|
1
|
Sobkowiak K, Kohzaki M, Böhm R, Mailler J, Huber F, Emamzadah S, Tropia L, Hiller S, Halazonetis TD. REV7 functions with REV3 as a checkpoint protein delaying mitotic entry until DNA replication is completed. Cell Rep 2025; 44:115431. [PMID: 40106439 DOI: 10.1016/j.celrep.2025.115431] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2024] [Revised: 12/18/2024] [Accepted: 02/24/2025] [Indexed: 03/22/2025] Open
Abstract
REV7, also named MAD2B or MAD2L2, is a subunit of the DNA translesion polymerase zeta and also part of the 53BP1-shieldin complex, which is present at sites of DNA double-strand breaks. REV7 has high sequence similarity to the MAD2 spindle assembly checkpoint protein, prompting us to examine whether REV7 has a checkpoint function. We observed that, in chicken and human cells exposed to agents that induce DNA replication stress, REV7 inhibits mitotic entry; this effect is most evident when the canonical DNA replication stress checkpoint, mediated by ATR, is inhibited. Similar to MAD2, REV7 undergoes conformational changes upon ligand binding, and its checkpoint function depends on its ability to homodimerize and bind its ligands. Notably, even in unchallenged cells, deletion of the REV7 gene leads to premature mitotic entry, raising the possibility that the REV7 checkpoint monitors ongoing DNA replication.
Collapse
Affiliation(s)
- Katarzyna Sobkowiak
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland
| | - Masaoki Kohzaki
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland.
| | - Raphael Böhm
- Biozentrum, University of Basel, 4056 Basel, Switzerland
| | - Jonathan Mailler
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland
| | - Florian Huber
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland
| | - Soheila Emamzadah
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland
| | - Laurence Tropia
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland
| | | | - Thanos D Halazonetis
- Department of Molecular and Cellular Biology, University of Geneva, 1205 Geneva, Switzerland.
| |
Collapse
|
|
2
|
Pain C. FRET-FLIM for the Study of Protein-Protein Interactions Underpinning Mitosis Checkpoints. Methods Mol Biol 2025; 2874:87-97. [PMID: 39614049 DOI: 10.1007/978-1-0716-4236-8_8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2024]
Abstract
Cell division is a key cellular process that ensures the continuation of life on Earth. In order to protect the genetic integrity of organisms, cell division must happen accurately, ensuring each daughter cell receives a complete copy of the original genome. The accuracy of this process is, in part, preserved by various cell cycle checkpoints. These checkpoints rely on the physical interactions of their components to ensure proper function. The spindle assembly checkpoint (SAC), for example, produces an inhibitory complex of BUBR1-BUB3 and MAD2 bound to CDC20. Many of these cell cycle checkpoint components have been identified in plants, but it has not yet been established whether plants have a mitotic checkpoint architecture that is similar to mammalian cells. To understand the function of plant cell cycle homologues, it is imperative to characterize their interactions in vivo. FRET-FLIM (Förster resonance energy transfer-fluorescence lifetime imaging microscopy), is a rapidly expanding technique that can be used to rapidly and simply characterize protein-protein interactions.
Collapse
Affiliation(s)
- Charlotte Pain
- Endomembrane Structure and Function Research Group, Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK.
| |
Collapse
|
|
3
|
Zhang J, Yuan HJ, Zhu J, Gong S, Luo MJ, Tan JH. Topoisomerase II dysfunction causes metaphase I arrest by activating aurora B, SAC and MPF and prevents PB1 abscission in mouse oocytes†. Biol Reprod 2022; 106:900-909. [PMID: 35084021 DOI: 10.1093/biolre/ioac011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2021] [Revised: 11/02/2021] [Accepted: 01/13/2022] [Indexed: 11/14/2022] Open
Abstract
Oocyte aneuploidy is caused mainly by chromosome nondisjunction and/or unbalanced sister chromatid pre-division. Although studies in somatic cells have shown that topoisomerase II (TOP2) plays important roles in chromosome condensation and timely separation of centromeres, little is known about its role during oocyte meiosis. Furthermore, because VP-16, which is a TOP2 inhibitor and induces DNA double strand breaks, is often used for ovarian cancer chemotherapy, its effects on oocytes must be studied for ovarian cancer patients to recover ovarian function following chemotherapy. This study showed that inhibiting TOP2 with either ICRF-193 or VP-16 during meiosis I impaired chromatin condensation, chromosome alignment, TOP2α localization and caused metaphase I (MI) arrest and first polar body (PB1) abscission failure. Inhibiting or neutralizing either spindle assembly checkpoint (SAC), Aurora B or maturation-promoting factor (MPF) significantly abolished the effect of ICRF-193 or VP-16 on MI arrest. Treatment with ICRF-193 or VP-16 significantly activated MPF and SAC but the effect disappeared when Aurora B was inhibited. Most of the oocytes matured in the presence of ICRF-193 or VP-16 were arrested at MI, and only 11% to 27% showed PB1 protrusion. Furthermore, most of the PB1 protrusions formed in the presence of ICRF-193 or VP-16 were retracted after further culture for 7 h. In conclusion, TOP2 dysfunction causes MI arrest by activating Aurora B, SAC and MPF and it prevents PB1 abscission by promoting chromatin bridges.
Collapse
Affiliation(s)
- Jie Zhang
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an City 271018, P. R. China
| | - Hong-Jie Yuan
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an City 271018, P. R. China
| | - Jiang Zhu
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an City 271018, P. R. China
| | - Shuai Gong
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an City 271018, P. R. China
| | - Ming-Jiu Luo
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an City 271018, P. R. China
| | - Jing-He Tan
- Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an City 271018, P. R. China
| |
Collapse
|
|
4
|
Maiato H. Mitosis under the macroscope. Semin Cell Dev Biol 2021; 117:1-5. [PMID: 34172396 DOI: 10.1016/j.semcdb.2021.06.014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Affiliation(s)
- Helder Maiato
- Chromosome Instability & Dynamics Group, i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal; Cell Division Group, Experimental Biology Unit, Department of Biomedicine, Faculdade de Medicina, Universidade do Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal.
| |
Collapse
|
|
5
|
Chi HY, Chan V, Li C, Hsieh JH, Lin PH, Tsai YH, Chen Y. Fabrication of polylactic acid/paclitaxel nano fibers by electrospinning for cancer therapeutics. BMC Chem 2020; 14:63. [PMID: 33111062 PMCID: PMC7585315 DOI: 10.1186/s13065-020-00711-4] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Accepted: 09/17/2020] [Indexed: 12/19/2022] Open
Abstract
Polylactic acid (PLA) is a thermoplastic and biodegradable polyester, largely derived from renewable resources such as corn starch, cassava starch and sugarcane. However, PLA is only soluble in a narrow range of solvents such as tetrahydrofuran, dioxane, chlorinated solvents and heated benzene. The limited choices of solvent for PLA dissolution have imposed significant challenges in the development of specifically engineered PLA nanofibers with electrospinning techniques. Generally, the electrospun polymeric materials have been rendered with unique properties such as high porosity and complex geometry while maintaining its biodegradability and biocompatibility for emerging biomedical applications. In this study, a new anticancer drug delivery system composed of PLA nanofibers with encapsulated paclitaxel was developed by the electrospinning of the respective nanofibers on top of a spin-coated thin film with the same chemical compositions. Our unique approach is meant for promoting strong bonding between PLA-based nanofibers and their respective films in order to improve the prolonged release properties and composite film stability within a fluctuative physiochemical environment during cell culture. PLA/paclitaxel nanofiber supported on respective polymeric films were probed by scanning electronic microscope, Fourier transform infrared spectrometer and water contact measurement for determining their surface morphologies, fibers’ diameters, molecular vibrational modes, and wettability, respectively. Moreover, PLA/paclitaxel nanofibers supported on respective spin-coated films at different loadings of paclitaxel were evaluated for their abilities in killing human colorectal carcinoma cells (HCT-116). More importantly, MTT assays showed that regardless of the concentrations of paclitaxel, the growth of HCT-116 was effectively inhibited by the prolonged release of paclitaxel from PLA/paclitaxel nanofibers. An effective prolonged delivery system of paclitaxel based on PLA nanofiber-based film has demonstrated exciting potentials for emerging applications as implantable drug delivery patch in post-surgical cancer eradication.
Collapse
Affiliation(s)
- H Y Chi
- Division of Cardiovascular Surgery, Department of Surgery, Taoyuan Armed Forces General Hospital, Taoyuan, 32551 Taiwan.,Department of Biomedical Engineering, National Yang Ming University, Taipei, 11221 Taiwan
| | - Vincent Chan
- Department of Biomedical Engineering, Khalifa University, PO Box 127788, Abu Dhabi, United Arab Emirates
| | - Chuan Li
- Department of Biomedical Engineering, National Yang Ming University, Taipei, 11221 Taiwan
| | - J H Hsieh
- Department of Materials Engineering, Ming Chi University of Technology, Taishan, New Taipei City, 24301 Taiwan
| | - P H Lin
- Department of Biomedical Engineering, National Yang Ming University, Taipei, 11221 Taiwan
| | - Ya-Hui Tsai
- Department of Surgery, Far Eastern Memorial Hospital, Banqiao, New Taipei City, 22060 Taiwan
| | - Yun Chen
- Department of Surgery, Far Eastern Memorial Hospital, Banqiao, New Taipei City, 22060 Taiwan
| |
Collapse
|
|
6
|
Pérez-Benavente B, Nasresfahani AF, Farràs R. Ubiquitin-Regulated Cell Proliferation and Cancer. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2020; 1233:3-28. [PMID: 32274751 DOI: 10.1007/978-3-030-38266-7_1] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Ubiquitin ligases (E3) play a crucial role in the regulation of different cellular processes such as proliferation and differentiation via recognition, interaction, and ubiquitination of key cellular proteins in a spatial and temporal regulated manner. The type of ubiquitin chain formed determines the fate of the substrates. The ubiquitinated substrates can be degraded by the proteasome, display altered subcellular localization, or can suffer modifications on their interaction with functional protein complexes. Deregulation of E3 activities is frequently found in various human pathologies, including cancer. The illegitimated or accelerated degradation of oncosuppressive proteins or, inversely, the abnormally high accumulation of oncoproteins, contributes to cell proliferation and transformation. Anomalies in protein abundance may be related to mutations that alter the direct or indirect recognition of proteins by the E3 enzymes or alterations in the level of expression or activity of ubiquitin ligases. Through a few examples, we illustrate here the complexity and diversity of the molecular mechanisms related to protein ubiquitination involved in cell cycle regulation. We will discuss the role of ubiquitin-dependent degradation mediated by the proteasome, the role of non-proteolytic ubiquitination during cell cycle progression, and the consequences of this deregulation on cellular transformation. Finally, we will highlight the novel opportunities that arise from these studies for therapeutic intervention.
