|
1
|
Tan T, Wu C, Wang R, Pan BF, Hawke D, Yin F, Su Z, Liu B, Lin SH, Zhang W, Kuang J. Revisiting phosphoregulation of Cdc25C during M-phase induction. iScience 2025; 28:111603. [PMID: 39834856 PMCID: PMC11743101 DOI: 10.1016/j.isci.2024.111603] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2023] [Revised: 07/23/2024] [Accepted: 11/11/2024] [Indexed: 01/22/2025] Open
Abstract
Cdc25C undergoes a sudden and substantial gel mobility shift at M-phase onset, correlating with abrupt activation of both Cdc25C and Cdk1 activities. A positive feedback loop between Cdk1 and Cdc25C has been used to explain this hallmark phenomenon. Here, we demonstrate that the M-phase supershift and robust activation of Cdc25C are due to the site-comprehensive phosphorylation of its long intrinsically disordered regulatory domain without requiring Cdk1 or other major mitotic kinase activities. The phosphorylation process involves substrate-mediated assembly of phosphorylation machinery that catalyzes multisite phosphorylation continuously without substrate dissociation. In contrast to the site-comprehensive phosphorylation of Cdc25C occurring at M-phase onset, the site-specific phosphorylation of Cdc25C by Cdk1 or other major mitotic kinases generates slight gel mobility shifts and modest activation of Cdc25C prior to M-phase onset. These findings suggest a two-stage framework consisting of site-specific phosphorylation followed by site-comprehensive phosphorylation for Cdc25C regulation during M-phase induction.
Collapse
Affiliation(s)
- Tan Tan
- Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
- Department of Biochemistry and Molecular Biology, University of South China, Hengyang, Hunan 421001, China
| | - Chuanfen Wu
- Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Ruoning Wang
- Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Bih-Fang Pan
- Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - David Hawke
- Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Fumin Yin
- Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Zehao Su
- Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Boye Liu
- Key Laboratory for Biodiversity and Ecological Engineering of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Sue-Hwa Lin
- Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Wei Zhang
- Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing 100875, China
| | - Jian Kuang
- Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| |
Collapse
|
|
2
|
Santoni M, Meneau F, Sekhsoukh N, Castella S, Le T, Miot M, Daldello EM. Unraveling the interplay between PKA inhibition and Cdk1 activation during oocyte meiotic maturation. Cell Rep 2024; 43:113782. [PMID: 38358892 DOI: 10.1016/j.celrep.2024.113782] [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: 09/03/2023] [Revised: 12/19/2023] [Accepted: 01/25/2024] [Indexed: 02/17/2024] Open
Abstract
Oocytes are arrested in prophase I. In vertebrates, meiotic resumption is triggered by hormonal stimulation that results in cAMP-dependent protein kinase (PKA) downregulation leading to Cdk1 activation. Yet the pathways connecting PKA to Cdk1 remain unclear. Here, we identify molecular events triggered by PKA downregulation occurring upstream of Cdk1 activation. We describe a two-step regulation controlling cyclin B1 and Mos accumulation, which depends on both translation and stabilization. Cyclin B1 accumulation is triggered by PKA inhibition upstream of Cdk1 activation, while its translation requires Cdk1 activity. Conversely, Mos translation initiates in response to the hormone, but the protein accumulates only downstream of Cdk1. Furthermore, two successive translation waves take place, the first controlled by PKA inhibition and the second by Cdk1 activation. Notably, Arpp19, an essential PKA effector, does not regulate the early PKA-dependent events. This study elucidates how PKA downregulation orchestrates multiple pathways that converge toward Cdk1 activation and induce the oocyte G2/M transition.
Collapse
Affiliation(s)
- Martina Santoni
- Sorbonne Université-CNRS, Laboratoire de Biologie du Développement Institut de Biologie Paris Seine, LBD-IBPS, 75005 Paris, France
| | - Ferdinand Meneau
- Sorbonne Université-CNRS, Laboratoire de Biologie du Développement Institut de Biologie Paris Seine, LBD-IBPS, 75005 Paris, France
| | - Nabil Sekhsoukh
- Sorbonne Université-CNRS, Laboratoire de Biologie du Développement Institut de Biologie Paris Seine, LBD-IBPS, 75005 Paris, France
| | - Sandrine Castella
- Sorbonne Université-CNRS, Laboratoire de Biologie du Développement Institut de Biologie Paris Seine, LBD-IBPS, 75005 Paris, France
| | - Tran Le
- Sorbonne Université-CNRS, Laboratoire de Biologie du Développement Institut de Biologie Paris Seine, LBD-IBPS, 75005 Paris, France
| | - Marika Miot
- Sorbonne Université-CNRS, Laboratoire de Biologie du Développement Institut de Biologie Paris Seine, LBD-IBPS, 75005 Paris, France
| | - Enrico Maria Daldello
- Sorbonne Université-CNRS, Laboratoire de Biologie du Développement Institut de Biologie Paris Seine, LBD-IBPS, 75005 Paris, France.
| |
Collapse
|
|
3
|
Liu J, Zhang C. Xenopus cell-free extracts and their applications in cell biology study. BIOPHYSICS REPORTS 2023; 9:195-205. [PMID: 38516620 PMCID: PMC10951473 DOI: 10.52601/bpr.2023.230016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2023] [Accepted: 12/05/2023] [Indexed: 03/23/2024] Open
Abstract
Xenopus has proven to be a remarkably versatile model organism in the realm of biological research for numerous years, owing to its straightforward maintenance in laboratory settings and its abundant provision of ample-sized oocytes, eggs, and embryos. The cell cycle of these oocytes, eggs, and early embryos exhibits synchrony, and extracts derived from these cells serve various research purposes. Many fundamental concepts in biochemistry, cell biology, and development have been elucidated through the use of cell-free extracts derived from Xenopus cells. Over the past few decades, a wide array of cell-free extracts has been prepared from oocytes, eggs, and early embryos of different Xenopus species at varying cell cycle stages. Each of these extracts possesses distinct characteristics. This review provides a concise overview of the Xenopus species employed in laboratory research, the diverse types of cell-free extracts available, and their respective properties. Furthermore, this review delves into the extensive investigation of spindle assembly in Xenopus egg extracts, underscoring the versatility and potency of these cell-free systems in the realm of cell biology.
Collapse
Affiliation(s)
- Junjun Liu
- Department of Biological Sciences, California State Polytechnic University, Pomona, CA 91768, USA
| | - Chuanmao Zhang
- The Academy for Cell and Life Health, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
- The Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, China
| |
Collapse
|
|
4
|
El Dika M, Dudka D, Kloc M, Kubiak JZ. CDC6 as a Key Inhibitory Regulator of CDK1 Activation Dynamics and the Timing of Mitotic Entry and Progression. BIOLOGY 2023; 12:855. [PMID: 37372141 DOI: 10.3390/biology12060855] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/14/2023] [Revised: 06/08/2023] [Accepted: 06/09/2023] [Indexed: 06/29/2023]
Abstract
Timely mitosis is critically important for early embryo development. It is regulated by the activity of the conserved protein kinase CDK1. The dynamics of CDK1 activation must be precisely controlled to assure physiologic and timely entry into mitosis. Recently, a known S-phase regulator CDC6 emerged as a key player in mitotic CDK1 activation cascade in early embryonic divisions, operating together with Xic1 as a CDK1 inhibitor upstream of the Aurora A and PLK1, both CDK1 activators. Herein, we review the molecular mechanisms that underlie the control of mitotic timing, with special emphasis on how CDC6/Xic1 function impacts CDK1 regulatory network in the Xenopus system. We focus on the presence of two independent mechanisms inhibiting the dynamics of CDK1 activation, namely Wee1/Myt1- and CDC6/Xic1-dependent, and how they cooperate with CDK1-activating mechanisms. As a result, we propose a comprehensive model integrating CDC6/Xic1-dependent inhibition into the CDK1-activation cascade. The physiological dynamics of CDK1 activation appear to be controlled by the system of multiple inhibitors and activators, and their integrated modulation ensures concomitantly both the robustness and certain flexibility of the control of this process. Identification of multiple activators and inhibitors of CDK1 upon M-phase entry allows for a better understanding of why cells divide at a specific time and how the pathways involved in the timely regulation of cell division are all integrated to precisely tune the control of mitotic events.
