| Research in our group focuses on the control of cell function by reversible protein phosphorylation. As a model system, we studied the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) and the control of its activity by protein kinases. This led to the study of insulin signalling and the AMP-activated protein kinase (AMPK) cascade.
Our research projects are centered on the AMPK signalling cascade. AMPK is a serine/threonine protein kinase involved in the control of cellular energy homeostasis. It stimulates ATP-producing pathways and inhibits energy consuming processes. We contributed to this field by finding new AMPK targets. We demonstrated that the activation of PFK-2 by AMPK participates in the stimulation of heart glycolysis by ischaemia. Similarly we showed that the inactivation of elongation factor 2 (eEF2) by AMPK contributes to the inhibition of protein synthesis during anoxia. Recently, the metabolic effects of AMPK were proposed to have implications for the treatment of type 2 diabetes. AMPK activation stimulates glucose uptake independently of insulin and AMPK activation in muscle during exercise is beneficial for diabetic patients. In addition, Metformin, a drug used for the treatment of type 2 diabetes activates AMPK. Lastly, the control of food intake by the hypothalamus might also be mediated by AMPK.
Various aspects of AMPK action are studied in our group. More precisely, we are now engaged in (i) studying mechanisms of control upstream of AMPK, (ii) the identification of new downstream targets, (iii) the study of effects of AMPK on mitochondrial respiration, (iv) the implication of AMPK in the mode of action of metformin (v) the involvement of hypothalamic AMPK in the control of food intake and (vi) assessing the role of AMPK in animals subjected to metabolic stress. Control of heart glycolysis by insulin and ischaemia
Over the last years we have been involved in studying the molecular mechanisms responsible for the activation of heart PFK-2 by insulin and ischaemia. Insulin activates heart PFK-2 via a signalling pathway downstream of phosphatidylinositol 3-kinase (PI 3-kinase) and phosphoinositide-dependent protein kinase-1 (PDK1). Our attention is currently focused on the relative contribution of protein kinase B (PKB), protein kinase Czeta (PKCzeta) and the serum- and glucocorticoid-induced kinase-3 (SGK3) to the insulin-induced activation of PFK-2.
Work from our laboratory demonstrated that the stimulation of heart glycolysis by anoxia or ischaemia (Pasteur Effect) resulted, in part, from the phosphorylation and activation of PFK-2 mediated by AMPK. Like insulin-stimulated protein kinases, AMPK was found to phosphorylate Ser466 in heart PFK-2 which is responsible for increasing the Vmax of the enzyme. New pathways under control by AMPK and new AMPK targets
We studied the well-known inhibition of protein synthesis during anoxia and showed that AMPK was implicated in this phenomenon. AMPK activation led to the phosphorylation and inactivation of elongation factor-2 (eEF2) via activation of its upstream eEF2 kinase. In addition, stimulation of the mTOR pathway by amino acids and insulin leading to the activation of p70 ribosomal protein S6 kinase (p70S6K) was antagonized following AMPK activation. Lastly we showed that in certain cells, prolonged AMPK activation induces apoptosis via activation of the c-Jun kinase pathway. Our current research aims at identifying new targets for AMPK using a phosphoproteomics approach (see below).
Regulation upstream of AMPK
AMPK is activated in response to a rise in the intracellular AMP:ATP ratio. The rise in AMP induces a conformational change allowing phosphorylation of AMPK at Thr172 in the activation loop of its catalytic alpha-subunits by an upstream AMPK kinase (AMPKK). One AMPKK phosphorylating Thr172 has been identified as the Peutz-Jehger’s protein, LKB1. However, we identified new phosphorylation sites in the alpha-subunits, namely Thr258 and Ser485 that were not directly involved in AMPK activation, implying the existence of other upstream kinases. We showed that, in heart, insulin antagonizes AMPK activation by ischaemia via a PI 3-kinase-dependent pathway, whereby PKB phosphorylates Ser485 and in so doing antagonizes phosphorylation of Thr172 by LKB1 and thus AMPK activation.
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Protein kinases of signalling pathways that
converge on heart PFK-2. Numbering of
residues refers to the bovine H1 isoform. |
Cross talk between the insulin/PKB/mTOR and
AMPK signalling pathways with respect to the
control of protein synthesis |
Mass spectrometry The establishment of mass spectrometry facilities within our group has not only helped our own research, principally for phosphorylation site identification, but has also assisted other groups within this Institute/University and other national/international laboratories. Collaborations have involved protein identification, analysis of chemical modification of proteins and the study of repair mechanisms for protein glycation. We are currently developping new strategies for phosphoproteomics. Our group has built up a number of national and international collaborations and is currently part of the EU Program FP6 consortium ‘EXGENESIS’ on the beneficial effects of exercise in counteracting the metabolic syndrome.
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