Collapse
Affiliation(s)
| | | | - Rosa Farràs
- Oncogenic Signaling Laboratory, Centro de Investigación Príncipe Felipe, Valencia, Spain.
| |
Collapse
|
|
7
|
Choi HJ, Zhu BT. Upregulated cyclin B1/CDK1 mediates apoptosis following 2-methoxyestradiol-induced mitotic catastrophe: Role of Bcl-X L phosphorylation. Steroids 2019; 150:108381. [PMID: 30797877 DOI: 10.1016/j.steroids.2019.02.014] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/06/2018] [Revised: 01/15/2019] [Accepted: 02/18/2019] [Indexed: 12/13/2022]
Abstract
2-Methoxyestradiol is an endogenous nonpolar metabolite of 17β-estradiol with a strong antitubulin activity. Earlier we showed that 2-methoxyestradiol increases the level and activity of cyclin B1/CDK1, which subsequently induces mitotic prometaphase arrest. In the present study, we demonstrate that upregulation of cyclin B1/CDK1 is responsible for the increased phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-XL in 2-methoxyestradiol-induced, mitotically-arrested cancer cells. Additional analysis shows that only the increase in phosphorylation of Bcl-XL, but not Bcl-2, is associated with activation of the mitochondrial cell death pathway. We find that MAD2 is an important upstream mediator of the antitubulin function of 2-methoxyestradiol, resulting in activation of the MKK4-JNK1 pathway. JNK1 activation then leads to cyclin B1/CDK1 upregulation, which further increases Bcl-2 and Bcl-XL phosphorylation. Together, these results indicate that cyclin B1/CDK1 upregulation in cancer cells undergoing 2-methoxyestradiol-induced mitotic catastrophe causes apoptosis via Bcl-XL phosphorylation.
Collapse
Affiliation(s)
- Hye Joung Choi
- School of Life and Health Sciences and School of Science and Engineering, The Chinese University of Hong Kong, Shenzhen, Guangdong 518172, China
| | - Bao Ting Zhu
- School of Life and Health Sciences and School of Science and Engineering, The Chinese University of Hong Kong, Shenzhen, Guangdong 518172, China.
| |
Collapse
|
|
8
|
French BT, Straight AF. The Power of Xenopus Egg Extract for Reconstitution of Centromere and Kinetochore Function. PROGRESS IN MOLECULAR AND SUBCELLULAR BIOLOGY 2019; 56:59-84. [PMID: 28840233 DOI: 10.1007/978-3-319-58592-5_3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Faithful transmission of genetic information during cell division requires attachment of chromosomes to the mitotic spindle via the kinetochore. In vitro reconstitution studies are beginning to uncover how the kinetochore is assembled upon the underlying centromere, how the kinetochore couples chromosome movement to microtubule dynamics, and how cells ensure the site of kinetochore assembly is maintained from one generation to the next. Here we give special emphasis to advances made in Xenopus egg extract, which provides a unique, biochemically tractable in vitro system that affords the complexity of cytoplasm and nucleoplasm to permit reconstitution of the dynamic, cell cycle-regulated functions of the centromere and kinetochore.
Collapse
Affiliation(s)
- Bradley T French
- Department of Biochemistry, Stanford University, 279 Campus Drive, Beckman 409, Stanford, CA, 94305, USA
| | - Aaron F Straight
- Department of Biochemistry, Stanford University, 279 Campus Drive, Beckman 409, Stanford, CA, 94305, USA.
| |
Collapse
|
|
9
|
Allegri L, Baldan F, Roy S, Aubé J, Russo D, Filetti S, Damante G. The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells. Sci Rep 2019; 9:7374. [PMID: 31089242 PMCID: PMC6517587 DOI: 10.1038/s41598-019-43894-0] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2018] [Accepted: 01/29/2019] [Indexed: 02/07/2023] Open
Abstract
Hu antigen R (HuR) is indeed one of the most studied RNA-binding protein (RBP) since its fundamental role both in tumorigenesis and cancer progression. For this reason, downregulation in HuR protein levels or inhibition of HuR biological function are, nowadays, attractive goals in cancer research. Here, we examined the antitumor effects of CMLD-2 in four thyroid cancer cell lines (SW1736, 8505 C, BCPAP and K1). Indeed, CMLD-2 competitively binds HuR protein disrupting its interaction with RNA-targets. 35 μM CLMD-2 produced a significant downregulation in thyroid cancer cell viability, coupled to an increase in apoptosis. Moreover, CMLD-2 treatment hindered both migration and colony formation ability. MAD2 is a microtubules-associated protein known to be greatly overexpressed in cancer and correlating with tumor aggressiveness. Furthermore, MAD2 is known to be a HuR target. CMLD-2 treatment induced a strong MAD2 downregulation and rescue experiments depicted it as a key effector in HuR-mediated in cancer. Altogether, these data contributed to foster HuR inhibition as valid antineoplastic treatment in thyroid cancer, highlighting MAD2 as a novel therapeutic target.
Collapse
Affiliation(s)
- Lorenzo Allegri
- Department of Medical Area, University of Udine, 33100, Udine, Italy
| | - Federica Baldan
- Department of Translational and Precision Medicine, University of Roma 'Sapienza', 06100, Roma, Italy.
| | - Sudeshna Roy
- Department of BioMelecular Sciences, School of Pharmacy, University of Mississippi, 413 Faser Hall, Mississippi, 38677-1848, USA
| | - Jeffrey Aubé
- Division of Chemical Biology and Medical Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina, 27599-7363, USA
| | - Diego Russo
- Department of Health Sciences, University of Catanzaro "Magna Graecia", 88100, Catanzaro, Italy
| | - Sebastiano Filetti
- Department of Translational and Precision Medicine, University of Roma 'Sapienza', 06100, Roma, Italy
| | - Giuseppe Damante
- Department of Medical Area, University of Udine, 33100, Udine, Italy
| |
Collapse
|
|
10
|
Alfonso-Pérez T, Hayward D, Holder J, Gruneberg U, Barr FA. MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling. J Cell Biol 2019; 218:1108-1117. [PMID: 30674583 PMCID: PMC6446853 DOI: 10.1083/jcb.201808015] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2018] [Revised: 11/19/2018] [Accepted: 01/07/2019] [Indexed: 12/22/2022] Open
Abstract
Cyclin B-dependent kinase (CDK1-CCNB1) promotes entry into mitosis. Additionally, it inhibits mitotic exit by activating the spindle checkpoint. This latter role is mediated through phosphorylation of the checkpoint kinase MPS1 and other spindle checkpoint proteins. We find that CDK1-CCNB1 localizes to unattached kinetochores and like MPS1 is lost from these structures upon microtubule attachment. This suggests that CDK1-CCNB1 is an integral component and not only an upstream regulator of the spindle checkpoint pathway. Complementary proteomic and cell biological analysis demonstrate that the spindle checkpoint protein MAD1 is one of the major components of CCNB1 complexes, and that CCNB1 is recruited to unattached kinetochores in an MPS1-dependent fashion through interaction with the first 100 amino acids of MAD1. This MPS1 and MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop necessary for timely recruitment of MPS1 to kinetochores during mitotic entry and for sustained spindle checkpoint arrest. CDK1-CCNB1 is therefore an integral component of the spindle checkpoint, ensuring the fidelity of mitosis.
Collapse
Affiliation(s)
| | - Daniel Hayward
- Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
| | - James Holder
- Department of Biochemistry, University of Oxford, Oxford, UK
| | - Ulrike Gruneberg
- Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
| | - Francis A Barr
- Department of Biochemistry, University of Oxford, Oxford, UK
| |
Collapse
|
|
11
|
Naaz A, Ahad S, Rai A, Surolia A, Panda D. BubR1 depletion delays apoptosis in the microtubule-depolymerized cells. Biochem Pharmacol 2018; 162:177-190. [PMID: 30468712 DOI: 10.1016/j.bcp.2018.11.015] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2018] [Accepted: 11/19/2018] [Indexed: 10/27/2022]
Abstract
We investigated the role of a spindle assembly checkpoint protein, BubR1, in determining the mechanism of cell killing of an anti-microtubule agent CXI-benzo-84. CXI-benzo-84 dampened microtubule dynamics in live MCF-7 cells. The compound arrested MCF-7 cells in mitosis and induced apoptosis in these cells. Though CXI-benzo-84 efficiently depolymerized microtubules in the BubR1-depleted MCF-7 cells, it did not arrest the BubR1-depleted cells at mitosis. Interestingly, apoptosis occurred in the BubR1-depleted MCF-7 cells in the absence of a mitotic block suggesting that the mitotic block is not a prerequisite for the induction of apoptosis by anti-microtubule agents. In the presence of CXI-Benzo-84, the level of apoptosis was initially found to be lesser in the BubR1-depleted MCF-7 cells than the control cells; however, the BubR1-depleted cells displayed a similar level of apoptosis as the control cells at 72 h of drug treatment. The depletion of BubR1 enhanced DNA damage in MCF-7 cells upon microtubule depolymerization. In addition, CXI-benzo-84 in combination with cisplatin induced more cell death in BubR1-depleted cells than the BubR1-expressing MCF-7 cells. The results indicated a possibility that the BubR1-compromised cancer patients can be treated with combination therapy.
