Jonathan S. Oakhill

AMPK Is a Direct Adenylate Charge-Regulated Protein Kinase

The cellular energy sensor adenosine monophosphate–activated protein kinase also binds and is regulated by adenosine diphosphate. The adenosine monophosphate (AMP)–activated protein kinase (AMPK) regulates whole-body and cellular energy balance in response to energy demand and supply. AMPK is an αβγ heterotrimer activated by decreasing concentrations of adenosine triphosphate (ATP) and increasing AMP concentrations. AMPK activation depends on phosphorylation of the α catalytic subunit on threonine-172 (Thr172) by kinases LKB1 or CaMKKβ, and this is promoted by AMP binding to the γ subunit. AMP sustains activity by inhibiting dephosphorylation of α-Thr172, whereas ATP promotes dephosphorylation. Adenosine diphosphate (ADP), like AMP, bound to γ sites 1 and 3 and stimulated α-Thr172 phosphorylation. However, in contrast to AMP, ADP did not directly activate phosphorylated AMPK. In this way, both ADP/ATP and AMP/ATP ratios contribute to AMPK regulation.

β-Subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase (AMPK)

The AMP-activated protein kinase (AMPK) is an αβγ heterotrimer that acts as a master metabolic regulator to maintain cellular energy balance following increased energy demand and increases in the AMP/ATP ratio. This regulation provides dynamic control of energy metabolism, matching energy supply with demand that is essential for the function and survival of organisms. AMPK is inactive unless phosphorylated on Thr172 in the α-catalytic subunit activation loop by upstream kinases (LKB1 or calcium-calmodulin-dependent protein kinase kinase β). How a rise in AMP levels triggers AMPK α-Thr172 phosphorylation and activation is incompletely understood. Here we demonstrate unequivocally that AMP directly stimulates α-Thr172 phosphorylation provided the AMPK β-subunit is myristoylated. Loss of the myristoyl group abolishes AMP activation and reduces the extent of α-Thr172 phosphorylation. Once AMPK is phosphorylated, AMP further activates allosterically but this activation does not require β-subunit myristoylation. AMP and glucose deprivation also promote membrane association of myristoylated AMPK, indicative of a myristoyl-switch mechanism. Our results show that AMP regulates AMPK activation at the initial phosphorylation step, and that β-subunit myristoylation is important for transducing the metabolic stress signal.

Thienopyridone Drugs Are Selective Activators of AMP-Activated Protein Kinase β1-Containing Complexes

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that plays a pivotal role in regulating cellular and whole-body metabolism. Activation of AMPK reverses many of the metabolic defects associated with obesity and type 2 diabetes, and therefore AMPK is considered a promising target for drugs to treat these diseases. Recently, the thienopyridone A769662 has been reported to directly activate AMPK by an unexpected mechanism. Here we show that A769662 activates AMPK by a mechanism involving the beta subunit carbohydrate-binding module and residues from the gamma subunit but not the AMP-binding sites. Furthermore, A769662 exclusively activates AMPK heterotrimers containing the beta1 subunit. Our findings highlight the regulatory role played by the beta subunit in modulating AMPK activity and the possibility of developing isoform specific therapeutic activators of this important metabolic regulator.

AMPK-independent pathways regulate skeletal muscle fatty acid oxidation.

