scholarly journals A Wee1 checkpoint inhibits anaphase onset

2013 ◽  
Vol 201 (6) ◽  
pp. 843-862 ◽  
Author(s):  
Noel Lianga ◽  
Elizabeth C. Williams ◽  
Erin K. Kennedy ◽  
Carole Doré ◽  
Sophie Pilon ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Regulatory Subunit ◽  
Anaphase Promoting Complex ◽  
Mitotic Entry ◽  
Anaphase Onset ◽  
Phosphatase 2A ◽  
Wee1 Kinase ◽  
Morphogenesis Checkpoint

Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APCCdc20). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25+) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2ACdc55 triggers anaphase onset.

scholarly journals Protein Phosphatase 2A Stabilizes Human Securin, Whose Phosphorylated Forms Are Degraded via the SCF Ubiquitin Ligase

2006 ◽  
Vol 26 (11) ◽  
pp. 4017-4027 ◽  
Author(s):  
Ana M. Gil-Bernabé ◽  
Francisco Romero ◽  
M. Cristina Limón-Mortés ◽  
María Tortolero
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Chromatid Segregation ◽  
Phosphatase 2A ◽  
F Box Protein

ABSTRACT Sister chromatid segregation is triggered at the metaphase-to-anaphase transition by the activation of the protease separase. For most of the cell cycle, separase activity is kept in check by its association with the inhibitory chaperone securin. Activation of separase occurs at anaphase onset, when securin is targeted for destruction by the anaphase-promoting complex or cyclosome E3 ubiquitin protein ligase. This results in the release of the cohesins from chromosomes, which in turn allows the segregation of sister chromatids to opposite spindle poles. Here we show that human securin (hSecurin) forms a complex with enzymatically active protein phosphatase 2A (PP2A) and that it is a substrate of the phosphatase, both in vitro and in vivo. Treatment of cells with okadaic acid, a potent inhibitor of PP2A, results in various hyperphosphorylated forms of hSecurin which are extremely unstable, due to the action of the Skp1/Cul1/F-box protein complex ubiquitin ligase. We propose that PP2A regulates hSecurin levels by counteracting its phosphorylation, which promotes its degradation. Misregulation of this process may lead to the formation of tumors, in which overproduction of hSecurin is often observed.

scholarly journals Loss of a Protein Phosphatase 2A Regulatory Subunit (Cdc55p) Elicits Improper Regulation of Swe1p Degradation

2000 ◽  
Vol 20 (21) ◽  
pp. 8143-8156 ◽  
Author(s):  
Haifeng Yang ◽  
Wei Jiang ◽  
Matthew Gentry ◽  
Richard L. Hallberg
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Cell Types ◽  
Regulatory Subunit ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Phosphatase 2A

ABSTRACT CDC55 encodes a Saccharomyces cerevisiaeprotein phosphatase 2A (PP2A) regulatory subunit.cdc55-null cells growing at low temperature exhibit a failure of cytokinesis and produce abnormally elongated buds, butcdc55-null cells producing the cyclin-dependent kinase Cdc28-Y19F, which is unable to be inhibited by Y19 phosphorylation, show a loss of the abnormal morphology. Furthermore,cdc55-null cells exhibit a hyperphosphorylation of Y19. For these reasons, we have examined in wild-type and cdc55-null cells the levels and activities of the kinase (Swe1p) and phosphatase (Mih1p) that normally regulate the extent of Cdc28 Y19 phosphorylation. We find that Mih1p levels are comparable in the two strains, and an estimate of the in vivo and in vitro phosphatase activity of this enzyme in the two cell types indicates no marked differences. By contrast, while Swe1p levels are similar in unsynchronized and S-phase-arrested wild-type and cdc55-null cells, Swe1 kinase is found at elevated levels in mitosis-arrestedcdc55-null cells. This excess Swe1p incdc55-null cells is the result of ectopic stabilization of this protein during G2 and M, thereby accounting for the accumulation of Swe1p in mitosis-arrested cells. We also present evidence indicating that, in cdc55-null cells, misregulated PP2A phosphatase activity is the cause of both the ectopic stabilization of Swe1p and the production of the morphologically abnormal phenotype.

scholarly journals Swm1/Apc13 Is an Evolutionarily Conserved Subunit of the Anaphase-Promoting Complex Stabilizing the Association of Cdc16 and Cdc27