Collapse
Affiliation(s)
- Mohammed El Dika
- Department of Biochemistry, Larner College of Medicine, UVM Cancer Center, University of Vermont, Burlington, VT 05405, USA
| | - Damian Dudka
- Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Malgorzata Kloc
- The Houston Methodist Research Institute, Transplant Immunology, Houston, TX 77030, USA
- Department of Surgery, The Houston Methodist Hospital, Houston, TX 77030, USA
- Department of Genetics, MD Anderson Cancer Center, The University of Texas, Houston, TX 77030, USA
| | - Jacek Z Kubiak
- Laboratory of Molecular Oncology and Innovative Therapies, Military Institute of Medicine-National Research Institute (WIM-PIB), Szaserow 128, 04-141 Warsaw, Poland
- Dynamics and Mechanics of Epithelia Group, Faculty of Medicine, Institute of Genetics and Development of Rennes, University of Rennes, CNRS, UMR 6290, 35043 Rennes, France
| |
Collapse
|
|
5
|
Kalous J, Aleshkina D. Multiple Roles of PLK1 in Mitosis and Meiosis. Cells 2023; 12:cells12010187. [PMID: 36611980 PMCID: PMC9818836 DOI: 10.3390/cells12010187] [Citation(s) in RCA: 27] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2022] [Revised: 12/22/2022] [Accepted: 12/23/2022] [Indexed: 01/05/2023] Open
Abstract
Cells are equipped with a diverse network of signaling and regulatory proteins that function as cell cycle regulators and checkpoint proteins to ensure the proper progression of cell division. A key regulator of cell division is polo-like kinase 1 (PLK1), a member of the serine/threonine kinase family that plays an important role in regulating the mitotic and meiotic cell cycle. The phosphorylation of specific substrates mediated by PLK1 controls nuclear envelope breakdown (NEBD), centrosome maturation, proper spindle assembly, chromosome segregation, and cytokinesis. In mammalian oogenesis, PLK1 is essential for resuming meiosis before ovulation and for establishing the meiotic spindle. Among other potential roles, PLK1 regulates the localized translation of spindle-enriched mRNAs by phosphorylating and thereby inhibiting the translational repressor 4E-BP1, a downstream target of the mTOR (mammalian target of rapamycin) pathway. In this review, we summarize the functions of PLK1 in mitosis, meiosis, and cytokinesis and focus on the role of PLK1 in regulating mRNA translation. However, knowledge of the role of PLK1 in the regulation of meiosis remains limited.
Collapse
|
|
6
|
Dong MZ, Ouyang YC, Gao SC, Ma XS, Hou Y, Schatten H, Wang ZB, Sun QY. PPP4C facilitates homologous recombination DNA repair by dephosphorylating PLK1 during early embryo development. Development 2022; 149:dev200351. [PMID: 35546066 DOI: 10.1242/dev.200351] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2021] [Accepted: 04/24/2022] [Indexed: 12/17/2023]
Abstract
Mammalian early embryo cells have complex DNA repair mechanisms to maintain genomic integrity, and homologous recombination (HR) plays the main role in response to double-strand DNA breaks (DSBs) in these cells. Polo-like kinase 1 (PLK1) participates in the HR process and its overexpression has been shown to occur in a variety of human cancers. Nevertheless, the regulatory mechanism of PLK1 remains poorly understood, especially during the S and G2 phase. Here, we show that protein phosphatase 4 catalytic subunit (PPP4C) deletion causes severe female subfertility due to accumulation of DNA damage in oocytes and early embryos. PPP4C dephosphorylated PLK1 at the S137 site, negatively regulating its activity in the DSB response in early embryonic cells. Depletion of PPP4C induced sustained activity of PLK1 when cells exhibited DNA lesions that inhibited CHK2 and upregulated the activation of CDK1, resulting in inefficient loading of the essential HR factor RAD51. On the other hand, when inhibiting PLK1 in the S phase, DNA end resection was restricted. These results demonstrate that PPP4C orchestrates the switch between high-PLK1 and low-PLK1 periods, which couple the checkpoint to HR.
Collapse
Affiliation(s)
- Ming-Zhe Dong
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100101, China
| | - Ying-Chun Ouyang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Shi-Cai Gao
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100101, China
| | - Xue-Shan Ma
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Yi Hou
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Heide Schatten
- Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA
| | - Zhen-Bo Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100101, China
| | - Qing-Yuan Sun
- Fertility Preservation Lab, Guangdong-Hong Kong Metabolism & Reproduction Joint Laboratory, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou 510317, China
| |
Collapse
|
|
7
|
Van Essen D, Alcaraz AJG, Miller JGP, Jones PD, Doering JA, Wiseman S. The brominated flame retardant, TBCO, impairs oocyte maturation in zebrafish (Danio rerio). AQUATIC TOXICOLOGY (AMSTERDAM, NETHERLANDS) 2021; 238:105929. [PMID: 34375885 DOI: 10.1016/j.aquatox.2021.105929] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/12/2021] [Revised: 07/15/2021] [Accepted: 07/27/2021] [Indexed: 06/13/2023]
Abstract
The brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), has been shown to decrease fecundity in Japanese medaka (Oryzias latipes) and there is indirect evidence from analysis of the transcriptome and proteome that this effect might be due to impaired oogenesis. An assay for disruption of oocyte maturation by chemical stressors has not been developed in Japanese medaka. Thus, using zebrafish (Danio rerio) as a model, objectives of the present study were to determine whether exposure to TBCO has effects on maturation of oocytes and to investigate potential mechanisms. Sexually mature female zebrafish were given a diet of 35.3 or 628.8 μg TBCO / g food for 14 days after which, stage IV oocytes were isolated to assess maturation in response to maturation inducing hormone. To explore potential molecular mechanisms, abundances of mRNAs of a suite of genes that regulate oocyte maturation were quantified by use of quantitative real-time PCR, and abundances of microRNAs were determined by use of miRNAseq. Ex vivo maturation of oocytes from fish exposed to TBCO was significantly less than maturation of oocytes from control fish. The percentage of oocytes which matured from control fish and those exposed to low and high TBCO were 89, 71, and 67%, respectively. Among the suite of genes known to regulate oocyte maturation, mRNA abundance of insulin like growth factor-3 was decreased by 1.64- and 3.44-fold in stage IV oocytes from females given the low and high concentrations of TBCO, respectively, compared to the control group. Abundances of microRNAs regulating the expression of proteins that regulate oocyte maturation, including processes related to insulin-like growth factor, were significantly different in stage IV oocytes from fish exposed to TBCO. Overall, results of this study indicated that impaired oocyte maturation might be a mechanism of reduced reproductive performance in TBCO-exposed fish. Results also suggested that effects of TBCO on oocyte maturation might be due to molecular perturbations on insulin-like growth factor signaling and expression of microRNAs.
Collapse
Affiliation(s)
- Darren Van Essen
- Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada
| | | | - Justin G P Miller
- Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada
| | - Paul D Jones
- Toxicology Centre, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada; School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, S7N 5B3, Canada
| | - Jon A Doering
- Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada; Intersectoral Centre for Endocrine Disruptor Analysis (ICEDA), Institut National de la Recherche Scientifique (INRS), Centre Eau Terre Environnement, Québec City, QC, G1K 9A9, Canada
| | - Steve Wiseman
- Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada; Intersectoral Centre for Endocrine Disruptor Analysis (ICEDA), Institut National de la Recherche Scientifique (INRS), Centre Eau Terre Environnement, Québec City, QC, G1K 9A9, Canada; Water Institute for Sustainable Environments, University of Lethbridge, Lethbridge, AB, T1K 3M4, Canada.
| |
Collapse
|
|
8
|
Abstract
For work in Xenopus, frog-specific antibodies must usually be raised, although a few antibodies against mammalian proteins cross-react. To produce an immunogen for antibody production, human embryonic kidney (HEK) expression systems can be used as described here. For most laboratories, the actual method of raising the antibody is determined by local ethical regulations controlling the adjuvant and injection protocols used. Because these steps are often outsourced, they are not included in this protocol.
Collapse
Affiliation(s)
- Maya Z Piccinni
- European Xenopus Resource Centre, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, Hampshire PO1 2DY, United Kingdom
| | - Matthew J Guille
- European Xenopus Resource Centre, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, Hampshire PO1 2DY, United Kingdom
| |
Collapse
|
|
9
|
Lemonnier T, Dupré A, Jessus C. The G2-to-M transition from a phosphatase perspective: a new vision of the meiotic division. Cell Div 2020; 15:9. [PMID: 32508972 PMCID: PMC7249327 DOI: 10.1186/s13008-020-00065-2] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2020] [Accepted: 05/12/2020] [Indexed: 12/15/2022] Open
Abstract
Cell division is orchestrated by the phosphorylation and dephosphorylation of thousands of proteins. These post-translational modifications underlie the molecular cascades converging to the activation of the universal mitotic kinase, Cdk1, and entry into cell division. They also govern the structural events that sustain the mechanics of cell division. While the role of protein kinases in mitosis has been well documented by decades of investigations, little was known regarding the control of protein phosphatases until the recent years. However, the regulation of phosphatase activities is as essential as kinases in controlling the activation of Cdk1 to enter M-phase. The regulation and the function of phosphatases result from post-translational modifications but also from the combinatorial association between conserved catalytic subunits and regulatory subunits that drive their substrate specificity, their cellular localization and their activity. It now appears that sequential dephosphorylations orchestrated by a network of phosphatase activities trigger Cdk1 activation and then order the structural events necessary for the timely execution of cell division. This review discusses a series of recent works describing the important roles played by protein phosphatases for the proper regulation of meiotic division. Many breakthroughs in the field of cell cycle research came from studies on oocyte meiotic divisions. Indeed, the meiotic division shares most of the molecular regulators with mitosis. The natural arrests of oocytes in G2 and in M-phase, the giant size of these cells, the variety of model species allowing either biochemical or imaging as well as genetics approaches explain why the process of meiosis has served as an historical model to decipher signalling pathways involved in the G2-to-M transition. The review especially highlights how the phosphatase PP2A-B55δ critically orchestrates the timing of meiosis resumption in amphibian oocytes. By opposing the kinase PKA, PP2A-B55δ controls the release of the G2 arrest through the dephosphorylation of their substrate, Arpp19. Few hours later, the inhibition of PP2A-B55δ by Arpp19 releases its opposing kinase, Cdk1, and triggers M-phase. In coordination with a variety of phosphatases and kinases, the PP2A-B55δ/Arpp19 duo therefore emerges as the key effector of the G2-to-M transition.
