Allosteric regulation by AMP and ATP

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AMPK is allosterically activated by AMP, which binds to the regulatory g subunit, resulting  in a 2 to 5 fold increase in activity compared to basal activity (Hardie et al., 1999). The degree of activation by AMP is markedly affected by nature of the catalytic a and regulatory g iso-forms constituting the AMPK complex, illustrating the complexity of AMPK signaling regulation. The greatest scale of activation is observed in AMPK complexes containing the a2 and g2 subunits, while complexes containing the g3 isoform are only weakly activated by AMP (Cheung et al., 2000). Binding of AMP to the g subunit causes direct allosteric activation of the kinase and also induces a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr-172 (Riek et al., 2008; Sanders et al., 2007; Suter et al., 2006), favoring accumulation of the phosphorylated active form of AMPK (see below). Interestingly, it has been demonstrated that high concentrations of ATP oppose activation of the AMPK complex by AMP, suggesting that the allosteric sites bind AMP and ATP in a mutually exclusive manner (Corton et al., 1995). Thus, AMPK can be considered more as a sensor of the intracellular AMP/ATP ratio, rather than a direct sensor of AMP levels. AMP and ATP vary reciprocally in cells, due to the action of adenylate kinase (AMP + ATP <-> 2ADP [adenosine 5'-diphosphate]), thus, AMP: ATP ratio may be a more sensitive indicator of cellular energy status than ADP: ATP ratio. The finding that AMPK activation is altered in contracting muscles from adenylate kinase-deficient mice also supports a role for adenylate kinase in generation of an AMPK activating signal (Hancock et al., 2006).

The crystal structures of mammalian g and yeast homologue revealed the structural and conformational elements required for binding the regulatory nucleotides AMP and ATP (Townley and Shapiro, 2007; Xiao et al., 2007). Out of all the CBS domains present, two CBS domains appear to bind AMP or ATP reversibly and may correspond to the two regulatory sites identified from previous binding studies (Scott et al., 2004). A third CBS domain binds AMP very strongly and does not readily exchange with ATP, but its physiological role is unclear. The fourth CBS domain remains unoccupied even in the presence of high concentrations of AMP or ATP (Xiao et al., 2007). Several naturally occurring point mutations in the CBS domains of human g2 isoform have been reported to cause an inherited syndrome of hypertrophic cardiomyopathy of varying severity associated with excessive glycogen storage in cardio myocytes, accompanied with Wolff–Parkinson–White syndrome, a pre-excitation disorder (Arad et al., 2007). Biochemical studies demonstrate that some of these mutations interfere with the binding of AMP and allosteric activation (Scott et al., 2004) of AMPK. This supports  the evidence that Bateman domains (CBS domains) constitute the regulatory binding sites for AMP. Interestingly, although mutations in the g2 subunit reduces binding of the activating nucleotide AMP, they also appear to increase the basal activity associated with elevated Thr-172 phosphorylation (Arad et al., 2002; Burwinkel et al., 2005). This is presumably due to a concomitant reduction in binding of the inhibitory nucleotide ATP and consequent reduction in phosphatase activity, thus hindering kinase inactivation (see below). Therefore, g2 mutants, unoccupied by ATP, behave in a partially active conformation and this “gain-of-function” effect could explain the dominant nature of g subunit mutations (Burwinkel et al., 2005; Hamilton et al., 2001). Hence, AMPK dysregulation could be connected with impaired binding or interaction of both AMP and ATP on the g subunit.

AMPK is also allosterically inhibited by physiological concentrations of phosphocreatine (Ponticos et al., 1998), consistent with the proposed physiological role of the kinase as a sensor of cellular energy status. As it decreases during muscle contraction, phosphocreatine, rather than AMP, may be the key regulator of the AMPK system during short-term exercise.