2004 ◽  
Vol 24 (8) ◽  
pp. 3562-3576 ◽  
Author(s):  
Martin Schwickart ◽  
Jan Havlis ◽  
Bianca Habermann ◽  
Aliona Bogdanova ◽  
Alain Camasses ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Small Subunit ◽  
Cell Cycle Regulators ◽  
Anaphase Promoting Complex ◽  
Ubiquitin Ligase Activity ◽  
Evolutionarily Conserved ◽  
Stable Association

ABSTRACT The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.

scholarly journals A mechanism for how Cdr1/Nim1 kinase promotes mitotic entry by inhibiting Wee1

2019 ◽  
Vol 30 (25) ◽  
pp. 3015-3023 ◽  
Author(s):  
Hannah E. Opalko ◽  
Isha Nasa ◽  
Arminja N. Kettenbach ◽  
James B. Moseley
Keyword(s):  
Kinase Domain ◽  
Mitotic Entry ◽  
Activity Inhibition ◽  
Cell Cycle Inhibitor ◽  
Step Model ◽  
Family Protein ◽  
Wee1 Kinase ◽  
In Cells

To enter into mitosis, cells must shut off the cell cycle inhibitor Wee1. SAD family protein kinases regulate Wee1 signaling in yeast and humans. In Schizosaccharomyces pombe, two SAD kinases (Cdr1/Nim1 and Cdr2) act as upstream inhibitors of Wee1. Previous studies found that S. pombe Cdr1/Nim1 directly phosphorylates and inhibits Wee1 in vitro, but different results were obtained for budding yeast and human SAD kinases. Without a full understanding of Cdr1 action on Wee1, it has been difficult to assess the in vivo relevance and conservation of this mechanism. Here, we show that both Cdr1 and Cdr2 promote Wee1 phosphorylation in cells, but only Cdr1 inhibits Wee1 kinase activity. Inhibition occurs when Cdr1 phosphorylates a cluster of serine residues linking α-helices G and H of the Wee1 kinase domain. This region is highly divergent among different Wee1 proteins, consistent with distinct regulatory mechanisms. A wee(4A) mutant that impairs phosphorylation by Cdr1 delays mitotic entry and causes elongated cells. By disrupting and retargeting Cdr1 localization, we show that Cdr1 inhibition of Wee1 occurs in cells at cortical nodes formed by Cdr2. On the basis of our results, we propose a two-step model for inhibition of Wee1 by Cdr1 and Cdr2 at nodes.

scholarly journals Pseudosubstrate Inhibition of the Anaphase-Promoting Complex by Acm1: Regulation by Proteolysis and Cdc28 Phosphorylation

2008 ◽  
Vol 28 (15) ◽  
pp. 4653-4664 ◽  
Author(s):  
Denis Ostapenko ◽  
Janet L. Burton ◽  
Ruiwen Wang ◽  
Mark J. Solomon
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Anaphase Promoting Complex ◽  
Inhibitory Protein ◽  
Ubiquitin Ligase Activity ◽  
Dependent Protein ◽  
Apc Mutation

ABSTRACT The ubiquitin ligase activity of the anaphase-promoting complex (APC)/cyclosome needs to be tightly regulated for proper cell cycle progression. Substrates are recruited to the APC by the Cdc20 and Cdh1 accessory proteins. The Cdh1-APC interaction is inhibited through phosphorylation of Cdh1 by Cdc28, the major cyclin-dependent protein kinase in budding yeast. More recently, Acm1 was reported to be a Cdh1-binding and -inhibitory protein in budding yeast. We found that although Acm1 is an unstable protein and contains the KEN-box and D-box motifs typically found in APC substrates, Acm1 itself is not an APC substrate. Rather, it uses these motifs to compete with substrates for Cdh1 binding, thereby inhibiting their recruitment to the APC. Mutation of these motifs prevented Acm1-Cdh1 binding in vivo and rendered Acm1 inactive both in vitro and in vivo. Acm1 stability was critically dependent on phosphorylation by Cdc28, as Acm1 was destabilized following inhibition of Cdc28, mutation of consensus Cdc28 phosphorylation sites in Acm1, or deletion of the Bmh1 and Bmh2 phosphoprotein-binding proteins. Thus, Cdc28 serves dual roles in inhibiting Cdh1-dependent APC activity during the cell cycle: stabilization of the Cdh1 inhibitor Acm1 and direct phosphorylation of Cdh1 to prevent its association with the APC.

