Separase cooperates with Zds1 and Zds2 to activate Cdc14 phosphatase in early anaphase

Completion of mitotic exit and cytokinesis requires the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. A key enzyme that counteracts Cdk during budding yeast mitotic exit is the Cdc14 phosphatase. Cdc14 is inactive for much of the cell cycle, sequestered by its inhibitor Net1 in the nucleolus. At anaphase onset, separase-dependent down-regulation of PP2ACdc55 allows phosphorylation of Net1 and consequent Cdc14 release. How separase causes PP2ACdc55 down-regulation is not known. Here, we show that two Cdc55-interacting proteins, Zds1 and Zds2, contribute to timely Cdc14 activation during mitotic exit. Zds1 and Zds2 are required downstream of separase to facilitate nucleolar Cdc14 release. Ectopic Zds1 expression in turn is sufficient to down-regulate PP2ACdc55 and promote Net1 phosphorylation. These findings identify Zds1 and Zds2 as new components of the mitotic exit machinery, involved in activation of the Cdc14 phosphatase at anaphase onset. Our results suggest that these proteins may act as separase-regulated PP2ACdc55 inhibitors

PP2A Functions during Mitosis and Cytokinesis in Yeasts

Protein phosphorylation is a common mechanism for the regulation of cell cycle progression. The opposing functions of cell cycle kinases and phosphatases are crucial for accurate chromosome segregation and exit from mitosis. Protein phosphatases 2A are heterotrimeric complexes that play essential roles in cell growth, proliferation, and regulation of the cell cycle. Here, we review the function of the protein phosphatase 2A family as the counteracting force for the mitotic kinases. We focus on recent findings in the regulation of mitotic exit and cytokinesis by PP2A phosphatases in S. cerevisiae and other fungal species

PP2A-Cdc55 phosphatase regulates actomyosin ring contraction and septum formation during cytokinesis

Eukaryotic cells divide and separate all their components after chromosome segregation by a process called cytokinesis to complete cell division. Cytokinesis is highly regulated by the recruitment of the components to the division site and through post-translational modifications such as phosphorylations. The budding yeast mitotic kinases Cdc28-Clb2, Cdc5, and Dbf2-Mob1 phosphorylate several cytokinetic proteins contributing to the regulation of cytokinesis. The PP2A-Cdc55 phosphatase regulates mitosis counteracting Cdk1- and Cdc5-dependent phosphorylation. This prompted us to propose that PP2A-Cdc55 could also be counteracting the mitotic kinases during cytokinesis. Here we show that in the absence of Cdc55, AMR contraction and the primary septum formation occur asymmetrically to one side of the bud neck supporting a role for PP2A-Cdc55 in cytokinesis regulation. In addition, by in vivo and in vitro assays, we show that PP2A-Cdc55 dephosphorylates the chitin synthase II (Chs2 in budding yeast) a component of the Ingression Progression Complexes (IPCs) involved in cytokinesis. Interestingly, the non-phosphorylable version of Chs2 rescues the asymmetric AMR contraction and the defective septa formation observed in cdc55 increment mutant cells. Therefore, timely dephosphorylation of the Chs2 by PP2A-Cdc55 is crucial for proper actomyosin ring contraction. These findings reveal a new mechanism of cytokinesis regulation by the PP2A-Cdc55 phosphatase and extend our knowledge of the involvement of multiple phosphatases during cytokinesis

Dual Regulation of the Mitotic Exit Network (MEN) by PP2A-Cdc55 Phosphatase

Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2A(Cdc55) phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2A(Cdc55) phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2A(Cdc55) regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2A(Cdc55) initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2A(Cdc55) functions affect the regulation of various MEN components, contributing to mitotic exit

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

Background: Protein phosphatase 2A (PP2A) is a family of conserved serine/threonine phosphatases involved in several essential aspects of cell growth and proliferation. PP2A(Cdc55) phosphatase has been extensively related to cell cycle events in budding yeast; however, few PP2A(Cdc55) substrates have been identified. Here, we performed a quantitative mass spectrometry approach to reveal new substrates of PP2A(Cdc55) phosphatase and new PP2A-related processes in mitotic arrested cells. Results: We identified 62 statistically significant PP2A(Cdc55) substrates involved mainly in actin-cytoskeleton organization. In addition, we validated new PP2A(Cdc55) 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. Conclusions: We used phosphoproteomics of Cdc55-deficient cells to uncover new PP2A(Cdc55) 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 PP2A(Cdc55) counteracts and regulates other kinases distinct from Cdk1. Indeed, Pkc1 emerged as a novel node of PP2A(Cdc55) regulation, highlighting a major role of PP2A(Cdc55) in actin cytoskeleton and cytokinesis, gene ontology terms significantly enriched in the PP2A(Cdc55)-dependent phosphoproteome

Disruption of NIPBL/Scc2 in Cornelia de Lange Syndrome provokes cohesin genome-wide redistribution with an impact in the transcriptome

Cornelia de Lange syndrome (CdLS) is a rare disease affecting multiple organs and systems during development. Mutations in the cohesin loader, NIPBL/Scc2, were first described and are the most frequent in clinically diagnosed CdLS patients. The molecular mechanisms driving CdLS phenotypes are not understood. In addition to its canonical role in sister chromatid cohesion, cohesin is implicated in the spatial organization of the genome. Here, we investigate the transcriptome of CdLS patient-derived primary fibroblasts and observe the downregulation of genes involved in development and system skeletal organization, providing a link to the developmental alterations and limb abnormalities characteristic of CdLS patients. Genome-wide distribution studies demonstrate a global reduction of NIPBL at the NIPBL-associated high GC content regions in CdLS-derived cells. In addition, cohesin accumulates at NIPBL-occupied sites at CpG islands potentially due to reduced cohesin translocation along chromosomes, and fewer cohesin peaks colocalize with CTCF

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

18 páginas, 5 figuras, 1 tablaBackground: 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. Results: 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. Conclusions: 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.Work in our laboratory is supported by the Spanish Ministry of Science and Innovation (BFU2011–27568), Spanish Ministry of Economy and Competitively (BFU2013–43132-P and BFU2016–77975-R AEI/FEDER, UE cofounded by FEDER funds/European Regional Development Fund- a way to build Europe). MRL was supported by the Lundbeck foundation (Junior Group Leader Fellowship). This work was supported by a generous grant from the VILLUM Foundation to the VILLUM Centre for Bioanalytical Sciences at the University of Southern Denmark. SBB is a recipient of ISCIII grant 13FIS037. IDIBELL Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, and is supported by grant PT13/0001/0033.Peer reviewe

The Mitotic Exit Network: Methods and protocols

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