Protein chemistry & metabolism

AMP-activated protein kinase (AMPK), is an αβγ heterotrimeric serine/threonine kinase that is directly responsible for sensing cellular metabolic stress. Under conditions of energy deprivation (low glucose) or high-energy demand (vigorous exercise) cellular adenylate charge ([ATP + 0.5 ADP]/[ATP = + ADP + AMP]) is reduced ie levels of ATP decrease and ADP, AMP levels rise and this switches on AMPK. The AMPK catalytic subunit contains an N-terminal kinase domain and a C-terminal subunit-binding domain that associates with the subunit’s C-terminal tail. The subunit consists of four CBS repeat sequences of which sites 1, 3 and 4 are responsible for binding adenine nucleotides. AMPK is inactive unless phosphorylated on T172 in the subunit activation loop by upstream kinases (eg LKB1, CaMKK). We have found that AMP binding to the subunit sites 1 and 3 stimulates phosphorylation of T172 by LKB1 and CaMKK. Stimulation of T172 phosphorylation depends on the N-terminal myristoylation of the subunit so that AMP fails to stimulate AMPK T172 phosphorylation if the subunit is non-myristoylated.  These finding indicate that AMP stimulation of T172 phosphorylation may involve a “myristoyl switch” mechanism whereby AMP triggers the release of the myristoyl group.. AMP has two additional regulatory functions once AMPK is phosphorylated on T172 but these secondary regulatory effects do not require subunit myristoylation. AMP suppresses the dephosphorylation of T172 by protein phosphatases as well as stimulating AMPK activity allosterically.  ADP also stimulates T172 phosphorylation and suppresses dephosphorylation but does not act as an allosteric activator.

Maintaining energy balance is a fundamental property of every living organism. A key component in the regulation of cellular and whole-body energy homeostasis is the AMP-activated protein kinase (AMPK), which functions as a fuel sensor co-ordinating metabolic processes to balance nutrient supply with energy demand. AMPK is at the hub of metabolic control in response to diet and exercise and, for this reason, is considered one of the most important targets for new drugs to treat obesity, Type 2 diabetes and cardiovascular disease. Multiple AMPK activating drugs have been found but none have progressed to the Clinic and their mechanisms of activation are poorly understood. We have found that the AMPK activator A769662 is specific for AMPK heterotrimers that contain the b1 isoform and do not activate b2 containing heterotrimers. The structure of A769662 bound to the enzyme has been solved by the Camblin & Carling laboratories in the UK showing A769662 is positioned in a pocket between the b-subunit CBM domain and the small lobe of the a-subunit kinase. We have found that A769662 is capable of activating AMPK independent of upstream kinase phosphorylation of Thr172. A769662 dependent activation of AMPK is negligible but in the presence of AMP there is remarkable synergy leading to a 1,000-fold activation.  Our results raise the possibility of combining direct activating drugs with drugs that elevate AMP to exploit this synergy. A potential example is the widely used type 2 diabetes drug metformin, which increases cellular AMP with drugs like A769662.

AMPK phosphorylates and modulates key enzymes in all branches of metabolism, as well as transcription factors that regulate their expression, to reposition cellular metabolism away from anabolic, ATP-consuming pathways, to catabolic pathways. Given its pivotal role in cellular energy metabolism, the regulatory events surrounding AMPK activation are of great interest. In order to better understand AMPK’s physiological roles we have generated mice lacking either 1 or 2 subunits as well as mice containing Ala in place of the AMPK phosphorylation sites in some of its classical substrates, including acetyl-CoA carboxylase 1 and 2 and HMG-CoA reductase. b1 null mice are fertile and show no development phenotypes but have a 90% reduction in hepatic AMPK activity due to loss of the catalytic subunits. There is a modest reduction of AMPK activity in the hypothalamus and white adipose tissue and no change in skeletal muscle or heart. On a low fat or an obesity-inducing high fat diet, b1 null mice had reduced food intake, reduced adiposity, and reduced total body mass. Metabolic rate, physical activity, adipose tissue lipolysis, and lipogenesis were similar to wild type littermates. The reduced appetite and body mass of b1 null mice were associated with protection from high fat diet-induced hyperinsulinemia, hepatic steatosis, and insulin resistance. Thus loss of b1 reduces food intake and protects against the deleterious effects of an obesity-inducing diet. b2 KO mice are viable and breed normally but have reduced skeletal muscle AMPK α1 and α2 expression. During treadmill running β2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Deletion of AMPK β2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and worsening of diet-induced obesity and glucose intolerance.  The combined b1b2 muscle specific double KO mice have a 96% reduction in exercise capacity highlighting the importance of AMPK in maintaining exercise capacity.

CaMKKβ belongs to the Ca2+ and calmodulin (CaM) dependent protein kinase (CaMK) subfamily and along with CaMKKα were initially identified as the upstream kinases responsible for phosphorylating and activating CaMK-IV and CaMK-I respectively. It is also an activating upstream kinase for the important metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) and the deacetylase Sirtuin 1 (Sirt1). Genetic loss of CaMKKβ leads to increased atherosclerotic lesions, as well as impaired long term memory formation. A mutation in human CaMKKβ (Thr85Ser) is associated with clinical anxiety, whereas a single nucleotide polymorphism (SNP; rs1063843) that causes decreased CaMKKβ expression is linked with schizophrenia. Consistent with these findings, we have found that mice lacking CaMKKβ exhibit anxiety and schizophrenic-like behaviour. We have shown that multisite phosphorylation of CaMKKβ is critical for regulating Ca2+/CaM dependence. We have identified an autophosphorylation site (Thr85) that is required for sustaining CaMKKβ activity after Ca2+ withdrawal. Autophosphorylation of the Thr85Ser mutant causes a sustained inhibition of CaMKKβ after Ca2+ withdrawal.