title: Identifying protein kinase-specific effectors of the osmostress response in yeast.
abstract: The budding yeastSaccharomyces cerevisiaereacts 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 theScerevisiaephosphoproteome. In addition, we analyzed how deletion ofRCK2, 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.
authors.affiliation: Department for Biochemistry, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria.
authors.forename: Natalie
authors.identifier: http://orcid.org/0000-0003-3749-2654
authors.initials: N
authors.lastname: Romanov
authors.affiliation: Department for Biochemistry, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria.
authors.forename: David Maria
authors.identifier: http://orcid.org/0000-0002-1977-1556
authors.initials: DM
authors.lastname: Hollenstein
authors.affiliation: Department for Biochemistry, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria.
authors.forename: Marion
authors.identifier: http://orcid.org/0000-0003-1151-0228
authors.initials: M
authors.lastname: Janschitz
authors.affiliation: Department for Biochemistry, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria.
authors.forename: Gustav
authors.initials: G
authors.lastname: Ammerer
authors.affiliation: Mass Spectrometry Facility, Max F. Perutz Laboratories, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Anrather
authors.affiliation: Department for Biochemistry, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria. wolfgang.l.reiter@univie.ac.at.
authors.forename: Wolfgang
authors.identifier: http://orcid.org/0000-0003-1266-8975
authors.initials: W
authors.lastname: Reiter
mesh_terms: D013058:Mass Spectrometry / Q000379:methods; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics
chemical_list: D010750:Phosphoproteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; C407464:RCK2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C088589:HOG1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases