SILAC-based phosphoproteomics reveals new PP2A-Cdc55-regulated processes in budding yeast

• Published in: Gigascience Vol. 7; no. 5
• Main Authors: Baro, Barbara, Játiva, Soraya, Calabria, Inés, Vinaixa, Judith, Bech-Serra, Joan-Josep, de LaTorre, Carolina, Rodrigues, João, Hernáez, María Luisa, Gil, Concha, Barceló-Batllori, Silvia, Larsen, Martin R, Queralt, Ethel
• Format: Journal Article
• Language: English
• Published: United States Oxford University Press 01.05.2018
• Subjects: Actin; Amino Acid Sequence; Anaphase; Cell cycle; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - metabolism; Cell walls; Cytokinesis; Cytoskeleton; Endocytosis; Gene Ontology; Isotope Labeling - methods; Kinases; Mass spectrometry; Mass spectroscopy; Metaphase; Mitosis; Molecular Docking Simulation; Phosphatase; Phosphoprotein phosphatase; Phosphoproteins - metabolism; Phosphorylation; Protein Binding; Protein Interaction Maps; Protein phosphatase; Protein Phosphatase 2 - chemistry; Protein Phosphatase 2 - metabolism; Proteins; Proteome - metabolism; Proteomics - methods; Reproducibility of Results; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Saccharomycetales - metabolism; Statistical analysis; Substrate Specificity; Substrates; Threonine; Yeast
• ISSN: 2047-217X; 2047-217X
• DOI: 10.1093/gigascience/giy047

Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2ACdc55 phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2ACdc55 substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2ACdc55 phosphatase and new PP2A-related processes in mitotic arrested cells. We identified 62 statistically significant PP2ACdc55 substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2ACdc55 substrates such as Slk19 and Lte1, involved in early and late anaphase pathways, and Zeo1, a component of the cell wall integrity pathway. Finally, we constructed docking models of Cdc55 and its substrate Mob1. We found that the predominant interface on Cdc55 is mediated by a protruding loop consisting of residues 84-90, thus highlighting the relevance of these aminoacids for substrate interaction. We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2ACdc55 substrates and functions in mitosis. As expected, several hyperphosphorylated proteins corresponded to Cdk1-dependent substrates, although other kinases' consensus motifs were also enriched in our dataset, suggesting that PP2ACdc55 counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2ACdc55 regulation, highlighting a major role of PP2ACdc55 in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2ACdc55-dependent phosphoproteome.