Collapse
Affiliation(s)
- Afsana Naaz
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India
| | - Shazia Ahad
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India
| | - Ankit Rai
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India
| | - Avadhesha Surolia
- Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India
| | - Dulal Panda
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India.
| |
Collapse
|
|
12
|
Kim DH, Han JS, Ly P, Ye Q, McMahon MA, Myung K, Corbett KD, Cleveland DW. TRIP13 and APC15 drive mitotic exit by turnover of interphase- and unattached kinetochore-produced MCC. Nat Commun 2018; 9:4354. [PMID: 30341343 PMCID: PMC6195577 DOI: 10.1038/s41467-018-06774-1] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2018] [Accepted: 09/13/2018] [Indexed: 12/18/2022] Open
Abstract
The mitotic checkpoint ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), a soluble inhibitor of the anaphase-promoting complex/cyclosome (APC/C) produced by unattached kinetochores. MCC is also assembled during interphase by Mad1/Mad2 bound at nuclear pores, thereby preventing premature mitotic exit prior to kinetochore maturation and checkpoint activation. Using degron tagging to rapidly deplete the AAA+ ATPase TRIP13, we show that its catalytic activity is required to maintain a pool of open-state Mad2 for MCC assembly, thereby supporting mitotic checkpoint activation, but is also required for timely mitotic exit through catalytic disassembly of MCC. Strikingly, combining TRIP13 depletion with elimination of APC15-dependent Cdc20 ubiquitination/degradation results in a complete inability to exit mitosis, even when MCC assembly at unattached kinetochores is prevented. Thus, mitotic exit requires MCC produced either in interphase or mitosis to be disassembled by TRIP13-catalyzed removal of Mad2 or APC15-driven ubiquitination/degradation of its Cdc20 subunit.
Collapse
Affiliation(s)
- Dong Hyun Kim
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, 92093, USA.,Department of Cellular and Molecular Medicine, University of California-San Diego, La Jolla, CA, 92093, USA
| | - Joo Seok Han
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan, 44919, Republic of Korea
| | - Peter Ly
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, 92093, USA.,Department of Cellular and Molecular Medicine, University of California-San Diego, La Jolla, CA, 92093, USA
| | - Qiaozhen Ye
- Department of Cellular and Molecular Medicine, University of California-San Diego, La Jolla, CA, 92093, USA
| | - Moira A McMahon
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, 92093, USA.,Department of Cellular and Molecular Medicine, University of California-San Diego, La Jolla, CA, 92093, USA.,Ionis Pharmaceuticals, 2855 Gazelle Ct, Carlsbad, CA, 92010, USA
| | - Kyungjae Myung
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan, 44919, Republic of Korea.,School of Life Sciences, Ulsan National Institute for Science and Technology (UNIST), Ulsan, 44919, Republic of Korea
| | - Kevin D Corbett
- Department of Cellular and Molecular Medicine, University of California-San Diego, La Jolla, CA, 92093, USA. .,Department of Chemistry, University of California-San Diego, La Jolla, CA, 92093, USA.
| | - Don W Cleveland
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, 92093, USA. .,Department of Cellular and Molecular Medicine, University of California-San Diego, La Jolla, CA, 92093, USA.
| |
Collapse
|
|
13
|
Wei B, Huang C, Liu B, Wang Y, Xia N, Fan Q, Chen GQ, Cheng J. Mitotic Phosphorylation of SENP3 Regulates DeSUMOylation of Chromosome-Associated Proteins and Chromosome Stability. Cancer Res 2018; 78:2171-2178. [DOI: 10.1158/0008-5472.can-17-2288] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2017] [Revised: 12/13/2017] [Accepted: 02/05/2018] [Indexed: 11/16/2022]
|
|
14
|
Wang R, Burton JL, Solomon MJ. Transcriptional and post-transcriptional regulation of Cdc20 during the spindle assembly checkpoint in S. cerevisiae. Cell Signal 2017; 33:41-48. [PMID: 28189585 DOI: 10.1016/j.cellsig.2017.02.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2016] [Revised: 01/21/2017] [Accepted: 02/06/2017] [Indexed: 01/26/2023]
Abstract
The anaphase-promoting complex (APC) is a ubiquitin ligase responsible for promoting the degradation of many cell cycle regulators. One of the activators and substrate-binding proteins for the APC is Cdc20. It has been shown previously that Cdc20 can promote its own degradation by the APC in normal cycling cells mainly through a cis-degradation mode (i.e. via an intramolecular mechanism). However, how Cdc20 is degraded during the spindle assembly checkpoint (SAC) is still not fully clear. In this study, we used a dual-Cdc20 system to investigate this issue and found that the cis-degradation mode is also the major pathway responsible for Cdc20 degradation during the SAC. In addition, we found that there is an inverse relationship between APCCdc20 activity and the transcriptional activity of the CDC20 promoter, which likely occurs through feedback regulation by APCCdc20 substrates, such as the cyclins Clb2 and Clb5. These findings contribute to our understanding of how the inhibition of APCCdc20 activity and enhanced Cdc20 degradation are required for proper spindle checkpoint arrest.
Collapse
Affiliation(s)
- Ruiwen Wang
- Institute of Life Sciences, College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian Province 350108, China.
| | - Janet L Burton
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA
| | - Mark J Solomon
- Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
| |
Collapse
|
|
15
|
Corbett KD. Molecular Mechanisms of Spindle Assembly Checkpoint Activation and Silencing. PROGRESS IN MOLECULAR AND SUBCELLULAR BIOLOGY 2017; 56:429-455. [PMID: 28840248 DOI: 10.1007/978-3-319-58592-5_18] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
In eukaryotic cell division, the Spindle Assembly Checkpoint (SAC) plays a key regulatory role by monitoring the status of chromosome-microtubule attachments and allowing chromosome segregation only after all chromosomes are properly attached to spindle microtubules. While the identities of SAC components have been known, in some cases, for over two decades, the molecular mechanisms of the SAC have remained mostly mysterious until very recently. In the past few years, advances in biochemical reconstitution, structural biology, and bioinformatics have fueled an explosion in the molecular understanding of the SAC. This chapter seeks to synthesize these recent advances and place them in a biological context, in order to explain the mechanisms of SAC activation and silencing at a molecular level.
Collapse
Affiliation(s)
- Kevin D Corbett
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, USA.
- Departments of Cellular & Molecular Medicine and Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, USA.
| |
Collapse
|
|
16
|
Abstract
The mitotic checkpoint is a specialized signal transduction pathway that contributes to the fidelity of chromosome segregation. The signaling of the checkpoint originates from defective kinetochore-microtubule interactions and leads to formation of the mitotic checkpoint complex (MCC), a highly potent inhibitor of the Anaphase Promoting Complex/Cyclosome (APC/C)—the E3 ubiquitin ligase essential for anaphase onset. Many important questions concerning the MCC and its interaction with APC/C have been intensively investigated and debated in the past 15 years, such as the exact composition of the MCC, how it is assembled during a cell cycle, how it inhibits APC/C, and how the MCC is disassembled to allow APC/C activation. These efforts have culminated in recently reported structure models for human MCC:APC/C supra-complexes at near-atomic resolution that shed light on multiple aspects of the mitotic checkpoint mechanisms. However, confusing statements regarding the MCC are still scattered in the literature, making it difficult for students and scientists alike to obtain a clear picture of MCC composition, structure, function and dynamics. This review will comb through some of the most popular concepts or misconceptions about the MCC, discuss our current understandings, present a synthesized model on regulation of CDC20 ubiquitination, and suggest a few future endeavors and cautions for next phase of MCC research.
Collapse
Affiliation(s)
- Song-Tao Liu
- Department of Biological Sciences, University of Toledo, 2801 West Bancroft St., Toledo, OH 43606, USA
| | - Hang Zhang
- Department of Biological Sciences, University of Toledo, 2801 West Bancroft St., Toledo, OH 43606, USA
| |
Collapse
|
|
17
|
Abstract
The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2.
