Phosphoproteomic study on osmotic shock in Saccharomyces cerevisiae over sub-minute and half- hour timescales
Protein phosphorylation is an important cellular regulatory mechanism. Kinases and phosphatases (KP) are vital for the signalling transductions in cells. Signals received by the cell must be transmitted effectively to ensure an appropriate adaptive response is achieved. Monitoring the phosphorylation activity on proteins helps us gain a better understanding to how cells work. In our study we have exposed Saccharomyces cerevisiae to high osmolarity using 0.4M NaCl, and caused a hyper osmotic shock. Protein phosphorylation was measured using a mass spectrometry technique called Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC). This is a quantitative proteomic technique that allows the comparison between cells that are exposed to NaCl and a control group, where cells are not exposed to NaCl. This comparison between the two groups allows us to deduce dynamic regulation of proteins specific to the stimulus applied. We have used a sub-minute and a half-hour timescales to examine the effects of osmotic shock in S. cerevisiae. In this memoir, I have examined the MAPK-Hog1 pathway during the half-hour timescale and have compared it to the sub-minute timescale, which was previously published in Kanshin & Bergeron-Sandoval et al., 2015 [26]. Moreover, the entire data had 161 dynamic phosphopeptide from 100 unique proteins that were present on both timescales. Dynamic phosphopeptides were also compared to another phosphoproteomic study that had used cold/hot stress as a stimulus (Kanshin et al., 2015 [25]). There were remarkable overlaps between osmotic shock and in particular heat stress. This led us to examine the possibility that TORC1&2 involvement in the osmoadaptation process. Furthermore, cells temporarily stop cell cycling during the osmoadaptation process. Consistent with this observation, 68% of the proteins involved in the G1/S transition underwent changes in phosphorylation, perhaps reflecting their regulation, during the first 30 minutes of osmotic shock.  Kinases and phosphatases are known to heavily regulate themselves. A network of  interconnected active KPs revealed Hog1 to be the most connected kinase in the sub-  . This is indicative of how cells adapt to high osmolarity; Hog1 being responsible for the fast sensing and adaptation, and Cdc28 for stopping and regulating the cell cycle. In summary, I have argued that it is possible to extract dynamic phosphopeptides that are stimulus-specific within the half-hour timescale. I have compared dynamic phosphopeptides from the sub-minute timescale to the half-hour timescale and have examined biological processes that play a part in the osmoadaptive process.
Kinase-phosphatase network Saccharomyces cerevisiae Dynamic phosphorylation Network hierarchical structure Cell cycle Osmoadaptation Mammalian target of rapamycin Mitogen-activated protein kinase