The AMP-activated protein kinase (AMPK) is a sensor of cellular energy stress that, once activated, promotes ATP-producing process while it switches off ATP-consuming pathways, in order to restore the cellular energetic balance under conditions of stress. Activation of AMPK is dependent on the phosphorylation of the residue Thr172 in its α subunit. This phosphorylation is generally mediated by the known tumour suppressor LKB1, but also CaMKKβ has been shown to phosphorylate AMPK. As its name suggests, AMPK is also activated by the binding of AMP to its γ subunit. This binding causes a >10 fold allosteric stimulation, promotes phosphorylation of Thr172 by upstream kinases and protects AMPK from dephosphorylation of Thr172 by protein phosphatase(s).
In 2010 it was reported that oxidative stress mediated by H2
activated AMPK by increasing the cellular AMP:ATP and ADP:ATP ratios (Hawley et al, 2010). However, the same year another work suggested that the mechanism of activation of AMPK by H2
was direct, independent of AMP and involved the oxidation of two cysteine residues in the α subunit of AMPK (Zmijewski et al, 2010). Given this discrepancy, here we provided evidence that H2
, generated by addition of glucose oxidase in the cell medium, activates AMPK mostly through an increase of AMP:ATP and ADP:ATP ratios, as previously suggested in our laboratory. However, it seems that there might be a second, minor mechanism of activation that is independent of the changes in cellular nucleotides. This second mechanism was not identified in our previous work because we were not aware of how rapidly a single bolus of H2
can be metabolized by the antioxidant defences of the cell. We could not identify the alternative mechanism of activation by H2
but showed that H2
could protect Thr172 from dephosphorylation, which might suggest a direct effect of H2
on the phosphatase(s) dephosphorylating AMPK. However, since the identity of this phosphatase(s) remains unclear, we could not rule out the possibility that the protection from dephosphorylation that we observed could still be mediated by the increase in AMP:ATP and ADP:ATP ratios. Moreover, it remains still possible that a direct effect of H2
on AMPK might be responsible for the small but significant activation we detected in cell expressing a nucleotides-insensitive mutant of AMPK.
Recently, a new crystal structure of AMPK obtained by Xiao et al (2013) provided new insights about AMPK structure and regulation. In particular, the authors identified a new binding pocket located at the interface between the N-lobe of the α-kinase domain and the β-CBM of AMPK, which appeared to be the binding site for two direct activators of AMPK: A769662 and 991. Here we confirm that this novel binding pocket is indeed the binding site for both A769662 and 991, and provide evidence that another direct activator of AMPK, MT63-78, also binds at the same site. Mutation of two important residues in this pocket (Lys29 and Lys31 of the α2 subunit) abolished the allosteric stimulation of AMPK by A769662, 991 and MT63-78 while it had no effect on allosteric stimulation by AMP. However, we also showed that the same mutation abolished protection against Thr172 dephosphorylation not only by A769662, 991 and MT63-78, but also by phenformin and H2O2, which are known to activate AMPK by increasing the AMP:ATP and ADP:ATP ratios. These data show that the integrity of this pocket is important for the effect of AMP to protect against Thr172 dephosphorylation, but not for its ability to cause allosteric stimulation. Moreover, in HEK-293 cell stably expressing an α2 subunit carrying the mutation of both Lys29 and Lys31, the basal activity of AMPK due to Thr172 phosphorylation was almost 6-fold less than in cells expressing wild-type α2. This result pointed out for the first time that there might be a natural ligand binding in the newly discovered binding pocket that is not able to bind to the double mutant, explaining the difference in activity observed. However the identity of this possible natural ligand remains unclear and more studies will be necessary to uncover it.
|Date of Award
|Grahame Hardie (Supervisor)