title: Activation and inhibition of Snf1 kinase activity by phosphorylation within the activation loop.
abstract: The AMP-activated protein kinase is a metabolic regulator that transduces information about energy and nutrient availability. In yeast, the AMP-activated protein kinase, called Snf1, is activated when energy and nutrients are scarce. Earlier studies have demonstrated that activation of Snf1 requires the phosphorylation of the activation loop on threonine 210. Here we examined the regulation of Snf1 kinase activity in response to phosphorylation at other sites. Phosphoproteomic studies have identified numerous phosphorylation sites within the Snf1 kinase enzyme. We made amino acid substitutions in the Snf1 protein that were either non-phosphorylatable (serine to alanine) or phospho-mimetic (serine to glutamate) and examined the effects of these changes on Snf1 kinase function in vivo and on its catalytic activity in vitro. We found that changes to most of the phosphorylation sites had no effect on Snf1 kinase function. However, changes to serine 214, a site within the kinase activation loop, inhibited Snf1 kinase activity. Snf1-activating kinase 1 still phosphorylates Snf1-S214E on threonine 210 but the S214E enzyme is non-functional in vivo and catalytically inactive in vitro. We conclude that yeast have developed two distinct pathways for down-regulating Snf1 activity. The first is through direct dephosphorylation of the conserved activation loop threonine. The second is through phosphorylation of serine 214.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: Rhonda R
authors.initials: RR
authors.lastname: McCartney
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: Leopold
authors.initials: L
authors.lastname: Garnar-Wortzel
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: Dakshayini G
authors.initials: DG
authors.lastname: Chandrashekarappa
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Electronic address: mcs2@pitt.edu.
authors.forename: Martin C
authors.initials: MC
authors.lastname: Schmidt
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D000072417:Protein Domains; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D013912:Threonine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010750:Phosphoproteins; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases
keywords: AMP-activated protein kinase; Activation loop; Phosphorylation; Snf1 kinase