running mice: role of vascular signalling kinases,2009.

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