Collapse
|
|
18
|
Skoneczna A, Kaniak A, Skoneczny M. Genetic instability in budding and fission yeast-sources and mechanisms. FEMS Microbiol Rev 2015; 39:917-67. [PMID: 26109598 PMCID: PMC4608483 DOI: 10.1093/femsre/fuv028] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/26/2015] [Indexed: 12/17/2022] Open
Abstract
Cells are constantly confronted with endogenous and exogenous factors that affect their genomes. Eons of evolution have allowed the cellular mechanisms responsible for preserving the genome to adjust for achieving contradictory objectives: to maintain the genome unchanged and to acquire mutations that allow adaptation to environmental changes. One evolutionary mechanism that has been refined for survival is genetic variation. In this review, we describe the mechanisms responsible for two biological processes: genome maintenance and mutation tolerance involved in generations of genetic variations in mitotic cells of both Saccharomyces cerevisiae and Schizosaccharomyces pombe. These processes encompass mechanisms that ensure the fidelity of replication, DNA lesion sensing and DNA damage response pathways, as well as mechanisms that ensure precision in chromosome segregation during cell division. We discuss various factors that may influence genome stability, such as cellular ploidy, the phase of the cell cycle, transcriptional activity of a particular region of DNA, the proficiency of DNA quality control systems, the metabolic stage of the cell and its respiratory potential, and finally potential exposure to endogenous or environmental stress. The stability of budding and fission yeast genomes is influenced by two contradictory factors: (1) the need to be fully functional, which is ensured through the replication fidelity pathways of nuclear and mitochondrial genomes through sensing and repairing DNA damage, through precise chromosome segregation during cell division; and (2) the need to acquire changes for adaptation to environmental challenges.
Collapse
Affiliation(s)
- Adrianna Skoneczna
- Laboratory of Mutagenesis and DNA Repair, Institute of Biochemistry and Biophysics, Polish Academy of Science, 02-106 Warsaw, Poland
| | - Aneta Kaniak
- Laboratory of Mutagenesis and DNA Repair, Institute of Biochemistry and Biophysics, Polish Academy of Science, 02-106 Warsaw, Poland
| | - Marek Skoneczny
- Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Science, 02-106 Warsaw, Poland
| |
Collapse
|
|
19
|
Krefman NI, Drubin DG, Barnes G. Control of the spindle checkpoint by lateral kinetochore attachment and limited Mad1 recruitment. Mol Biol Cell 2015; 26:2620-39. [PMID: 26023090 PMCID: PMC4501360 DOI: 10.1091/mbc.e15-05-0276] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2015] [Accepted: 05/18/2015] [Indexed: 01/06/2023] Open
Abstract
The spindle checkpoint proteins Mad1 and Bub1 are dynamically recruited after induced de novo kinetochore assembly. Detached kinetochores compete with alternate binding sites in the nucleus to recruit Mad1 and Bub1 from very limited pools. Lateral kinetochore attachment to microtubules licenses Mad1 removal from kinetochores. We observed the dynamic recruitment of spindle checkpoint proteins Mad1 and Bub1 to detached kinetochores in budding yeast using real-time live-cell imaging and quantified recruitment in fixed cells. After induced de novo kinetochore assembly at one pair of sister centromeres, Mad1 appeared after the kinetochore protein Mtw1. Detached kinetochores were not associated with the nuclear envelope, so Mad1 does not anchor them to nuclear pore complexes (NPCs). Disrupting Mad1's NPC localization increased Mad1 recruitment to detached sister kinetochores. Conversely, increasing the number of detached kinetochores reduced the amount of Mad1 per detached kinetochore. Bub1 also relocalized completely from the spindle to detached sister centromeres after kinetochore assembly. After their capture by microtubules, Mad1 and Bub1 progressively disappeared from kinetochores. Sister chromatids that arrested with a lateral attachment to one microtubule exhibited half the Mad1 of fully detached sisters. We propose that detached kinetochores compete with alternate binding sites in the nucleus to recruit Mad1 and Bub1 from available pools that are small enough to be fully depleted by just one pair of detached kinetochores and that lateral attachment licenses Mad1 removal from kinetochores after a kinetic delay.
Collapse
Affiliation(s)
- Nathaniel I Krefman
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
| | - David G Drubin
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
| | - Georjana Barnes
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720
| |
Collapse
|
|
20
|
Lian HY, Jiao GZ, Wang HL, Tan XW, Wang TY, Zheng LL, Kong QQ, Tan JH. Role of cytoskeleton in regulating fusion of nucleoli: a study using the activated mouse oocyte model. Biol Reprod 2014; 91:56. [PMID: 25061094 DOI: 10.1095/biolreprod.114.120188] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca²⁺-free CZB medium containing 10 mM SrCl₂) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion, and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF.
Collapse
Affiliation(s)
- Hua-Yu Lian
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, P.R. China
| | - Guang-Zhong Jiao
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, P.R. China
| | - Hui-Li Wang
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, P.R. China
| | - Xiu-Wen Tan
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, P.R. China
| | - Tian-Yang Wang
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, P.R. China
| | - Liang-Liang Zheng
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, P.R. China
| | - Qiao-Qiao Kong
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, P.R. China
| | - Jing-He Tan
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, P.R. China
| |
Collapse
|
|
21
|
Chen Y, Chen CF, Polci R, Wei R, Riley DJ, Chen PL. Increased Nek1 expression in renal cell carcinoma cells is associated with decreased sensitivity to DNA-damaging treatment. Oncotarget 2014; 5:4283-94. [PMID: 24970796 PMCID: PMC4147323 DOI: 10.18632/oncotarget.2005] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2014] [Accepted: 05/24/2014] [Indexed: 01/09/2023] Open
Abstract
Renal cell carcinoma (RCC) is a heterogeneous disease with resistance to systemic chemotherapy. Elevated expression of multiple drug resistance (MDR) has been suggested to be one of the mechanisms for this resistance. Here, we provide an alternative mechanism to explain RCC's resistance to chemotherapy-induced apoptosis. Never-in mitosis A-related protein kinase 1 (Nek1) plays an important role in DNA damage response and proper checkpoint activation. The association of Nek1 with the voltage-dependent anion channel (VDAC1) is a critical determinant of cell survival following DNA-damaging treatment. We report here that Nek1 is highly expressed in RCC tumor and cultured RCC cells compared to that of normal renal tubular epithelial cells (RTE). The association between Nek1 and VDAC1 is genotoxic dependent: prolonged Nek1/VDAC1 dissociation will lead to VDAC1 dephosphorylation and initiate apoptosis. Down-regulation of Nek1 expression in RCC cells enhanced their sensitivity to DNA-damaging treatment. Collectively, these results suggest that the increased Nek1 expression in RCC cells maintain persistent VDAC1 phosphorylation, closing its channel and preventing the onset of apoptosis under genotoxic insults. Based on these results, we believe that Nek1 can serve as a potential therapeutic target for drug development in the treatment of RCC.
Collapse
Affiliation(s)
- Yumay Chen
- Department of Medicine, Division of Endocrinology, University of California at Irvine
| | - Chi-Fen Chen
- Department of Biological Chemistry, University of California at Irvine
| | - Rosaria Polci
- Department of Medicine, Division of Nephrology, The University of Texas Health Science Center at San Antonio
| | - Randy Wei
- Department of Biological Chemistry, University of California at Irvine
| | - Daniel J. Riley
- Department of Medicine, Division of Nephrology, The University of Texas Health Science Center at San Antonio
- Department of Surgery, The University of Texas Health Science Center at San Antonio
| | - Phang-Lang Chen
- Department of Biological Chemistry, University of California at Irvine
| |
Collapse
|
|
22
|
Rajanala K, Sarkar A, Jhingan GD, Priyadarshini R, Jalan M, Sengupta S, Nandicoori VK. Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function. J Cell Sci 2014; 127:3505-20. [PMID: 24938596 DOI: 10.1242/jcs.149112] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
A major constituent of the nuclear basket region of the nuclear pore complex (NPC), nucleoporin Tpr, plays roles in regulating multiple important processes. We have previously established that Tpr is phosphorylated in both a MAP-kinase-dependent and MAP-kinase-independent manner, and that Tpr acts as both a substrate and as a scaffold for ERK2 (also known as MAPK1). Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as additional ERK-independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue and that the site is hyperphosphorylated during mitosis. Furthermore, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during telophase. Abrogation of S2059 phosphorylation abolishes the interaction of Tpr with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, resulting in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study allow better understanding of Tpr functions.
Collapse
Affiliation(s)
- Kalpana Rajanala
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
| | - Anshuk Sarkar
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
| | - Gagan Deep Jhingan
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
| | - Raina Priyadarshini
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
| | - Manisha Jalan
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
| | - Sagar Sengupta
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
| | | |
Collapse
|
|
23
|
Wang J, Beauchemin M, Bertrand R. Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis. Cell Cycle 2014; 13:1313-26. [PMID: 24621501 PMCID: PMC4014433 DOI: 10.4161/cc.28293] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2014] [Revised: 02/13/2014] [Accepted: 02/19/2014] [Indexed: 01/16/2023] Open
Abstract
Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).
Collapse
Affiliation(s)
- Jianfang Wang
- Centre de recherche; Centre hospitalier de l’Université de Montréal (CRCHUM) and Institut du Cancer de Montréal; Montréal, Québec, Canada
| | - Myriam Beauchemin
- Centre de recherche; Centre hospitalier de l’Université de Montréal (CRCHUM) and Institut du Cancer de Montréal; Montréal, Québec, Canada
| | - Richard Bertrand
- Centre de recherche; Centre hospitalier de l’Université de Montréal (CRCHUM) and Institut du Cancer de Montréal; Montréal, Québec, Canada
- Département de médecine; Université de Montréal; Montréal, Québec, Canada
| |
Collapse
|
|
24
|
Abstract
The propagation of all organisms depends on the accurate and orderly segregation of chromosomes in mitosis and meiosis. Budding yeast has long served as an outstanding model organism to identify the components and underlying mechanisms that regulate chromosome segregation. This review focuses on the kinetochore, the macromolecular protein complex that assembles on centromeric chromatin and maintains persistent load-bearing attachments to the dynamic tips of spindle microtubules. The kinetochore also serves as a regulatory hub for the spindle checkpoint, ensuring that cell cycle progression is coupled to the achievement of proper microtubule-kinetochore attachments. Progress in understanding the composition and overall architecture of the kinetochore, as well as its properties in making and regulating microtubule attachments and the spindle checkpoint, is discussed.
