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Attiogbe MKI, Huang TT, Zhao HY, Wang HY, Cao L, Yan PP, Zhang SQ, Cao YX. EGFR tyrosine kinase inhibitor ZZC4 overcomes acquired resistance to gefitinib. Toxicol Appl Pharmacol 2025; 497:117280. [PMID: 39999922 DOI: 10.1016/j.taap.2025.117280] [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: 12/02/2024] [Revised: 02/21/2025] [Accepted: 02/21/2025] [Indexed: 02/27/2025]
Abstract
Despite the tremendous progress of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) development, acquired resistance mechanisms have limited their efficacy in treating non-small cell lung cancer (NSCLC). To overcome these limitations, novel EGFR-TKIs are needed. In our previous study, we presented ZZC4 as a potent EGFR-TKI. In this study, we developed NSCLC cells resistant to EGFR-TKI gefitinib and osimertinib and assessed the effect and mechanism of action of ZZC4 on those cells. HCC827 cells were cultured with gefitinib in a concentration-escalation manner to achieve HCC827 gefitinib-resistant (HCC827-GR) cells after 6 months of treatment. Then, the effect of ZZC4 was assessed at the cellular and animal levels. To understand ZZC4's mechanism of action, the proteome alteration induced by ZZC4 on the resistant cell line was compared to the parental HCC827 cells using comparative proteomics. The result showed that gefitinib's IC50 on HCC827 was 533 nM, approximately 80 times its IC50 on normal cells (7.6 nM), confirming its resistance to HCC827 cells. The obtained resistant cells were treated with ZZC4, which potently suppressed the resistant cells' proliferation with an IC50 of 0.1 nM. In tumor-bearing mice, ZZC4 also suppressed the growth of HCC827-GR cell tumors with an inhibition ratio of 82 % at ZZC4 4 mg/kg. Further, the proteomic analysis revealed that ZZC4 inhibited HCC827-GR cell growth by upregulating CDKN1B and downregulating CCNA2 and CHEK1. In conclusion, ZZC4 overcomes resistance to gefitinib by altering the cell cycle pathway.
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Affiliation(s)
- Mawusse K I Attiogbe
- Department of Pharmacology, School of Basic Medical Science, Xi'an Jiaotong University Health Science Center, Xi'an 710061, Shaanxi, China
| | - Ting-Ting Huang
- Department of Pharmacology, School of Basic Medical Science, Xi'an Jiaotong University Health Science Center, Xi'an 710061, Shaanxi, China
| | - Hong-Yi Zhao
- Department of Medicinal Chemistry, School of Pharmacy, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Hong-Ying Wang
- Department of Medicinal Chemistry, School of Pharmacy, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Lei Cao
- Precision Medical Institute, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, Shaanxi, China
| | - Ping-Ping Yan
- Department of Pharmacology, School of Basic Medical Science, Xi'an Jiaotong University Health Science Center, Xi'an 710061, Shaanxi, China
| | - San-Qi Zhang
- Department of Medicinal Chemistry, School of Pharmacy, Xi'an Jiaotong University, Xi'an 710061, Shaanxi, China
| | - Yong-Xiao Cao
- Department of Pharmacology, School of Basic Medical Science, Xi'an Jiaotong University Health Science Center, Xi'an 710061, Shaanxi, China; College of Medicine, Xi'an International University, Xi'an 710077, Shaanxi, China.
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Wang Y, Huang J, Song Z, Zhang S, Guo H, Leng Q, Fang N, Ji S, Yang J. c-Jun promotes neuroblastoma cell differentiation by inhibiting APC formation via CDC16 and reduces neuroblastoma malignancy. Biol Direct 2025; 20:37. [PMID: 40149013 PMCID: PMC11948754 DOI: 10.1186/s13062-025-00630-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2024] [Accepted: 03/11/2025] [Indexed: 03/29/2025] Open
Abstract
Neuroblastoma is a pediatric embryonal malignancy characterized by impaired neuronal differentiation. Differentiation status in neuroblastoma strongly affects the clinical outcome, thus, enforcement of differentiation becomes a treatment strategy for this disease. However, the molecular mechanisms that control neuroblastoma differentiation are poorly understood. As an extensively studied protein of the activator protein-1 (AP-1) complex, c-Jun is involved in numerous cell regulations such as proliferation, survival and differentiation. In the current study, we demonstrated that c-Jun expression was upregulated by retinoic acid (RA) and flow cytometry assay indicated c-Jun overexpression arrested cell cycle to G1 phase, which, in turn, promoted the initiation of neuroblastoma cell differentiation. Co-immunoprecipitation (co-IP) assay showed that c-Jun competitively interacted with CDC16, a key subunit in anaphase-promoting complex (APC), resulting in reduced APC formation and inhibition of cell cycle progression. Furthermore, EdU proliferation assay and transwell experiment showed that c-Jun overexpression inhibited neuroblastoma cell proliferation and migration via interacting and sequestering CDC16. These findings identify c-Jun as a key regulator of neuroblastoma cell cycle and differentiation and may represent a promising therapeutic target to induce neuroblastoma differentiation via the interaction between c-Jun and CDC16.
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Affiliation(s)
- Yunyun Wang
- Department of Biochemistry and Molecular Biology, Cell Signal Transduction Laboratory, School of Basic Medical Science, Henan University, Kaifeng, Henan Province, 475004, China.
| | - Jingjing Huang
- Department of Biochemistry and Molecular Biology, Cell Signal Transduction Laboratory, School of Basic Medical Science, Henan University, Kaifeng, Henan Province, 475004, China
| | - Zhenhua Song
- Department of Biochemistry and Molecular Biology, Cell Signal Transduction Laboratory, School of Basic Medical Science, Henan University, Kaifeng, Henan Province, 475004, China
| | - Shuo Zhang
- Department of Biochemistry and Molecular Biology, Cell Signal Transduction Laboratory, School of Basic Medical Science, Henan University, Kaifeng, Henan Province, 475004, China
| | - Haojie Guo
- Department of Biochemistry and Molecular Biology, Cell Signal Transduction Laboratory, School of Basic Medical Science, Henan University, Kaifeng, Henan Province, 475004, China
| | - Qi Leng
- Department of Biochemistry and Molecular Biology, Cell Signal Transduction Laboratory, School of Basic Medical Science, Henan University, Kaifeng, Henan Province, 475004, China
| | - Na Fang
- Department of Biochemistry and Molecular Biology, Cell Signal Transduction Laboratory, School of Basic Medical Science, Henan University, Kaifeng, Henan Province, 475004, China
| | - Shaoping Ji
- Department of Biochemistry and Molecular Biology, Cell Signal Transduction Laboratory, School of Basic Medical Science, Henan University, Kaifeng, Henan Province, 475004, China.
- Zhengzhou Shuqing Medical College, Zhengzhou, Henan Province, 450064, China.
| | - Jian Yang
- College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, S7N 5E5, Canada.
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Lara-Gonzalez P, Variyar S, Moghareh S, Nguyen ACN, Kizhedathu A, Budrewicz J, Schlientz A, Varshney N, Bellaart A, Oegema K, Bardwell L, Desai A. Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses. J Cell Biol 2024; 223:e202308034. [PMID: 39105756 PMCID: PMC11303871 DOI: 10.1083/jcb.202308034] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2023] [Revised: 04/27/2024] [Accepted: 07/18/2024] [Indexed: 08/07/2024] Open
Abstract
Mitosis in early embryos often proceeds at a rapid pace, but how this pace is achieved is not understood. Here, we show that cyclin B3 is the dominant driver of rapid embryonic mitoses in the C. elegans embryo. Cyclins B1 and B2 support slow mitosis (NEBD to anaphase ∼600 s), but the presence of cyclin B3 dominantly drives the approximately threefold faster mitosis observed in wildtype. Multiple mitotic events are slowed down in cyclin B1 and B2-driven mitosis, and cyclin B3-associated Cdk1 H1 kinase activity is ∼25-fold more active than cyclin B1-associated Cdk1. Addition of cyclin B1 to fast cyclin B3-only mitosis introduces an ∼60-s delay between completion of chromosome alignment and anaphase onset; this delay, which is important for segregation fidelity, is dependent on inhibitory phosphorylation of the anaphase activator Cdc20. Thus, cyclin B3 dominance, coupled to a cyclin B1-dependent delay that acts via Cdc20 phosphorylation, sets the rapid pace and ensures mitotic fidelity in the early C. elegans embryo.
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Affiliation(s)
- Pablo Lara-Gonzalez
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
| | - Smriti Variyar
- Department of Cell and Developmental Biology, University of California, San Diego, San Diego, CA, USA
- Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, CA, USA
| | - Shabnam Moghareh
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA
| | - Anh Cao Ngoc Nguyen
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA
| | - Amrutha Kizhedathu
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA
| | | | - Aleesa Schlientz
- Department of Cell and Developmental Biology, University of California, San Diego, San Diego, CA, USA
- Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, CA, USA
| | - Neha Varshney
- Department of Cell and Developmental Biology, University of California, San Diego, San Diego, CA, USA
- Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, CA, USA
| | - Andrew Bellaart
- Department of Cell and Developmental Biology, University of California, San Diego, San Diego, CA, USA
- Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, CA, USA
| | - Karen Oegema
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
- Department of Cell and Developmental Biology, University of California, San Diego, San Diego, CA, USA
- Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, CA, USA
| | - Lee Bardwell
- Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA
| | - Arshad Desai
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
- Department of Cell and Developmental Biology, University of California, San Diego, San Diego, CA, USA
- Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, CA, USA
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Liao Y, Zhang W, Liu Y, Zhu C, Zou Z. The role of ubiquitination in health and disease. MedComm (Beijing) 2024; 5:e736. [PMID: 39329019 PMCID: PMC11424685 DOI: 10.1002/mco2.736] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2024] [Revised: 08/23/2024] [Accepted: 08/26/2024] [Indexed: 09/28/2024] Open
Abstract
Ubiquitination is an enzymatic process characterized by the covalent attachment of ubiquitin to target proteins, thereby modulating their degradation, transportation, and signal transduction. By precisely regulating protein quality and quantity, ubiquitination is essential for maintaining protein homeostasis, DNA repair, cell cycle regulation, and immune responses. Nevertheless, the diversity of ubiquitin enzymes and their extensive involvement in numerous biological processes contribute to the complexity and variety of diseases resulting from their dysregulation. The ubiquitination process relies on a sophisticated enzymatic system, ubiquitin domains, and ubiquitin receptors, which collectively impart versatility to the ubiquitination pathway. The widespread presence of ubiquitin highlights its potential to induce pathological conditions. Ubiquitinated proteins are predominantly degraded through the proteasomal system, which also plays a key role in regulating protein localization and transport, as well as involvement in inflammatory pathways. This review systematically delineates the roles of ubiquitination in maintaining protein homeostasis, DNA repair, genomic stability, cell cycle regulation, cellular proliferation, and immune and inflammatory responses. Furthermore, the mechanisms by which ubiquitination is implicated in various pathologies, alongside current modulators of ubiquitination are discussed. Enhancing our comprehension of ubiquitination aims to provide novel insights into diseases involving ubiquitination and to propose innovative therapeutic strategies for clinical conditions.