The activation of AMP‐activated protein kinase (AMPK) and phosphorylation/inhibition of acetyl‐CoA carboxylase 2 (ACC2) is believed to be the principal pathway regulating fatty acid oxidation. However, during exercise AMPK activity and ACC Ser‐221 phosphorylation does not always correlate with rates of fatty acid oxidation. To address this issue we have investigated the requirement for skeletal muscle AMPK in controlling aminoimidazole‐4‐carboxymide‐1‐β‐d‐ribofuranoside (AICAR) and contraction‐stimulated fatty acid oxidation utilizing transgenic mice expressing a muscle‐specific kinase dead (KD) AMPK α2. In wild‐type (WT) mice, AICAR and contraction increased AMPK α2 and α1 activities, the phosphorylation of ACC2 and rates of fatty acid oxidation while tending to reduce malonyl‐CoA levels. Despite no activation of AMPK in KD mice, ACC2 phosphorylation was maintained, malonyl‐CoA levels were reduced and rates of fatty acid oxidation were comparable between genotypes. During treadmill exercise both KD and WT mice had similar values of respiratory exchange ratio. These studies suggested the presence of an alternative ACC2 kinase(s). Using a phosphoproteomics‐based approach we identified 18 Ser/Thr protein kinases whose phosphorylation was increased by greater than 25% in contracted KD relative to WT muscle. Utilizing bioinformatics we predicted that extracellular regulated protein‐serine kinase (ERK1/2), inhibitor of nuclear factor (NF)‐κB protein‐serine kinase β (IKKβ) and protein kinase D (PKD) may phosphorylate ACC2 at Ser‐221 but during in vitro phosphorylation assays only AMPK phosphorylated ACC2. These data demonstrate that AMPK is not essential for the regulation of fatty acid oxidation by AICAR or muscle contraction.

AMPK functions as an adenylate charge-regulated protein kinase

The energy sensor AMP-activated protein kinase (AMPK) is activated by metabolic stress and restores ATP levels in cells by switching off anabolic and switching on catabolic pathways. Recent discoveries demonstrate that AMPK is activated primarily by rising ADP levels and not, as previously thought, by AMP. AMPK activation is dependent on ADP-controlled phosphorylation of Thr172 on its activation loop, a mechanism of protein regulation that represents an example of an allosterically regulated modification (ARM). AMPK embodies many features of an adenylate charge regulatory system envisaged by Atkinson, where anabolic and catabolic pathway regulation is modulated by adenine nucleotide ratios. Here we discuss the current state of AMPK regulation by adenine nucleotides and we propose that AMPK functions as an adenylate charge-regulated protein kinase.

Small molecule drug A-769662 and AMP synergistically activate naive AMPK independent of upstream kinase signaling

The AMP-activated protein kinase (AMPK) is a metabolic stress-sensing αβγ heterotrimer responsible for energy homeostasis, making it a therapeutic target for metabolic diseases such as type 2 diabetes and obesity. AMPK signaling is triggered by phosphorylation on the AMPK α subunit activation loop Thr172 by upstream kinases. Dephosphorylated, naive AMPK is thought to be catalytically inactive and insensitive to allosteric regulation by AMP and direct AMPK-activating drugs such as A-769662. Here we show that A-769662 activates AMPK independently of α-Thr172 phosphorylation, provided β-Ser108 is phosphorylated. Although neither A-769662 nor AMP individually stimulate the activity of dephosphorylated AMPK, together they stimulate >1,000-fold, bypassing the requirement for β-Ser108 phosphorylation. Consequently A-769662 and AMP together activate naive AMPK entirely allosterically and independently of upstream kinase signaling. These findings have important implications for development of AMPK-targeting therapeutics and point to possible combinatorial therapeutic strategies based on AMP and AMPK drugs.

Subcellular Localization of Cyclic AMP-Responsive Element Binding Protein-Regulated Transcription Coactivator 2 Provides a Link between Obesity and Breast Cancer in Postmenopausal Women

