AMPK β Subunit Targets Metabolic Stress Sensing to Glycogen

163)) is most closely related to isoamylase domains [8] found in glycoside hydrolase family 13, which includes glycogen and starch branching enzymes [9], and is more * families 20 and 21 (http://afmb.cnrs-mrs.fr/CAZY/) (Figure 1B). Department of Medicine Bacterially expressed ␤-GBD bound glycogen in a saturating manner (Figure 2A) (at 75 ␮g/ml in the pres-University of Melbourne 41 Victoria Parade ence of 5 mg/ml glycogen). ␤-GBD binding to glycogen was inhibited by increasing concentrations of ␤-cyclodex-Fitzroy, Victoria 3065 Australia trin (Figure 2B), and half maximal inhibition occurred at ‫5.1ف‬ mM ␤-cyclodextrin, as is the case for starch bind-3 Endocrine-Metabolism Division Departments of Medicine and Biochemistry ing proteins [10]. Isoamylase domains, although found in glycogen and starch branching enzymes, have not Dartmouth Medical School Hanover, New Hampshire 03755-3833 been shown formally to associate with either carbohydrate , but we demonstrate that ␤-GBD belongs to this family and show that it binds glycogen. Thus, ␤-GBD represents a new subfamily of glycogen binding Summary modules. The ␤-GBD sequence is highly conserved across the AMP-activated protein kinase (AMPK) is a multisub-strate enzyme activated by increases in AMP during AMPK/SNF1 ␤ subfamily (Figure 3A). We constructed a 3D homology model of ␤-GBD (Figure 3B) based on metabolic stress caused by exercise, hypoxia, lack of cell nutrients [1], as well as hormones, including aligning residues 68–163 of the rat AMPK ␤1 subunit with the three known isoamylase domain structures [8, adiponectin and leptin [2, 3]. Furthermore, metformin and rosiglitazone, frontline drugs used for the treat-11, 12]. Inspection of the model revealed two surface-exposed tryptophans and a lysine (W100, K126, and ment of type II diabetes, activate AMPK [4]. Mamma-lian AMPK is an ␣␤␥ heterotrimer with multiple iso-W133) that together formed a putative carbohydrate binding site (Figure 3C) that is strikingly similar to bind-forms of each subunit comprising ␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3, which have varying tissue and subcellular ing site 1 in the glucoamylase and cyclodextrin glycosyl-transferase starch binding domains [13, 14]. When mal-expression [5, 6]. Mutations in the AMPK ␥ subunit cause glycogen storage disease in humans [7], but totriose was modeled into the glycogen binding site, the apolar faces of two sugars stacked against the aromatic the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. rings of W100 and W133 (Figure 3C). The dual specificity phosphatase laforin (Figure 1B) contains a glycogen We show that the AMPK …