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

Abstract

BackgroundProtein 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.ResultsWe 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.ConclusionsWe 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.

Yeast Sphingolipid Phospholipase Gene ISC1 Regulates the Spindle Checkpoint by a CDC55-Dependent Mechanism

“…reported a SILAC based phosphoproteomics study of a PP2A-Cdc55 mutant in which they identified 62 potential Cdc55 substrates [27]. By comparing their results to our list of Isc1 and Cdc55-dependent phosphoproteins, we found a significant overlap of ~29%, strongly supporting the validity of our analysis.…”

Cdc14 and PP2A Phosphatases Cooperate to Shape Phosphoproteome Dynamics during Mitotic Exit

“…Proteome-wide surveys uncovered a plethora of PP2A Cdc55 substrates. Consistent with its biochemical characteristics, PP2A Cdc55 preferentially targets threonine residues, thereby establishing a temporal order of serine before threonine phosphorylation as cells progress from S phase to mitosis (Baro et al., 2018, Godfrey et al., 2017). Little is known about the possible roles of PP2A Rts3 .…”

Ser/Thr protein phosphatases in fungi: structure, regulation and function

“…Recent phosphoproteomics studies suggest additional roles for PP2A Cdc55 in sensing several internal and environmental cues [144] being important for the septin ring dynamics and morphogenesis checkpoint ( Figure 8 ). Thus, PP2A Cdc55 downregulates Swe1 after septin rings are properly organized, promoting mitotic entry, and it is also involved in the regulation of the septin ring disassembly process [107].…”