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Affiliation(s)
- Yan Liao
- Faculty of Anesthesiology Changhai Hospital Naval Medical University Shanghai China
- School of Anesthesiology Naval Medical University Shanghai China
| | - Wangzheqi Zhang
- Faculty of Anesthesiology Changhai Hospital Naval Medical University Shanghai China
- School of Anesthesiology Naval Medical University Shanghai China
| | - Yang Liu
- Faculty of Anesthesiology Changhai Hospital Naval Medical University Shanghai China
- School of Anesthesiology Naval Medical University Shanghai China
| | - Chenglong Zhu
- Faculty of Anesthesiology Changhai Hospital Naval Medical University Shanghai China
- School of Anesthesiology Naval Medical University Shanghai China
| | - Zui Zou
- Faculty of Anesthesiology Changhai Hospital Naval Medical University Shanghai China
- School of Anesthesiology Naval Medical University Shanghai China
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He Q, Qu M, Xu C, Wu L, Xu Y, Su J, Bao H, Shen T, He Y, Cai J, Xu D, Zeng LH, Wu X. Smoking-induced CCNA2 expression promotes lung adenocarcinoma tumorigenesis by boosting AT2/AT2-like cell differentiation. Cancer Lett 2024; 592:216922. [PMID: 38704137 DOI: 10.1016/j.canlet.2024.216922] [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: 10/26/2023] [Revised: 04/25/2024] [Accepted: 04/26/2024] [Indexed: 05/06/2024]
Abstract
Lung adenocarcinoma (LUAD), a type of non-small cell lung cancer (NSCLC), originates from not only bronchial epithelial cells but also alveolar type 2 (AT2) cells, which could differentiate into AT2-like cells. AT2-like cells function as cancer stem cells (CSCs) of LUAD tumorigenesis to give rise to adenocarcinoma. However, the mechanism underlying AT2 cell differentiation into AT2-like cells in LUAD remains unknown. We analyze genes differentially expressed and genes with significantly different survival curves in LUAD, and the combination of these two analyses yields 147 differential genes, in which 14 differentially expressed genes were enriched in cell cycle pathway. We next analyze the protein levels of these genes in LUAD and find that Cyclin-A2 (CCNA2) is closely associated with LUAD tumorigenesis. Unexpectedly, high CCNA2 expression in LUAD is restrictedly associated with smoking and independent of other driver mutations. Single-cell sequencing analyses reveal that CCNA2 is predominantly involved in AT2-like cell differentiation, while inhibition of CCNA2 significantly reverses smoking-induced AT2-like cell differentiation. Mechanistically, CCNA2 binding to CDK2 phosphorylates the AXIN1 complex, which in turn induces ubiquitination-dependent degradation of β-catenin and inhibits the WNT signaling pathway, thereby failing AT2 cell maintenance. These results uncover smoking-induced CCNA2 overexpression and subsequent WNT/β-catenin signaling inactivation as a hitherto uncharacterized mechanism controlling AT2 cell differentiation and LUAD tumorigenesis.
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Affiliation(s)
- Qiangqiang He
- Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou 310058, China
| | - Meiyu Qu
- Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou 310058, China
| | - Chengyun Xu
- Department of Pharmacology, Hangzhou City University, Hangzhou 310015, China
| | - Lichao Wu
- Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou 310058, China
| | - Yana Xu
- Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou 310058, China
| | - Jiakun Su
- Technology Center, China Tobacco Jiangxi Industrial Co. Ltd., Nanchang 330096, China
| | - Hangyang Bao
- Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou 310058, China
| | - Tingyu Shen
- Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou 310058, China
| | - Yangxun He
- Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou 310058, China
| | - Jibao Cai
- Technology Center, China Tobacco Jiangxi Industrial Co. Ltd., Nanchang 330096, China
| | - Da Xu
- Technology Center, China Tobacco Jiangxi Industrial Co. Ltd., Nanchang 330096, China
| | - Ling-Hui Zeng
- Department of Pharmacology, Hangzhou City University, Hangzhou 310015, China.
| | - Ximei Wu
- Department of Pharmacology, Zhejiang University School of Medicine, Hangzhou 310058, China; Shulan International Medical College, Zhejiang Shuren University, Hangzhou 310015, China.
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Iglesias-Romero AB, Soto T, Flor-Parra I, Salas-Pino S, Ruiz-Romero G, Gould KL, Cansado J, Daga RR. MAPK-dependent control of mitotic progression in S. pombe. BMC Biol 2024; 22:71. [PMID: 38523261 PMCID: PMC10962199 DOI: 10.1186/s12915-024-01865-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2023] [Accepted: 03/08/2024] [Indexed: 03/26/2024] Open
Abstract
BACKGROUND Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress. RESULTS We show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20Slp1 during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20Slp1 N-terminus through a canonical MAPK docking site. Most important, the Cdc20Slp1 pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit. CONCLUSIONS Our data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20Slp1 turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.
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Affiliation(s)
| | - Terersa Soto
- Yeast Physiology Group, Department of Genetics and Microbiology, Facultad de Biología, Universidad de Murcia, Murcia, 30071, Spain
| | - Ignacio Flor-Parra
- Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Seville, 41013, Spain
| | - Silvia Salas-Pino
- Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Seville, 41013, Spain
| | - Gabriel Ruiz-Romero
- Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Seville, 41013, Spain
| | - Kathleen L Gould
- Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, 37240, USA
| | - José Cansado
- Yeast Physiology Group, Department of Genetics and Microbiology, Facultad de Biología, Universidad de Murcia, Murcia, 30071, Spain.
| | - Rafael R Daga
- Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Seville, 41013, Spain.
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Hertz EPT, Vega IAD, Kruse T, Wang Y, Hendriks IA, Bizard AH, Eugui-Anta A, Hay RT, Nielsen ML, Nilsson J, Hickson ID, Mailand N. The SUMO-NIP45 pathway processes toxic DNA catenanes to prevent mitotic failure. Nat Struct Mol Biol 2023; 30:1303-1313. [PMID: 37474739 PMCID: PMC10497417 DOI: 10.1038/s41594-023-01045-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2022] [Accepted: 06/26/2023] [Indexed: 07/22/2023]
Abstract
SUMOylation regulates numerous cellular processes, but what represents the essential functions of this protein modification remains unclear. To address this, we performed genome-scale CRISPR-Cas9-based screens, revealing that the BLM-TOP3A-RMI1-RMI2 (BTRR)-PICH pathway, which resolves ultrafine anaphase DNA bridges (UFBs) arising from catenated DNA structures, and the poorly characterized protein NIP45/NFATC2IP become indispensable for cell proliferation when SUMOylation is inhibited. We demonstrate that NIP45 and SUMOylation orchestrate an interphase pathway for converting DNA catenanes into double-strand breaks (DSBs) that activate the G2 DNA-damage checkpoint, thereby preventing cytokinesis failure and binucleation when BTRR-PICH-dependent UFB resolution is defective. NIP45 mediates this new TOP2-independent DNA catenane resolution process via its SUMO-like domains, promoting SUMOylation of specific factors including the SLX4 multi-nuclease complex, which contributes to catenane conversion into DSBs. Our findings establish that SUMOylation exerts its essential role in cell proliferation by enabling resolution of toxic DNA catenanes via nonepistatic NIP45- and BTRR-PICH-dependent pathways to prevent mitotic failure.
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Affiliation(s)
- Emil P T Hertz
- Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.
| | - Ignacio Alonso-de Vega
- Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
| | - Thomas Kruse
- Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
| | - Yiqing Wang
- Center for Chromosome Stability, University of Copenhagen, Copenhagen, Denmark
- Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark
| | - Ivo A Hendriks
- Proteomics Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
| | - Anna H Bizard
- Center for Chromosome Stability, University of Copenhagen, Copenhagen, Denmark
- Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark
| | - Ania Eugui-Anta
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
| | - Ronald T Hay
- Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
| | - Michael L Nielsen
- Proteomics Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
| | - Jakob Nilsson
- Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
| | - Ian D Hickson
- Center for Chromosome Stability, University of Copenhagen, Copenhagen, Denmark
- Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark
| | - Niels Mailand
- Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.
- Center for Chromosome Stability, University of Copenhagen, Copenhagen, Denmark.
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Lara-Gonzalez P, Variyar S, Budrewicz J, Schlientz A, Varshney N, Bellaart A, Moghareh S, Nguyen ACN, Oegema K, Desai A. Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.11.553011. [PMID: 37609212 PMCID: PMC10441424 DOI: 10.1101/2023.08.11.553011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/24/2023]
Abstract
In many species, early embryonic mitoses proceed at a very rapid pace, but how this pace is achieved is not understood. Here we show that in the early C. elegans embryo, cyclin B3 is the dominant driver of rapid embryonic mitoses. Metazoans typically have three cyclin B isoforms that associate with and activate Cdk1 kinase to orchestrate mitotic events: the related cyclins B1 and B2 and the more divergent cyclin B3. We show that whereas embryos expressing cyclins B1 and B2 support slow mitosis (NEBD to Anaphase ~ 600s), the presence of cyclin B3 dominantly drives the ~3-fold faster mitosis observed in wildtype embryos. CYB-1/2-driven mitosis is longer than CYB-3-driven mitosis primarily because the progression of mitotic events itself is slower, rather than delayed anaphase onset due to activation of the spindle checkpoint or inhibitory phosphorylation of the anaphase activator CDC-20. Addition of cyclin B1 to cyclin B3-only mitosis introduces an ~60s delay between the completion of chromosome alignment and anaphase onset, which likely ensures segregation fidelity; this delay is mediated by inhibitory phosphorylation on CDC-20. Thus, the dominance of cyclin B3 in driving mitotic events, coupled to introduction of a short cyclin B1-dependent delay in anaphase onset, sets the rapid pace and ensures fidelity of mitoses in the early C. elegans embryo.
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Affiliation(s)
- Pablo Lara-Gonzalez
- Department of Developmental and Cell Biology, University of California Irvine, CA 92697
- Ludwig Institute for Cancer Research, La Jolla CA 92093
| | - Smriti Variyar
- Department of Cell & Developmental Biology, University of California San Diego, CA 92093
- Department of Cellular & Molecular Medicine, University of California San Diego, CA 92093
| | - Jacqueline Budrewicz
- Ludwig Institute for Cancer Research, La Jolla CA 92093
- Current address: Department of Molecular and Medical Genetics, Oregon Health & Science University (OHSU), OR 97239
- Current address: Division of Reproductive & Developmental Sciences, Oregon National Primate Research Center (ONPRC), Beaverton, Oregon
| | - Aleesa Schlientz
- Department of Cell & Developmental Biology, University of California San Diego, CA 92093
- Department of Cellular & Molecular Medicine, University of California San Diego, CA 92093
| | - Neha Varshney
- Department of Cell & Developmental Biology, University of California San Diego, CA 92093
- Department of Cellular & Molecular Medicine, University of California San Diego, CA 92093
| | - Andrew Bellaart
- Department of Cell & Developmental Biology, University of California San Diego, CA 92093
- Department of Cellular & Molecular Medicine, University of California San Diego, CA 92093
| | - Shabnam Moghareh
- Department of Developmental and Cell Biology, University of California Irvine, CA 92697
| | - Anh Cao Ngoc Nguyen
- Department of Developmental and Cell Biology, University of California Irvine, CA 92697
| | - Karen Oegema
- Ludwig Institute for Cancer Research, La Jolla CA 92093
- Department of Cell & Developmental Biology, University of California San Diego, CA 92093
- Department of Cellular & Molecular Medicine, University of California San Diego, CA 92093
| | - Arshad Desai
- Ludwig Institute for Cancer Research, La Jolla CA 92093
- Department of Cell & Developmental Biology, University of California San Diego, CA 92093
- Department of Cellular & Molecular Medicine, University of California San Diego, CA 92093
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Sahibdad I, Khalid S, Chaudhry GR, Salim A, Begum S, Khan I. Zinc enhances the cell adhesion, migration, and self-renewal potential of human umbilical cord derived mesenchymal stem cells. World J Stem Cells 2023; 15:751-767. [PMID: 37545753 PMCID: PMC10401417 DOI: 10.4252/wjsc.v15.i7.751] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/08/2023] [Revised: 05/15/2023] [Accepted: 06/06/2023] [Indexed: 07/25/2023] Open
Abstract
BACKGROUND Zinc (Zn) is the second most abundant trace element after Fe, present in the human body. It is frequently reported in association with cell growth and proliferation, and its deficiency is considered to be a major disease contributing factor.
