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Nucleotide regulation of AMP-activated protein kinase

Underwood, EA; (2013) Nucleotide regulation of AMP-activated protein kinase. Doctoral thesis , UCL (University College London). Green open access

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Abstract

AMP-activated protein kinase (AMPK) acts as the cell’s master energy regulator, sensing and maintaining the concentration of ATP in a narrow range irrespective of energy demand. This kinase has received significant attention as a drug-target for type-2 diabetes, obesity and cancer. Although historically the AMP:ATP ratio has been considered the signal for AMPK activation, we have recently demonstrated that ADP is likely to be an important physiological regulator of AMPK in both mammals and yeast. The binding of adenine nucleotides and staurosporine to the full-length alpha1beta1gamma1 heterotrimer, both phosphorylated and unphosphorylated, is described. Binding was monitored through displacement of fluorescently labelled nucleotides (coumarin-AXP), either via direct coumarin excitation or Forster Resonance Energy Transfer (FRET) in which tryptophan residues were excited. Mg.ATP was found to bind more weakly than ADP, a feature which is likely key to AMPK regulation. A Nicotinamide adenine dinucleotide (NADH) coupled spectrophotometric assay was used to monitor AMPK kinetics and its regulation by nucleotides. NADH binds at Site-1, within the gamma-subunit, and competes with allosteric activation by AMP, but not the protective effect of AMP/ADP against alpha-T172 dephosphorylation. Therefore it seems that AMP binding at Site-1 mediates allostery whilst AMP/ADP binding at Site-3 affords protection against dephosphorylation. In order to explore this idea further, AXP binding constants were used to model binding site occupancies over the concentration ranges used in vitro. The modelling demonstrates that, in vitro, Site-1 is occupied by AMP and Site-3 by AMP/ADP in a manner consistent with their assigned regulatory functions. This modelling study was also extended to consider in vivo binding site occupancy. It was important to verify that coumarin-ADP bound in a homologous fashion to ADP, specifically in the same exchangeable binding pockets. X-ray crystallography was used to determine the structure of a truncated form of AMPK in complex with coumarin-ADP. This structure is compared to an ADP-bound form. SNF1 is the Saccharomyces cerevisiae AMPK ortholog. The binding of nucleotides to SNF1, and its regulation by ADP, was also characterised. As with the mammalian enzyme AXP bound at two exchangeable sites, and interacted with Mg.ATP more weakly than ADP.

Type: Thesis (Doctoral)
Title: Nucleotide regulation of AMP-activated protein kinase
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright restricted material has been removed from the ethesis
Keywords: AMPK, SNF1, xray crystallography, biophysics
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: https://discovery.ucl.ac.uk/id/eprint/1382013
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