Protein Chemistry and Metabolism - Research Units - Protein Chemistry and Metabolism - Research themes
Control of AMPK signalling by adenylate charge
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/[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. We are currently investigating where the myristoyl group is bound on AMPK and whether AMP stimulates membrane binding in response to exposure 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. Oakhill JS, Chen ZP, Scott, JW, Steel R, Castelli LA, Ling N, Macaulay SL and Kemp BE. β-Subunit Myristoylation is the Gatekeeper for Initiating Metabolic Stress Sensing by AMPK. Proc Nat Acad Sci 2010 (in press)
Development of AMPK isoform specific drugs
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. Despite considerable interest in this area, progress in identifying therapeutic activators of AMPK has been modest. We have found that the AMPK activator A769662 is specific for AMPK heterotrimers that contain the 1 isoform and do not activate 2 containing heterotrimers. We have discovered a group of benzopyridone compounds that directly activate or inhibit AMPK depending on the b subunit isoform present in the heterotrimer complex. This is significant because it highlights that developing direct isoform-specific modulators of AMPK is feasible, and raises the possibility of being able to target specific AMPK complexes in particular tissues. For example 2 specific activators may be useful in promoting skeletal muscle glucose uptake whereas 1 specific activators may inhibit lipid synthesis in the liver. Drugs like A769662 are able to activate AMPK in the absence of promoting T172 phosphorylation in contrast to AMP, which stimulates T172 phosphorylation by the upstream kinases LKB1 and CaMKK. Understanding the underlying mechanism for drug activation of AMPK is an important goal of this work. Scott JW, van Denderen BJ, Jorgensen SB, Honeyman JE, Steinberg GR, Oakhill JS, Iseli TJ, Koay A, Gooley PR, Stapleton D, Kemp BE. Thienopyridone drugs are selective activators of AMP-activated protein kinase beta1-containing complexes. Chem Biol. 2008 Nov 24; 15(11): 1220-30.
Genetic modification of AMPK signaling pathways
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. 1 null mice are fertile and show now 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, 1 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 1 null mice were associated with protection from high fat diet-induced hyperinsulinemia, hepatic steatosis, and insulin resistance. Thus loss of 1 reduces food intake and protects against the deleterious effects of an obesity-inducing diet. β2 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.