title: Proteome-wide search for PP2A substrates in fission yeast.
abstract: PP2A (protein phosphatase 2A) is a major phosphatase in eukaryotic cells that plays an essential role in many processes. PP2A mutations in Schizosaccharomyces pombe result in defects of cell cycle control, cytokinesis and morphogenesis. Which PP2A substrates are responsible for these changes is not known. In this work, we searched for PP2A substrates in S. pombe using two approaches, 2D-DIGE analysis of PP2A complex mutants and identification of PP2A interacting proteins. In both cases, we used MS to identify proteins of interest. In the DIGE experiment, we compared proteomes of wild-type S. pombe, deletion of pta2, the phosphoactivator of the PP2A catalytic subunit, and pab1-4, a mutant of B-type PP2A regulatory subunit. A total of 1742 protein spots were reproducibly resolved by 2D-DIGE and 51 spots demonstrated significant changes between PP2A mutants and the wild-type control. MS analysis of these spots identified 27 proteins that include key regulators of glycerol synthesis, carbon metabolism, amino acid biosyntesis, vitamin production, and protein folding. Importantly, we independently identified a subset of these proteins as PP2A binding partners by affinity precipitation, suggesting they may be direct targets of PP2A. We have validated our approach by demonstrating that phosphorylation of Gpd1, a key enzyme in glycerol biogenesis, is regulated by PP2A and that ability of cells to respond to osmotic stress by synthesizing glycerol is compromised in the PP2A mutants. Our work contributes to a better understanding of PP2A function and identifies potential PP2A substrates.
authors.affiliation: Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-Consejo Superior de Investigaciones Científicas, Junta de Andalucía, Sevilla, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Bernal
authors.forename: Jacob
authors.initials: J
authors.lastname: Zhurinsky
authors.forename: Ana B
authors.initials: AB
authors.lastname: Iglesias-Romero
authors.forename: Maria A
authors.initials: MA
authors.lastname: Sanchez-Romero
authors.forename: Ignacio
authors.initials: I
authors.lastname: Flor-Parra
authors.forename: Laura
authors.initials: L
authors.lastname: Tomas-Gallardo
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Perez-Pulido
authors.forename: Juan
authors.initials: J
authors.lastname: Jimenez
authors.forename: Rafael R
authors.initials: RR
authors.lastname: Daga
mesh_terms: D009154:Mutation; D009997:Osmotic Pressure; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D057825:Two-Dimensional Difference Gel Electrophoresis / Q000379:methods
chemical_list: D020543:Proteome; D029702:Schizosaccharomyces pombe Proteins; D054648:Protein Phosphatase 2
keywords: 2D-DIGE; Glycerol; PP2A; Phosphorylation; Schizosaccharomyces pombe; Technology