The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress

Cell Signal. 2016 Dec;28(12):1881-1893. doi: 10.1016/j.cellsig.2016.08.016. Epub 2016 Aug 31.

Abstract

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms.

Keywords: AMP-activated protein kinase; Saccharomyces cerevisiae; Snf1 kinase; β subunit.

MeSH terms

  • AMP-Activated Protein Kinases / metabolism*
  • Alkalies / pharmacology*
  • Cell Nucleus / drug effects
  • Cell Nucleus / metabolism
  • Conserved Sequence
  • Enzyme Activation
  • Glucose / pharmacology
  • Histidine / metabolism
  • Isoenzymes / metabolism
  • Kinetics
  • Mutant Proteins / metabolism
  • Phosphorylation / drug effects
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism*
  • Protein Transport / drug effects
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / metabolism*
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / enzymology*
  • Stress, Physiological / drug effects*
  • Substrate Specificity / drug effects

Substances

  • Alkalies
  • Isoenzymes
  • Mutant Proteins
  • Protein Subunits
  • Histidine
  • SNF1-related protein kinases
  • Protein-Serine-Threonine Kinases
  • AMP-Activated Protein Kinases
  • Glucose