scholarly journals Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis

PLoS Genetics ◽  
2020 ◽  
Vol 16 (11) ◽  
pp. e1009184
Author(s):  
Damien Garrido ◽  
Mohammed Bourouh ◽  
Éric Bonneil ◽  
Pierre Thibault ◽  
Andrew Swan ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Regulatory Subunit ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Loss Of Function ◽  
Metaphase Arrest ◽  
Phosphatase 2A ◽  
Cyclin B3 ◽  
Mitosis And Meiosis

In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, we found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, we found that CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. Our results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis.

scholarly journals Protein Phosphatase 2A Subunit PR70 Interacts with pRb and Mediates Its Dephosphorylation

2007 ◽  
Vol 28 (2) ◽  
pp. 873-882 ◽  
Author(s):  
Alessandra Magenta ◽  
Pasquale Fasanaro ◽  
Sveva Romani ◽  
Valeria Di Stefano ◽  
Maurizio C. Capogrossi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Phosphatase ◽  
Molecular Mechanisms ◽  
Protein Phosphatase 2A ◽  
Phosphate Removal ◽  
Regulatory Subunit ◽  
Phosphatase 2A ◽  
Retinoblastoma Tumor

ABSTRACT The retinoblastoma tumor suppressor protein (pRb) regulates cell proliferation and differentiation via phosphorylation-sensitive interactions with specific targets. While the role of cyclin/cyclin-dependent kinase complexes in the modulation of pRb phosphorylation has been extensively studied, relatively little is known about the molecular mechanisms regulating phosphate removal by phosphatases. Protein phosphatase 2A (PP2A) is constituted by a core dimer bearing catalytic activity and one variable B regulatory subunit conferring target specificity and subcellular localization. We previously demonstrated that PP2A core dimer binds pRb and dephosphorylates pRb upon oxidative stress. In the present study, we identified a specific PP2A-B subunit, PR70, that was associated with pRb both in vitro and in vivo. PR70 overexpression caused pRb dephosphorylation; conversely, PR70 knockdown prevented both pRb dephosphorylation and DNA synthesis inhibition induced by oxidative stress. Moreover, we found that intracellular Ca2+ mobilization was necessary and sufficient to trigger pRb dephosphorylation and PP2A phosphatase activity of PR70 was Ca2+ induced. These data underline the importance of PR70-Ca2+ interaction in the signal transduction mechanisms triggered by redox imbalance and leading to pRb dephosphorylation.

scholarly journals EZH2-mediated PP2A inactivation confers resistance to HER2-targeted breast cancer therapy

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yi Bao ◽  
Gokce Oguz ◽  
Wee Chyan Lee ◽  
Puay Leng Lee ◽  
Kakaly Ghosh ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Clonal Selection ◽  
Acquired Resistance ◽  
Epigenetic Mechanism ◽  
Regulatory Subunit ◽  
Breast Cancers ◽  
Her2 Breast Cancer ◽  
Phosphatase 2A

AbstractHER2-targeted therapy has yielded a significant clinical benefit in patients with HER2+ breast cancer, yet disease relapse due to intrinsic or acquired resistance remains a significant challenge in the clinic. Here, we show that the protein phosphatase 2A (PP2A) regulatory subunit PPP2R2B is a crucial determinant of anti-HER2 response. PPP2R2B is downregulated in a substantial subset of HER2+ breast cancers, which correlates with poor clinical outcome and resistance to HER2-targeted therapies. EZH2-mediated histone modification accounts for the PPP2R2B downregulation, resulting in sustained phosphorylation of PP2A targets p70S6K and 4EBP1 which leads to resistance to inhibition by anti-HER2 treatments. Genetic depletion or inhibition of EZH2 by a clinically-available EZH2 inhibitor restores PPP2R2B expression, abolishes the residual phosphorylation of p70S6K and 4EBP1, and resensitizes HER2+ breast cancer cells to anti-HER2 treatments both in vitro and in vivo. Furthermore, the same epigenetic mechanism also contributes to the development of acquired resistance through clonal selection. These findings identify EZH2-dependent PPP2R2B suppression as an epigenetic control of anti-HER2 resistance, potentially providing an opportunity to mitigate anti-HER2 resistance with EZH2 inhibitors.

