Investigating the regulation of AMP-activated protein kinase

Abstract

AMP-activated protein kinase (AMPK) is a highly evolutionarily conserved heterotrimeric kinase, characterised as a critical sensor of energetic stress. AMPK is being investigated as a therapeutic target for metabolic diseases including type II diabetes, obesity and cancer. The development of direct pharmacological activators has revealed a novel binding site on AMPK, termed the allosteric drug and metabolite (ADaM) site. It has been suggested that the ADaM site is the receptor for an additional endogenous regulator of AMPK. Cell lines to enable screening for a condition that regulates AMPK through this novel mode of action have been generated, and a screen of compounds using an in vitro fluorescence polarisation assay is described. There is potential for isoform specific roles of the AMPK subunits, which are differentially expressed in tissues. The effect of the γ isoform on activation by direct ADaM site ligands has not previously been described. In this study, the γ2 subunit is shown to exhibit enhanced protection from dephosphorylation in vitro. The N-terminus of the γ2 subunit is shown to enhance activation of AMPK complexes in cells via increased Thr172 phosphorylation in response to treatment with the direct activator 991. The effect of enhanced phosphorylation of the γ2 subunit is shown to be independent of β2 Ser108 phosphorylation. The recent suggestion that dual allosteric activation by ADaM site activators and AMP stimulates the non-phosphorylated form of AMPK to physiologically significant levels of activity was investigated, both in vitro and in mammalian cells. Using cell lines in which the upstream kinases liver kinase B1 (LKB1) and Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ) are absent, AMPK activation was essentially abolished even in the presence of dual sources of allosteric activation. The results presented in this thesis provide further understanding of the regulation of AMPK by nucleotides and by direct pharmacological activators.