Regulation of Snf1 kinase. Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit.
The yeast Snf1 kinase and its metazoan orthologues, the AMP-activated protein kinases, are activated in response to nutrient limitation. Activation requires the phosphorylation of a conserved threonine residue in the activation loop of the catalytic subunit. A phosphopeptide antibody was generated that specifically recognizes Snf1 protein that is phosphorylated in its activation loop on threonine 210. Using this reagent, we show that phosphorylation of threonine 210 correlates with Snf1 activity, since it is detected in cells subjected to glucose limitation but not in cells grown in abundant glucose. A Snf1 mutant completely lacking kinase activity was phosphorylated normally on threonine 210 in glucose-starved cells, eliminating the possibility that the threonine 210 modification is due to an autophosphorylation event. Cells lacking the Reg1 protein, a regulatory subunit for the Glc7 phosphatase, showed constitutive phosphorylation of Snf1 threonine 210. Exposure of cells to high concentrations of sodium chloride also induced phosphorylation of Snf1. Interestingly, Mig1, a downstream target of Snf1 kinase, is phosphorylated in glucose-stressed but not sodium-stressed cells. Finally, cells lacking the gamma subunit of the Snf1 kinase complex encoded by the SNF4 gene exhibited normal regulation of threonine 210 phosphorylation in response to glucose limitation but are unable to phosphorylate Mig1 efficiently. Our data indicate that activation of the Snf1 kinase complex involves two steps, one that requires a distinct upstream kinase and one that is mediated by the gamma subunit of the kinase itself.
McCartney
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Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Schmidt
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MC
D055372:AMP-Activated Protein Kinases; D000483:Alleles; D015153:Blotting, Western; D002352:Carrier Proteins; D020134:Catalytic Domain; D004789:Enzyme Activation; D000939:Epitopes / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005838:Genotype; D005947:Glucose / Q000378:metabolism; D007477:Ions; D008954:Models, Biological; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D017354:Point Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D012964:Sodium / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D002352:Carrier Proteins; D000939:Epitopes; D005656:Fungal Proteins; D007477:Ions; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D010769:Phosphothreonine; D013912:Threonine; D012965:Sodium Chloride; D012964:Sodium; D011494:Protein Kinases; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; C078651:SNF4 protein, S cerevisiae; D055372:AMP-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C070829:REG1 protein, S cerevisiae; D005947:Glucose