Collapse
Affiliation(s)
- Tom Lemonnier
- Laboratoire de Biologie du Développement-Institut de Biologie Paris Seine, LBD-IBPS, Sorbonne Université, CNRS, 75005 Paris, France
| | - Aude Dupré
- Laboratoire de Biologie du Développement-Institut de Biologie Paris Seine, LBD-IBPS, Sorbonne Université, CNRS, 75005 Paris, France
| | - Catherine Jessus
- Laboratoire de Biologie du Développement-Institut de Biologie Paris Seine, LBD-IBPS, Sorbonne Université, CNRS, 75005 Paris, France
| |
Collapse
|
|
10
|
Raab M, Strebhardt K, Rudd CE. Immune adaptor SKAP1 acts a scaffold for Polo-like kinase 1 (PLK1) for the optimal cell cycling of T-cells. Sci Rep 2019; 9:10462. [PMID: 31320682 PMCID: PMC6639320 DOI: 10.1038/s41598-019-45627-9] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2018] [Accepted: 06/06/2019] [Indexed: 02/06/2023] Open
Abstract
While the immune cell adaptor protein SKAP1 mediates LFA-1 activation induced by antigen-receptor (TCR/CD3) ligation on T-cells, it is unclear whether the adaptor interacts with other mediators of T-cell function. In this context, the serine/threonine kinase, polo-like kinase (PLK1) regulates multiple steps in the mitotic and cell cycle progression of mammalian cells. Here, we show that SKAP1 is phosphorylated by and binds to PLK1 for the optimal cycling of T-cells. PLK1 binds to the N-terminal residue serine 31 (S31) of SKAP1 and the interaction is needed for optimal PLK1 kinase activity. Further, siRNA knock-down of SKAP1 reduced the rate of T-cell division concurrent with a delay in the expression of PLK1, Cyclin A and pH3. Reconstitution of these KD cells with WT SKAP1, but not the SKAP1 S31 mutant, restored normal cell division. SKAP1-PLK1 binding is dynamically regulated during the cell cycle of T-cells. Our findings identify a novel role for SKAP1 in the regulation of PLK1 and optimal cell cycling needed for T-cell clonal expansion in response to antigenic activation.
Collapse
Affiliation(s)
- Monika Raab
- Department of Obstetrics and Gynaecology, School of Medicine, J.W. Goethe-University, Theodor-Stern-Kai 7, 60590, Frankfurt, Germany.
- Cell Signaling Section, Department of Pathology, Tennis Court Road, University of Cambridge, CB2 1Q, Cambridge, UK.
| | - Klaus Strebhardt
- Department of Obstetrics and Gynaecology, School of Medicine, J.W. Goethe-University, Theodor-Stern-Kai 7, 60590, Frankfurt, Germany
| | - Christopher E Rudd
- Cell Signaling Section, Department of Pathology, Tennis Court Road, University of Cambridge, CB2 1Q, Cambridge, UK.
- Centre de Recherch-Hopital Maisonneuve-Rosemont (CR-HMR), Montreal, Quebec, H1T 2M4, Canada.
- Département de Medicine, Université de Montréal, Montreal, Quebec, H3C 3J7, Canada.
| |
Collapse
|
|
11
|
Bulbake U, Kommineni N, Bryszewska M, Ionov M, Khan W. Cationic liposomes for co-delivery of paclitaxel and anti-Plk1 siRNA to achieve enhanced efficacy in breast cancer. J Drug Deliv Sci Technol 2018. [DOI: 10.1016/j.jddst.2018.09.017] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
|
|
12
|
Pintard L, Archambault V. A unified view of spatio-temporal control of mitotic entry: Polo kinase as the key. Open Biol 2018; 8:180114. [PMID: 30135239 PMCID: PMC6119860 DOI: 10.1098/rsob.180114] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2018] [Accepted: 07/29/2018] [Indexed: 12/18/2022] Open
Abstract
The Polo kinase is an essential regulator of cell division. Its ability to regulate multiple events at distinct subcellular locations and times during mitosis is remarkable. In the last few years, a much clearer mechanistic understanding of the functions and regulation of Polo in cell division has emerged. In this regard, the importance of coupling changes in activity with changes in localization is striking, both for Polo itself and for its upstream regulators. This review brings together several new pieces of the puzzle that are gradually revealing how Polo is regulated, in space and time, to enable its functions in the early stages of mitosis in animal cells. As a result, a unified view of how mitotic entry is spatio-temporally regulated is emerging.
Collapse
Affiliation(s)
- Lionel Pintard
- Cell Cycle and Development Team, Institut Jacques Monod, UMR7592 CNRS-Université Paris Diderot, Sorbonne Paris Cité, Ligue contre le Cancer, Paris, France
- Equipe labellisée, Ligue contre le Cancer, Paris, France
| | - Vincent Archambault
- Institut de recherche en immunologie et en cancérologie, Université de Montréal, Montréal, Québec, Canada
| |
Collapse
|
|
13
|
Molecular Mechanisms of Prophase I Meiotic Arrest Maintenance and Meiotic Resumption in Mammalian Oocytes. Reprod Sci 2018; 26:1519-1537. [DOI: 10.1177/1933719118765974] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Mechanisms of meiotic prophase I arrest maintenance (germinal vesicle [GV] stage) and meiotic resumption (germinal vesicle breakdown [GVBD] stage) in mammalian oocytes seem to be very complicated. These processes are regulated via multiple molecular cascades at transcriptional, translational, and posttranslational levels, and many of them are interrelated. There are many molecular cascades of meiosis maintaining and meiotic resumption in oocyte which are orchestrated by multiple molecules produced by pituitary gland and follicular cells. Furthermore, many of these molecular cascades are duplicated, thus ensuring the stability of the entire system. Understanding mechanisms of oocyte maturation is essential to assess the oocyte status, develop effective protocols of oocyte in vitro maturation, and design novel contraceptive drugs. Mechanisms of meiotic arrest maintenance at prophase I and meiotic resumption in mammalian oocytes are covered in the present article.
Collapse
|
|
14
|
Schaefer-Ramadan S, Hubrack S, Machaca K. Transition metal dependent regulation of the signal transduction cascade driving oocyte meiosis. J Cell Physiol 2017; 233:3164-3175. [DOI: 10.1002/jcp.26157] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2017] [Accepted: 08/14/2017] [Indexed: 12/19/2022]
Affiliation(s)
| | - Satanay Hubrack
- Department of Physiology and Biophysics; Weill Cornell Medicine-Qatar; Doha Qatar
| | - Khaled Machaca
- Department of Physiology and Biophysics; Weill Cornell Medicine-Qatar; Doha Qatar
| |
Collapse
|
|
15
|
Gheghiani L, Loew D, Lombard B, Mansfeld J, Gavet O. PLK1 Activation in Late G2 Sets Up Commitment to Mitosis. Cell Rep 2017; 19:2060-2073. [PMID: 28591578 DOI: 10.1016/j.celrep.2017.05.031] [Citation(s) in RCA: 136] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2016] [Revised: 04/08/2017] [Accepted: 05/09/2017] [Indexed: 11/15/2022] Open
Abstract
Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. While we have previously established that the timely activation of CyclinB1-Cdk1 in late G2 triggers mitotic entry, the upstream regulatory mechanisms remain unclear. Here, we report that Polo-like kinase 1 (Plk1) is required for entry into mitosis during an unperturbed cell cycle and is rapidly activated shortly before CyclinB1-Cdk1. We determine that Plk1 associates with the Cdc25C1 phosphatase and induces its phosphorylation before mitotic entry. Plk1-dependent Cdc25C1 phosphosites are sufficient to promote mitotic entry, even when Plk1 activity is inhibited. Furthermore, we find that activation of Plk1 during G2 relies on CyclinA2-Cdk activity levels. Our findings thus elucidate a critical role for Plk1 in CyclinB1-Cdk1 activation and mitotic entry and outline how CyclinA2-Cdk, an S-promoting factor, poises cells for commitment to mitosis.
