Tight regulation of the MAP kinase Hog1 is crucial for survival under changing osmotic conditions. Interestingly, we found that Hog1 phosphorylates multiple upstream components, implying feedback regulation within the signaling cascade. Taking advantage of an unexpected link between glucose availability and Hog1 activity, we used quantitative single cell measurements and computational modeling to unravel feedback regulation operating in addition to the well-known adaptation feedback triggered by glycerol accumulation. Indeed, we found that Hog1 phosphorylates its activating kinase Ssk2 on several sites, and cells expressing a non-phosphorylatable Ssk2 mutant are partially defective for feedback regulation and proper control of basal Hog1 activity. Together, our data suggest that Hog1 activity is controlled by intertwined regulatory mechanisms operating with varying kinetics, which together tune the Hog1 response to balance basal Hog1 activity and its steady-state level after adaptation to high osmolarity.

F.L. is supported by a HFSPO post-doctoral fellowship. The Pelet and Peter laboratories are supported by grants of the Swiss National Science Foundation (SNF), and work in the Peter laboratory is funded by the European Research Council (ERC), the Swiss Initiative in Systems Biology SystemsX (RTD project YeastX) and the ETH Zurich. In addition grants from the Spanish Ministry of Economy and Competitiveness (BFU2012-33503 and FEDER), 2014 SGR (Generalitat de Catalunya) and the Fundación Marcelino Botín (FB). F.P. is a recipient of an ICREA Acadèmia award (Generalitat de Catalunya).