The Role of Cdh1p in Maintaining Genomic Stability in Budding Yeast

Abstract Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G1 transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1Δ and mad2Δ single mutants, the mad2Δ cdh1Δ double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2Δ cdh1Δ and pds1Δ cdh1Δ strains were rescued by overexpressing Swe1p, a G2/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1Δ mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.

Download Full-text

Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity

The Journal of Cell Biology ◽

10.1083/jcb.201305049 ◽

2014 ◽

Vol 204 (6) ◽

pp. 891-900 ◽

Cited By ~ 24

Author(s):

Ibtissem Nabti ◽

Petros Marangos ◽

Jenny Bormann ◽

Nobuaki R. Kudo ◽

John Carroll

Keyword(s):

Protein Kinase ◽

Spindle Assembly Checkpoint ◽

Mitogen Activated Protein Kinase ◽

Cyclin Dependent Kinase ◽

Anaphase Promoting Complex ◽

Female Meiosis ◽

Dual Mode ◽

Mitogen Activated Protein ◽

Meiosis I

Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. In this paper, we find that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates essential for meiosis I. Furthermore, inhibition of MAPK around the onset of APC/C activity at the transition from meiosis I to meiosis II led to accelerated completion of meiosis I and an increase in aneuploidy at metaphase II. These effects appear to be mediated via a Cdk1/MAPK-dependent stabilization of the spindle assembly checkpoint, which when inhibited leads to increased APC/C activity. These findings demonstrate new roles for MAPK in the regulation of meiosis in mammalian oocytes.

Download Full-text

Intermediates in the assembly of mitotic checkpoint complexes and their role in the regulation of the anaphase-promoting complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1524551113 ◽

2016 ◽

Vol 113 (4) ◽

pp. 966-971 ◽

Cited By ~ 10

Author(s):

Sharon Kaisari ◽

Danielle Sitry-Shevah ◽

Shirly Miniowitz-Shemtov ◽

Avram Hershko

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Mitotic Checkpoint ◽

Anaphase Promoting Complex ◽

Checkpoint Proteins ◽

Sister Chromatids ◽

Unknown Species ◽

Mitotic Checkpoint Complex

The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. Kinetochores that are not attached properly to the mitotic spindle produce an inhibitory signal that prevents progression into anaphase. The checkpoint system acts on the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, which targets for degradation inhibitors of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which assembles when the checkpoint is activated. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20. The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some subcomplexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. We used purified subcomplexes of mitotic checkpoint proteins to examine these problems. Our results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. We also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. A previously unknown species of MCC, which consists of Mad2, BubR1, and two molecules of Cdc20, contributes to the inhibition of APC/C by the mitotic checkpoint system.

Download Full-text

FANCA Controls Mitotic Phosphosignaling Networks To Ensure Genome Stability During Cell Division

Blood ◽

10.1182/blood.v122.21.801.801 ◽

2013 ◽

Vol 122 (21) ◽

pp. 801-801

Author(s):

Rikki Enzor ◽

Zahi Abdul Sater ◽

Donna Cerabona ◽

Zejin Sun ◽

Su-jung Park ◽

...

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Chromosome Segregation ◽

Fanconi Anemia ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Signaling Network ◽

Mitotic Apparatus ◽

Risk Of Cancer

Abstract Fanconi anemia (FA) is a heterogenous genome instability syndrome with a high risk of cancer. The FA proteins are essential for interphase DNA damage repair. However, it is incompletely understood why FA-deficient cells also develop gross aneuploidy and multinucleation, which are symptoms of error-prone chromosome segregation. Emerging evidence indicates that the FA signaling network functions as a guardian of the genome throughout the cell cycle, including chromosome segregation during mitosis. However, the mechanistic aspects of the critical role of the FA signaling in mitosis remain poorly understood. We have recently shown that the FA signaling network localizes to the mitotic apparatus to control the spindle assembly checkpoint and centrosome maintenance (J Clin Invest 2013, in press). The spindle assembly checkpoint (SAC) is a complex tumor suppressor signaling network that prevents premature separation of sister chromatids by delaying the metaphase-to-anaphase transition until all the kinetochores are properly attached to the mitotic spindle. Since weakened SAC promotes stochastic chromosome segregation, mutagenesis and cancer, these findings shed new light on the role of FA signaling in maintenance of genomic stability. We found the subcellular localization of FA proteins to the mitotic apparatus is spatiotemporally regulated as cells divide. Our new data revealed the pathways connecting the FANCA protein with canonical mitotic phosphosignaling networks. We have employed unbiased kinome-wide phospho-mass spectrometry to compare the landscape of abnormalities of mitotic signaling pathways in primary FANCA-/- patient cells and gene-corrected isogenic cells. These experiments led us to identify and quantify a wide range of phosphorylation abnormalities of multiple FANCA-dependent centrosome-, kinetochore- and chromosome-associated regulators of mitosis. Our data illuminated the role for FA signaling in three critical stages of cell division: (1) the spindle assembly checkpoint, (2) anaphase and (3) cytokinesis. Thus, we employed live phase-contrast imaging of primary FANCA-/- patient cells in comparison to gene-corrected cells to separately quantify aberrations in (1) chromosome congression and metaphase-anaphase transition (SAC malfunction), (2) execution of anaphase and (3) completion of cytokinesis. Our findings further our understanding of human cell cycle control and provide new insights into the origins of genomic instability in Fanconi anemia by establishing mechanistic connection between the FANCA protein and key mitotic signaling networks. The identification of cell division pathways regulated by FANCA has implications for future targeted drug development in Fanconi anemia and FA-deficient malignancies in the general population. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Saccharomyces cerevisiae Anaphase-Promoting Complex Interacts with Multiple Histone-Modifying Enzymes To Regulate Cell Cycle Progression