Collapse
Affiliation(s)
- Lilia Gheghiani
- Sorbonne Universités, UPMC University Paris 06, UFR927, 75005 Paris, France; CNRS UMR 8200, 94805 Villejuif, France; Gustave Roussy Cancer Campus, 94805 Villejuif, France
| | - Damarys Loew
- Institut Curie, PSL Research University, LSMP, 75248 Paris, France
| | | | - Jörg Mansfeld
- Cell Cycle, Biotechnology Center, Technische Universität Dresden, 01307 Dresden, Germany
| | - Olivier Gavet
- Sorbonne Universités, UPMC University Paris 06, UFR927, 75005 Paris, France; CNRS UMR 8200, 94805 Villejuif, France; Gustave Roussy Cancer Campus, 94805 Villejuif, France.
| |
Collapse
|
|
16
|
Yuan X, Sun X, Shi X, Jiang C, Yu D, Zhang W, Ding Y. USP39 regulates the growth of SMMC-7721 cells via FoxM1. Exp Ther Med 2017; 13:1506-1513. [PMID: 28413501 PMCID: PMC5377580 DOI: 10.3892/etm.2017.4115] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2015] [Accepted: 12/01/2016] [Indexed: 01/17/2023] Open
Abstract
The present study investigated ubiquitin specific peptidase 39 (USP39) gene knockdown on SMMC-7721 cells in vitro and in vivo, and the role of USP39 in regulating the growth of hepatocellular carcinoma (HCC). Two small interfering RNAs (siRNA) were constructed, which targeted the USP39 gene and control sequences were synthesized and inserted into a pGCSIL-GFP lentiviral vector. The full length of USP39 cDNA was amplified by polymerase chain reaction (PCR) and cloned into pEGFP-N2, and the recombinant plasmids were transfected into cells. Knockdown efficiency and upregulation of USP39 was determined by reverse transcription-quantitative PCR and western blotting. The impact of USP39 on the growth of SMMC-7721 cells in vitro was examined using an MTT assay, colony formation, flow cytometry (FCM) and immunohistochemical staining. The impact of USP39 on the growth of SMMC-7721 cells in vivo was examined by assessing tumorigenicity in nude mice. Western blotting was performed to examine the mechanism of USP39 regulation on SMMC-7721 cell growth. Recombinant vectors containing specific and scrambled USP39 siRNA sequences were constructed and transfected into SMMC-7721 cells. USP39 knockdown inhibited cell proliferation and colony formation in SMMC-7721 cells, while upregulation of USP39 promoted the growth of tumor cells. FCM indicated that USP39 knockdown led to G2/M arrest and induced apoptosis in SMMC-7721 cells. USP39 knockdown inhibited xenograft tumor growth in nude mice and led to the downregulation of the transcription factor Forkhead Box M1 (FoxM1). Gene expression of FoxM1 targets, including polo-like kinase 1, cyclin B1 and centromere protein A also decreased following USP39 knockdown. The results suggest that knockdown of USP39 inhibits the growth of HCC in vitro and in vivo, potentially through the induction of G2/M arrest by regulating the pre-mRNA splicing of FoxM1.
Collapse
Affiliation(s)
- Xianwen Yuan
- Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China
- Institute of Hepatobiliary Surgery, Nanjing University, Nanjing, Jiangsu 210008, P.R. China
| | - Xitai Sun
- Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China
- Center of Minimally Invasive Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China
| | - Xiaolei Shi
- Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China
- Institute of Hepatobiliary Surgery, Nanjing University, Nanjing, Jiangsu 210008, P.R. China
| | - Chunping Jiang
- Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China
- Institute of Hepatobiliary Surgery, Nanjing University, Nanjing, Jiangsu 210008, P.R. China
| | - Decai Yu
- Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China
- Institute of Hepatobiliary Surgery, Nanjing University, Nanjing, Jiangsu 210008, P.R. China
| | - Weiwei Zhang
- Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China
- Institute of Hepatobiliary Surgery, Nanjing University, Nanjing, Jiangsu 210008, P.R. China
| | - Yitao Ding
- Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, P.R. China
- Institute of Hepatobiliary Surgery, Nanjing University, Nanjing, Jiangsu 210008, P.R. China
| |
Collapse
|
|
17
|
Abstract
Sesquiterpene lactones (STLs) are a large and structurally diverse group of plant metabolites generally found in the Asteraceae family. STLs exhibit a wide spectrum of biological activities and it is generally accepted that their major mechanism of action is the alkylation of the thiol groups of biological molecules. The guaianolides is one of various groups of STLs. Anti-tumour and anti-migraine effects, an allergenic agent, an inhibitor of smooth muscle cells and of meristematic cell proliferation are only a few of the most commonly reported activities of STLs. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under stimulus with progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. There are previous records of the inhibitory effect of dehydroleucodin (DhL), a guaianolide lactone, on the progression of meiosis. It has been also shown that DhL and its 11,13-dihydroderivative (2H-DhL; a mixture of epimers at C-11) act as blockers of the resumption of meiosis in fully grown ovarian oocytes from the amphibian Rhinella arenarum (formerly classified as Bufo arenarum). The aim of this study was to analyze the effect of four closely related guaianolides, i.e., DhL, achillin, desacetoxymatricarin and estafietin as possible inhibitors of meiosis in oocytes of amphibians in vitro and discuss some structure-activity relationships. It was found that the inhibitory effect on meiosis resumption is greater when the lactone has two potentially reactive centres, either a α,β-α',β'-diunsaturated cyclopentanone moiety or an epoxide group plus an exo-methylene-γ-lactone function.
Collapse
|
|
18
|
Gillespie PJ, Neusiedler J, Creavin K, Chadha GS, Blow JJ. Cell Cycle Synchronization in Xenopus Egg Extracts. Methods Mol Biol 2016; 1342:101-47. [PMID: 26254920 DOI: 10.1007/978-1-4939-2957-3_6] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Many important discoveries in cell cycle research have been made using cell-free extracts prepared from the eggs of the South African clawed frog Xenopus laevis. These extracts efficiently support the key nuclear functions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. Here, we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei. We detail how these extracts can be used to study the key transitions of the eukaryotic cell cycle and describe conditions under which these transitions can be manipulated by addition of drugs that either retard or advance passage. In addition, we describe in detail essential techniques that provide a practical starting point for investigating the function of proteins involved in the operation of the eukaryotic cell cycle.
Collapse
Affiliation(s)
- Peter J Gillespie
- Centre for Gene Regulation & Expression, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
| | | | | | | | | |
Collapse
|
|
19
|
Jia JL, Han YH, Kim HC, Ahn M, Kwon JW, Luo Y, Gunasekaran P, Lee SJ, Lee KS, Kyu Bang J, Kim NH, Namgoong S. Structural basis for recognition of Emi2 by Polo-like kinase 1 and development of peptidomimetics blocking oocyte maturation and fertilization. Sci Rep 2015; 5:14626. [PMID: 26459104 PMCID: PMC4602232 DOI: 10.1038/srep14626] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2015] [Accepted: 09/01/2015] [Indexed: 11/08/2022] Open
Abstract
In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr(152) and Thr(176)) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.
Collapse
Affiliation(s)
- Jia-Lin Jia
- Department of Animal Sciences, Chungbuk National University, Republic of Korea
| | - Young-Hyun Han
- Department of Animal Sciences, Chungbuk National University, Republic of Korea
| | - Hak-Cheol Kim
- Department of Animal Sciences, Chungbuk National University, Republic of Korea
| | - Mija Ahn
- Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Korea
| | - Jeong-Woo Kwon
- Department of Animal Sciences, Chungbuk National University, Republic of Korea
| | - Yibo Luo
- Department of Animal Sciences, Chungbuk National University, Republic of Korea
| | | | - Soo-Jae Lee
- College of Pharmacy, Chungbuk National University, Republic of Korea
| | - Kyung S. Lee
- National Cancer Institute, National Institute of Health, Rockville, Maryland, United States
| | - Jeong Kyu Bang
- Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Korea
| | - Nam-Hyung Kim
- Department of Animal Sciences, Chungbuk National University, Republic of Korea
| | - Suk Namgoong
- Department of Animal Sciences, Chungbuk National University, Republic of Korea
| |
Collapse
|
|
20
|
Arquint C, Gabryjonczyk AM, Nigg EA. Centrosomes as signalling centres. Philos Trans R Soc Lond B Biol Sci 2015; 369:rstb.2013.0464. [PMID: 25047618 DOI: 10.1098/rstb.2013.0464] [Citation(s) in RCA: 120] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Centrosomes-as well as the related spindle pole bodies (SPBs) of yeast-have been extensively studied from the perspective of their microtubule-organizing roles. Moreover, the biogenesis and duplication of these organelles have been the subject of much attention, and the importance of centrosomes and the centriole-ciliary apparatus for human disease is well recognized. Much less developed is our understanding of another facet of centrosomes and SPBs, namely their possible role as signalling centres. Yet, many signalling components, including kinases and phosphatases, have been associated with centrosomes and spindle poles, giving rise to the hypothesis that these organelles might serve as hubs for the integration and coordination of signalling pathways. In this review, we discuss a number of selected studies that bear on this notion. We cover different processes (cell cycle control, development, DNA damage response) and organisms (yeast, invertebrates and vertebrates), but have made no attempt to be comprehensive. This field is still young and although the concept of centrosomes and SPBs as signalling centres is attractive, it remains primarily a concept-in need of further scrutiny. We hope that this review will stimulate thought and experimentation.
