David Grahame Hardie

Characterization of the AMP-activated Protein Kinase Kinase from Rat Liver and Identification of Threonine 172 as the Major Site at Which It Phosphorylates AMP-activated Protein Kinase

We have developed a sensitive assay for the AMP-activated protein kinase kinase, the upstream component in the AMP-activated protein kinase cascade. Phosphorylation and activation of the downstream kinase by the upstream kinase absolutely requires AMP and is antagonized by high (millimolar) concentrations of ATP. We have purified the upstream kinase >1000-fold from rat liver; a variety of evidence indicates that the catalytic subunit may be a polypeptide of 58 kDa. The physical properties of the downstream and upstream kinases, e.g. catalytic subunit masses (63 versus 58 kDa) and native molecular masses (190 versus 195 kDa), are very similar. However, unlike the downstream kinase, the upstream kinase is not inactivated by protein phosphatases. The upstream kinase phosphorylates the downstream kinase at a single major site on the α subunit, i.e. threonine 172, which lies in the “activation segment” between the DFG and APE motifs. This site aligns with activating phosphorylation sites on many other protein kinases, including Thr177 on calmodulin-dependent protein kinase I. As well as suggesting a mechanism of activation of AMP-activated protein kinase, this finding is consistent with our recent report that the AMP-activated protein kinase kinase can slowly phosphorylate and activate calmodulin-dependent protein kinase I, at least in vitro (Hawley, S. A., Selbert, M. A., Goldstein, E. G., Edelman, A. M., Carling, D., and Hardie, D. G. (1995) J. Biol. Chem. 270, 27186-27191).

The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase

In addition to acetyl-CoA carboxylase and HMG-CoA reductase, the AMP-activated protein kinase phosphorylates glycogen synthase, phosphorylase kinase, hormone-sensitive lipase and casein. A number of other substrates for the cyclic AMP-dependent protein kinase, e.g., L-pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, are not phosphorylated at significant rates. Examination of the sites phosphorylated on acetyl-CoA carboxylase, hormone-sensitive lipase, glycogen synthase and phosphorylase kinase suggests a consensus recognition sequence in which the serine residue phosphorylated by the AMP-activated protein kinase has a hydrophobic residue on the N-terminal side (i.e., at -1) and at least one arginine residue at -2, -3 or -4. Substrates for cyclic AMP-dependent protein kinase which lack the hydrophobic residue at -1 are not substrates for the AMP-activated protein kinase.

Histological evaluation of AMPK signalling in primary breast cancer

BackgroundAMP-activated protein kinase (AMPK) acts as a cellular fuel gauge that responds to energy stress by suppressing cell growth and biosynthetic processes, thus ensuring that energy-consuming processes proceed only if there are sufficient metabolic resources. Malfunction of the AMPK pathway may allow cancer cells to undergo uncontrolled proliferation irrespective of their molecular energy levels. The aim of this study was to examine the state of AMPK phosphorylation histologically in primary breast cancer in relation to clinical and pathological parameters.MethodsImmunohistochemistry was performed using antibodies to phospho-AMPK (pAMPK), phospho-Acetyl Co-A Carboxylase (pACC) an established target for AMPK, HER2, ERα, and Ki67 on Tissue Micro-Array (TMA) slides of two cohorts of 117 and 237 primary breast cancers. The quick score method was used for scoring and patterns of protein expression were compared with clinical and pathological data, including a minimum 5 years follow up.ResultsReduced signal, compared with the strong expression in normal breast epithelium, using a pAMPK antibody was demonstrated in 101/113 (89.4%) and 217/236 (91.9%) of two cohorts of patients. pACC was significantly associated with pAMPK expression (p = 0.007 & p = 0.014 respectively). For both cohorts, reduced pAMPK signal was significantly associated with higher histological grade (p = 0.010 & p = 0.021 respectively) and axillary node metastasis (p = 0.061 & p = 0.039 respectively). No significant association was found between pAMPK and any of HER2, ERα, or Ki67 expression, disease-free survival or overall survival.ConclusionThis study extends in vitro evidence through immunohistochemistry to confirm that AMPK is dysfunctional in primary breast cancer. Reduced signalling via the AMPK pathway, and the inverse relationship with histological grade and axillary node metastasis, suggests that AMPK re-activation could have therapeutic potential in breast cancer.