Collapse
|
|
25
|
Wang TY, Li Q, Li Q, Li H, Zhu J, Cui W, Jiao GZ, Tan JH. Non-frozen preservation protocols for mature mouse oocytes dramatically extend their developmental competence by reducing oxidative stress. Mol Hum Reprod 2013; 20:318-29. [DOI: 10.1093/molehr/gat088] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
|
|
26
|
Abstract
During mitosis and meiosis, the spindle assembly checkpoint acts to maintain genome stability by delaying cell division until accurate chromosome segregation can be guaranteed. Accuracy requires that chromosomes become correctly attached to the microtubule spindle apparatus via their kinetochores. When not correctly attached to the spindle, kinetochores activate the spindle assembly checkpoint network, which in turn blocks cell cycle progression. Once all kinetochores become stably attached to the spindle, the checkpoint is inactivated, which alleviates the cell cycle block and thus allows chromosome segregation and cell division to proceed. Here we review recent progress in our understanding of how the checkpoint signal is generated, how it blocks cell cycle progression and how it is extinguished.
Collapse
Affiliation(s)
- Pablo Lara-Gonzalez
- Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK
| | | | | |
Collapse
|
|
27
|
Usukura K, Kasamatsu A, Okamoto A, Kouzu Y, Higo M, Koike H, Sakamoto Y, Ogawara K, Shiiba M, Tanzawa H, Uzawa K. Tripeptidyl peptidase II in human oral squamous cell carcinoma. J Cancer Res Clin Oncol 2013; 139:123-30. [PMID: 22986808 DOI: 10.1007/s00432-012-1307-y] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2012] [Accepted: 09/03/2012] [Indexed: 01/08/2023]
Abstract
PURPOSE Tripeptidyl peptidase II (TPP2), a member of the family of eukaryotic serine peptidase, has been implicated in DNA repair, cellular division, and apoptosis. The aim of this study was to examine TPP2 expression and its functional mechanisms in oral squamous cell carcinoma (OSCC). METHODS TPP2 mRNA and protein expression in seven OSCC-derived cells (Ca9-22, HSC-2, HSC-3, HSC-4, HO-1-N-1, H1, and Sa3) was analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunoblotting analyses. Since previous studies indicated that TPP2 might control chromosomal division, we investigated cellular proliferation and spindle assembly checkpoint (SAC) molecules, MAD2 and CCNB1. In addition, we evaluated the correlation between TPP2 expression levels in primary OSCCs (n = 108 specimens) and the clinicopathologic status by immunohistochemistry (IHC). RESULTS TPP2 mRNA and protein were significantly (P < 0.05) up-regulated in OSCC-derived cells compared with human normal oral keratinocytes. Suppression of TPP2 expression with shRNA significantly (P < 0.05) inhibited cellular proliferation compared with the control cells. In addition, appropriate localization of MAD2 and up-regulation of CCNB1 were observed in TPP2 knockdown OSCC cells. IHC showed that TPP2 expression in primary OSCCs was significantly (P < 0.001) greater than that in the normal oral counterparts, and the TPP2-positive cases were significantly (P < 0.05) correlated with tumor size. CONCLUSION The current study showed that overexpression of TPP2 occurs frequently during oral carcinogenesis and might be associated with OSCC progression via SAC activation.
Collapse
Affiliation(s)
- Katsuya Usukura
- Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
28
|
Schuyler SC, Wu YF, Kuan VJW. The Mad1-Mad2 balancing act--a damaged spindle checkpoint in chromosome instability and cancer. J Cell Sci 2012; 125:4197-206. [PMID: 23093575 DOI: 10.1242/jcs.107037] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Cancer cells are commonly aneuploid. The spindle checkpoint ensures accurate chromosome segregation by controlling cell cycle progression in response to aberrant microtubule-kinetochore attachment. Damage to the checkpoint, which is a partial loss or gain of checkpoint function, leads to aneuploidy during tumorigenesis. One form of damage is a change in levels of the checkpoint proteins mitotic arrest deficient 1 and 2 (Mad1 and Mad2), or in the Mad1:Mad2 ratio. Changes in Mad1 and Mad2 levels occur in human cancers, where their expression is regulated by the tumor suppressors p53 and retinoblastoma 1 (RB1). By employing a standard assay, namely the addition of a mitotic poison at mitotic entry, it has been shown that checkpoint function is normal in many cancer cells. However, in several experimental systems, it has been observed that this standard assay does not always reveal checkpoint aberrations induced by changes in Mad1 or Mad2, where excess Mad1 relative to Mad2 can lead to premature anaphase entry, and excess Mad2 can lead to a delay in entering anaphase. This Commentary highlights how changes in the levels of Mad1 and Mad2 result in a damaged spindle checkpoint, and explores how these changes cause chromosome instability that can lead to aneuploidy during tumorigenesis.
Collapse
Affiliation(s)
- Scott C Schuyler
- Department of Biomedical Sciences, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan, 333 Taiwan, Republic of China.
| | | | | |
Collapse
|
|
29
|
Choi HJ, Zhu BT. Role of cyclin B1/Cdc2 in mediating Bcl-XL
phosphorylation and apoptotic cell death following nocodazole-induced mitotic arrest. Mol Carcinog 2012; 53:125-37. [DOI: 10.1002/mc.21956] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2012] [Revised: 07/18/2012] [Accepted: 08/01/2012] [Indexed: 12/21/2022]
Affiliation(s)
- Hye Joung Choi
- Department of Pharmacology, Toxicology and Therapeutics, School of Medicine; University of Kansas Medical Center; Kansas City Kansas
| | - Bao Ting Zhu
- Department of Pharmacology, Toxicology and Therapeutics, School of Medicine; University of Kansas Medical Center; Kansas City Kansas
| |
Collapse
|
|
30
|
Chao WCH, Kulkarni K, Zhang Z, Kong EH, Barford D. Structure of the mitotic checkpoint complex. Nature 2012; 484:208-13. [PMID: 22437499 DOI: 10.1038/nature10896] [Citation(s) in RCA: 245] [Impact Index Per Article: 18.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2011] [Accepted: 01/25/2012] [Indexed: 12/23/2022]
Abstract
In mitosis, the spindle assembly checkpoint (SAC) ensures genome stability by delaying chromosome segregation until all sister chromatids have achieved bipolar attachment to the mitotic spindle. The SAC is imposed by the mitotic checkpoint complex (MCC), whose assembly is catalysed by unattached chromosomes and which binds and inhibits the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome segregation. Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. Mad3 and p31(comet) (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31(comet) disrupts MCC assembly to antagonize the SAC. This study shows how APC/C inhibition is coupled to degron recognition by co-activators.
Collapse
Affiliation(s)
- William C H Chao
- Division of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB, UK
| | | | | | | | | |
Collapse
|
|
31
|
Cui W, Zhang J, Lian HY, Wang HL, Miao DQ, Zhang CX, Luo MJ, Tan JH. Roles of MAPK and spindle assembly checkpoint in spontaneous activation and MIII arrest of rat oocytes. PLoS One 2012; 7:e32044. [PMID: 22384134 PMCID: PMC3288063 DOI: 10.1371/journal.pone.0032044] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2011] [Accepted: 01/18/2012] [Indexed: 11/18/2022] Open
Abstract
Rat oocytes are well known to undergo spontaneous activation (SA) after leaving the oviduct, but the SA is abortive with oocytes being arrested in metaphase III (MIII) instead of forming pronuclei. This study was designed to investigate the mechanism causing SA and MIII arrest. Whereas few oocytes collected from SD rats at 13 h after hCG injection that showed 100% of mitogen-activated protein kinase (MAPK) activities activated spontaneously, all oocytes recovered 19 h post hCG with MAPK decreased to below 75% underwent SA during in vitro culture. During SA, MAPK first declined to below 45% and then increased again to 80%; the maturation-promoting factor (MPF) activity fluctuated similarly but always began to change ahead of the MAPK activity. In SA oocytes with 75% of MAPK activities, microtubules were disturbed with irregularly pulled chromosomes dispersed over the spindle and the spindle assembly checkpoint (SAC) was activated. When MAPK decreased to 45%, the spindle disintegrated and chromosomes surrounded by microtubules were scattered in the ooplasm. SA oocytes entered MIII and formed several spindle-like structures by 6 h of culture when the MAPK activity re-increased to above 80%. While SA oocytes showed one Ca2+ rise, Sr2+-activated oocytes showed several. Together, the results suggested that SA stimuli triggered SA in rat oocytes by inducing a premature MAPK inactivation, which led to disturbance of spindle microtubules. The microtubule disturbance impaired pulling of chromosomes to the spindle poles, caused spindle disintegration and activated SAC. The increased SAC activity reactivated MPF and thus MAPK, leading to MIII arrest.
Collapse
Affiliation(s)
| | | | | | | | | | | | | | - Jing-He Tan
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
- * E-mail:
| |
Collapse
|
|
32
|
Abstract
Aneuploidy is a common feature of cancer cells, and is believed to play a critical role in tumorigenesis and cancer progression. Most cancer cells also exhibit high rates of mitotic chromosome mis-segregation, a phenomenon known as chromosomal instability, which leads to high variability of the karyotype. Here, we describe the nature, nuances, and implications of cancer karyotypic diversity. Moreover, we summarize recent studies aimed at identifying the mitotic defects that may be responsible for inducing chromosome mis-segregation in cancer cells. These include kinetochore attachment errors, spindle assembly checkpoint dysfunction, mitotic spindle defects, and other cell division inaccuracies. Finally, we discuss how such mitotic errors generate karyotypic diversity in cancer cells.