Collapse
Affiliation(s)
- Christian Arquint
- Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland
| | | | - Erich A Nigg
- Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland
| |
Collapse
|
|
21
|
Targeting Echinococcus multilocularis stem cells by inhibition of the Polo-like kinase EmPlk1. PLoS Negl Trop Dis 2014; 8:e2870. [PMID: 24901228 PMCID: PMC4046951 DOI: 10.1371/journal.pntd.0002870] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2014] [Accepted: 04/01/2014] [Indexed: 11/19/2022] Open
Abstract
Background Alveolar echinococcosis (AE) is a life-threatening disease caused by larvae of the fox-tapeworm Echinococcus multilocularis. Crucial to AE pathology is continuous infiltrative growth of the parasite's metacestode stage, which is driven by a population of somatic stem cells, called germinative cells. Current anti-AE chemotherapy using benzimidazoles is ineffective in eliminating the germinative cell population, thus leading to remission of parasite growth upon therapy discontinuation. Methodology/Principal findings We herein describe the characterization of EmPlk1, encoded by the gene emplk1, which displays significant homologies to members of the Plk1 sub-family of Polo-like kinases that regulate mitosis in eukaryotic cells. We demonstrate germinative cell-specific expression of emplk1 by RT-PCR, transcriptomics, and in situ hybridization. We also show that EmPlk1 can induce germinal vesicle breakdown when heterologously expressed in Xenopus oocytes, indicating that it is an active kinase. This activity was significantly suppressed in presence of BI 2536, a Plk1 inhibitor that has been tested in clinical trials against cancer. Addition of BI 2536 at concentrations as low as 20 nM significantly blocked the formation of metacestode vesicles from cultivated Echinococcus germinative cells. Furthermore, low concentrations of BI 2536 eliminated the germinative cell population from mature metacestode vesicles in vitro, yielding parasite tissue that was no longer capable of proliferation. Conclusions/Significance We conclude that BI 2536 effectively inactivates E. multilocularis germinative cells in parasite larvae in vitro by direct inhibition of EmPlk1, thus inducing mitotic arrest and germinative cell killing. Since germinative cells are decisive for parasite proliferation and metastasis formation within the host, BI 2536 and related compounds are very promising compounds to complement benzimidazoles in AE chemotherapy. The lethal disease AE is characterized by continuous and infiltrative growth of the metacestode larva of the tapeworm E. multilocularis within host organs. This cancer-like progression is exclusively driven by a population of parasite stem cells (germinative cells) that have to be eliminated for an effective cure of the disease. Current treatment options, using benzimidazoles, are parasitostatic only, and thus obviously not effective in germinative cell killing. We herein describe a novel, druggable parasite enzyme, EmPlk1, that specifically regulates germinative cell proliferation. We show that a compound, BI 2536, originally designed to inhibit the human ortholog of EmPlk1, can also inhibit the parasite protein at low doses. Furthermore, low doses of BI 2536 eliminated germinative cells from Echinococcus larvae in vitro and prevented parasite growth and development. We propose that BI 2536 and related compounds are promising drugs to complement current benzimidazole treatment for achieving parasite killing.
Collapse
|
|
22
|
Sheppard CL, Lee LCY, Hill EV, Henderson DJP, Anthony DF, Houslay DM, Yalla KC, Cairns LS, Dunlop AJ, Baillie GS, Huston E, Houslay MD. Mitotic activation of the DISC1-inducible cyclic AMP phosphodiesterase-4D9 (PDE4D9), through multi-site phosphorylation, influences cell cycle progression. Cell Signal 2014; 26:1958-74. [PMID: 24815749 DOI: 10.1016/j.cellsig.2014.04.023] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2014] [Revised: 04/28/2014] [Accepted: 04/29/2014] [Indexed: 10/25/2022]
Abstract
In Rat-1 cells, the dramatic decrease in the levels of both intracellular cyclic 3'5' adenosine monophosphate (cyclic AMP; cAMP) and in the activity of cAMP-activated protein kinase A (PKA) observed in mitosis was paralleled by a profound increase in cAMP hydrolyzing phosphodiesterase-4 (PDE4) activity. The decrease in PKA activity, which occurs during mitosis, was attributable to PDE4 activation as the PDE4 selective inhibitor, rolipram, but not the phosphodiesterase-3 (PDE3) inhibitor, cilostamide, specifically ablated this cell cycle-dependent effect. PDE4 inhibition caused Rat-1 cells to move from S phase into G2/M more rapidly, to transit through G2/M more quickly and to remain in G1 for a longer period. Inhibition of PDE3 elicited no observable effects on cell cycle dynamics. Selective immunopurification of each of the four PDE4 sub-families identified PDE4D as being selectively activated in mitosis. Subsequent analysis uncovered PDE4D9, an isoform whose expression can be regulated by Disrupted-In-Schizophrenia 1 (DISC1)/activating transcription factor 4 (ATF4) complex, as the sole PDE4 species activated during mitosis in Rat-1 cells. PDE4D9 becomes activated in mitosis through dual phosphorylation at Ser585 and Ser245, involving the combined action of ERK and an unidentified 'switch' kinase that has previously been shown to be activated by H2O2. Additionally, in mitosis, PDE4D9 also becomes phosphorylated at Ser67 and Ser81, through the action of MK2 (MAPKAPK2) and AMP kinase (AMPK), respectively. The multisite phosphorylation of PDE4D9 by all four of these protein kinases leads to decreased mobility (band-shift) of PDE4D9 on SDS-PAGE. PDE4D9 is predominantly concentrated in the perinuclear region of Rat-1 cells but with a fraction distributed asymmetrically at the cell margins. Our investigations demonstrate that the diminished levels of cAMP and PKA activity that characterise mitosis are due to enhanced cAMP degradation by PDE4D9. PDE4D9, was found to locate primarily not only in the perinuclear region of Rat-1 cells but also at the cell margins. We propose that the sequestration of PDE4D9 in a specific complex together with AMPK, ERK, MK2 and the H2O2-activatable 'switch' kinase allows for its selective multi-site phosphorylation, activation and regulation in mitosis.
Collapse
Affiliation(s)
- Catherine L Sheppard
- Institute of Neuroscience and Psychology, Wolfson Link and Davidson Buildings, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK
| | - Louisa C Y Lee
- Institute of Neuroscience and Psychology, Wolfson Link and Davidson Buildings, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK
| | - Elaine V Hill
- Institute of Neuroscience and Psychology, Wolfson Link and Davidson Buildings, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK
| | - David J P Henderson
- Institute of Neuroscience and Psychology, Wolfson Link and Davidson Buildings, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK
| | - Diana F Anthony
- Institute of Neuroscience and Psychology, Wolfson Link and Davidson Buildings, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK
| | - Daniel M Houslay
- Institute of Neuroscience and Psychology, Wolfson Link and Davidson Buildings, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK
| | - Krishna C Yalla
- Institute of Neuroscience and Psychology, Wolfson Link and Davidson Buildings, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK
| | - Lynne S Cairns
- Institute of Neuroscience and Psychology, Wolfson Link and Davidson Buildings, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK
| | - Allan J Dunlop
- Institute of Neuroscience and Psychology, Wolfson Link and Davidson Buildings, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK
| | - George S Baillie
- Institute of Cardiovascular and Medical Sciences, Wolfson Link and Davidson Buildings, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK
| | - Elaine Huston
- Institute of Pharmaceutical Science, King's College London, 5th Floor, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK
| | - Miles D Houslay
- Institute of Pharmaceutical Science, King's College London, 5th Floor, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK.
| |
Collapse
|
|
23
|
Feine O, Hukasova E, Bruinsma W, Freire R, Fainsod A, Gannon J, Mahbubani HM, Lindqvist A, Brandeis M. Phosphorylation-mediated stabilization of Bora in mitosis coordinates Plx1/Plk1 and Cdk1 oscillations. Cell Cycle 2014; 13:1727-36. [PMID: 24675888 PMCID: PMC4111719 DOI: 10.4161/cc.28630] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2014] [Accepted: 03/23/2014] [Indexed: 11/19/2022] Open
Abstract
Cdk1 and Plk1/Plx1 activation leads to their inactivation through negative feedback loops. Cdk1 deactivates itself by activating the APC/C, consequently generating embryonic cell cycle oscillations. APC/C inhibition by the mitotic checkpoint in somatic cells and the cytostatic factor (CSF) in oocytes sustain the mitotic state. Plk1/Plx1 targets its co-activator Bora for degradation, but it remains unclear how embryonic oscillations in Plx1 activity are generated, and how Plk1/Plx1 activity is sustained during mitosis. We show that Plx1-mediated degradation of Bora in interphase generates oscillations in Plx1 activity and is essential for development. In CSF extracts, phosphorylation of Bora on the Cdk consensus site T52 blocks Bora degradation. Upon fertilization, Calcineurin dephosphorylates T52, triggering Plx1 oscillations. Similarly, we find that GFP-Bora is degraded when Plk1 activity spreads to somatic cell cytoplasm before mitosis. Interestingly, GFP-Bora degradation stops upon mitotic entry when Cdk1 activity is high. We hypothesize that Cdk1 controls Bora through an incoherent feedforward loop synchronizing the activities of mitotic kinases.