Eukaryotic Cell ◽

10.1128/ec.00097-10 ◽

2010 ◽

Vol 9 (10) ◽

pp. 1418-1431 ◽

Cited By ~ 14

Author(s):

Emma L. Turner ◽

Mackenzie E. Malo ◽

Marnie G. Pisclevich ◽

Megan D. Dash ◽

Gerald F. Davies ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Chromatin Assembly ◽

Temperature Sensitive ◽

Anaphase Promoting Complex ◽

Cycle Progression ◽

Ubiquitin Ligase Complex ◽

Genes Encoding ◽

Dependent Cell ◽

Histone Modifying Enzymes

ABSTRACT The anaphase-promoting complex (APC), a large evolutionarily conserved ubiquitin ligase complex, regulates cell cycle progression through mitosis and G1. Here, we present data suggesting that APC-dependent cell cycle progression relies on a specific set of posttranslational histone-modifying enzymes. Multiple APC subunit mutants were impaired in total and modified histone H3 protein content. Acetylated H3K56 (H3K56Ac) levels were as reduced as those of total H3, indicating that loading histones with H3K56Ac is unaffected in APC mutants. However, under restrictive conditions, H3K9Ac and dimethylated H3K79 (H3K79me2) levels were more greatly reduced than those of total H3. In a screen for histone acetyltransferase (HAT) and histone deacetylase (HDAC) mutants that genetically interact with the apc5 CA (chromatin assembly) mutant, we found that deletion of GCN5 or ELP3 severely hampered apc5 CA temperature-sensitive (ts) growth. Further analyses showed that (i) the elp3Δ gcn5Δ double mutant ts defect was epistatic to that observed in apc5 CA cells; (ii) gcn5Δ and elp3Δ mutants accumulate in mitosis; and (iii) turnover of the APC substrate Clb2 is not impaired in elp3Δ gcn5Δ cells. Increased expression of ELP3 and GCN5, as well as genes encoding the HAT Rtt109 and the chromatin assembly factors Msi1 and Asf1, suppressed apc5 CA defects, while increased APC5 expression partially suppressed elp3Δ gcn5Δ growth defects. Finally, we demonstrate that Gcn5 is unstable during G1 and following G1 arrest and is stabilized in APC mutants. We present our working model in which Elp3/Gcn5 and the APC work together to facilitate passage through mitosis and G1. To progress into S, we propose that at least Gcn5 must then be targeted for degradation in an APC-dependent fashion.

Download Full-text

Molecular Evolution Allows Bypass of the Requirement for Activation Loop Phosphorylation of the Cdc28 Cyclin-Dependent Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.18.5.2923 ◽

1998 ◽

Vol 18 (5) ◽

pp. 2923-2931 ◽

Cited By ~ 33

Author(s):

Frederick R. Cross ◽

Kristi Levine

Keyword(s):

Cell Cycle ◽

Biological Activity ◽

Cell Cycle Progression ◽

Dna Amplification ◽

Protein Kinase Activity ◽

Activation Loop ◽

Cyclin Dependent Kinase ◽

High Biological Activity ◽

Cycle Progression ◽

Growth Defects

ABSTRACT Many protein kinases are regulated by phosphorylation in the activation loop, which is required for enzymatic activity. Glutamic acid can substitute for phosphothreonine in some proteins activated by phosphorylation, but this substitution (T169E) at the site of activation loop phosphorylation in the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28p blocks biological function and protein kinase activity. Using cycles of error-prone DNA amplification followed by selection for successively higher levels of function, we identified mutant versions of Cdc28p-T169E with high biological activity. The enzymatic and biological activity of the mutant Cdc28p was essentially normally regulated by cyclin, and the mutants supported normal cell cycle progression and regulation. Therefore, it is not a requirement for control of the yeast cell cycle that Cdc28p be cyclically phosphorylated and dephosphorylated. TheseCDC28 mutants allow viability in the absence of Cak1p, the essential kinase that phosphorylates Cdc28p-T169, demonstrating that T169 phosphorylation is the only essential function of Cak1p. Some growth defects remain in suppressed cak1 cdc28 strains carrying the mutant CDC28 genes, consistent with additional nonessential roles for CAK1.

