Quantitative proteomic analysis of kinase and phosphatase substrates in Candida albicans

• Main Author: Kaneva, Iliyana
• Other Authors: Sudbery, Peter ; Dickman, Mark
• Published: University of Sheffield 2017
• Subjects: 572
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.718822

The ability of the fungus Candida albicans to switch interchangeably between yeast and hyphal forms of growth contributes significantly to its pathogenesis. This morphogenetic shift occurs in response to environmental changes and it is accomplished by a complex network of signal transduction pathways. Protein kinases and phosphatases are important messengers in these pathways and several of them have been directly implicated in controlling C. albicans morphogenesis. Kinases and phosphatases (KP) are enzymes that modulate the function of their substrates via reversible phosphorylation and dephosphorylation, respectively. While the specificity of KP is tightly controlled, some enzymes can target a huge number of proteins and have a master regulatory role over various cell processes. The function of KP in C. albicans is poorly understood and methods for global analysis of KP interactions have not been adapted to this organism. This study developed a protocol for large scale analysis of protein interactions in C. albicans using immunoprecipitation and SILAC in conjunction with quantitative mass spectrometry analysis. The protocol was successfully applied for identification of Cdc14 interactors using the substrate-trapping mutant Cdc14C275S. Cdc14 is a phosphatase required for proper hyphal formation, cytoskeletal organisation and cell separation at the end of mitosis. This study reveals over 100 potential substrates of Cdc14 and new roles of the phosphatase in DNA damage repair, DNA replication, chromosome segregation and transcription regulation. In addition, experiments were performed separately with both yeast and hyphae allowing for direct comparison of Cdc14 interactome between both forms. Many of the identified proteins have unknown function and the significance of these putative interactions remains to be found.