Collapse
Affiliation(s)
- Oren Feine
- Department of Genetics; The Hebrew University of Jerusalem; Jerusalem, Israel
| | - Elvira Hukasova
- Department of Cell and Molecular Biology; Karolinska Institute; Stockholm, Sweden
| | - Wytse Bruinsma
- Department of Cell Biology; The Netherlands Cancer Institute; Amsterdam, Netherlands
| | - Raimundo Freire
- Unidad de Investigación; Hospital Universitario de Canarias; Instituto de Tecnologias Biomedicas; Tenerife, Spain
| | - Abraham Fainsod
- Department of Developmental Biology and Cancer Research; The Hebrew University of Jerusalem; Jerusalem, Israel
| | - Julian Gannon
- Cancer Research UK; Clare Hall Laboratories; South Mimms, Hertfordshire, UK
| | - Hiro M Mahbubani
- Cancer Research UK; Clare Hall Laboratories; South Mimms, Hertfordshire, UK
| | - Arne Lindqvist
- Department of Cell and Molecular Biology; Karolinska Institute; Stockholm, Sweden
| | - Michael Brandeis
- Department of Genetics; The Hebrew University of Jerusalem; Jerusalem, Israel
| |
Collapse
|
|
24
|
Kinsey WH. SRC-family tyrosine kinases in oogenesis, oocyte maturation and fertilization: an evolutionary perspective. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2014; 759:33-56. [PMID: 25030759 DOI: 10.1007/978-1-4939-0817-2_3] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The oocyte is a highly specialized cell poised to respond to fertilization with a unique set of actions needed to recognize and incorporate a single sperm, complete meiosis, reprogram maternal and paternal genomes and assemble them into a unique zygotic genome, and finally initiate the mitotic cell cycle. Oocytes accomplish this diverse series of events through an array of signal transduction pathway components that include a characteristic collection of protein tyrosine kinases. The src-family protein kinases (SFKs) figure importantly in this signaling array and oocytes characteristically express certain SFKs at high levels to provide for the unique actions that the oocyte must perform. The SFKs typically exhibit a distinct pattern of subcellular localization in oocytes and perform critical functions in different subcellular compartments at different steps during oocyte maturation and fertilization. While many aspects of SFK signaling are conserved among oocytes from different species, significant differences exist in the extent to which src-family-mediated pathways are used by oocytes from species that fertilize externally vs those which are fertilized internally. The observation that several oocyte functions which require SFK signaling appear to represent common points of failure during assisted reproductive techniques in humans, highlights the importance of these signaling pathways for human reproductive health.
Collapse
Affiliation(s)
- William H Kinsey
- Department of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS, 66160, USA,
| |
Collapse
|
|
25
|
Juanes MA, Khoueiry R, Kupka T, Castro A, Mudrak I, Ogris E, Lorca T, Piatti S. Budding yeast greatwall and endosulfines control activity and spatial regulation of PP2A(Cdc55) for timely mitotic progression. PLoS Genet 2013; 9:e1003575. [PMID: 23861665 PMCID: PMC3701715 DOI: 10.1371/journal.pgen.1003575] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2013] [Accepted: 05/02/2013] [Indexed: 12/29/2022] Open
Abstract
Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2ACdc55 phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2) bind to PP2ACdc55 in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2ACdc55 activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like PP2ACdc55, timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2ACdc55, which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms. In all eukaryotic cells chromosome partition during mitosis requires a number of processes, including the formation of the mitotic spindle, i.e. the machinery that drives chromosome segregation to the daughter cells. Mitotic entry requires a delicate balance between protein phosphorylation, driven by cyclin-dependent kinases (CDKs), and protein dephosphorylation, carried out by specific phosphatases that counteract CDK activity. A critical threshold in CDK activity is indeed required for mitotic entry. In the past few years the Greatwall kinase has also been implicated in mitotic entry through phosphorylation of proteins of the endosulfine family, which in turn inhibit the activity of the PP2A phosphatase that would otherwise dephosphorylate CDK targets. Whether Greatwall and endosulfines have a mitotic function in budding yeast, where PP2A promotes, rather than inhibits, mitotic entry has not been established. Here we show that the Greatwall-endosulfine-PP2A regulatory module is conserved also in budding yeast and that endosulfines from different species are interchangeable for their mitotic function. However, in budding yeast cells endosulfines contribute to full activation and proper localization of PP2A, suggesting that they act as both inhibitors and activators of PP2A. Our data emphasize how the same regulatory module is adapted to meet specific mitotic features in different organisms.
Collapse
Affiliation(s)
| | - Rita Khoueiry
- Centre de Recherche en Biochimie Macromoléculaire, Montpellier, France
| | - Thomas Kupka
- Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria
| | - Anna Castro
- Centre de Recherche en Biochimie Macromoléculaire, Montpellier, France
| | - Ingrid Mudrak
- Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria
| | - Egon Ogris
- Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria
| | - Thierry Lorca
- Centre de Recherche en Biochimie Macromoléculaire, Montpellier, France
| | - Simonetta Piatti
- Centre de Recherche en Biochimie Macromoléculaire, Montpellier, France
- * E-mail:
| |
Collapse
|
|
26
|
Abstract
Conjugation of ubiquitin (ubiquitination) to substrate proteins is a widespread modification that ensures fidelity of many cellular processes. During mitosis, different dynamic morphological transitions have to be coordinated in a temporal and spatial manner to allow for precise partitioning of the genetic material into two daughter cells, and ubiquitination of key mitotic factors is believed to provide both directionality and fidelity to this process. While directionality can be achieved by a proteolytic type of ubiquitination signal, the fidelity is often determined by various types of ubiquitin conjugation that does not target substrates for proteolysis by the proteasome. An additional level of complexity is provided by various ubiquitin-interacting proteins that act downstream of the ubiquitinated substrate and can serve as "decoders" for the ubiquitin signal. They may, specifically reverse ubiquitin attachment (deubiquitinating enzymes, DUBs) or, act as a receptor for transfer of the ubiquitinated substrate toward downstream signaling components and/or subcellular compartments (ubiquitin-binding proteins, UBPs). In this review, we aim at summarizing the knowledge and emerging concepts about the role of ubiquitin decoders, DUBs, and UBPs that contribute to faithful regulation of mitotic division.
Collapse
Affiliation(s)
- Sadek Fournane
- Institute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch, France
| | | | | | | |
Collapse
|
|
27
|
Khan PP, Maitra S. Participation of cAMP-dependent protein kinase and MAP kinase pathways during Anabas testudineus oocyte maturation. Gen Comp Endocrinol 2013; 181:88-97. [PMID: 23174698 DOI: 10.1016/j.ygcen.2012.10.016] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/03/2012] [Revised: 10/10/2012] [Accepted: 10/12/2012] [Indexed: 11/17/2022]
Abstract
Possible involvement of cyclic nucleotide dependent protein kinase (PKA) and MAP kinase (MAPK) pathways during oocyte maturation in Anabas testudineus was investigated. Pre-incubation with phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX), inhibited 17α, 20β-DHP-induced GVBD dose dependently. PKA inhibitor, H89 could induce resumption of meiosis independent of 17α, 20β-DHP, in dose and duration dependent manner. The maximum response was obtained with the dose of 10 μM of H89 and 95% of cells underwent GVBD within 18 h. Moreover, stimulation with 17α, 20β-DHP inhibited endogenous PKA activity significantly within first hour and this effect was attenuated by PDE inhibitor IBMX at all time points. The pattern of PKA inhibition corresponded well with kinetics of histone H1 kinase activation and p34cdc2 phosphorylation. These results suggest physiological relevance of cAMP/PKA signaling in perch oocytes undergoing G2/M transition. MAPK was demonstrated as two distinct isoforms (ERK1 and ERK2) which resolved in the range of 42-44 kDa in immunoblot. Though total protein content did not show significant variation, H89 stimulation was able to stimulate phosphorylation of ERK1/2 from 5h onwards and the strongest response was observed between 10 and 18 h. MEK inhibitor, U0126 completely blocked PKA inhibition induced MAPK activation and GVBD. In addition, inhibition of endogenous PKA by a more selective peptide inhibitor [PKI-(6-22)-amide] was sufficient to resume GVBD and MAPK activation in intact perch oocytes. Also, significant ERK1/2 phosphorylation could be stimulated in cell-free extracts of perch oocytes supplemented with PKI-(6-22)-amide. The results suggest an interaction between cAMP/PKA and MAPK pathways in mediating meiosis resumption in perch oocyte.
Collapse
Affiliation(s)
- P P Khan
- Department of Zoology, Visva-Bharati University, Santiniketan, India
| | | |
Collapse
|
|
28
|
Affiliation(s)
- James L Maller
- Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
| |
Collapse
|
|
29
|
Pfeuty B, Bodart JF, Blossey R, Lefranc M. A dynamical model of oocyte maturation unveils precisely orchestrated meiotic decisions. PLoS Comput Biol 2012; 8:e1002329. [PMID: 22238511 PMCID: PMC3252271 DOI: 10.1371/journal.pcbi.1002329] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2011] [Accepted: 11/11/2011] [Indexed: 12/04/2022] Open
Abstract
Maturation of vertebrate oocytes into haploid gametes relies on two consecutive meioses without intervening DNA replication. The temporal sequence of cellular transitions driving eggs from G2 arrest to meiosis I (MI) and then to meiosis II (MII) is controlled by the interplay between cyclin-dependent and mitogen-activated protein kinases. In this paper, we propose a dynamical model of the molecular network that orchestrates maturation of Xenopus laevis oocytes. Our model reproduces the core features of maturation progression, including the characteristic non-monotonous time course of cyclin-Cdks, and unveils the network design principles underlying a precise sequence of meiotic decisions, as captured by bifurcation and sensitivity analyses. Firstly, a coherent and sharp meiotic resumption is triggered by the concerted action of positive feedback loops post-translationally activating cyclin-Cdks. Secondly, meiotic transition is driven by the dynamic antagonism between positive and negative feedback loops controlling cyclin turnover. Our findings reveal a highly modular network in which the coordination of distinct regulatory schemes ensures both reliable and flexible cell-cycle decisions. In the life cycle of sexual organisms, a specialized cell division -meiosis- reduces the number of chromosomes in gametes or spores while fertilization or mating restores the original number. The essential feature that distinguishes meiosis from mitosis (the usual division) is the succession of two rounds of division following a single DNA replication, as well as the arrest at the second division in the case of oocyte maturation. The fact that meiosis and mitosis are similar but different raises several interesting questions: What is the meiosis-specific dynamics of cell-cycle regulators? Are there mechanisms which guarantee the occurence of two and only two rounds of division despite the presence of intrinsic and extrinsic noises ? The study of a model of the molecular network that underlies the meiotic maturation process in Xenopus oocytes provides unexpected answers to these questions. On the one hand, the modular organization of this network ensures separate controls of the first and second divisions. On the other hand, regulatory synergies ensure that these two stages are precisely and reliably sequenced during meiosis. We conclude that cells have evolved a sophisticated regulatory network to achieve a robust, albeit flexible, meiotic dynamics.