Download Full-text

Intricate Regulatory Mechanisms of the Anaphase-Promoting Complex/Cyclosome and Its Role in Chromatin Regulation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.687515 ◽

2021 ◽

Vol 9 ◽

Author(s):

Tatyana Bodrug ◽

Kaeli A. Welsh ◽

Megan Hinkle ◽

Michael J. Emanuele ◽

Nicholas G. Brown

Keyword(s):

Cell Cycle ◽

Signaling Pathways ◽

Biological Process ◽

Conformational Dynamics ◽

Intimate Relationship ◽

Regulatory Mechanisms ◽

Cell Cycle Regulators ◽

Chromatin Regulation ◽

Anaphase Promoting Complex

The ubiquitin (Ub)-proteasome system is vital to nearly every biological process in eukaryotes. Specifically, the conjugation of Ub to target proteins by Ub ligases, such as the Anaphase-Promoting Complex/Cyclosome (APC/C), is paramount for cell cycle transitions as it leads to the irreversible destruction of cell cycle regulators by the proteasome. Through this activity, the RING Ub ligase APC/C governs mitosis, G1, and numerous aspects of neurobiology. Pioneering cryo-EM, biochemical reconstitution, and cell-based studies have illuminated many aspects of the conformational dynamics of this large, multi-subunit complex and the sophisticated regulation of APC/C function. More recent studies have revealed new mechanisms that selectively dictate APC/C activity and explore additional pathways that are controlled by APC/C-mediated ubiquitination, including an intimate relationship with chromatin regulation. These tasks go beyond the traditional cell cycle role historically ascribed to the APC/C. Here, we review these novel findings, examine the mechanistic implications of APC/C regulation, and discuss the role of the APC/C in previously unappreciated signaling pathways.

Download Full-text

New role of human ribosomal protein S3: Regulation of cell cycle via phosphorylation by cyclin-dependent kinase 2

Oncology Letters ◽

10.3892/ol.2017.5906 ◽

2017 ◽

Vol 13 (5) ◽

pp. 3681-3687 ◽

Cited By ~ 8

Author(s):

Se Hee Han ◽

Ji Hyung Chung ◽

Joon Kim ◽

Key-Sun Kim ◽

Ye Sun Han

Keyword(s):

Cell Cycle ◽

Ribosomal Protein ◽

Cyclin Dependent Kinase ◽

Ribosomal Protein S3 ◽

Human Ribosomal Protein ◽

Regulation Of Cell Cycle

Download Full-text

Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

Molecular Biology of the Cell ◽

10.1091/mbc.01-11-0535 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2289-2300 ◽

Cited By ~ 92

Author(s):

Brett K. Kaiser ◽

Zachary A. Zimmerman ◽

Harry Charbonneau ◽

Peter K. Jackson

Keyword(s):

Cell Cycle ◽

Phosphatase Activity ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Genomic Stability ◽

Mitotic Exit ◽

Protein Abundance ◽

Cyclin Dependent Kinase ◽

Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

Download Full-text

Review of rationale and progress toward targeting cyclin-dependent kinase 2 (CDK2) for male contraception†

Biology of Reproduction ◽

10.1093/biolre/ioaa107 ◽

2020 ◽

Vol 103 (2) ◽

pp. 357-367

Author(s):

Erik B Faber ◽

Nan Wang ◽

Gunda I Georg

Keyword(s):

Cell Cycle ◽

Binding Site ◽

Knockout Mice ◽

Male Contraception ◽

Cyclin Dependent Kinase ◽

Compensatory Mechanisms ◽

Binding Partner ◽

Male Contraceptive ◽

Clinical Stages

Abstract Cyclin-dependent kinase 2 (CDK2) is a member of the larger cell cycle regulating CDK family of kinases, activated by binding partner cyclins as its name suggests. Despite its canonical role in mitosis, CDK2 knockout mice are viable but sterile, suggesting compensatory mechanisms for loss of CDK2 in mitosis but not meiosis. Here, we review the literature surrounding the role of CDK2 in meiosis, particularly a cyclin-independent role in complex with another activator, Speedy 1 (SPY1). From this evidence, we suggest that CDK2 could be a viable nonhormonal male contraceptive target. Finally, we review the literature of pertinent CDK2 inhibitors from the preclinical to clinical stages, mostly developed to treat various cancers. To date, there is no potent yet selective CDK2 inhibitor that could be repurposed as a contraceptive without appreciable off-target toxicity. To achieve selectivity for CDK2 over closely related kinases, developing compounds that bind outside the conserved adenosine triphosphate-binding site may be necessary.

Download Full-text