AIM To determine the effect of Zn on in vitro growth and proliferation of human umbilical cord (hUC)-derived mesenchymal stem cells (MSCs).
METHODS hUC-MSCs were isolated from human umbilical cord tissue and characterized based on immunocytochemistry, immunophenotyping, and tri-lineage differentiation. The impact of Zn on cytotoxicity and proliferation was determined by MTT and Alamar blue assay. To determine the effect of Zn on population doubling time (PDT), hUC-MSCs were cultured in media with and without Zn for several passages. An in vitro scratch assay was performed to analyze the effect of Zn on the wound healing and migration capability of hUC-MSCs. A cell adhesion assay was used to test the surface adhesiveness of hUC-MSCs. Transcriptional analysis of genes involved in the cell cycle, proliferation, migration, and self-renewal of hUC-MSCs was performed by quantitative real-time polymerase chain reaction. The protein expression of Lin28, a pluripotency marker, was analyzed by immunocytochemistry.
RESULTS Zn at lower concentrations enhanced the rate of proliferation but at higher concentrations (> 100 µM), showed concentration dependent cytotoxicity in hUC-MSCs. hUC-MSCs treated with Zn exhibited a significantly greater healing and migration rate compared to untreated cells. Zn also increased the cell adhesion rate, and colony forming efficiency (CFE). In addition, Zn upregulated the expression of genes involved in the cell cycle (CDC20, CDK1, CCNA2, CDCA2), proliferation (transforming growth factor β1, GDF5, hypoxia-inducible factor 1α), migration (CXCR4, VCAM1, VEGF-A), and self-renewal (OCT4, SOX2, NANOG) of hUC-MSCs. Expression of Lin28 protein was significantly increased in cells treated with Zn.
CONCLUSION Our findings suggest that zinc enhances the proliferation rate of hUC-MSCs decreasing the PDT, and maintaining the CFE. Zn also enhances the cell adhesion, migration, and self-renewal of hUC-MSCs. These results highlight the essential role of Zn in cell growth and development.
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Affiliation(s)
- Iqra Sahibdad
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
| | - Shumaila Khalid
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
| | - G Rasul Chaudhry
- Department of Biological Sciences, Oakland University, Rochester, MI 48309, United States
| | - Asmat Salim
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
| | - Sumreen Begum
- Stem Cell Research Laboratory (SCRL), Sindh Institute of Urology and Transplantation (SIUT), Karachi 74200, Sindh, Pakistan
| | - Irfan Khan
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
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10
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Houston J, Ohta M, Gómez-Cavazos JS, Deep A, Corbett KD, Oegema K, Lara-Gonzalez P, Kim T, Desai A. BUB-1-bound PLK-1 directs CDC-20 kinetochore recruitment to ensure timely embryonic mitoses. Curr Biol 2023; 33:2291-2299.e10. [PMID: 37137308 PMCID: PMC10270731 DOI: 10.1016/j.cub.2023.04.021] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2022] [Revised: 03/13/2023] [Accepted: 04/12/2023] [Indexed: 05/05/2023]
Abstract
During mitosis, chromosomes assemble kinetochores to dynamically couple with spindle microtubules.1,2 Kinetochores also function as signaling hubs directing mitotic progression by recruiting and controlling the fate of the anaphase promoting complex/cyclosome (APC/C) activator CDC-20.3,4,5 Kinetochores either incorporate CDC-20 into checkpoint complexes that inhibit the APC/C or dephosphorylate CDC-20, which allows it to interact with and activate the APC/C.4,6 The importance of these two CDC-20 fates likely depends on the biological context. In human somatic cells, the major mechanism controlling mitotic progression is the spindle checkpoint. By contrast, progression through mitosis during the cell cycles of early embryos is largely checkpoint independent.7,8,9,10 Here, we first show that CDC-20 phosphoregulation controls mitotic duration in the C. elegans embryo and defines a checkpoint-independent temporal mitotic optimum for robust embryogenesis. CDC-20 phosphoregulation occurs at kinetochores and in the cytosol. At kinetochores, the flux of CDC-20 for local dephosphorylation requires an ABBA motif on BUB-1 that directly interfaces with the structured WD40 domain of CDC-20.6,11,12,13 We next show that a conserved "STP" motif in BUB-1 that docks the mitotic kinase PLK-114 is necessary for CDC-20 kinetochore recruitment and timely mitotic progression. The kinase activity of PLK-1 is required for CDC-20 to localize to kinetochores and phosphorylates the CDC-20-binding ABBA motif of BUB-1 to promote BUB-1-CDC-20 interaction and mitotic progression. Thus, the BUB-1-bound pool of PLK-1 ensures timely mitosis during embryonic cell cycles by promoting CDC-20 recruitment to the vicinity of kinetochore-localized phosphatase activity.
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Affiliation(s)
- Jack Houston
- Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA; Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Ludwig Institute for Cancer Research, La Jolla, CA 92093, USA
| | - Midori Ohta
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Ludwig Institute for Cancer Research, La Jolla, CA 92093, USA
| | - J Sebastián Gómez-Cavazos
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Department of Cell and Developmental Biology, School of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA
| | - Amar Deep
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Kevin D Corbett
- Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA; Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Karen Oegema
- Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA; Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Ludwig Institute for Cancer Research, La Jolla, CA 92093, USA; Department of Cell and Developmental Biology, School of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA
| | - Pablo Lara-Gonzalez
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Ludwig Institute for Cancer Research, La Jolla, CA 92093, USA; Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697, USA
| | - Taekyung Kim
- Ludwig Institute for Cancer Research, La Jolla, CA 92093, USA; Department of Biology Education, Pusan National University, Busan 46241, Republic of Korea.
| | - Arshad Desai
- Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093, USA; Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Ludwig Institute for Cancer Research, La Jolla, CA 92093, USA; Department of Cell and Developmental Biology, School of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.
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11
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PRMT6-CDC20 facilitates glioblastoma progression via the degradation of CDKN1B. Oncogene 2023; 42:1088-1100. [PMID: 36792756 PMCID: PMC10063447 DOI: 10.1038/s41388-023-02624-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2022] [Revised: 01/30/2023] [Accepted: 02/01/2023] [Indexed: 02/17/2023]
Abstract
PRMT6, a type I arginine methyltransferase, di-methylates the arginine residues of both histones and non-histones asymmetrically. Increasing evidence indicates that PRMT6 plays a tumor mediator involved in human malignancies. Here, we aim to uncover the essential role and underlying mechanisms of PRMT6 in promoting glioblastoma (GBM) proliferation. Investigation of PRMT6 expression in glioma tissues demonstrated that PRMT6 is overexpressed, and elevated expression of PRMT6 is negatively correlated with poor prognosis in glioma/GBM patients. Silencing PRMT6 inhibited GBM cell proliferation and induced cell cycle arrest at the G0/G1 phase, while overexpressing PRMT6 had opposite results. Further, we found that PRMT6 attenuates the protein stability of CDKN1B by promoting its degradation. Subsequent mechanistic investigations showed that PRMT6 maintains the transcription of CDC20 by activating histone methylation mark (H3R2me2a), and CDC20 interacts with and destabilizes CDKN1B. Rescue experimental results confirmed that PRMT6 promotes the ubiquitinated degradation of CDKN1B and cell proliferation via CDC20. We also verified that the PRMT6 inhibitor (EPZ020411) could attenuate the proliferative effect of GBM cells. Our findings illustrate that PRMT6, an epigenetic mediator, promotes CDC20 transcription via H3R2me2a to mediate the degradation of CDKN1B to facilitate GBM progression. Targeting PRMT6-CDC20-CDKN1B axis might be a promising therapeutic strategy for GBM.
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12
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Zheng Q, Takei-Hoshi R, Okumura H, Ito M, Kawaguchi K, Otagaki S, Matsumoto S, Luo Z, Zhang Q, Shiratake K. Genome editing of SlMYB3R3, a cell cycle transcription factor gene of tomato, induces elongated fruit shape. JOURNAL OF EXPERIMENTAL BOTANY 2022; 73:7312-7325. [PMID: 36070755 PMCID: PMC9730800 DOI: 10.1093/jxb/erac352] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Accepted: 09/05/2022] [Indexed: 06/15/2023]
Abstract
Fruit shape is an important trait that attracts consumers, and the regulation of genes related to cell division is crucial for shaping multicellular organs. In Arabidopsis, MYB3R transcription factors, which harbor three imperfect repeats in the N-terminus, control organ growth by regulating cell division. However, the function of MYB3Rs in tomato remains unknown. Here, we characterized tomato SlMYB3R3, which was preferentially expressed in flowers and placed in a subclade with two Arabidopsis cell cycle suppressors (MYB3R3/5). slmyb3r3 knockout mutants were generated using the CRISPR/Cas9 system. Morphological observation of the slmyb3r3 mutants showed that fruits that were elongated and occasionally peanut-like in shape were formed, which was caused by significantly increased cell numbers in the longitudinal direction. Transcriptome and yeast one-hybrid assay results suggested that SlMYB3R3 acted as a suppressor of cell-cycle-related genes by binding to the mitosis-specific activator (MSA) motifs in their promoters. Taken together, knock out of the suppressor SlMYB3R3 leads to elongated fruit, which results from the altered cell division pattern at the ovary stage, by regulating cell-cycle-related genes in an MSA-dependent manner. Our results suggest that SlMYB3R3 and its orthologs have the potential to change fruit shape as part of the molecular breeding of fruit crops.
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Affiliation(s)
- Qingyou Zheng
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
- Key Laboratory of Horticultural Plant Biology, Huazhong Agricultural University, Wuhan 430070, China
| | - Rie Takei-Hoshi
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
| | - Hitomi Okumura
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
| | - Masaki Ito
- School of Biological Science and Technology, College of Science and Engineering, Kanazawa University, Kanazawa 920-1192, Japan
| | - Kohei Kawaguchi
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
| | - Shungo Otagaki
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
| | - Shogo Matsumoto
- Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
| | - Zhengrong Luo
- Key Laboratory of Horticultural Plant Biology, Huazhong Agricultural University, Wuhan 430070, China
| | - Qinglin Zhang
- Key Laboratory of Horticultural Plant Biology, Huazhong Agricultural University, Wuhan 430070, China
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13
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Liu NQ, Cao WH, Wang X, Chen J, Nie J. Cyclin genes as potential novel prognostic biomarkers and therapeutic targets in breast cancer. Oncol Lett 2022; 24:374. [PMID: 36238849 PMCID: PMC9494629 DOI: 10.3892/ol.2022.13494] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2022] [Accepted: 08/15/2022] [Indexed: 11/06/2022] Open
Affiliation(s)
- Nian-Qiu Liu
- Department of Breast Surgery, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Center, Kunming, Yunnan 650000, P.R. China
| | - Wei-Han Cao
- Department of Ultrasound, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650000, P.R. China
| | - Xing Wang
- Department of Breast Surgery, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Center, Kunming, Yunnan 650000, P.R. China
| | - Junyao Chen
- Department of Breast Surgery, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Center, Kunming, Yunnan 650000, P.R. China
| | - Jianyun Nie
- Department of Breast Surgery, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Center, Kunming, Yunnan 650000, P.R. China
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Molecular Regulation of Yak Preadipocyte Differentiation and Proliferation by LncFAM200B and ceRNA Regulatory Network Analysis. Cells 2022; 11:cells11152366. [PMID: 35954210 PMCID: PMC9368248 DOI: 10.3390/cells11152366] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2022] [Revised: 07/28/2022] [Accepted: 07/30/2022] [Indexed: 02/04/2023] Open
Abstract
The positive regulatory role of lncFAM200B in differentiation and lipid deposition in yak intramuscular preadipocytes has been demonstrated in our previous study. However, the regulatory mechanisms remain unclear. In this study, we aimed to produce complete mRNA and microRNA (miRNA) profiles after adenovirus-mediated lncFAM200B overexpression in yak preadipocytes using high-throughput sequencing. We constructed a competing endogenous RNA (ceRNA) network with lncFAM200B as the core and identified the functions of the selected target miRNA during cell proliferation and differentiation. We obtained 118 differentially expressed genes (DEGs) after lncFAM200B overexpression, 76 of which were up-regulated, including Notch signaling members NOTCH3, DTX3L, and HES4, and 42 DEGs were down-regulated, including genes related to the cell cycle (CCNA2, BUB1, CDC20, TOP2A, and KIF20A). Additionally, many ubiquitin-mediated proteolysis pathway members were also significantly up-regulated (BUA7, PML, TRIM21, and TRIM25). MiRNA sequencing showed that 13 miRNAs were significantly up-regulated, and 12 miRNAs were down-regulated. Among them, 29 targets of 10 differentially expressed miRNAs (DEMs) were differentially expressed, including miR-152-FBXO33, miR-6529a-TRIM21, miR-148c-NOTCH3, and the miR-6529b-HES4 axis. We further verified that overexpression and inhibition of miR-6529a can inhibit and promote, respectively, the proliferation and differentiation of preadipocytes. Taken together, our study not only revealed the regulatory network of lncFAM200B during yak preadipocytes differentiation but also laid a foundation for elucidating the cause for lower intramuscular fat content in yaks at the molecular level.