Collapse
Affiliation(s)
- Benjamin Pfeuty
- Laboratoire de Physique des Lasers, Atomes, et Molécules, CNRS, UMR8523, Université Lille 1 Sciences et Technologies, Villeneuve d'Ascq, France.
| | | | | | | |
Collapse
|
|
30
|
Caruso M, Valsasina B, Ballinari D, Bertrand J, Brasca MG, Caldarelli M, Cappella P, Fiorentini F, Gianellini LM, Scolaro A, Beria I. 5-(2-amino-pyrimidin-4-yl)-1H-pyrrole and 2-(2-amino-pyrimidin-4-yl)-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one derivatives as new classes of selective and orally available Polo-like kinase 1 inhibitors. Bioorg Med Chem Lett 2012; 22:96-101. [PMID: 22154349 DOI: 10.1016/j.bmcl.2011.11.065] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2011] [Revised: 11/17/2011] [Accepted: 11/18/2011] [Indexed: 12/26/2022]
Abstract
The discovery and characterization of two new chemical classes of potent and selective Polo-like kinase 1 (PLK1) inhibitors is reported. For the most interesting compounds, we discuss the biological activities, crystal structures and preliminary pharmacokinetic parameters. The more advanced compounds inhibit PLK1 in the enzymatic assay at the nM level and exhibit good activity in cell proliferation on A2780 cells. Furthermore, these compounds showed high levels of selectivity on a panel of unrelated kinases, as well as against PLK2 and PLK3 isoforms. Additionally, the compounds show acceptable oral bioavailability in mice making these inhibitors suitable candidates for further in vivo activity studies.
Collapse
Affiliation(s)
- Michele Caruso
- Nerviano Medical Sciences srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano, (MI), Italy
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
31
|
The Polo-like kinase PLKA in Aspergillus nidulans is not essential but plays important roles during vegetative growth and development. EUKARYOTIC CELL 2011; 11:194-205. [PMID: 22140227 DOI: 10.1128/ec.05130-11] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
The Polo-like kinases (Plks) are conserved, multifunctional cell cycle regulators that are induced in many forms of cancer and play additional roles in metazoan development. We previously identified plkA in Aspergillus nidulans, the only Plk investigated in filamentous fungi to date, and partially characterized its function through overexpression. Here, we report the plkA null phenotype. Surprisingly, plkA was not essential, unlike Plks in other organisms that contain a single homologue. A subset of cells lacking PLKA contained defects in spindle formation and chromosome organization, supporting some conservation in cell cycle function. However, septa were present, suggesting that PLKA, unlike other Plks, is not a central regulator of septation. Colonies lacking PLKA were compact with multibranched hyphae, implying a role for this factor in aspects of hyphal morphogenesis. These defects were suppressed by high temperature or low concentrations of benomyl, suggesting that PLKA may function during vegetative growth by influencing microtubule dynamics. However, the colonies also showed reduced conidiation and precocious formation of sexual Hülle cells in a benomyl- and temperature-insensitive manner. This result suggests that PLKA may influence reproduction through distinct mechanisms and represents the first example of a link between Plk function and development in fungi. Finally, filamentous fungal Plks have distinct features, and phylogenetic analyses reveal that they may group more closely with metazoan PLK4. In contrast, yeast Plks are more similar to metazoan proteins PLK1 to PLK3. Thus, A. nidulans PLKA shows some conservation in cell cycle function but may also play novel roles during hyphal morphogenesis and development.
Collapse
|
|
32
|
Gotoh T, Villa LM, Capelluto DGS, Finkielstein CV. Regulatory pathways coordinating cell cycle progression in early Xenopus development. Results Probl Cell Differ 2011; 53:171-99. [PMID: 21630146 DOI: 10.1007/978-3-642-19065-0_9] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
The African clawed frog, Xenopus laevis, is used extensively as a model organism for studying both cell development and cell cycle regulation. For over 20 years now, this model organism has contributed to answering fundamental questions concerning the mechanisms that underlie cell cycle transitions--the cellular components that synthesize, modify, repair, and degrade nucleic acids and proteins, the signaling pathways that allow cells to communicate, and the regulatory pathways that lead to selective expression of subsets of genes. In addition, the remarkable simplicity of the Xenopus early cell cycle allows for tractable manipulation and dissection of the basic components driving each transition. In this organism, early cell divisions are characterized by rapid cycles alternating phases of DNA synthesis and division. The post-blastula stages incorporate gap phases, lengthening progression, and allowing more time for DNA repair. Various cyclin/Cdk complexes are differentially expressed during the early cycles with orderly progression being driven by both the combined action of cyclin synthesis and degradation and the appropriate selection of specific substrates by their Cdk components. Like other multicellular organisms, chief developmental events in early Xenopus embryogenesis coincide with profound remodeling of the cell cycle, suggesting that cell proliferation and differentiation events are linked and coordinated through crosstalk mechanisms acting on signaling pathways involving the expression of cell cycle control genes.
Collapse
Affiliation(s)
- Tetsuya Gotoh
- Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Polytechnic Institute and State University, 1981 Kraft Drive, Blacksburg, VA 24061, USA
| | | | | | | |
Collapse
|
|
33
|
Raab M, Kappel S, Krämer A, Sanhaji M, Matthess Y, Kurunci-Csacsko E, Calzada-Wack J, Rathkolb B, Rozman J, Adler T, Busch DH, Esposito I, Fuchs H, Gailus-Durner V, Klingenspor M, Wolf E, Sänger N, Prinz F, Angelis MHD, Seibler J, Yuan J, Bergmann M, Knecht R, Kreft B, Strebhardt K. Toxicity modelling of Plk1-targeted therapies in genetically engineered mice and cultured primary mammalian cells. Nat Commun 2011; 2:395. [PMID: 21772266 PMCID: PMC3144583 DOI: 10.1038/ncomms1395] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2010] [Accepted: 06/16/2011] [Indexed: 01/08/2023] Open
Abstract
High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions.
Collapse
Affiliation(s)
- Monika Raab
- Department of Obstetrics and Gynecology, School of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
- Klinik und Poliklinik für Hals-, Nasen- und Ohrenheilkunde, UKE Hamburg, Martinistrasse 52, 20246 Hamburg, Germany
- These authors contributed equally to this work
| | - Sven Kappel
- Department of Obstetrics and Gynecology, School of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
- These authors contributed equally to this work
| | - Andrea Krämer
- Department of Obstetrics and Gynecology, School of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Mourad Sanhaji
- Department of Obstetrics and Gynecology, School of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Yves Matthess
- Department of Obstetrics and Gynecology, School of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Elisabeth Kurunci-Csacsko
- Department of Obstetrics and Gynecology, School of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Julia Calzada-Wack
- Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstaedter Landstrasse, 85764 Munich/Neuherberg, Germany
| | - Birgit Rathkolb
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstaedter Landstrasse, 85764 Munich/Neuherberg, Germany
- Chair for Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
| | - Jan Rozman
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstaedter Landstrasse, 85764 Munich/Neuherberg, Germany
- Molecular Nutritional Medicine, Else Kröner-Fresenius Center, Technische Universität München, Gregor-Mendel-Strasse 2, 85350 Freising-Weihenstephan, Germany
| | - Thure Adler
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstaedter Landstrasse, 85764 Munich/Neuherberg, Germany
- Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Trogerstrasse 30, 81675 Munich, Germany
| | - Dirk H. Busch
- Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Trogerstrasse 30, 81675 Munich, Germany
| | - Irene Esposito
- Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstaedter Landstrasse, 85764 Munich/Neuherberg, Germany
- Institute of Pathology, Technische Universität München, Ismaningerstrasse 22, 81675 Munich, Germany
| | - Helmut Fuchs
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstaedter Landstrasse, 85764 Munich/Neuherberg, Germany
| | - Valérie Gailus-Durner
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstaedter Landstrasse, 85764 Munich/Neuherberg, Germany
| | - Martin Klingenspor
- Molecular Nutritional Medicine, Else Kröner-Fresenius Center, Technische Universität München, Gregor-Mendel-Strasse 2, 85350 Freising-Weihenstephan, Germany
| | - Eckhard Wolf
- Chair for Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany
| | - Nicole Sänger
- Department of Obstetrics and Gynecology, School of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Florian Prinz
- Bayer Schering Pharma AG, Global Drug Discovery, Therapeutic Research Group Oncology, Müllerstrasse 178, 13353 Berlin, Germany
| | - Martin Hrabě de Angelis
- German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstaedter Landstrasse, 85764 Munich/Neuherberg, Germany
- Lehrstuhl für Experimentelle Genetik, Technische Universität München, Emil-Ramann-Strasse 8, 85350 Freising-Weihenstephan, Germany
| | - Jost Seibler
- TaconicArtemis GmbH, Neurather Ring 1, 51063 Köln, Germany
| | - Juping Yuan
- Department of Obstetrics and Gynecology, School of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Martin Bergmann
- Institute of Veterinary Anatomy, Histology and Embryology, University of Giessen, Frankfurterstrasse 98, 35392GiessenGermany
| | - Rainald Knecht
- Klinik und Poliklinik für Hals-, Nasen- und Ohrenheilkunde, UKE Hamburg, Martinistrasse 52, 20246 Hamburg, Germany
| | - Bertolt Kreft
- Bayer Schering Pharma AG, Global Drug Discovery, Therapeutic Research Group Oncology, Müllerstrasse 178, 13353 Berlin, Germany
| | - Klaus Strebhardt
- Department of Obstetrics and Gynecology, School of Medicine, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| |
Collapse
|
|
34
|
Yuan K, Huang Y, Yao X. Illumination of mitotic orchestra during cell division: a Polo view. Cell Signal 2011; 23:1-5. [PMID: 20633640 PMCID: PMC3118837 DOI: 10.1016/j.cellsig.2010.07.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2010] [Accepted: 07/06/2010] [Indexed: 11/27/2022]
Abstract
Protein kinase and phosphatase signaling cascade, coupled with other post-translational modifications, orchestrates temporal order of various events during cell division. Among the many mitotic kinases, Polo-like kinase 1 (PLK1) as a key regulator, participates in regulating mitosis from mitotic entry to cytokinesis. The advancement in optical reporter engineering and the recent development of specific chemical probes enable us to visualize spatiotemporal gradient of kinase activity at nano-scale. One of such tools is FRET-based optic sensor that allows us to delineate the PLK1 activity in space and time. In this review, we address the inter-relationships between PLK1 and other protein kinases/phosphatases, as well as the crosstalk between PLK1 phosphorylation and ubiquitination during cell division. In particular, we discuss the molecular mechanisms and steps underlying PLK1 kinase priming, activation and turn-off during cell division.