Effects of metformin on breast cancer cell proliferation, the AMPK pathway and the cell cycle

AimThe aim of this study was to compare the effects and mechanisms of action of metformin on estrogen receptor (ER)-positive and ER-negative breast cancer cell lines.MethodsThe anti-proliferative effects of metformin, and of the direct activator of adenosine monophosphate-activated protein kinase (AMPK), A-769662, on MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) breast cancer cell lines were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, a yellow tetrazole) assays. Fluorescence-activated cell sorting was also used to examine the effect of metformin on the cell cycle. Finally, phosphorylation of the metformin target AMPK, and of its potential downstream targets including acetyl-CoA carboxylase (ACC), p53, p70-S6K and Raptor, was examined using immunoblotting.ResultsMetformin and A-769662 caused significant, concentration-dependent suppression of cell proliferation with G1 cell cycle arrest in both MCF-7 and MDA-MB-231 cells. The proliferation suppression effect was more profound in MCF-7 cells. A concentration-dependent phosphorylation of AMPK was detected following metformin treatment, as was phosphorylation of ACC in both cell lines, but not p53, p70-S6k or Raptor.ConclusionMetformin acts as a growth inhibitor in both ER-positive and ER-negative breast cancer cells in vitro, and arrests cells in G1 phase, particularly in the ER-positive MCF-7 cells. The effect is likely to be mediated by AMPK activation, in part by inhibition of fatty acid synthesis via ACC phosphorylation.

Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms

AMPK complexes containing γ1, γ2 or γ3 subunit isoforms were generated by expression in human cells. They displayed differences in all three effects by which adenine nucleotides cause regulation, i.e. in allosteric activation, promotion of phosphorylation and inhibition of dephosphorylation

Pharmacological activators of AMP-activated protein kinase have different effects on Na+ transport processes across human lung epithelial cells

AMP‐activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride‐sensitive (Iamiloride) and ouabain‐sensitive basolateral (Iouabain) short circuit current in H441 lung epithelial cells.

Mechanisms underlying the metabolic actions of galegine that contribute to weight loss in mice

Background and purpose: Galegine and guanidine, originally isolated from Galega officinalis, led to the development of the biguanides. The weight‐reducing effects of galegine have not previously been studied and the present investigation was undertaken to determine its mechanism(s) of action.

Regulation of AMP-activated protein kinase by natural and synthetic activators

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function.

AMP-activated protein kinase – not just an energy sensor

Orthologues of AMP-activated protein kinase (AMPK) occur in essentially all eukaryotes as heterotrimeric complexes comprising catalytic α subunits and regulatory β and γ subunits. The canonical role of AMPK is as an energy sensor, monitoring levels of the nucleotides AMP, ADP, and ATP that bind competitively to the γ subunit. Once activated, AMPK acts to restore energy homeostasis by switching on alternate ATP-generating catabolic pathways while switching off ATP-consuming anabolic pathways. However, its ancestral role in unicellular eukaryotes may have been in sensing of glucose rather than energy. In this article, we discuss a few interesting recent developments in the AMPK field. Firstly, we review recent findings on the canonical pathway by which AMPK is regulated by adenine nucleotides. Secondly, AMPK is now known to be activated in mammalian cells by glucose starvation by a mechanism that occurs in the absence of changes in adenine nucleotides, involving the formation of complexes with Axin and LKB1 on the surface of the lysosome. Thirdly, in addition to containing the nucleotide-binding sites on the γ subunits, AMPK heterotrimers contain a site for binding of allosteric activators termed the allosteric drug and metabolite (ADaM) site. A large number of synthetic activators, some of which show promise as hypoglycaemic agents in pre-clinical studies, have now been shown to bind there. Fourthly, some kinase inhibitors paradoxically activate AMPK, including one (SU6656) that binds in the catalytic site. Finally, although downstream targets originally identified for AMPK were mainly concerned with metabolism, recently identified targets have roles in such diverse areas as mitochondrial fission, integrity of epithelial cell layers, and angiogenesis.