Investigating the regulation of AMP-activated protein kinase and SNF1

Abstract

AMP-activated protein kinase (AMPK) has long been known to play a critical role in the maintenance of energy homeostasis through direct interaction with or altering gene and protein expression of key players in diverse metabolic pathways. AMPK has been implicated in a number of diseases with roots in metabolic dysregulation, including obesity, type 2 diabetes and cancer. Elucidating the regulation of AMPK is an important part in understanding the progression of these diseases, and for developing small molecule modulators of AMPK activity which could have therapeutic applications. AMPK activity is determined by the phosphorylation status of T172 in the activation loop of the α subunit kinase domain. Binding of AMP to the γ subunit also increases its activity, primarily by preventing dephosphorylation of T172 but also by direct allosteric activation of the complex. The overall aim of this study was to investigate nucleotide regulation of AMPK. Site-directed mutagenesis studies showed that loss of highly conserved residues in γ1 disrupts regulation of both dephosphorylation and allosteric activation of AMPK by AMP. However, my studies revealed that these mutations do not have site-specific effects. The role of ADP in AMPK regulation was also investigated following the observation that this nucleotide also prevented dephosphorylation and inactivation of AMPK. The action of ADP on AMPK activity was characterised in wild-type complexes and insights from new structures of the active AMPK complex has provided insight into the molecular mechanism underlying AMP/ADP protection and dephosphorylation of T172. The yeast homologue of AMPK, SNF1, plays a central role in responding to glucose limitation and adaption to alternative carbon sources. Recombinant SNF1 complexes were used to show that ADP is the long-sought metabolite responsible for transmitting this low glucose signal and activates SNF1 by a similar mechanism to that seen in AMPK, preventing dephosphorylation and inactivation. Together these studies identify an important activator of both AMPK and SNF1, drawing parallels between these two systems and characterising a highly conserved regulatory mechanism, suggesting that ADP may represent a unifying trigger for activation of AMPK homologues in diverse species. Finally a potential link between AMPK and redox metabolism was identified in the form of NADH, opening new avenues of research in this field.