Collapse
|
|
33
|
Westhorpe FG, Tighe A, Lara-Gonzalez P, Taylor SS. p31comet-mediated extraction of Mad2 from the MCC promotes efficient mitotic exit. J Cell Sci 2011; 124:3905-16. [PMID: 22100920 PMCID: PMC3225272 DOI: 10.1242/jcs.093286] [Citation(s) in RCA: 113] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 07/25/2011] [Indexed: 12/19/2022] Open
Abstract
Accurate chromosome segregation requires the spindle assembly checkpoint to be active at the onset of mitosis, before being silenced following chromosome alignment. p31(comet) is a checkpoint antagonist in that its inhibition delays mitotic exit, whereas its overexpression overrides the checkpoint. How exactly p31(comet) antagonises the checkpoint is unclear. A prevalent model is that p31(comet) acts as a 'cap' by inhibiting recruitment of the open conformation form of Mad2 (O-Mad2) to the kinetochore-bound complex of Mad1-C-Mad2 (closed conformation Mad2), an essential step that is required for checkpoint activation. Here, we show that although p31(comet) localises to kinetochores in mitosis, modulation of its activity has no effect on recruitment of O-Mad2 to kinetochores. Rather, our observations support a checkpoint-silencing role for p31(comet) downstream of kinetochores. We show that p31(comet) binds Mad2 when it is bound to the mitotic checkpoint complex (MCC) components BubR1 and Cdc20. Furthermore, RNAi-mediated inhibition of p31(comet) results in more Mad2 bound to BubR1-Cdc20, and conversely, overexpression of p31(comet) results in less Mad2 bound to BubR1-Cdc20. Addition of recombinant p31(comet) to checkpoint-arrested extracts removes Mad2 from the MCC, whereas a p31(comet) mutant that cannot bind Mad2 has no effect. Significantly, expression of a Mad2 mutant that cannot bind p31(comet) prolongs the metaphase to anaphase transition. Taken together, our data support the notion that p31(comet) negatively regulates the spindle assembly checkpoint by extracting Mad2 from the MCC.
Collapse
Affiliation(s)
- Frederick G. Westhorpe
- Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK
| | - Anthony Tighe
- Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK
| | - Pablo Lara-Gonzalez
- Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK
| | - Stephen S. Taylor
- Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK
| |
Collapse
|
|
34
|
Tipton AR, Tipton M, Yen T, Liu ST. Closed MAD2 (C-MAD2) is selectively incorporated into the mitotic checkpoint complex (MCC). Cell Cycle 2011; 10:3740-50. [PMID: 22037211 DOI: 10.4161/cc.10.21.17919] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
The mitotic checkpoint is a specialized signal transduction pathway that monitors kinetochore-microtubule attachment to achieve faithful chromosome segregation. MAD2 is an evolutionarily conserved mitotic checkpoint protein that exists in open (O) and closed (C) conformations. The increase of intracellular C-MAD2 level during mitosis, through O→C-MAD2 conversion as catalyzed by unattached kinetochores, is a critical signaling event for the mitotic checkpoint. However, it remains controversial whether MAD2 is an integral component of the effector of the mitotic checkpoint--the Mitotic Checkpoint Complex (MCC). We show here that endogenous human MCC is assembled by first forming a BUBR1:BUB3:CDC20 complex in G2 and then selectively incorporating C-MAD2 during mitosis. Nevertheless, MCC can be induced to form in G1/S cells by expressing a C-conformation locked MAD2 mutant, indicating intracellular level of C-MAD2 as a major limiting factor for MCC assembly. In addition, a recombinant MCC containing C-MAD2 exhibits effective inhibitory activity towards APC/C isolated from mitotic HeLa cells, while a recombinant BUBR1:BUB3:CDC20 ternary complex is ineffective at comparable concentrations despite association with APC/C. These results help establish a direct connection between a major signal transducer (C-MAD2) and the potent effector (MCC) of the mitotic checkpoint, and provide novel insights into protein-protein interactions during assembly of a functional MCC.
Collapse
Affiliation(s)
- Aaron R Tipton
- Department of Biological Sciences, University of Toledo, Toledo, OH, USA
| | | | | | | |
Collapse
|
|
35
|
Choi HJ, Fukui M, Zhu BT. Role of cyclin B1/Cdc2 up-regulation in the development of mitotic prometaphase arrest in human breast cancer cells treated with nocodazole. PLoS One 2011; 6:e24312. [PMID: 21918689 PMCID: PMC3168870 DOI: 10.1371/journal.pone.0024312] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2011] [Accepted: 08/04/2011] [Indexed: 12/23/2022] Open
Abstract
BACKGROUND During a normal cell cycle, the transition from G₂ phase to mitotic phase is triggered by the activation of the cyclin B1-dependent Cdc2 kinase. Here we report our finding that treatment of MCF-7 human breast cancer cells with nocodazole, a prototypic microtubule inhibitor, results in strong up-regulation of cyclin B1 and Cdc2 levels, and their increases are required for the development of mitotic prometaphase arrest and characteristic phenotypes. METHODOLOGY/PRINCIPAL FINDINGS It was observed that there was a time-dependent early increase in cyclin B1 and Cdc2 protein levels (peaking between 12 and 24 h post treatment), and their levels started to decline after the initial increase. This early up-regulation of cyclin B1 and Cdc2 closely matched in timing the nocodazole-induced mitotic prometaphase arrest. Selective knockdown of cyclin B1or Cdc2 each abrogated nocodazole-induced accumulation of prometaphase cells. The nocodazole-induced prometaphase arrest was also abrogated by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or with cycloheximide, a protein synthesis inhibitor that was found to suppress cyclin B1 and Cdc2 up-regulation. In addition, we found that MAD2 knockdown abrogated nocodazole-induced accumulation of cyclin B1 and Cdc2 proteins, which was accompanied by an attenuation of nocodazole-induced prometaphase arrest. CONCLUSIONS/SIGNIFICANCE These observations demonstrate that the strong early up-regulation of cyclin B1 and Cdc2 contributes critically to the rapid and selective accumulation of prometaphase-arrested cells, a phenomenon associated with exposure to microtubule inhibitors.
Collapse
Affiliation(s)
- Hye Joung Choi
- Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, Kansas, United States of America
| | - Masayuki Fukui
- Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, Kansas, United States of America
| | - Bao Ting Zhu
- Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, Kansas, United States of America
- * E-mail:
| |
Collapse
|
|
36
|
Orth M, Mayer B, Rehm K, Rothweiler U, Heidmann D, Holak TA, Stemmann O. Shugoshin is a Mad1/Cdc20-like interactor of Mad2. EMBO J 2011; 30:2868-80. [PMID: 21666598 DOI: 10.1038/emboj.2011.187] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2010] [Accepted: 05/18/2011] [Indexed: 12/24/2022] Open
Abstract
Mammalian centromeric cohesin is protected from phosphorylation-dependent displacement in mitotic prophase by shugoshin-1 (Sgo1), while shugoshin-2 (Sgo2) protects cohesin from separase-dependent cleavage in meiosis I. In higher eukaryotes, progression and faithful execution of both mitosis and meiosis are controlled by the spindle assembly checkpoint, which delays anaphase onset until chromosomes have achieved proper attachment to microtubules. According to the so-called template model, Mad1-Mad2 complexes at unattached kinetochores instruct conformational change of soluble Mad2, thus catalysing Mad2 binding to its target Cdc20. Here, we show that human Sgo2, but not Sgo1, specifically interacts with Mad2 in a manner that strongly resembles the interactions of Mad2 with Mad1 or Cdc20. Sgo2 contains a Mad1/Cdc20-like Mad2-interaction motif and competes with Mad1 and Cdc20 for binding to Mad2. NMR and biochemical analyses show that shugoshin binding induces similar conformational changes in Mad2 as do Mad1 or Cdc20. Mad2 binding regulates fine-tuning of Sgo2's sub-centromeric localization. Mad2 binding is conserved in the only known Xenopus laevis shugoshin homologue and, compatible with a putative meiotic function, the interaction occurs in oocytes.
Collapse
Affiliation(s)
- Michael Orth
- Department of Genetics, University of Bayreuth, Bayreuth, Germany
| | | | | | | | | | | | | |
Collapse
|
|
37
|
Ma HT, Poon RYC. Orderly inactivation of the key checkpoint protein mitotic arrest deficient 2 (MAD2) during mitotic progression. J Biol Chem 2011; 286:13052-9. [PMID: 21335556 DOI: 10.1074/jbc.m110.201897] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Anaphase is promoted by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) only when all the chromosomes have achieved bipolar attachment to the mitotic spindles. Unattached kinetochores or the absence of tension between the paired kinetochores activates a surveillance mechanism termed the spindle-assembly checkpoint. A fundamental principle of the checkpoint is the activation of mitotic arrest deficient 2 (MAD2). MAD2 then forms a diffusible complex called mitotic checkpoint complex (designated as MAD2(MCC)) before it is recruited to APC/C (designated as MAD2(APC/C)). Large gaps in our knowledge remain on how MAD2 is inactivated after the checkpoint is satisfied. In this study, we have investigated the regulation of MAD2-containing complexes during mitotic progression. Using selective immunoprecipitation of checkpoint components and gel filtration chromatography, we found that MAD2(MCC) and MAD2(APC/C) were regulated very differently during mitotic exit. Temporally, MAD2(MCC) was broken down ahead of MAD2(APC/C). The inactivation of the two complexes also displayed different requirements of proteolysis; although APC/C and proteasome activities were dispensable for MAD2(MCC) inactivation, they are required for MAD2(APC/C) inactivation. In fact, the degradation of CDC20 is inextricably linked to the breakdown of MAD2(APC/C). These data extended our understanding of the checkpoint complexes during checkpoint silencing.
