Analysis of the Candida albicans Phosphoproteome

Abstract

dCandida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many contexts, including morphological transitions, mating competence, biofilm formation, stress resistance, and cell wall synthesis. Analysis of kinase-and phosphatase-deficient mutants has made it clear that protein phosphorylation plays an important role in the regulation of these pathways. In this study, to further our understanding of phosphorylation in C. albicans regulation, we performed a deep analysis of the phosphoproteome in C. albicans. We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine, and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in Gene Ontology (GO) terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional coregulatory protein complex. A targeted analysis of the phosphosites in Mediator complex proteins confirmed the large-scale studies, and further in vitro assays identified a subset of these phosphorylations that were catalyzed by Cdk8 (Ssn3), a kinase within the Mediator complex. These data represent the deepest single analysis of a fungal phosphoproteome and lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins.

Complex Haploinsufficiency-Based Genetic Analysis of the NDR/Lats Kinase Cbk1 Provides Insight into Its Multiple Functions in Candida albicans

“…Since cbk1D/D does not form filaments, lysates from yeast phase cells were used for these experiments. Consistent with the extensive phosphorylation of Rgd3 apparent in the data set reported by Willger et al (2015), multiple species of Rgd3 with a wide range of isoelectric points are apparent in blots derived from WT cells ( Figure 7B). In contrast, Rgd3-GFP isolated from cbk1D/D cells shows a much less complex pattern, suggesting that significant changes in Rgd3 phosphorylation result from loss of Cbk1 activity.…”

“…Rgd3 contains a large number of predicted protein kinase phosphorylation motifs and, consistent with that analysis, phosphoproteomics data recently reported by Willger et al (2015) indicate that Rgd3 contains a total of 27 phosphorylated serine or threonine residues. We wondered whether loss of Cbk1 function may affect the phosphorylation state of Rgd3.…”

“…Specifically, Luca and Winey (1998) showed that double mutants of mps1-1 and mob2D, the binding partner of Cbk1, showed increased ploidy relative to each single mutant. Inspection of the protein sequence of Cbk1 revealed the presence of a consensus Mps1 phosphorylation site (E/D/N/Q at the 22 position relative to S or T; Gogl et al 2015) at S98 (EMS) and data from a recent phosphoproteomic study indicate that this position is phosphorylated in C. albicans (Willger et al 2015). In addition, C. albicans Mob2 contains five consensus Mps1 sites that are also phosphorylated (NLS 35 ; NLS 44 ; QDS 83 ; QSS 86 ; and QSS 93 ) according to this study (Dou et al 2011).…”

“…Both Rga2 and Bem3 are substrates for CDK1/Hgc1 during hyphal development (Zheng et al 2007). Rdg3 also contains consensus CDK phosphorylation sites and the mass spectroscopy data reported by Willger et al (2015) indicate at least of one these sites (T 761 PSR) is phosphorylated. The identification of a third GAP for C. albicans is not surprising.…”

Candida albicans responds to glycostructure damage by Ace2‐mediated feedback regulation of Cek1 signaling

“…Corresponding to this notion, ACE2 overexpression restored the sensitivity phenotypes of all mutants in the Cek1 pathway except in the cek1 mutant, indicating that Ace2 activation requires phosphorylation by Cek1 and that an increase in Ace2 levels obviates the necessity for Ace2 activation by upstream components. Ace2 was found to contain 18 phosphorylated residues (Willger et al ., ), which may be due to the action of Cek1 and other kinases, including Cst20 (discussed below). Previously, it was shown that ACE2 overexpression is able to suppress the sensitivity phenotypes of the pmt1 mutant (Cantero and Ernst, ), which probably is mediated by induced expression of PMT2/PMT4 genes.…”