Investigating the regulation of AMP-activated protein kinase
File(s)
Author(s)
Willows, Robin
Type
Thesis
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.
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.
Version
Open Access
Date Issued
2017-09
Date Awarded
2018-02
Advisor
Carling, David
Sponsor
Medical Research Council (Great Britain)
Publisher Department
Institute of Clinical Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)