Collapse
Affiliation(s)
- Hoi Tang Ma
- Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
| | | |
Collapse
|
|
38
|
Abstract
The mitotic checkpoint protein Mad2 halts cell division by interfering with MKlp2-mediated relocation of the chromosome passenger complex from centromeres to the mitotic spindle. We identified the mitotic kinesin-like protein 2 (MKlp2), a kinesin required for chromosome passenger complex (CPC)–mediated cytokinesis, as a target of the mitotic checkpoint protein Mad2. MKlp2 possesses a consensus Mad2-binding motif required for Mad2 binding. Mad2 prevents MKlp2 from loading onto the mitotic spindle, a prerequisite step for its function as a mitotic kinesin. Furthermore, Mad2 inhibits the ability of MKlp2 to relocate the CPC from centromeres, an essential step to promote cytokinesis. An MKlp2 mutant that is refractory to Mad2-mediated inhibition prematurely translocates to the mitotic spindle and mislocalizes the CPC component Aurora B from the midbody of dividing cells. This correlates with an increased incidence of cytokinesis failure. Together, these findings reveal that MKlp2 is a novel mitotic target of Mad2 necessary for proper mitotic progression and cytokinesis.
Collapse
Affiliation(s)
- Sang Hyun Lee
- Program in Cancer and Stem Cell Biology, Graduate Medical School, Duke-National University of Singapore, 169857 Singapore.
| | | | | |
Collapse
|
|
39
|
Mad2 haploinsufficiency protects hematopoietic progenitor cells subjected to cell-cycle stress in vivo and to inhibition of redox function of Ape1/Ref-1 in vitro. Exp Hematol 2011; 39:415-23. [PMID: 21216274 DOI: 10.1016/j.exphem.2010.12.012] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2010] [Revised: 12/30/2010] [Accepted: 12/31/2010] [Indexed: 11/21/2022]
Abstract
OBJECTIVE Cell-cycle checkpoints guarantee movement through the cell cycle. Mitotic arrest deficiency 2 (Mad2), a mitotic checkpoint protein, appears crucial for generating the wait anaphase signal to prevent onset of anaphase. We evaluated effects of Mad2 haploinsufficiency on hematopoietic stem (HSC) and progenitor (HPC) function in response to stress. MATERIALS AND METHODS We studied effects of Mad2(+/-) on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2(+/-) mice. RESULTS Mad2(+/-) HPCs were protected from cytotoxic effects in vivo of a cell-cycle-specific agent, Ara-C, events consistent with Mad2(+/-) HPCs being in a slow or noncycling state, but not from recovery of functional HPC after treatment with non-cycle-specific cyclophosphamide or sublethal irradiation. There were no differences in phenotyped HSCs in Mad2(+/-) &Mad2(+/+) mice, information confirmed by no changes in short- or long-term repopulating HSC assay. To better understand Mad2(+/-) HPC function, E3330, a cytostatic agent, was used to assess redox function of Ape1/Ref-1; colony growth was examined under 5% and 20% O(2) tension. Mad2(+/-) HPCs were less responsive to E3330 than Mad2(+/+) HPCs, and E3330 was more effective under lowered O(2) tension. Mad2(+/-) HPCs were not enhanced at lowered oxygen, as were Mad2(+/+) HPCs. CONCLUSIONS Our studies have unexpectedly found that Mad2 haploinsufficiency is protective in the presence of a cycle-specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro.
Collapse
|
|
40
|
Wu YG, Zhou P, Lan GC, Gao D, Li Q, Wei DL, Wang HL, Tan JH. MPF governs the assembly and contraction of actomyosin rings by activating RhoA and MAPK during chemical-induced cytokinesis of goat oocytes. PLoS One 2010; 5:e12706. [PMID: 20856880 PMCID: PMC2938347 DOI: 10.1371/journal.pone.0012706] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2010] [Accepted: 08/19/2010] [Indexed: 01/09/2023] Open
Abstract
The interplay between maturation-promoting factor (MPF), mitogen-activated protein kinase (MAPK) and Rho GTPase during actin-myosin interactions has yet to be determined. The mechanism by which microtubule disrupters induce the formation of ooplasmic protrusion during chemical-assisted enucleation of mammalian oocytes is unknown. Moreover, a suitable model is urgently needed for the study of cytokinesis. We have established a model of chemical-induced cytokinesis and have studied the signaling events leading to cytokinesis using this model. The results suggested that microtubule inhibitors activated MPF, which induced actomyosin assembly (formation of ooplasmic protrusion) by activating RhoA and thus MAPK. While MAPK controlled actin recruitment on its own, MPF promoted myosin enrichment by activating RhoA and MAPK. A further chemical treatment of oocytes with protrusions induced constriction of the actomyosin ring by inactivating MPF while activating RhoA. In conclusion, the present data suggested that the assembly and contraction of the actomyosin ring were two separable steps: while an increase in MPF activity promoted the assembly through RhoA-mediated activation of MAPK, a decrease in MPF activity triggered contraction of the ring by activating RhoA.
Collapse
Affiliation(s)
- Yan-Guang Wu
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Ping Zhou
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Guo-Cheng Lan
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Da Gao
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Qing Li
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - De-Li Wei
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Hui-Li Wang
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| | - Jing-He Tan
- College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China
| |
Collapse
|
|
41
|
A quantitative systems view of the spindle assembly checkpoint. EMBO J 2009; 28:2162-73. [PMID: 19629044 PMCID: PMC2722251 DOI: 10.1038/emboj.2009.186] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2009] [Accepted: 06/16/2009] [Indexed: 12/04/2022] Open
Abstract
The idle assembly checkpoint acts to delay chromosome segregation until all duplicated sister chromatids are captured by the mitotic spindle. This pathway ensures that each daughter cell receives a complete copy of the genome. The high fidelity and robustness of this process have made it a subject of intense study in both the experimental and computational realms. A significant number of checkpoint proteins have been identified but how they orchestrate the communication between local spindle attachment and global cytoplasmic signalling to delay segregation is not yet understood. Here, we propose a systems view of the spindle assembly checkpoint to focus attention on the key regulators of the dynamics of this pathway. These regulators in turn have been the subject of detailed cellular measurements and computational modelling to connect molecular function to the dynamics of spindle assembly checkpoint signalling. A review of these efforts reveals the insights provided by such approaches and underscores the need for further interdisciplinary studies to reveal in full the quantitative underpinnings of this cellular control pathway.
Collapse
|
|
42
|
Wang L, Yin F, Du Y, Du W, Chen B, Zhang Y, Wu K, Ding J, Liu J, Fan D. MAD2 as a key component of mitotic checkpoint: A probable prognostic factor for gastric cancer. Am J Clin Pathol 2009; 131:793-801. [PMID: 19461085 DOI: 10.1309/ajcpbmhhd0hfcy8w] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
We studied the subcellular localization of MAD2 in normal human tissues and gastric cancers. MAD2 showed nuclear and cytoplasmic localization in normal tissues such as muscle, testis, thyroid gland, cerebrum, trachea, and skin; blood vessels in some organs were also MAD2+. In normal stomach, MAD2 was expressed mainly in cytoplasm but showed nuclear staining in the majority of gastric cancers. MAD2 was significantly overexpressed in gastric cancer compared with matched adjacent tissues (P < .001), and expression was related to differentiation and other clinical parameters of cancer (P < .001). The cancer/adjacent normal tissue (C/N) ratio of MAD2 expression was higher than 2 and more frequently observed in patients with lymph gland metastasis (P < .05) and related to cancer differentiation. Our findings suggest that the steady-state amount of MAD2 inside cells may serve as a molecular switch in mitotic checkpoint control and that the subcellular localizations of this spindle protein undergo a shift during malignant transformation. The change of MAD2 expression may be involved mainly in gastric carcinogenesis and associated with the prognosis of gastric cancer; a C/N of more than 2 may be associated with the worse prognosis for survival in gastric carcinoma.