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15
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Wang X, Li F, Zhu J, Feng D, Shi Y, Qu L, Li Y, Guo K, Zhang Y, Wang Q, Wang N, Wang X, Ge S. Upregulation of Cell Division Cycle 20 Expression Alters the Morphology of Neuronal Dendritic Spines in the Nucleus Accumbens by Promoting FMRP Ubiquitination. J Neurochem 2022; 162:166-189. [PMID: 35621027 DOI: 10.1111/jnc.15649] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Revised: 05/04/2022] [Accepted: 05/23/2022] [Indexed: 11/26/2022]
Abstract
The nucleus accumbens (NAc) is the key area of the reward circuit, but its heterogeneity has been poorly studied. Using single-cell RNA sequencing, we revealed a subcluster of GABAergic neurons characterized by cell division cycle 20 (Cdc20) mRNA expression in the NAc of adult rats. We studied the coexpression of Cdc20 and Gad1 mRNA in the NAc neurons of adult rats and assessed Cdc20 protein expression in the NAc during rat development. Moreover, we microinjected AAV2/9-hSyn-Cdc20 with or without the dual-AAV system into the bilateral NAc for sparse labelling to observe changes in the synaptic morphology of mature neurons and assessed rat behaviours in open field and elevated plus maze tests. Furthermore, we performed the experiments with a Cdc20 inhibitor, Cdc20 overexpression AAV vector, and Cdc20 conditional knockout primary striatal neurons to understand the ubiquitination-dependent degradation of fragile X mental retardation protein (FMRP) in vitro and in vivo. We confirmed the mRNA expression of Cdc20 in the NAc GABAergic neurons of adult rats, and its protein level was decreased significantly 3 weeks post-birth. Upregulated Cdc20 expression in the bilateral NAc decreased the dendritic spine density in mature neurons and induced anxiety-like behaviour in rats. Cdc20-APC triggered FMRP degradation through K48-linked polyubiquitination in Neuro-2a cells and primary striatal neurons and downregulated FMRP expression in the NAc of adult rats. These data revealed that upregulation of Cdc20 in the bilateral NAc reduced dendritic spine density and led to anxiety-like behaviours, possibly by enhancing FMRP degradation via K48-linked polyubiquitination.
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Affiliation(s)
- Xin Wang
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Fei Li
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Jun Zhu
- College of Acupuncture and Massage, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi, China
| | - Dayun Feng
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Yingwu Shi
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Liang Qu
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Yang Li
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Kang Guo
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Yue Zhang
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Qiang Wang
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Naigeng Wang
- Second Clinical Medical College, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi, China
| | - Xuelian Wang
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
| | - Shunnan Ge
- Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China
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16
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Cell cycle involvement in cancer therapy; WEE1 kinase, a potential target as therapeutic strategy. Mutat Res 2022; 824:111776. [PMID: 35247630 DOI: 10.1016/j.mrfmmm.2022.111776] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2021] [Revised: 02/07/2022] [Accepted: 02/08/2022] [Indexed: 12/22/2022]
Abstract
Mitosis is the process of cell division and is regulated by checkpoints in the cell cycle. G1-S, S, and G2-M are the three main checkpoints that prevent initiation of the next phase of the cell cycle phase until previous phase has completed. DNA damage leads to activation of the G2-M checkpoint, which can trigger a downstream DNA damage response (DDR) pathway to induce cell cycle arrest while the damage is repaired. If the DNA damage cannot be repaired, the replication stress response (RSR) pathway finally leads to cell death by apoptosis, in this case called mitotic catastrophe. Many cancer treatments (chemotherapy and radiotherapy) cause DNA damages based on SSBs (single strand breaks) or DSBs (double strand breaks), which cause cell death through mitotic catastrophe. However, damaged cells can activate WEE1 kinase (as a part of the DDR and RSR pathways), which prevents apoptosis and cell death by inducing cell cycle arrest at G2 phase. Therefore, inhibition of WEE1 kinase could sensitize cancer cells to chemotherapeutic drugs. This review focuses on the role of WEE1 kinase (as a biological macromolecule which has a molecular mass of 96 kDa) in the cell cycle, and its interactions with other regulatory pathways. In addition, we discuss the potential of WEE1 inhibition as a new therapeutic approach in the treatment of various cancers, such as melanoma, breast cancer, pancreatic cancer, cervical cancer, etc.
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17
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Li Z, Zhang Y, Zhou Y, Wang F, Yin C, Ding L, Zhang S. Tanshinone IIA suppresses the progression of lung adenocarcinoma through regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway. Sci Rep 2021; 11:23681. [PMID: 34880385 PMCID: PMC8654884 DOI: 10.1038/s41598-021-03166-2] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2021] [Accepted: 11/29/2021] [Indexed: 12/26/2022] Open
Abstract
Lung adenocarcinoma (LUAD) belongs to a subgroup of non-small cell lung cancer (NSCLC) with an increasing incidence all over the world. Tanshinone IIA (TSA), an active compound of Salvia miltiorrhiza Bunge., has been found to have anti-tumor effects on many tumors, but its anti-LUAD effect and its mechanism have not been reported yet. In this study, bio-information analysis was applied to characterize the potential mechanism of TSA on LUA, biological experiments were used to verify the mechanisms involved. TCGA, Pubchem, SwissTargetPrediction, Venny2.1.0, STRING, DAVID, Cytoscape 3.7.2, Omicshare, GEPIA, RSCBPDB, Chem Draw, AutoDockTools, and PyMOL were utilized for analysis in the bio-information analysis and network pharmacology. Our experiments in vitro focused on the anti-LUAD effects and mechanisms of TSA on LUAD cells (A549 and NCI-H1975 cells) via MTT, plate cloning, Annexin V-FITC and PI dual staining, flow cytometry, and western blot assays. A total of 64 differentially expressed genes (DEGs) of TSA for treatment of LUAD were screened out. Gene ontology and pathway analysis revealed characteristic of the DEGs network. After GEPIA-based DEGs confirmation, 46 genes were considered having significant differences. Further, 10 key DEGs (BTK, HSD11B1, ADAM33, TNNC1, THRA, CCNA2, AURKA, MIF, PLK1, and SORD) were identified as the most likely relevant genes from overall survival analysis. Molecular Docking results showed that CCNA2, CDK2 and PLK1 had the lowest docking energy. MTT and plate cloning assays results showed that TSA inhibited the proliferation of LUAD cells in a concentration-dependent manner. Annexin V-FITC and PI dual staining and flow cytometry assays results told that TSA promoted the apoptosis of the two LUAD cells in different degrees, and induced cycle arrest in the G1/S phase. Western blot results showed that TSA significantly down-regulated the expression of CCNA2, CDK2, AURKA, PLK1, and p-ERK. In summary, TSA could suppress the progression of LUAD by inducing cell apoptosis and arresting cell cycle, and these were done by regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway. These findings are the first to demonstrate the molecular mechanism of TSA in treatment of LUAD combination of network bio-information analysis and biological experiments in vitro.
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Affiliation(s)
- Ziheng Li
- Faculty of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430065, China
| | - Ying Zhang
- Faculty of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430065, China
| | - Yuan Zhou
- Faculty of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430065, China
| | - Fuqian Wang
- Faculty of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430065, China
| | - Chao Yin
- Faculty of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430065, China
| | - Li Ding
- Faculty of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430065, China.
| | - Shunbo Zhang
- Faculty of Pharmacy, Hubei University of Chinese Medicine, Wuhan, 430065, China.
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18
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Hedström U, Öberg L, Vaarala O, Dellgren G, Silverborn M, Bjermer L, Westergren-Thorsson G, Hallgren O, Zhou X. Impaired Differentiation of Chronic Obstructive Pulmonary Disease Bronchial Epithelial Cells Grown on Bronchial Scaffolds. Am J Respir Cell Mol Biol 2021; 65:201-213. [PMID: 33882260 PMCID: PMC8399573 DOI: 10.1165/rcmb.2019-0395oc] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation, small airway remodeling, and emphysema. Airway remodeling in patients with COPD involves both the airway epithelium and the subepithelial extracellular matrix (ECM). However, it is currently unknown how epithelial remodeling in COPD airways depends on the relative influence from inherent defects in the epithelial cells and alterations in the ECM. To address this, we analyzed global gene expression in COPD human bronchial epithelial cells (HBEC) and normal HBEC after repopulation on decellularized bronchial scaffolds derived from patients with COPD or donors without COPD. COPD HBEC grown on bronchial scaffolds showed an impaired ability to initiate ciliated-cell differentiation, which was evident on all scaffolds regardless of their origin. In addition, although normal HBEC were less affected by the disease state of the bronchial scaffolds, COPD HBEC showed a gene expression pattern indicating increased proliferation and a retained basal-cell phenotype when grown on COPD bronchial scaffolds compared with normal bronchial scaffolds. By using mass spectrometry, we identified 13 matrisome proteins as being differentially abundant between COPD bronchial scaffolds and normal bronchial scaffolds. These observations are consistent with COPD pathology and suggest that both epithelial cells and the ECM contribute to epithelial-cell remodeling in COPD airways.
