Identification and functional analysis of protein tyrosine nitration in MAP kinase signaling pathway in Saccharomyces cerevisiae

Abstract

Identification and characterization of a novel posttranslational modification is crucial for understanding of accurate signaling regulation mechanism. As many signaling pathways are conserved in eukaryotes from yeast to human, yeast could be a good model system for studying the mechanism of posttranslational modifications in cellular signaling pathways. Protein tyrosine nitration is a selective posttranslational modification that is involved in many diseases caused by oxidative stress. Recently, it is suggested that many signaling proteins are supposed to be nitrated on specific tyrosine residues and these tyrosine nitration regulate protein activity or localization and signaling flux. However, due to low abundance or tyrosine nitration and relevance of specific disease, protein tyrosine nitration in yeast was not studied well. In this study, we profiled and investigated the role of protein tyrosine nitration in yeast S. cerevisiae. Introducing enrichment methods using chemical and immunoprecipitation into LC-MS/MS, we successfully identified tyrosine nitrated proteins in yeast in vivo. This is the first nitroproteome study in yeast during signal transduction. 23 proteins were identified as nitrated in enrichment methods, and the overall level of nitration was increased after pheromone stimulation. Sequence and structural analysis showed that most tyrosine residues, surrounded by acidic residues and located in solvent accessible site, could be easily nitrated. These results imply that the environment of tyrosine residue is important to allow of tyrosine nitration, thus protein tyrosine nitration is a selective and reversible modification regulated during mating signal transduction. In addition, we showed that tyrosine nitration of Ste7 was increased during mating signaling and predicted Tyr 381 as a putative nitration residue. Substitution of Tyr 381 to Phe reduced mating signaling flux by inhibition of Ste7 activity, and this mechanism seems to be conserved in other MAPK signaling pathway in yeast such as Hog signaling pathway. Taken together, we suggest that tyrosine nitration is a novel modification which is regulated during MAPK signaling pathway in yeast. Also, proteins identified in this study could provide a clue for searching nitration targets in other signaling pathway or other species.