scholarly journals Phosphorylation sites in BubR1 that regulate kinetochore attachment, tension, and mitotic exit

2008 ◽  
Vol 183 (4) ◽  
pp. 667-680 ◽  
Author(s):  
Haomin Huang ◽  
James Hittle ◽  
Francesca Zappacosta ◽  
Roland S. Annan ◽  
Avram Hershko ◽  
...  
Keyword(s):  
Mitotic Checkpoint ◽  
Phosphorylation Sites ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Conserved Residue ◽  
Vitro Data ◽  
In Vitro Data ◽  
Combined Data

BubR1 kinase is essential for the mitotic checkpoint and also for kinetochores to establish microtubule attachments. In this study, we report that BubR1 is phosphorylated in mitosis on four residues that differ from sites recently reported to be phosphorylated by Plk1 (Elowe, S., S. Hummer, A. Uldschmid, X. Li, and E.A. Nigg. 2007. Genes Dev. 21:2205–2219; Matsumura, S., F. Toyoshima, and E. Nishida. 2007. J. Biol. Chem. 282:15217–15227). S670, the most conserved residue, is phosphorylated at kinetochores at the onset of mitosis and dephosphorylated before anaphase onset. Unlike the Plk1-dependent S676 phosphorylation, S670 phosphorylation is sensitive to microtubule attachments but not to kinetochore tension. Functionally, phosphorylation of S670 is essential for error correction and for kinetochores with end-on attachments to establish tension. Furthermore, in vitro data suggest that the phosphorylation status of BubR1 is important for checkpoint inhibition of the anaphase-promoting complex/cyclosome. Finally, RNA interference experiments show that Mps1 is a major but not the exclusive kinase that specifies BubR1 phosphorylation in vivo. The combined data suggest that BubR1 may be an effector of multiple kinases that are involved in discrete aspects of kinetochore attachments and checkpoint regulation.

scholarly journals Cell Cycle–regulated Proteolysis of Mitotic Target Proteins

1999 ◽  
Vol 10 (11) ◽  
pp. 3927-3941 ◽  
Author(s):  
Holger Bastians ◽  
Leana M. Topper ◽  
Gary L. Gorbsky ◽  
Joan V. Ruderman
Keyword(s):  
Cell Cycle ◽  
Metaphase Plate ◽  
S Phase ◽  
Dominant Negative ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Mitotic Cyclin ◽  
Ubiquitin Conjugating Enzyme

The ubiquitin-dependent proteolysis of mitotic cyclin B, which is catalyzed by the anaphase-promoting complex/cyclosome (APC/C) and ubiquitin-conjugating enzyme H10 (UbcH10), begins around the time of the metaphase–anaphase transition and continues through G1 phase of the next cell cycle. We have used cell-free systems from mammalian somatic cells collected at different cell cycle stages (G0, G1, S, G2, and M) to investigate the regulated degradation of four targets of the mitotic destruction machinery: cyclins A and B, geminin H (an inhibitor of S phase identified in Xenopus), and Cut2p (an inhibitor of anaphase onset identified in fission yeast). All four are degraded by G1 extracts but not by extracts of S phase cells. Maintenance of destruction during G1 requires the activity of a PP2A-like phosphatase. Destruction of each target is dependent on the presence of an N-terminal destruction box motif, is accelerated by additional wild-type UbcH10 and is blocked by dominant negative UbcH10. Destruction of each is terminated by a dominant activity that appears in nuclei near the start of S phase. Previous work indicates that the APC/C–dependent destruction of anaphase inhibitors is activated after chromosome alignment at the metaphase plate. In support of this, we show that addition of dominant negative UbcH10 to G1 extracts blocks destruction of the yeast anaphase inhibitor Cut2p in vitro, and injection of dominant negative UbcH10 blocks anaphase onset in vivo. Finally, we report that injection of dominant negative Ubc3/Cdc34, whose role in G1–S control is well established and has been implicated in kinetochore function during mitosis in yeast, dramatically interferes with congression of chromosomes to the metaphase plate. These results demonstrate that the regulated ubiquitination and destruction of critical mitotic proteins is highly conserved from yeast to humans.

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