Intrasteric control of AMPK via the γ1 subunit AMP allosteric regulatory site

Abstract

AMP‐activated protein kinase (AMPK) is a αβγ heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the α subunit and by AMP allosteric control previously thought to be mediated by both α and γ subunits. Here we present evidence that adjacent γ subunit pairs of CBS repeat sequences (after C ystathionine B eta S ynthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the γ 1 CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast γ homolog, snf4 contains a His151Gly substitution, and when this is introduced into γ 1, AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in γ 1 corresponds to the site of mutation in human γ 2 and pig γ 3 genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the α and γ subunits and that AMP functions to derepress AMPK activity.