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Affiliation(s)
- Ulf Hedström
- Department of Bioscience COPD/IPF, and.,Division of Lung Biology, Department of Experimental Medical Science, and
| | - Lisa Öberg
- Department of Translational Science and Experimental Medicine, Research and Early Development, Respiratory and Immunology, BioPharmaceuticals Research and Development, AstraZeneca, Gothenburg, Sweden
| | | | - Göran Dellgren
- Transplant Institute and.,Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Martin Silverborn
- Transplant Institute and.,Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden
| | - Leif Bjermer
- Division of Respiratory Medicine and Allergology, Department of Clinical Sciences, Lund University, Lund, Sweden; and
| | | | - Oskar Hallgren
- Division of Lung Biology, Department of Experimental Medical Science, and.,Division of Respiratory Medicine and Allergology, Department of Clinical Sciences, Lund University, Lund, Sweden; and
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Cao X, Zao X, Xue B, Chen H, Zhang J, Li S, Li X, Zhu S, Guo R, Li X, Ye Y. The mechanism of TiaoGanYiPi formula for treating chronic hepatitis B by network pharmacology and molecular docking verification. Sci Rep 2021; 11:8402. [PMID: 33863948 PMCID: PMC8052433 DOI: 10.1038/s41598-021-87812-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Accepted: 03/30/2021] [Indexed: 12/14/2022] Open
Abstract
The Chinese herbal formula TiaoGanYiPi (TGYP) showed effective against chronic hepatitis B (CHB) caused by hepatitis B virus (HBV) infection. Hence, we aimed to clarify the mechanisms and potential targets between TGYP and CHB. The active compounds and related putative targets of TGYP, and disease targets of CHB were obtained from the public databases. The key targets between TGYP and CHB were identified through the network construction and module analysis. The expression of the key targets was detected in Gene Expression Omnibus (GEO) dataset and normal hepatocyte cell line LO2. We first obtained 11 key targets which were predominantly enriched in the Cancer, Cell cycle and HBV-related pathways. And the expression of the key targets was related to HBV infection and liver inflammation verified in GSE83148 database. Furthermore, the results of real-time quantitative PCR and CCK-8 assay indicated that TGYP could regulate the expression of key targets including CCNA2, ABL1, CDK4, CDKN1A, IGFR and MAP2K1, and promote proliferation of LO2 cells. In coclusion, we identified the active compounds and key targets btween TGYP and CHB, and found that the TGYP might exhibite curative effect on CHB via promoting hepatocyte proliferation and inhibiting the liver inflammatory processes.
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Affiliation(s)
- Xu Cao
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Xiaobin Zao
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China
| | - Baiquan Xue
- The First People's Hospital of Jinzhou District, Dalian, 116100, China
| | - Hening Chen
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Jiaxin Zhang
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Shuo Li
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Xiaobin Li
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Shun Zhu
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China.,Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Rui Guo
- Beijing University of Chinese Medicine, Beijing, 100102, China
| | - Xiaoke Li
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China. .,Institute of Liver Diseases, Beijing University of Chinese Medicine, Beijing, 100700, China.
| | - Yong'an Ye
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, 100700, China. .,Institute of Liver Diseases, Beijing University of Chinese Medicine, Beijing, 100700, China.
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20
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Hein JB, Garvanska DH, Nasa I, Kettenbach AN, Nilsson J. Coupling of Cdc20 inhibition and activation by BubR1. J Cell Biol 2021; 220:211939. [PMID: 33819340 PMCID: PMC8025235 DOI: 10.1083/jcb.202012081] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Revised: 02/18/2021] [Accepted: 02/25/2021] [Indexed: 12/25/2022] Open
Abstract
Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.
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Affiliation(s)
- Jamin B Hein
- Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, Copenhagen, Denmark
| | - Dimitriya H Garvanska
- Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, Copenhagen, Denmark
| | - Isha Nasa
- Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH
| | - Arminja N Kettenbach
- Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth College, Hanover, NH
| | - Jakob Nilsson
- Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Science, Copenhagen, Denmark
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21
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Silva Cascales H, Burdova K, Middleton A, Kuzin V, Müllers E, Stoy H, Baranello L, Macurek L, Lindqvist A. Cyclin A2 localises in the cytoplasm at the S/G2 transition to activate PLK1. Life Sci Alliance 2021; 4:e202000980. [PMID: 33402344 PMCID: PMC7812317 DOI: 10.26508/lsa.202000980] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Revised: 12/22/2020] [Accepted: 12/23/2020] [Indexed: 01/23/2023] Open
Abstract
Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.
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Affiliation(s)
| | - Kamila Burdova
- Laboratory of Cancer Cell Biology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
| | - Anna Middleton
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Vladislav Kuzin
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Erik Müllers
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Henriette Stoy
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Laura Baranello
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
| | - Libor Macurek
- Laboratory of Cancer Cell Biology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
| | - Arne Lindqvist
- Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
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22
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Abstract
Accurate chromosome segregation is required for cell survival and organismal development. During mitosis, the spindle assembly checkpoint acts as a safeguard to maintain the high fidelity of mitotic chromosome segregation by monitoring the attachment of kinetochores to the mitotic spindle. Bub1 is a conserved kinase critical for the spindle assembly checkpoint. Bub1 also facilitates chromosome alignment and contributes to the regulation of mitotic duration. Here, focusing on the spindle assembly checkpoint and on chromosome alignment, we summarize the primary literature on Bub1, discussing its structure and functional domains, as well its regulation and roles in mitosis. In addition, we discuss recent evidence for roles of Bub1 beyond mitosis regulation in TGFβ signaling and telomere replication. Finally, we discuss the involvement of Bub1 in human diseases, especially in cancer, and the potential of using Bub1 as a drug target for therapeutic applications.
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Affiliation(s)
- Taekyung Kim
- Department of Biology Education, Pusan National University, Busan, Korea
| | - Anton Gartner
- IBS Center for Genomic Integrity, Ulsan, Korea.,School of Life Sciences, Ulsan National Institute of Science and Technology
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23
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Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis. PLoS Genet 2020; 16:e1009184. [PMID: 33137813 PMCID: PMC7660922 DOI: 10.1371/journal.pgen.1009184] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2020] [Revised: 11/12/2020] [Accepted: 10/08/2020] [Indexed: 11/19/2022] Open
Abstract
In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, we found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, we found that CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. Our results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis.
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24
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Bancroft J, Holder J, Geraghty Z, Alfonso-Pérez T, Murphy D, Barr FA, Gruneberg U. PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20. Mol Biol Cell 2020; 31:2315-2330. [PMID: 32755477 PMCID: PMC7851957 DOI: 10.1091/mbc.e20-04-0252] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Revised: 07/22/2020] [Accepted: 07/29/2020] [Indexed: 12/30/2022] Open
Abstract
Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase-promoting complex/cyclosome and its coactivator CDC20 (APC/CCDC20) form the main ubiquitin E3 ligase for these two proteins. APC/CCDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylation. Here, we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilizes cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1 phosphorylation-defective CDC206A mutants. These CDC206A cells show a normal spindle checkpoint response and rapidly destroy cyclin B once all chromosomes have aligned and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase transition by promoting APC/CCDC20-dependent destruction of cyclin B in human cells.
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Affiliation(s)
- James Bancroft
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK
| | - James Holder
- Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK
| | - Zoë Geraghty
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK
| | | | - Daniel Murphy
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK
| | - Francis A. Barr
- Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK
| | - Ulrike Gruneberg
- Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK
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25
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Cunha-Silva S, Conde C. From the Nuclear Pore to the Fibrous Corona: A MAD Journey to Preserve Genome Stability. Bioessays 2020; 42:e2000132. [PMID: 32885448 DOI: 10.1002/bies.202000132] [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: 05/31/2020] [Revised: 07/22/2020] [Indexed: 11/09/2022]
Abstract
The relationship between kinetochores and nuclear pore complexes (NPCs) is intimate but poorly understood. Several NPC components and associated proteins are relocated to mitotic kinetochores to assist in different activities that ensure faithful chromosome segregation. Such is the case of the Mad1-c-Mad2 complex, the catalytic core of the spindle assembly checkpoint (SAC), a surveillance pathway that delays anaphase until all kinetochores are attached to spindle microtubules. Mad1-c-Mad2 is recruited to discrete domains of unattached kinetochores from where it promotes the rate-limiting step in the assembly of anaphase-inhibitory complexes. SAC proficiency further requires Mad1-c-Mad2 to be anchored at NPCs during interphase. However, the mechanistic relevance of this arrangement for SAC function remains ill-defined. Recent studies uncover the molecular underpinnings that coordinate the release of Mad1-c-Mad2 from NPCs with its prompt recruitment to kinetochores. Here, current knowledge on Mad1-c-Mad2 function and spatiotemporal regulation is reviewed and the critical questions that remain unanswered are highlighted.
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Affiliation(s)
- Sofia Cunha-Silva
- i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, 4200-135, Portugal.,IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, 4200-135, Portugal.,Programa Doutoral em Biologia Molecular e Celular (MCbiology), Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, 4050-313, Portugal
| | - Carlos Conde
- i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, 4200-135, Portugal.,IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, 4200-135, Portugal
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26
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Bouhaddou M, Memon D, Meyer B, White KM, Rezelj VV, Correa Marrero M, Polacco BJ, Melnyk JE, Ulferts S, Kaake RM, Batra J, Richards AL, Stevenson E, Gordon DE, Rojc A, Obernier K, Fabius JM, Soucheray M, Miorin L, Moreno E, Koh C, Tran QD, Hardy A, Robinot R, Vallet T, Nilsson-Payant BE, Hernandez-Armenta C, Dunham A, Weigang S, Knerr J, Modak M, Quintero D, Zhou Y, Dugourd A, Valdeolivas A, Patil T, Li Q, Hüttenhain R, Cakir M, Muralidharan M, Kim M, Jang G, Tutuncuoglu B, Hiatt J, Guo JZ, Xu J, Bouhaddou S, Mathy CJP, Gaulton A, Manners EJ, Félix E, Shi Y, Goff M, Lim JK, McBride T, O'Neal MC, Cai Y, Chang JCJ, Broadhurst DJ, Klippsten S, De Wit E, Leach AR, Kortemme T, Shoichet B, Ott M, Saez-Rodriguez J, tenOever BR, Mullins RD, Fischer ER, Kochs G, Grosse R, García-Sastre A, Vignuzzi M, Johnson JR, Shokat KM, Swaney DL, Beltrao P, Krogan NJ. The Global Phosphorylation Landscape of SARS-CoV-2 Infection. Cell 2020; 182:685-712.e19. [PMID: 32645325 PMCID: PMC7321036 DOI: 10.1016/j.cell.2020.06.034] [Citation(s) in RCA: 748] [Impact Index Per Article: 149.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 06/09/2020] [Accepted: 06/23/2020] [Indexed: 02/07/2023]
Abstract
The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.