Epidemiologic evidence supports a correlation between obesity and breast cancer in women. AMP-activated protein kinase plays an important role in energy homeostasis and inhibits the actions of cyclic AMP-responsive element binding protein-regulated transcription coactivator 2 (CRTC2). In postmenopausal women, the cyclic AMP-responsive element binding protein-dependent regulation of aromatase is a determinant of breast tumor formation through local production of estrogens. The present work aimed to examine the effect of adipokines on aromatase expression and identify additional mechanisms by which prostaglandin E(2) causes increased aromatase expression in human breast adipose stromal cells. Treatment of human adipose stromal cells with forskolin and phorbol 12-myristate 13-acetate (PMA), to mimic prostaglandin E(2), resulted in nuclear translocation of CRTC2. Aromatase promoter II (PII) activity assays showed that CRTC2 in addition to forskolin/PMA treatment significantly increased PII-induced activity. CRTC2 binding to PII was examined by chromatin immunoprecipitation, and forskolin/PMA treatment was associated with increased binding to PII. Treatment of human adipose stromal cells with leptin significantly up-regulated aromatase expression associated with nuclear translocation of CRTC2 and increased binding of CRTC2 to PII. Adiponectin treatment significantly decreased forskolin/PMA-stimulated aromatase expression, consistent with the decreased nuclear translocation of CRTC2 and the decreased binding of CRTC2 to PII. The expression and activity of the AMP-activated protein kinase LKB1 was examined and found to be significantly decreased following either forskolin/PMA or leptin treatment. In contrast, adiponectin significantly increased LKB1 expression and activity. In conclusion, the regulation of aromatase by CRTC2, in response to the altered hormonal milieu associated with menopause and obesity, provides a critical link between obesity and breast cancer.

Metformin and salicylate synergistically activate liver AMPK, inhibit lipogenesis and improve insulin sensitivity.

Metformin is the mainstay therapy for type 2 diabetes (T2D) and many patients also take salicylate-based drugs [i.e., aspirin (ASA)] for cardioprotection. Metformin and salicylate both increase AMP-activated protein kinase (AMPK) activity but by distinct mechanisms, with metformin altering cellular adenylate charge (increasing AMP) and salicylate interacting directly at the AMPK β1 drug-binding site. AMPK activation by both drugs results in phosphorylation of ACC (acetyl-CoA carboxylase; P-ACC) and inhibition of acetyl-CoA carboxylase (ACC), the rate limiting enzyme controlling fatty acid synthesis (lipogenesis). We find doses of metformin and salicylate used clinically synergistically activate AMPK in vitro and in vivo, resulting in reduced liver lipogenesis, lower liver lipid levels and improved insulin sensitivity in mice. Synergism occurs in cell-free assays and is specific for the AMPK β1 subunit. These effects are also observed in primary human hepatocytes and patients with dysglycaemia exhibit additional improvements in a marker of insulin resistance (proinsulin) when treated with ASA and metformin compared with either drug alone. These data indicate that metformin-salicylate combination therapy may be efficacious for the treatment of non-alcoholic fatty liver disease (NAFLD) and T2D.

Are genuine changes in protein expression being overlooked? Reassessing Western blotting.

This study used purified calsequestrin 1 and AMP kinase (AMPK) proteins to demonstrate how Western blotting outcomes can be influenced when either the density of proteins detected lie within a nonproportional region of a standard curve or a standard curve is not taken into account for data analyses. It outlines the likelihood of true changes being overlooked through the simple mistake of using band density alone and/or through analyzing too much sample. To demonstrate this, extrapolation of a typical linear, although nonproportional, standard curve resulted in approximately fourfold error. The standard curve method was used to estimate the concentration of AMPK beta1 in rat extensor digitorum longus muscle as being of the order of 60 microM. The article suggests that adopting a more sensitive Western blotting protocol will improve the reliability of quantitative Western blotting outcomes.

Structure and function of AMP‐activated protein kinase

AMP‐activated protein kinase (AMPK) regulates metabolism in response to energy demand and supply. AMPK is activated in response to rises in intracellular AMP or calcium‐mediated signalling and is responsible for phosphorylating a wide variety of substrates. Recent structural studies have revealed the architecture of the αβγ subunit interactions as well as the AMP binding pockets on the γ subunit. The α catalytic domain (1–280) is autoinhibited by a C‐terminal tail (313–335), which is proposed to interact with the small lobe of the catalytic domain by homology modelling with the MARK2 protein structure. Two direct activating drugs have been reported for AMPK, the thienopyridone compound A769662 and PTI, which may activate by distinct mechanisms.