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 (Thr(172)) by kinases LKB1 or CaMKKβ, and this is promoted by AMP binding to the γ subunit. AMP sustains activity by inhibiting dephosphorylation of α-Thr(172), whereas ATP promotes dephosphorylation. Adenosine diphosphate (ADP), like AMP, bound to γ sites 1 and 3 and stimulated α-Thr(172) 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.

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-calmodulindependent 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.myristome | signal transduction | adenylate charge | γ-subunit T he AMP-activated protein kinase (AMPK) is a key regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways in order to balance nutrient supply with energy demand. AMPK protects cells from physiological and pathological stresses (e.g., nutrient starvation, hypoxia/ischemia, and exercise) that lower cellular energy charge (increase AMP∕ ATP ratio) by directing metabolism toward ATP production and inhibiting anabolic pathways that utilize ATP∕NADPH (1, 2). This regulation is achieved by acute phosphorylation of key enzymes in major branches of metabolism including fat synthesis, protein synthesis, and carbohydrate metabolism, as well as phosphorylation of transcription factors to have longer-term regulatory effects. AMPK also functions in regulating whole-body energy homeostasis, food intake, and body weight in response to a variety of hormones including leptin, adiponectin, and ghrelin. These properties have made AMPK a promising drug target to treat the growing incidence of metabolic diseases including obesity, type 2 diabetes, cancer growth and metastasis, and cardiovascular disease (1, 2).AMPK is an αβγ heterotrimer consisting of an α catalytic subunit and β and γ regulatory subunits, with corresponding homologues in all eukaryotes. Multiple isoforms exist for each subunit in mammals (α1, α2, β1, β2, γ1, γ2, and γ3). The α-subunits consist of an N-terminal kinase catalytic domain followed by an autoinhibitory sequence and a C-terminal β-subunit binding domain (3). The β-subunits contain an internal carbohydrate-binding module and a conserved C-terminal sequence that functions to tether α-and γ-subunits (4, 5). ...

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.