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Affiliation(s)
- Mehdi Bouhaddou
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Danish Memon
- European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Bjoern Meyer
- Viral Populations and Pathogenesis Unit, CNRS UMR 3569, Institut Pasteur, 75724 Paris, Cedex 15, France
| | - Kris M White
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Veronica V Rezelj
- Viral Populations and Pathogenesis Unit, CNRS UMR 3569, Institut Pasteur, 75724 Paris, Cedex 15, France
| | - Miguel Correa Marrero
- European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Benjamin J Polacco
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - James E Melnyk
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute
| | - Svenja Ulferts
- Institute for Clinical and Experimental Pharmacology and Toxicology, University of Freiburg, Freiburg 79104, Germany
| | - Robyn M Kaake
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Jyoti Batra
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Alicia L Richards
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Erica Stevenson
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - David E Gordon
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Ajda Rojc
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Kirsten Obernier
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Jacqueline M Fabius
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Margaret Soucheray
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Lisa Miorin
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Elena Moreno
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA
| | - Cassandra Koh
- Viral Populations and Pathogenesis Unit, CNRS UMR 3569, Institut Pasteur, 75724 Paris, Cedex 15, France
| | - Quang Dinh Tran
- Viral Populations and Pathogenesis Unit, CNRS UMR 3569, Institut Pasteur, 75724 Paris, Cedex 15, France
| | - Alexandra Hardy
- Viral Populations and Pathogenesis Unit, CNRS UMR 3569, Institut Pasteur, 75724 Paris, Cedex 15, France
| | - Rémy Robinot
- Virus & Immunity Unit, Department of Virology, CNRS UMR 3569, Institut Pasteur, 75724 Paris, Cedex 15, France; Vaccine Research Institute, 94000 Creteil, France
| | - Thomas Vallet
- Viral Populations and Pathogenesis Unit, CNRS UMR 3569, Institut Pasteur, 75724 Paris, Cedex 15, France
| | | | - Claudia Hernandez-Armenta
- European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Alistair Dunham
- European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Sebastian Weigang
- Institute of Virology, Medical Center - University of Freiburg, Freiburg 79104, Germany
| | - Julian Knerr
- Institute for Clinical and Experimental Pharmacology and Toxicology, University of Freiburg, Freiburg 79104, Germany
| | - Maya Modak
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Diego Quintero
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Yuan Zhou
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Aurelien Dugourd
- Institute for Computational Biomedicine, Bioquant, Heidelberg University, Faculty of Medicine, and Heidelberg University Hospital, Heidelberg 69120, Germany
| | - Alberto Valdeolivas
- Institute for Computational Biomedicine, Bioquant, Heidelberg University, Faculty of Medicine, and Heidelberg University Hospital, Heidelberg 69120, Germany
| | - Trupti Patil
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Qiongyu Li
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Ruth Hüttenhain
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Merve Cakir
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Monita Muralidharan
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Minkyu Kim
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Gwendolyn Jang
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Beril Tutuncuoglu
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Joseph Hiatt
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Jeffrey Z Guo
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Jiewei Xu
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Sophia Bouhaddou
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA
| | - Christopher J P Mathy
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; Department of Bioengineering & Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Anna Gaulton
- European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Emma J Manners
- European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Eloy Félix
- European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Ying Shi
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute
| | - Marisa Goff
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Jean K Lim
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | | | | | | | | | | | | | - Emmie De Wit
- NIH/NIAID/Rocky Mountain Laboratories, Hamilton, MT 59840, USA
| | - Andrew R Leach
- European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK
| | - Tanja Kortemme
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; Department of Bioengineering & Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Brian Shoichet
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA
| | - Melanie Ott
- J. David Gladstone Institutes, San Francisco, CA 94158, USA
| | - Julio Saez-Rodriguez
- Institute for Computational Biomedicine, Bioquant, Heidelberg University, Faculty of Medicine, and Heidelberg University Hospital, Heidelberg 69120, Germany
| | - Benjamin R tenOever
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - R Dyche Mullins
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute
| | | | - Georg Kochs
- Institute of Virology, Medical Center - University of Freiburg, Freiburg 79104, Germany; Faculty of Medicine, University of Freiburg, Freiburg 79008, Germany
| | - Robert Grosse
- Institute for Clinical and Experimental Pharmacology and Toxicology, University of Freiburg, Freiburg 79104, Germany; Faculty of Medicine, University of Freiburg, Freiburg 79008, Germany; Centre for Integrative Biological Signalling Studies (CIBSS), Freiburg 79104, Germany.
| | - Adolfo García-Sastre
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
| | - Marco Vignuzzi
- Viral Populations and Pathogenesis Unit, CNRS UMR 3569, Institut Pasteur, 75724 Paris, Cedex 15, France.
| | - Jeffery R Johnson
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
| | - Kevan M Shokat
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute.
| | - Danielle L Swaney
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA.
| | - Pedro Beltrao
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK.
| | - Nevan J Krogan
- QBI COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
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27
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Jackman M, Marcozzi C, Barbiero M, Pardo M, Yu L, Tyson AL, Choudhary JS, Pines J. Cyclin B1-Cdk1 facilitates MAD1 release from the nuclear pore to ensure a robust spindle checkpoint. J Cell Biol 2020; 219:e201907082. [PMID: 32236513 PMCID: PMC7265330 DOI: 10.1083/jcb.201907082] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2019] [Revised: 02/05/2020] [Accepted: 03/06/2020] [Indexed: 11/22/2022] Open
Abstract
How the cell rapidly and completely reorganizes its architecture when it divides is a problem that has fascinated researchers for almost 150 yr. We now know that the core regulatory machinery is highly conserved in eukaryotes, but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated in space and time to remodel the cell in a matter of minutes remains a major question. Cyclin B1-Cdk is the primary kinase that drives mitotic remodeling; here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the kinetochore checkpoint protein, MAD1, where it coordinates NPC disassembly with kinetochore assembly. Localized cyclin B1-Cdk1 is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore so that it can be recruited to kinetochores before nuclear envelope breakdown to maintain genomic stability.
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28
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Martínez-Alonso D, Malumbres M. Mammalian cell cycle cyclins. Semin Cell Dev Biol 2020; 107:28-35. [PMID: 32334991 DOI: 10.1016/j.semcdb.2020.03.009] [Citation(s) in RCA: 97] [Impact Index Per Article: 19.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2020] [Revised: 03/27/2020] [Accepted: 03/31/2020] [Indexed: 12/23/2022]
Abstract
Proper progression throughout the cell division cycle depends on the expression level of a family of proteins known as cyclins, and the subsequent activation of cyclin-dependent kinases (Cdks). Among the numerous members of the mammalian cyclin family, only a few of them, cyclins A, B, C, D and E, are known to display critical roles in the cell cycle. These functions will be reviewed here with a special focus on their relevance in different cell types in vivo and their implications in human disease.
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Affiliation(s)
- Diego Martínez-Alonso
- Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO) Madrid, Spain.
| | - Marcos Malumbres
- Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO) Madrid, Spain.
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29
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Bansal S, Tiwari S. Mechanisms for the temporal regulation of substrate ubiquitination by the anaphase-promoting complex/cyclosome. Cell Div 2019; 14:14. [PMID: 31889987 PMCID: PMC6927175 DOI: 10.1186/s13008-019-0057-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2019] [Accepted: 12/04/2019] [Indexed: 12/16/2022] Open
Abstract
The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit, multifunctional ubiquitin ligase that controls the temporal degradation of numerous cell cycle regulatory proteins to direct the unidirectional cell cycle phases. Several different mechanisms contribute to ensure the correct order of substrate modification by the APC/C complex. Recent advances in biochemical, biophysical and structural studies of APC/C have provided a deep mechanistic insight into the working of this complex ubiquitin ligase. This complex displays remarkable conformational flexibility in response to various binding partners and post-translational modifications, which together regulate substrate selection and catalysis of APC/C. Apart from this, various features and modifications of the substrates also influence their recognition and affinity to APC/C complex. Ultimately, temporal degradation of substrates depends on the kind of ubiquitin modification received, the processivity of APC/C, and other extrinsic mechanisms. This review discusses our current understanding of various intrinsic and extrinsic mechanisms responsible for 'substrate ordering' by the APC/C complex.
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Affiliation(s)
- Shivangee Bansal
- School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067 India
| | - Swati Tiwari
- School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067 India
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30
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Clark DJ, Dhanasekaran SM, Petralia F, Pan J, Song X, Hu Y, da Veiga Leprevost F, Reva B, Lih TSM, Chang HY, Ma W, Huang C, Ricketts CJ, Chen L, Krek A, Li Y, Rykunov D, Li QK, Chen LS, Ozbek U, Vasaikar S, Wu Y, Yoo S, Chowdhury S, Wyczalkowski MA, Ji J, Schnaubelt M, Kong A, Sethuraman S, Avtonomov DM, Ao M, Colaprico A, Cao S, Cho KC, Kalayci S, Ma S, Liu W, Ruggles K, Calinawan A, Gümüş ZH, Geiszler D, Kawaler E, Teo GC, Wen B, Zhang Y, Keegan S, Li K, Chen F, Edwards N, Pierorazio PM, Chen XS, Pavlovich CP, Hakimi AA, Brominski G, Hsieh JJ, Antczak A, Omelchenko T, Lubinski J, Wiznerowicz M, Linehan WM, Kinsinger CR, Thiagarajan M, Boja ES, Mesri M, Hiltke T, Robles AI, Rodriguez H, Qian J, Fenyö D, Zhang B, Ding L, Schadt E, Chinnaiyan AM, Zhang Z, Omenn GS, Cieslik M, Chan DW, Nesvizhskii AI, Wang P, Zhang H. Integrated Proteogenomic Characterization of Clear Cell Renal Cell Carcinoma. Cell 2019; 179:964-983.e31. [PMID: 31675502 PMCID: PMC7331093 DOI: 10.1016/j.cell.2019.10.007] [Citation(s) in RCA: 432] [Impact Index Per Article: 72.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2019] [Revised: 07/15/2019] [Accepted: 10/07/2019] [Indexed: 02/07/2023]
Abstract
To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology.
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Affiliation(s)
- David J Clark
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | | | - Francesca Petralia
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Jianbo Pan
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Xiaoyu Song
- Department of Population Health Science and Policy, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Yingwei Hu
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | | | - Boris Reva
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Tung-Shing M Lih
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Hui-Yin Chang
- Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Weiping Ma
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Chen Huang
- Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030, USA
| | - Christopher J Ricketts
- Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Lijun Chen
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Azra Krek
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Yize Li
- Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Dmitry Rykunov
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Qing Kay Li
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Lin S Chen
- Department of Public Health Sciences, University of Chicago, Chicago, IL 60637, USA
| | - Umut Ozbek
- Department of Population Health Science and Policy, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Suhas Vasaikar
- Department of Translational Molecular Pathology, MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Yige Wu
- Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Seungyeul Yoo
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Shrabanti Chowdhury
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | | | - Jiayi Ji
- Department of Population Health Science and Policy, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Michael Schnaubelt
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Andy Kong
- Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
| | | | - Dmitry M Avtonomov
- Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Minghui Ao
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Antonio Colaprico
- Department of Public Health Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Song Cao
- Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Kyung-Cho Cho
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Selim Kalayci
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Shiyong Ma
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Wenke Liu
- Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA
| | - Kelly Ruggles
- Department of Medicine, New York University School of Medicine, New York, NY 10016, USA
| | - Anna Calinawan
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Zeynep H Gümüş
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Daniel Geiszler
- Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI 48109, USA
| | - Emily Kawaler
- Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA
| | - Guo Ci Teo
- Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Bo Wen
- Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030, USA
| | - Yuping Zhang
- Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Sarah Keegan
- Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA
| | - Kai Li
- Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030, USA
| | - Feng Chen
- Departments of Medicine and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Nathan Edwards
- Department of Biochemistry and Cellular Biology, Georgetown University, Washington, DC 20007, USA
| | - Phillip M Pierorazio
- Brady Urological Institute and Department of Urology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Xi Steven Chen
- Department of Public Health Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA; Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Christian P Pavlovich
- Brady Urological Institute and Department of Urology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - A Ari Hakimi
- Department of Surgery, Urology Service, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Gabriel Brominski
- Department of Urology, Poznań University of Medical Sciences, Szwajcarska 3, Poznań 61-285, Poland
| | - James J Hsieh
- Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Andrzej Antczak
- Department of Urology, Poznań University of Medical Sciences, Szwajcarska 3, Poznań 61-285, Poland
| | - Tatiana Omelchenko
- Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Jan Lubinski
- Department of Genetics and Pathology, Pomeranian Medical University, Szczecin 71-252, Poland
| | - Maciej Wiznerowicz
- International Institute for Molecular Oncology, Poznań 60-203, Poland; Poznań University of Medical Sciences, Poznan 60-701, Poland
| | - W Marston Linehan
- Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Christopher R Kinsinger
- Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USA
| | | | - Emily S Boja
- Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USA
| | - Mehdi Mesri
- Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USA
| | - Tara Hiltke
- Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USA
| | - Ana I Robles
- Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USA
| | - Henry Rodriguez
- Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892, USA
| | - Jiang Qian
- Department of Ophthalmology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - David Fenyö
- Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA
| | - Bing Zhang
- Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
| | - Li Ding
- Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Eric Schadt
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Sema4, Stamford, CT 06902, USA
| | - Arul M Chinnaiyan
- Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
| | - Zhen Zhang
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA
| | - Gilbert S Omenn
- Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI 48109, USA; Department of Internal Medicine, Human Genetics, and School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA
| | - Marcin Cieslik
- Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA.
