Abstract
The AMP-activated protein kinase (AMPK) is a sensor of cellular energy that is activated by increases in the intracellular AMP:ATP or ADP:ATP ratios. Once active, AMPK activates catabolic pathways and inhibits anabolic pathways to restore cellular energy homeostasis. AMPK activity can be increased by phosphorylation of Thr172, a conserved residue in the activation loop of the kinase domain, by either LKB1 or CaMKKß. Increases in Thr172 phosphorylation can be mediated by either protecting this site against dephosphorylation by protein phosphatases, or by promoting its phosphorylation by upstream kinases, both of which were proposed to be mediated by binding of AMP or ADP to the ?-subunit of AMPK. AMPK is also allosterically activated by binding of AMP, but not ADP.Recently, ADP has been proposed as the major regulator of AMPK and the role of AMP has been questioned. The reasons for this are: (i) while both AMP and ADP can increase phosphorylation of Thr172, ADP is present at higher concentrations in the cell and it was proposed that AMP would be unable to compete with ADP for binding at the ?-subunit; (ii) allosteric activation by AMP was reported to increase AMPK activity by less than 2-fold, whereas changes in the phosphorylation of Thr172 can increase AMPK activity by 100-fold.
Using cell-free systems and intact cells, the regulation of Thr172 phosphorylation and AMPK activity by AMP, ADP and ATP was re-investigated. AMP promoted Thr172 phosphorylation by LKB1 but not by CaMKKß, while ADP had no effect on Thr172 phosphorylation by either upstream kinase. Additionally, while both AMP and ADP could protect Thr172 against dephosphorylation, AMP was more potent. The allosteric activation of AMPK by AMP was demonstrated to be a significant component of the overall activation mechanism. Using phosphorylation of ACC (a downstream target of AMPK) as a marker, allosteric activation of AMPK was observed under conditions where there were no changes in Thr172 phosphorylation. The changes in Thr172 phosphorylation and AMPK activity observed in intact cells in response to AMPK activators were also examined, and demonstrated to be far lower than the maximal effects observed in cell-free systems. Taken together, these results demonstrate that AMP is a true physiological regulator of AMPK activity.
The regulation of LKB1 has also been the subject of much interest. Whilst it appears that LKB1 is constitutively active, several groups have reported that LKB1 activity is regulated by post-translational modifications, particularly phosphorylation. The role of phosphorylation of LKB1 on Ser31 by AMPK was investigated. While AMPK could phosphorylate LKB1 in a cell-free system, an effect lost in an LKB1 [S31A] mutant, this phosphorylation was not observed in intact cells. Additionally, mutation of Ser31 did not appear to alter LKB1 activity. These results suggest that phosphorylation of Ser31 of LKB1 by AMPK, whilst possible, may not be a physiologically relevant event.
The mTOR pathway is a key regulator of cell growth and protein synthesis in response to a number of stimuli, including growth factors, nutrients and energy status. The mTOR pathway is frequently deregulated in a number of cancers and cancer syndromes, and inhibition of mTOR is a promising therapeutic approach. The nature of the genetic lesion in tumours may determine the response of cells to mTOR inhibition. The LKB1-AMPK pathway, as well as being a negative regulator of the mTOR pathway, is also frequently inactivated in human cancers. The role that the LKB1-AMPK pathway plays in determining the cellular response to AZ4, a dual mTORC1 and mTORC2 inhibitor, was investigated. AZ4 induced apoptosis in a number of cell lines, confirming its potential as an anti-cancer therapeutic. However, the presence of an active LKB1-AMPK pathway protected cells against AZ4-induced apoptosis, suggesting that the status of this pathway may determine how cells respond to mTOR inhibition.
| Date of Award | 2014 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Grahame Hardie (Supervisor) |