Identifying protein kinase-specific effectors of the osmostress response in yeast

Sci Signal. 2017 Mar 7;10(469):eaag2435. doi: 10.1126/scisignal.aag2435.

Abstract

The budding yeast Saccharomyces cerevisiae reacts to increased external osmolarity by modifying many cellular processes. Adaptive signaling relies primarily on the high-osmolarity glycerol (HOG) pathway, which is closely related to the mammalian p38 mitogen-activated protein kinase (MAPK) pathway in core architecture. To identify target proteins of the MAPK Hog1, we designed a mass spectrometry-based high-throughput experiment to measure the impact of Hog1 activation or inhibition on the Scerevisiae phosphoproteome. In addition, we analyzed how deletion of RCK2, which encodes a known effector protein kinase target of Hog1, modulated osmotic stress-induced phosphorylation. Our results not only provide an overview of the diversity of cellular functions that are directly and indirectly affected by the activity of the HOG pathway but also enabled an assessment of the Hog1-independent events that occur under osmotic stress conditions. We extended the number of putative Hog1 direct targets by analyzing the modulation of motifs consisting of serine or threonine followed by a proline (S/T-P motif) and subsequently validated these with an in vivo interaction assay. Rck2 appears to act as a central hub for many Hog1-mediated secondary phosphorylation events. This study clarifies many of the direct and indirect effects of HOG signaling and its stress-adaptive functions.

MeSH terms

  • Mass Spectrometry / methods
  • Mitogen-Activated Protein Kinases / genetics
  • Mitogen-Activated Protein Kinases / metabolism*
  • Mutation
  • Osmolar Concentration
  • Osmotic Pressure*
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Proteome / metabolism
  • Proteomics / methods
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Signal Transduction / genetics

Substances

  • Phosphoproteins
  • Proteome
  • Saccharomyces cerevisiae Proteins
  • RCK2 protein, S cerevisiae
  • Protein-Serine-Threonine Kinases
  • HOG1 protein, S cerevisiae
  • Mitogen-Activated Protein Kinases