| | - Daniel W Chan
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
| | | | - Pei Wang
- Department of Genetics and Genomic Sciences and Icahn Institute for Data Science and Genomic Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
| | - Hui Zhang
- Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
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31
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Lara-Gonzalez P, Moyle MW, Budrewicz J, Mendoza-Lopez J, Oegema K, Desai A. The G2-to-M Transition Is Ensured by a Dual Mechanism that Protects Cyclin B from Degradation by Cdc20-Activated APC/C. Dev Cell 2019; 51:313-325.e10. [PMID: 31588029 DOI: 10.1016/j.devcel.2019.09.005] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2019] [Revised: 06/26/2019] [Accepted: 09/05/2019] [Indexed: 12/23/2022]
Abstract
In the eukaryotic cell cycle, a threshold level of cyclin B accumulation triggers the G2-to-M transition, and subsequent cyclin B destruction triggers mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is the E3 ubiquitin ligase that, together with its co-activator Cdc20, targets cyclin B for destruction during mitotic exit. Here, we show that two pathways act in concert to protect cyclin B from Cdc20-activated APC/C in G2, in order to enable cyclin B accumulation and the G2-to-M transition. The first pathway involves the Mad1-Mad2 spindle checkpoint complex, acting in a distinct manner from checkpoint signaling after mitotic entry but employing a common molecular mechanism-the promotion of Mad2-Cdc20 complex formation. The second pathway involves cyclin-dependent kinase phosphorylation of Cdc20, which is known to reduce Cdc20's affinity for the APC/C. Cooperation of these two mechanisms, which target distinct APC/C binding interfaces of Cdc20, enables cyclin B accumulation and the G2-to-M transition.
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Affiliation(s)
- Pablo Lara-Gonzalez
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
| | - Mark W Moyle
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Jacqueline Budrewicz
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Jose Mendoza-Lopez
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Karen Oegema
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Arshad Desai
- Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, CA, USA; Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
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32
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Chen ZH, Jing YJ, Yu JB, Jin ZS, Li Z, He TT, Su XZ. ESRP1 Induces Cervical Cancer Cell G1-Phase Arrest Via Regulating Cyclin A2 mRNA Stability. Int J Mol Sci 2019; 20:ijms20153705. [PMID: 31362365 PMCID: PMC6695732 DOI: 10.3390/ijms20153705] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2019] [Revised: 07/18/2019] [Accepted: 07/26/2019] [Indexed: 12/18/2022] Open
Abstract
Accumulating evidence indicates that epithelial splicing regulatory protein 1 (ESRP1) can inhibit the epithelial-to-mesenchymal transition (EMT), thus playing a central role in regulating the metastatic progression of tumors. However, it is still not clear whether ESRP1 directly influences the cell cycle, or what the possible underlying molecular mechanisms are. In this study, we showed that ESRP1 protein levels were significantly correlated with the Ki-67 proliferative index (r = −0.521; p < 0.01), and that ESRP1 overexpression can significantly inhibit cervical carcinoma cell proliferation and induced G1-phase arrest by downregulating cyclin A2 expression. Importantly, ESRP1 can bind to GGUGGU sequence in the 3′UTR of the cyclin A2 mRNA, and ESRP1 overexpression significantly decreases the stability of the cyclin A2 mRNA. In addition, our experimental results confirm that ESRP1 overexpression results in enhanced CDC20 expression, which is known to be responsible for cyclin A2 degradation. This study provides the first evidence that ESRP1 overexpression induces G1-phase cell cycle arrest via reducing the stability of the cyclin A2 mRNA, and inhibits cervical carcinoma cell proliferation. The findings suggest that the ESRP1/cyclin A2 regulatory axis may be essential as a regulator of cell proliferation, and may thus represent an attractive target for cervical cancer prevention and treatment.
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Affiliation(s)
- Zhi-Hong Chen
- School of Basic Medicine, Youjiang Medical University for Nationalities, No. 98 Chengxiang Road, Baise 533000, China.
- Heilongjiang Province Key Laboratory of Cancer Prevention and Treatment, Mudanjiang Medical University, No. 3, Tongxiang Street, Mudanjiang 157011, China.
| | - Ya-Jie Jing
- Sciences Research Center, Youjiang Medical University for Nationalities, No. 98 Chengxiang Road, Baise 533000, China
| | - Jian-Bo Yu
- Heilongjiang Province Key Laboratory of Cancer Prevention and Treatment, Mudanjiang Medical University, No. 3, Tongxiang Street, Mudanjiang 157011, China
| | - Zai-Shu Jin
- Heilongjiang Province Key Laboratory of Cancer Prevention and Treatment, Mudanjiang Medical University, No. 3, Tongxiang Street, Mudanjiang 157011, China
| | - Zhu Li
- Heilongjiang Province Key Laboratory of Cancer Prevention and Treatment, Mudanjiang Medical University, No. 3, Tongxiang Street, Mudanjiang 157011, China
| | - Ting-Ting He
- Sciences Research Center, Youjiang Medical University for Nationalities, No. 98 Chengxiang Road, Baise 533000, China
| | - Xiu-Zhen Su
- Sciences Research Center, Youjiang Medical University for Nationalities, No. 98 Chengxiang Road, Baise 533000, China
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33
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The RIF1-PP1 Axis Controls Abscission Timing in Human Cells. Curr Biol 2019; 29:1232-1242.e5. [DOI: 10.1016/j.cub.2019.02.037] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Revised: 12/12/2018] [Accepted: 02/14/2019] [Indexed: 01/10/2023]
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34
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Pintard L, Bowerman B. Mitotic Cell Division in Caenorhabditis elegans. Genetics 2019; 211:35-73. [PMID: 30626640 PMCID: PMC6325691 DOI: 10.1534/genetics.118.301367] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Accepted: 10/24/2018] [Indexed: 11/18/2022] Open
Abstract
Mitotic cell divisions increase cell number while faithfully distributing the replicated genome at each division. The Caenorhabditis elegans embryo is a powerful model for eukaryotic cell division. Nearly all of the genes that regulate cell division in C. elegans are conserved across metazoan species, including humans. The C. elegans pathways tend to be streamlined, facilitating dissection of the more redundant human pathways. Here, we summarize the virtues of C. elegans as a model system and review our current understanding of centriole duplication, the acquisition of pericentriolar material by centrioles to form centrosomes, the assembly of kinetochores and the mitotic spindle, chromosome segregation, and cytokinesis.
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Affiliation(s)
- Lionel Pintard
- Equipe labellisée Ligue contre le Cancer, Institut Jacques Monod, Team Cell Cycle and Development UMR7592, Centre National de la Recherche Scientifique - Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France
| | - Bruce Bowerman
- Institute of Molecular Biology, University of Oregon, Eugene, OR 97403
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35
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Heim A, Tischer T, Mayer TU. Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20. EMBO Rep 2018; 19:embr.201846433. [PMID: 30373936 DOI: 10.15252/embr.201846433] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2018] [Revised: 10/05/2018] [Accepted: 10/05/2018] [Indexed: 11/09/2022] Open
Abstract
Vertebrate oocytes await fertilization arrested at metaphase of the second meiotic division. Fertilization triggers a transient calcium wave, which induces the activation of the anaphase-promoting complex/cyclosome (APC/C) and its co-activator Cdc20 resulting in the destruction of cyclin B and hence meiotic exit. Two calcium-dependent enzymes are implicated in fertilization-induced APC/CC dc20 activation: calcium-/calmodulin-dependent kinase type II (CaMKII) and calcineurin (CaN). While the role of CaMKII in targeting the APC/C inhibitor XErp1/Emi2 for destruction is well-established, it remained elusive how CaN affects APC/CC dc20 activation. Here, we discover that CaN contributes to APC/CC dc20 activation in Xenopus laevis oocytes by two independent but interrelated mechanisms. First, it facilitates the degradation of XErp1 by dephosphorylating it at a site that is part of a phosphorylation-dependent recruiting motif for PP2A-B'56, which antagonizes inhibitory phosphorylation of XErp1. Second, it dephosphorylates Cdc20 at an inhibitory site, thereby supporting its APC/C-activating function. Thus, our comprehensive analysis reveals that CaN contributes to timely APC/C activation at fertilization by both negatively regulating the APC/C inhibitory activity of XErp1 and positively regulating the APC/C-activating function of Cdc20.
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Affiliation(s)
- Andreas Heim
- Department of Biology, University of Konstanz, Konstanz, Germany.,Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany
| | | | - Thomas U Mayer
- Department of Biology, University of Konstanz, Konstanz, Germany .,Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany
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36
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Shugoshin 1 is dislocated by KSHV-encoded LANA inducing aneuploidy. PLoS Pathog 2018; 14:e1007253. [PMID: 30212568 PMCID: PMC6136811 DOI: 10.1371/journal.ppat.1007253] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2018] [Accepted: 07/31/2018] [Indexed: 11/30/2022] Open
Abstract
Shugoshin-1 (Sgo1) protects the integrity of the centromeres, and H2A phosphorylation is critical for this process. The mitotic checkpoint kinase Bub1, phosphorylates H2A and ensures fidelity of chromosome segregation and chromosome number. Oncogenic KSHV induces genetic alterations through chromosomal instability (CIN), and its essential antigen LANA regulates Bub1. We show that LANA inhibits Bub1 phosphorylation of H2A and Cdc20, important for chromosome segregation and mitotic signaling. Inhibition of H2A phosphorylation at residue T120 by LANA resulted in dislocation of Sgo1, and cohesin from the centromeres. Arrest of Cdc20 phosphorylation also rescued degradation of Securin and Cyclin B1 at mitotic exit, and interaction of H2A, and Cdc20 with Bub1 was inhibited by LANA. The N-terminal nuclear localization sequence domain of LANA was essential for LANA and Bub1 interaction, reversed LANA inhibited phosphorylation of H2A and Cdc20, and attenuated LANA-induced aneuploidy and cell proliferation. This molecular mechanism whereby KSHV-induced CIN, demonstrated that the NNLS of LANA is a promising target for development of anti-viral therapies targeting KSHV associated cancers. KSHV is a known oncogenic herpes virus associated with human malignancies and lymphoproliferative disorders, which includes Kaposi’s sarcoma, Primary effusion lymphoma, and Multicentric Castleman’s disease. KSHV disrupts the G1 and G2/M checkpoints through multiple pathways. Whether KSHV can directly interfere with spindle checkpoints is not known. Impairment of the mitotic checkpoint protein Bub1 leads to CIN and oncogenesis through displacement of Shugoshin-1. KSHV associated diseases have genetic alterations which are driven by chromosomal instability (CIN), as seen in numerous viral-associated cancer cells. Here we examined the molecular mechanism behind KSHV-induced CIN. We showed that the latent antigen LANA, encoded by KSHV, inhibits Bub1 phosphorylation of H2A and Cdc20, and this led to the dislocation of Shugoshin-1. Our studies demonstrated the direct induction of aneuploidy by LANA. The NNLS domain of LANA serves as an anchor for LANA to promote its multiple functions. We also showed that the NNLS polypeptide can antagonize LANA’s inhibition on Bub1 kinase function, and so rescue the aneuploidy induced by LANA. Development of this property of NNLS is potentially useful for targeted elimination of KSHV-associated cancers.
