Phosphoproteomic analyses reveal novel cross-modulation mechanisms between two signaling pathways in yeast

Abstract

Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen-activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time-resolved phosphorylation site dynamics after pathway co-stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone-induced down-regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways.

Keywords: HOG pathway; cell signaling network; crosstalk; pheromone pathway; phosphoproteomics.

Figure 1

Model pathways and experimental workflow

Figure 2

Data overview and validation

Figure 3

Data representation and clustering

Figure 4

Classification of phosphopeptides (P-peps) according to how NaCl and pheromone affect the shape of their dynamic curves

Figure 5

Hog1 and Fus3 activation sites

Figure 6

Quantification of the NaCl- and the pheromone-induced influence on each phosphopeptide

Figure 7

Modulation of Hog1 activation by pheromone and NaCl

Figure 8

Hypothesis validation by logic modeling