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

Phosphoproteomics identifies direct and indirect targets of the kinase Hog1 during the yeast osmostress response. Defining the osmotic stress phosphoproteome The high-osmolarity glycerol (HOG) pathway is critical for the ability of the budding yeast Saccharomyces cerevisiae to respond to increased extracellular osmolarity. Hog1 is the mitogen-activated protein kinase (MAPK) homolog at the core of this pathway. Romanov et al. combined inhibition of Hog1 with mass spectrometry analysis to identify Hog1-dependent and Hog1-independent osmostress-induced changes in the yeast phosphoproteome. By examining cells at different time points after the induction of osmotic stress, the authors predicted which Hog1-dependent phosphorylation events were likely to be directly mediated by Hog1 and validated these targets in protein-protein proximity assays. The kinase Rck2, a direct target of Hog1, controlled the phosphorylation of many indirect targets of Hog1. Gene ontology analysis indicated that these direct and indirect targets of Hog1 affected diverse cellular functions and identified some processes not previously implicated in the HOG-mediated response to osmotic stress. 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 S. cerevisiae 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.

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