Collapse
Affiliation(s)
- Li Wang
- State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases; The Fourth Military Medical University, Xi’an, China
| | - Fang Yin
- State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases; The Fourth Military Medical University, Xi’an, China
| | - Yulei Du
- State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases; The Fourth Military Medical University, Xi’an, China
| | - Wenqi Du
- State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases; The Fourth Military Medical University, Xi’an, China
| | - Bei Chen
- State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases; The Fourth Military Medical University, Xi’an, China
| | - Yongguo Zhang
- State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases; The Fourth Military Medical University, Xi’an, China
| | - Kaichun Wu
- State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases; The Fourth Military Medical University, Xi’an, China
| | - Jie Ding
- State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases; The Fourth Military Medical University, Xi’an, China
| | - Jie Liu
- State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases; The Fourth Military Medical University, Xi’an, China
| | - Daiming Fan
- State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases; The Fourth Military Medical University, Xi’an, China
| |
Collapse
|
|
43
|
Yun M, Han YH, Yoon SH, Kim HY, Kim BY, Ju YJ, Kang CM, Jang SH, Chung HY, Lee SJ, Cho MH, Yoon G, Park GH, Kim SH, Lee KH. p31comet Induces cellular senescence through p21 accumulation and Mad2 disruption. Mol Cancer Res 2009; 7:371-82. [PMID: 19276188 DOI: 10.1158/1541-7786.mcr-08-0056] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Functional suppression of spindle checkpoint protein activity results in apoptotic cell death arising from mitotic failure, including defective spindle formation, chromosome missegregation, and premature mitotic exit. The recently identified p31(comet) protein acts as a spindle checkpoint silencer via communication with the transient Mad2 complex. In the present study, we found that p31(comet) overexpression led to two distinct phenotypic changes, cellular apoptosis and senescence. Because of a paucity of direct molecular link of spindle checkpoint to cellular senescence, however, the present report focuses on the relationship between abnormal spindle checkpoint formation and p31(comet)-induced senescence by using susceptible tumor cell lines. p31(comet)-induced senescence was accompanied by mitotic catastrophe with massive nuclear and chromosomal abnormalities. The progression of the senescence was completely inhibited by the depletion of p21(Waf1/Cip1) and partly inhibited by the depletion of the tumor suppressor protein p53. Notably, p21(Waf1/Cip1) depletion caused a dramatic phenotypic conversion of p31(comet)-induced senescence into cell death through mitotic catastrophe, indicating that p21(Waf1/Cip1) is a major mediator of p31(comet)-induced cellular senescence. In contrast to wild-type p31(comet), overexpression of a p31 mutant lacking the Mad2 binding region did not cause senescence. Moreover, depletion of Mad2 by small interfering RNA induced senescence. Here, we show that p31(comet) induces tumor cell senescence by mediating p21(Waf1/Cip1) accumulation and Mad2 disruption and that these effects are dependent on a direct interaction of p31(comet) with Mad2. Our results could be used to control tumor growth.
Collapse
Affiliation(s)
- Miyong Yun
- Laboratory of Radiation Molecular Cancer, Korea Institute of Radiological and Medical Sciences, Seoul, Korea
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
44
|
Lee SH, Sterling H, Burlingame A, McCormick F. Tpr directly binds to Mad1 and Mad2 and is important for the Mad1-Mad2-mediated mitotic spindle checkpoint. Genes Dev 2009; 22:2926-31. [PMID: 18981471 DOI: 10.1101/gad.1677208] [Citation(s) in RCA: 114] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The mitotic arrest-deficient protein Mad1 forms a complex with Mad2, which is required for imposing mitotic arrest on cells in which the spindle assembly is perturbed. By mass spectrometry of affinity-purified Mad2-associated factors, we identified the translocated promoter region (Tpr), a component of the nuclear pore complex (NPC), as a novel Mad2-interacting protein. Tpr directly binds to Mad1 and Mad2. Depletion of Tpr in HeLa cells disrupts the NPC localization of Mad1 and Mad2 during interphase and decreases the levels of Mad1-bound Mad2. Furthermore, depletion of Tpr decreases the levels of Mad1 at kinetochores during prometaphase, correlating with the inability of Mad1 to activate Mad2, which is required for inhibiting APC(Cdc20). These findings reveal an important role for Tpr in which Mad1-Mad2 proteins are regulated during the cell cycle and mitotic spindle checkpoint signaling.
Collapse
Affiliation(s)
- Sang Hyun Lee
- Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, San Francisco, California 94115, USA
| | | | | | | |
Collapse
|
|
45
|
Dupasquier S, Quittau-Prévostel C. A disrupted expression in cancers: multiple potential causes. C R Biol 2009; 332:1-14. [PMID: 19200921 DOI: 10.1016/j.crvi.2008.10.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2008] [Accepted: 10/03/2008] [Indexed: 10/21/2022]
Abstract
Tumor cells exhibit significant variations in the rate of pro- or anti-tumoral proteins that provide them a selective advantage of growth over normal cells. The control of these rates occurs at the three DNA, RNA and protein levels, and is determined by the structure of each of these three actors for the implementation of the molecular mechanisms involved in the control of the synthesis, maturation and stability of the mRNA and the protein itself. We give here an overview of the main events that can lead to a disruption of these mechanisms.
Collapse
Affiliation(s)
- Sébastien Dupasquier
- CNRS, UMR 5203, Institut de génomique fonctionnelle, 141 rue de la Cardonille, 34094 Montpellier cedex, France
| | | |
Collapse
|
|
46
|
Johnson MK, Cooksey AM, Wise DA. Localization of spindle checkpoint proteins in cells undergoing mitosis with unreplicated genomes. CELL MOTILITY AND THE CYTOSKELETON 2008; 65:890-5. [PMID: 18720400 DOI: 10.1002/cm.20310] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
CHO cells can be arrested with hydoxyurea at the beginning of the DNA synthesis phase of the cell cycle. Subsequent treatment with the xanthine, caffeine, induces cells to bypass the S-phase checkpoint and enter unscheduled mitosis [Schlegel and Pardee,1986, Science 232:1264-1266]. These treated cells build a normal spindle and distribute kinetochores, unattached to chromosomes, to their daughter cells [Brinkley et al.,1988, Nature 336:251-254; Zinkowski et al.,1991, J Cell Biol 113:1091-1110; Wise and Brinkley,1997, Cell Motil Cytoskeleton 36:291-302; Balczon et al.,2003, Chromosoma 112:96-102]. To investigate how these cells distribute kinetochores to daughter cells, we analyzed the spindle checkpoint components, Mad2, CENP-E, and the 3F3 phosphoepitope, using immunofluorescence and digital microscopy. Even though the kinetochores were unpaired and DNA was fragmented, the tension, alignment, and motor components of the checkpoint were found to be present and localized as predicted in prometaphase and metaphase. This unusual mitosis proves that a cell can successfully localize checkpoint proteins and divide even when kinetochores are unpaired and fragmented.
Collapse
|
|
47
|
Wang X, Di K, Zhang X, Han HY, Wong YC, Leung SCL, Ling MT. Id-1 promotes chromosomal instability through modification of APC/C activity during mitosis in response to microtubule disruption. Oncogene 2008; 27:4456-66. [PMID: 18372912 DOI: 10.1038/onc.2008.87] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Id-1 (Inhibitor of DNA binding/differential-1) plays a positive role in tumorigenesis through regulation of multiple signaling pathways. Recently, it is suggested that upregulation of Id-1 in cancer cells promotes chromosomal instability. However, the underlying molecular mechanism is not known. In this study, we report a novel function of Id-1 in regulation of mitosis through physical interaction with Cdc20 (cell division cycle protein 20) and Cdh1 (Cdc20 homolog 1). During early mitosis, Id-1 interacts with Cdc20 and RASSF1A (Ras association domain family 1A), leading to enhanced APC(Cdc20) activity, which in turn promotes cyclin B1/securin degradation and premature mitosis. During late mitosis, Id-1 binds to Cdh1 and disrupts the interaction between Cdh1 and APC, resulting in suppression of APC(Cdh1) activity. On the other hand, overexpression of Cdh1 leads to Id-1 protein degradation, suggesting that Id-1 may also act as a substrate of APC(Cdh1). The negative effect of Id-1 on APC(Cdh1) results in suppression of APC(Cdh1)-induced Aurora A and Cdc20 degradation, leading to failure in cytokinesis. As a result, overexpression of Id-1 in human prostate epithelial cells leads to polyploidy in response to microtubule disruption, and this effect is abolished when Id-1 expression is suppressed using antisense technology. These results demonstrate a novel function of Id-1 in promoting chromosomal instability through modification of APC/C activity during mitosis and provide a novel molecular mechanism accounted for the function of Id-1 as an oncogene.
Collapse
Affiliation(s)
- X Wang
- Cancer Biology Group, Department of Anatomy, Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.
| | | | | | | | | | | | | |
Collapse
|
|
48
|
Horner VL, Wolfner MF. Transitioning from egg to embryo: Triggers and mechanisms of egg activation. Dev Dyn 2008; 237:527-44. [DOI: 10.1002/dvdy.21454] [Citation(s) in RCA: 150] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
|
|
49
|
Díaz-Martínez LA, Yu H. Running on a treadmill: dynamic inhibition of APC/C by the spindle checkpoint. Cell Div 2007; 2:23. [PMID: 17650307 PMCID: PMC1947974 DOI: 10.1186/1747-1028-2-23] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2007] [Accepted: 07/24/2007] [Indexed: 12/05/2022] Open
Abstract
During mitosis, the genome duplicated during S-phase is synchronously and accurately segregated to the two daughter cells. The spindle checkpoint prevents premature sister-chromatid separation and mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is a key target of the spindle checkpoint. Upon checkpoint activation, the mitotic checkpoint complex (MCC) containing Mad2, Bub3, Mad3/BubR1 and Cdc20 inhibits APC/C. Two independent studies in budding yeast have now shed light on the mechanism by which MCC inhibits APC/C. These studies indicate that Mad3 binds to the mitotic activator of APC/C Cdc20 using peptide motifs commonly found in APC/C substrates and thus competes with APC/C substrates for APC/CCdc20 binding. In addition, Mad3 binding to APC/CCdc20 induces Cdc20 ubiquitination by APC/C, leading to the dissociation of MCC. Meanwhile, two other studies have shown that a deubiquitinating enzyme is required for the spindle checkpoint whereas APC/C-dependent ubiquitination is needed for checkpoint inactivation. Collectively, these studies suggest a dynamic model for APC/CCdc20 regulation by MCC in which APC/C- and Mad3-dependent ubiquitination of Cdc20 constitutes a self-regulated switch that rapidly inactivates the spindle checkpoint upon correct chromosome attachment.
Collapse
Affiliation(s)
- Laura A Díaz-Martínez
- Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041, USA
| | - Hongtao Yu
- Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041, USA
| |
Collapse
|
|
50
|
|