Quantitative comparative phosphoproteomics to decipher signal transduction

Adaptation to changing environmental conditions takes place rapidly in all living organisms: cells must respond to a wide variety of signals. Through successive protein phosphorylation and de-phosphorylation steps, signal transduction cascades mediate between the perception of the extracellular conditions and the adaptation of intracellular processes. The first event after the perception of the external signal is the auto-phosphorylation of a receptor kinase, which, on its turn, phosphorylates down-stream targets, mainly kinase cascades. The downstream effectors, mitogen-activated protein kinases (MAPKs) and other kinases, regulate the activity of transcription factors, metabolic enzymes and other factors, thereby initiating the fungal adaptive response. Understanding these adaptive responses requires deciphering early phosphorylation events. While global proteomic analyses have been improved over decades, the global study of protein (de)phosphorylation events in living organisms only became possible recently. Significant advances in mass spectrometry based phosphoproteomics have taken place, including phosphopeptide enrichment, detection and quantification, and phosphorylation site localization. Reports of fungal phosphoproteomic studies are increasing, but those dealing with filamentous ascomycetes are still rare. The first phosphoproteomic study of kinase dependent phosphorylation events has been reported for the model fungus Neurospora crassa. In Botrytis cinerea, phosphoproteomics have been used to study the global phosphoprotein content in axenic culture and the differential phosphorylation profiles between mycelia grown on glucose or tomato-cell wall as carbon-source. We have undertaken a comparative proteomics and phosphoproteomics study of the B. cinerea osmosensing pathway challenged by the fungicide fludioxonil. We compared mutants of the sensor histidine-kinase Bos1 and of the MAPK Sak1 to the parental wild-type. Strains were exposed (or not) to fludioxonil for 15 min. during exponential in vitro growth. Shotgun proteomics revealed considerable differences in protein content among the strains, but no treatment effect. These results indicate a strong transcriptional and/or translational regulation under Bos1 and Sak1 control, respectively, under standard conditions. Quantitative phosphoproteomics revealed a clear response to the fludioxonil treatment as well as (de)phosphorylation events controlled by Bos1, Sak1, or both.