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Saurin AT. Kinase and Phosphatase Cross-Talk at the Kinetochore. Front Cell Dev Biol 2018; 6:62. [PMID: 29971233 PMCID: PMC6018199 DOI: 10.3389/fcell.2018.00062] [Citation(s) in RCA: 91] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2018] [Accepted: 05/31/2018] [Indexed: 01/26/2023] Open
Abstract
Multiple kinases and phosphatases act on the kinetochore to control chromosome segregation: Aurora B, Mps1, Bub1, Plk1, Cdk1, PP1, and PP2A-B56, have all been shown to regulate both kinetochore-microtubule attachments and the spindle assembly checkpoint. Given that so many kinases and phosphatases converge onto two key mitotic processes, it is perhaps not surprising to learn that they are, quite literally, entangled in cross-talk. Inhibition of any one of these enzymes produces secondary effects on all the others, which results in a complicated picture that is very difficult to interpret. This review aims to clarify this picture by first collating the direct effects of each enzyme into one overarching schematic of regulation at the Knl1/Mis12/Ndc80 (KMN) network (a major signaling hub at the outer kinetochore). This schematic will then be used to discuss the implications of the cross-talk that connects these enzymes; both in terms of why it may be needed to produce the right type of kinetochore signals and why it nevertheless complicates our interpretations about which enzymes control what processes. Finally, some general experimental approaches will be discussed that could help to characterize kinetochore signaling by dissociating the direct from indirect effect of kinase or phosphatase inhibition in vivo. Together, this review should provide a framework to help understand how a network of kinases and phosphatases cooperate to regulate two key mitotic processes.
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Affiliation(s)
- Adrian T. Saurin
- Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom
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38
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Taming the Beast: Control of APC/C Cdc20-Dependent Destruction. COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 2017; 82:111-121. [PMID: 29133301 DOI: 10.1101/sqb.2017.82.033712] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit ubiquitin ligase that triggers the metaphase-to-anaphase transition in the cell cycle by targeting the substrates cyclin B and securin for destruction. APC/C activity toward these two key substrates requires the coactivator Cdc20. To ensure that cells enter mitosis and partition their duplicated genome with high accuracy, APC/CCdc20 activity must be tightly controlled. Here, we discuss the mechanisms that regulate APC/CCdc20 activity both before and during mitosis. We focus our discussion primarily on the chromosomal pathways that both accelerate and delay APC/C activation by targeting Cdc20 to opposing fates. The findings discussed provide an overview of how cells control the activation of this major cell cycle regulator to ensure both accurate and timely cell division.
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39
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Affiliation(s)
- Michelle Levine
- Johns Hopkins University, Department of Molecular Biology and Genetics, Baltimore, MD 21205, USA
| | - Andrew Holland
- Johns Hopkins University, Department of Molecular Biology and Genetics, Baltimore, MD 21205, USA.
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40
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Alfieri C, Zhang S, Barford D. Visualizing the complex functions and mechanisms of the anaphase promoting complex/cyclosome (APC/C). Open Biol 2017; 7:170204. [PMID: 29167309 PMCID: PMC5717348 DOI: 10.1098/rsob.170204] [Citation(s) in RCA: 113] [Impact Index Per Article: 14.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2017] [Accepted: 10/10/2017] [Indexed: 12/17/2022] Open
Abstract
The anaphase promoting complex or cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that orchestrates cell cycle progression by mediating the degradation of important cell cycle regulators. During the two decades since its discovery, much has been learnt concerning its role in recognizing and ubiquitinating specific proteins in a cell-cycle-dependent manner, the mechanisms governing substrate specificity, the catalytic process of assembling polyubiquitin chains on its target proteins, and its regulation by phosphorylation and the spindle assembly checkpoint. The past few years have witnessed significant progress in understanding the quantitative mechanisms underlying these varied APC/C functions. This review integrates the overall functions and properties of the APC/C with mechanistic insights gained from recent cryo-electron microscopy (cryo-EM) studies of reconstituted human APC/C complexes.
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Affiliation(s)
- Claudio Alfieri
- MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
| | - Suyang Zhang
- MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
| | - David Barford
- MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
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41
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Distinct kinetics of serine and threonine dephosphorylation are essential for mitosis. Nat Cell Biol 2017; 19:1433-1440. [DOI: 10.1038/ncb3634] [Citation(s) in RCA: 73] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2017] [Accepted: 09/19/2017] [Indexed: 12/13/2022]
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42
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Friedlander P, Wassmann K, Christenfeld AM, Fisher D, Kyi C, Kirkwood JM, Bhardwaj N, Oh WK. Whole-blood RNA transcript-based models can predict clinical response in two large independent clinical studies of patients with advanced melanoma treated with the checkpoint inhibitor, tremelimumab. J Immunother Cancer 2017; 5:67. [PMID: 28807052 PMCID: PMC5557000 DOI: 10.1186/s40425-017-0272-z] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2017] [Accepted: 08/01/2017] [Indexed: 12/15/2022] Open
Abstract
Background Tremelimumab is an antibody that blocks CTLA-4 and demonstrates clinical efficacy in a subset of advanced melanoma patients. An unmet clinical need exists for blood-based response-predictive gene signatures to facilitate clinically effective and cost-efficient use of such immunotherapeutic interventions. Methods Peripheral blood samples were collected in PAXgene® tubes from 210 treatment-naïve melanoma patients receiving tremelimumab in a worldwide, multicenter phase III study (discovery dataset). A central panel of radiologists determined objective response using RECIST criteria. Gene expression for 169 mRNA transcripts was measured using quantitative PCR. A 15-gene pre-treatment response-predictive classifier model was identified. An independent population (N = 150) of refractory melanoma patients receiving tremelimumab after chemotherapy enrolled in a worldwide phase II study (validation dataset). The classifier model, using the same genes, coefficients and constants for objective response and one-year survival after treatment, was applied to the validation dataset. Results A 15-gene pre-treatment classifier model (containing ADAM17, CDK2, CDKN2A, DPP4, ERBB2, HLA-DRA, ICOS, ITGA4, LARGE, MYC, NAB2, NRAS, RHOC, TGFB1, and TIMP1) achieved an area under the curve (AUC) of 0.86 (95% confidence interval 0.81 to 0.91, p < 0.0001) for objective response and 0.6 (95% confidence interval 0.54 to 0.67, p = 0.0066) for one-year survival in the discovery set. This model was validated in the validation set with AUCs of 0.62 (95% confidence interval 0.54 to 0.70 p = 0.0455) for objective response and 0.68 for one-year survival (95% confidence interval 0.59 to 0.75 p = 0.0002). Conclusions To our knowledge, this is the largest blood-based biomarker study of a checkpoint inhibitor, tremelimumab, which demonstrates a validated pre-treatment mRNA classifier model that predicts clinical response. The data suggest that the model captures a biological signature representative of genes needed for a robust anti-cancer immune response. It also identifies non-responders to tremelimumab at baseline prior to treatment. Electronic supplementary material The online version of this article (doi:10.1186/s40425-017-0272-z) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Philip Friedlander
- Division of Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, Mount Sinai Hospital, New York, NY, USA.
| | | | | | - David Fisher
- Department of Dermatology, Harvard Medical School, Boston, MA, USA
| | - Chrisann Kyi
- Division of Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, Mount Sinai Hospital, New York, NY, USA
| | - John M Kirkwood
- Departments of Medicine, Dermatology and Translational Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Nina Bhardwaj
- Division of Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, Mount Sinai Hospital, New York, NY, USA.,Parker Institute of Cancer Immunotherapy, San Francisco, CA, USA
| | - William K Oh
- Division of Hematology and Medical Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, Mount Sinai Hospital, New York, NY, USA
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43
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Kim T, Lara-Gonzalez P, Prevo B, Meitinger F, Cheerambathur DK, Oegema K, Desai A. Kinetochores accelerate or delay APC/C activation by directing Cdc20 to opposing fates. Genes Dev 2017; 31:1089-1094. [PMID: 28698300 PMCID: PMC5538432 DOI: 10.1101/gad.302067.117] [Citation(s) in RCA: 49] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2017] [Accepted: 06/12/2017] [Indexed: 11/30/2022]
Abstract
In this study, Kim et al. show that flux of Cdc20 through kinetochores accelerates mitotic exit by promoting its dephosphorylation by kinetochore-localized protein phosphatase 1, which allows Cdc20 to activate the APC/C. The microtubule attachment status of kinetochores therefore optimizes mitotic duration by controlling the balance between opposing Cdc20 fates. Mitotic duration is determined by activation of the anaphase-promoting complex/cyclosome (APC/C) bound to its coactivator, Cdc20. Kinetochores, the microtubule-interacting machines on chromosomes, restrain mitotic exit when not attached to spindle microtubules by generating a Cdc20-containing complex that inhibits the APC/C. Here, we show that flux of Cdc20 through kinetochores also accelerates mitotic exit by promoting its dephosphorylation by kinetochore-localized protein phosphatase 1, which allows Cdc20 to activate the APC/C. Both APC/C activation and inhibition depend on Cdc20 fluxing through the same binding site at kinetochores. The microtubule attachment status of kinetochores therefore optimizes mitotic duration by controlling the balance between opposing Cdc20 fates.
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Affiliation(s)
- Taekyung Kim
- Ludwig Institute for Cancer Research, San Diego, California 92093, USA.,Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA
| | - Pablo Lara-Gonzalez
- Ludwig Institute for Cancer Research, San Diego, California 92093, USA.,Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA
| | - Bram Prevo
- Ludwig Institute for Cancer Research, San Diego, California 92093, USA.,Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA
| | - Franz Meitinger
- Ludwig Institute for Cancer Research, San Diego, California 92093, USA.,Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA
| | - Dhanya K Cheerambathur
- Ludwig Institute for Cancer Research, San Diego, California 92093, USA.,Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA
| | - Karen Oegema
- Ludwig Institute for Cancer Research, San Diego, California 92093, USA.,Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA
| | - Arshad Desai
- Ludwig Institute for Cancer Research, San Diego, California 92093, USA.,Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA
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44
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Lee SJ, Rodriguez-Bravo V, Kim H, Datta S, Foley EA. The PP2A B56 phosphatase promotes the association of Cdc20 with APC/C in mitosis. J Cell Sci 2017; 130:1760-1771. [PMID: 28404789 DOI: 10.1242/jcs.201608] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2017] [Accepted: 03/28/2017] [Indexed: 12/20/2022] Open
Abstract
PP2A comprising B56 regulatory subunit isoforms (PP2AB56) is a serine/threonine phosphatase essential for mitosis. At the kinetochore, PP2AB56 both stabilizes microtubule binding and promotes silencing of the spindle assembly checkpoint (SAC) through its association with the SAC protein BubR1. Cells depleted of the B56 regulatory subunits of PP2A are delayed in activation of Cdc20-containing APC/C (APC/CCdc20), which is an essential step for mitotic exit. It has been hypothesized that this delay arises from increased production of the mitotic checkpoint complex (MCC), an APC/CCdc20 inhibitor formed at unattached kinetochores through SAC signaling. In contrast to this prediction, we show that depletion of B56 subunits does not increase the amount or stability of the MCC. Rather, delays in APC/CCdc20 activation in B56-depleted cells correlate with impaired Cdc20 binding to APC/C. Stimulation of APC/CCdc20 assembly does not require binding between PP2AB56 and BubR1, and thus this contribution of PP2AB56 towards mitotic exit is distinct from its functions at kinetochores. PP2AB56 associates with APC/C constitutively in a BubR1-independent manner. A mitotic phosphorylation site on Cdc20, known to be a substrate of PP2AB56, modulates APC/CCdc20 assembly. These results elucidate the contributions of PP2AB56 towards completion of mitosis.
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Affiliation(s)
- Sun Joo Lee
- Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | | | - Hyunjung Kim
- Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Sutirtha Datta
- Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Emily A Foley
- Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
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Abstract
Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.
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