title: Ligand binding to the AMP-activated protein kinase active site mediates protection of the activation loop from dephosphorylation.
abstract: The AMP-activated protein kinase (AMPK) is a conserved signaling molecule in a pathway that maintains adenosine triphosphate homeostasis. Recent studies have suggested that low energy adenylate ligands bound to one or more sites in the γ subunit of AMPK promote the formation of an active, phosphatase-resistant conformation. We propose an alternative model in which the kinase domain association with the heterotrimer core results in activation of the kinase catalytic activity, whereas low energy adenylate ligands bound in the kinase active site promote phosphatase resistance. Purified Snf1 α subunit with a conservative, single amino acid substitution in the kinase domain is protected from dephosphorylation by adenosine diphosphate in the complete absence of the β and γ subunits. Staurosporine, a compound known to bind to the active site of many protein kinases, mediates strong protection from dephosphorylation to yeast and mammalian AMPK enzymes. The analog-sensitive Snf1-I132G protein but not wild type Snf1 exhibits protection from dephosphorylation when bound by the adenosine analog 2NM-PP1 in vitro and in vivo. These data demonstrate that ligand binding to the Snf1 active site can mediate phosphatase resistance. Finally, Snf1 kinase with an amino acid substitution at the interface of the kinase domain and the heterotrimer core exhibits normal regulation of phosphorylation in vivo but greatly reduced Snf1 kinase activity, supporting a model in which kinase domain association with the heterotrimer core is needed for kinase activation.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Dakshayini G
authors.initials: DG
authors.lastname: Chandrashekarappa
authors.forename: Rhonda R
authors.initials: RR
authors.lastname: McCartney
authors.forename: Martin C
authors.initials: MC
authors.lastname: Schmidt
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000818:Animals; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D012568:Schizosaccharomyces / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008024:Ligands; D000244:Adenosine Diphosphate; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D019311:Staurosporine