Collapse
Affiliation(s)
- Kai Yuan
- Anhui Key Laboratory for Cellular Dynamics and Chemical Biology, and University of Science and Technology of China, Hefei 230027, China
- Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia 30310, USA
| | - Yuejia Huang
- Anhui Key Laboratory for Cellular Dynamics and Chemical Biology, and University of Science and Technology of China, Hefei 230027, China
- Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia 30310, USA
| | - Xuebiao Yao
- Anhui Key Laboratory for Cellular Dynamics and Chemical Biology, and University of Science and Technology of China, Hefei 230027, China
| |
Collapse
|
|
35
|
Shen W, Ahmad F, Hockman S, Ma J, Omi H, Raghavachari N, Manganiello V. Female infertility in PDE3A(-/-) mice: polo-like kinase 1 (Plk1) may be a target of protein kinase A (PKA) and involved in meiotic arrest of oocytes from PDE3A(-/-) mice. Cell Cycle 2010; 9:4720-34. [PMID: 21099356 PMCID: PMC3048038 DOI: 10.4161/cc.9.23.14090] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2010] [Revised: 10/29/2010] [Accepted: 11/02/2010] [Indexed: 01/01/2023] Open
Abstract
Mechanisms of cAMP/PKA-induced meiotic arrest in oocytes are not completely identified. In cultured, G2/M-arrested PDE3A(-/-) murine oocytes, elevated PKA activity was associated with inactivation of Cdc2 and Plk1, and inhibition of phosphorylation of histone H3 (S10) and of dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15). In cultured WT oocytes, PKA activity was transiently reduced and then increased to that observed in PDE3A(-/-) oocytes; Cdc2 and Plk1 were activated, phosphorylation of histone H3 (S10) and dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15) were observed. In WT oocytes, PKAc were rapidly translocated into nucleus, and then to the spindle apparatus, but in PDE3A(-/-) oocytes, PKAc remained in the cytosol. Plk1 was reactivated by incubation of PDE3A(-/-) oocytes with PKA inhibitor, Rp-cAMPS. PDE3A was co-localized with Plk1 in WT oocytes, and co-immunoprecipitated with Plk1 in WT ovary and Hela cells. PKAc phosphorylated rPlk1 and Hela cell Plk1 and inhibited Plk1 activity in vitro. Our results suggest that PKA-induced inhibition of Plk1 may be critical in oocyte meiotic arrest and female infertility in PDE3A(-/-) mice.
Collapse
Affiliation(s)
- Weixing Shen
- Translational Medicine Branch (TMB); National Heart, Lung and Blood Institute (NHLBI); National Institutes of Health; Bethesda, MD USA
| | - Faiyaz Ahmad
- Translational Medicine Branch (TMB); National Heart, Lung and Blood Institute (NHLBI); National Institutes of Health; Bethesda, MD USA
| | - Steven Hockman
- Translational Medicine Branch (TMB); National Heart, Lung and Blood Institute (NHLBI); National Institutes of Health; Bethesda, MD USA
| | - John Ma
- Translational Medicine Branch (TMB); National Heart, Lung and Blood Institute (NHLBI); National Institutes of Health; Bethesda, MD USA
| | - Hitoshi Omi
- Translational Medicine Branch (TMB); National Heart, Lung and Blood Institute (NHLBI); National Institutes of Health; Bethesda, MD USA
| | - Nalini Raghavachari
- Genomics Core Facility; Pulmonary and Vascular Medicine Branch (PVMB); National Heart, Lung and Blood Institute (NHLBI); National Institutes of Health; Bethesda, MD USA
| | - Vincent Manganiello
- Translational Medicine Branch (TMB); National Heart, Lung and Blood Institute (NHLBI); National Institutes of Health; Bethesda, MD USA
| |
Collapse
|
|
36
|
Lorca T, Bernis C, Vigneron S, Burgess A, Brioudes E, Labbé JC, Castro A. Constant regulation of both the MPF amplification loop and the Greatwall-PP2A pathway is required for metaphase II arrest and correct entry into the first embryonic cell cycle. J Cell Sci 2010; 123:2281-91. [PMID: 20554897 DOI: 10.1242/jcs.064527] [Citation(s) in RCA: 65] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Recent results indicate that regulating the balance between cyclin-B-Cdc2 kinase, also known as M-phase-promoting factor (MPF), and protein phosphatase 2A (PP2A) is crucial to enable correct mitotic entry and exit. In this work, we studied the regulatory mechanisms controlling the cyclin-B-Cdc2 and PP2A balance by analysing the activity of the Greatwall kinase and PP2A, and the different components of the MPF amplification loop (Myt1, Wee1, Cdc25) during the first embryonic cell cycle. Previous data indicated that the Myt1-Wee1-Cdc25 equilibrium is tightly regulated at the G2-M and M-G1 phase transitions; however, no data exist regarding the regulation of this balance during M phase and interphase. Here, we demonstrate that constant regulation of the cyclin-B-Cdc2 amplification loop is required for correct mitotic division and to promote correct timing of mitotic entry. Our results show that removal of Cdc25 from metaphase-II-arrested oocytes promotes mitotic exit, whereas depletion of either Myt1 or Wee1 in interphase egg extracts induces premature mitotic entry. We also provide evidence that, besides the cyclin-B-Cdc2 amplification loop, the Greatwall-PP2A pathway must also be tightly regulated to promote correct first embryonic cell division. When PP2A is prematurely inhibited in the absence of cyclin-B-Cdc2 activation, endogenous cyclin-A-Cdc2 activity induces irreversible aberrant mitosis in which there is, first, partial transient phosphorylation of mitotic substrates and, second, subsequent rapid and complete degradation of cyclin A and cyclin B, thus promoting premature and rapid exit from mitosis.
Collapse
Affiliation(s)
- Thierry Lorca
- Universités Montpellier 2 et 1, Centre de Recherche de Biochimie Macromoléculaire, CNRS UMR 5237, IFR 122, 1919 Route de Mende, 34293 Montpellier CEDEX 5, France.
| | | | | | | | | | | | | |
Collapse
|
|
37
|
Participation of MAPK, PKA and PP2A in the regulation of MPF activity in Bufo arenarum oocytes. ZYGOTE 2010; 19:181-9. [DOI: 10.1017/s0967199410000456] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
SummaryThe objectives of the present paper were to study the involvement and possible interactions of both cAMP-PKA and protein phosphatases in Bufo arenarum oocyte maturation and to determine if these pathways are independent or not of the MAP kinase (MAPK) cascade. Our results indicated that the inhibition of PKA by treatment with H-89, an inhibitor of the catalytic subunit of PKA, was capable of inducing GVBD in a dose-dependent manner by a pathway in which Cdc25 phosphatase but not the MAPK cascade is involved. The injection of 50 nl of H-89 10 μM produced GVBD percentages similar to those obtained with treatment with progesterone. In addition, the assays with okadaic acid (OA), a PP2A inhibitor, significantly enhanced the percentage of oocytes that resumed meiosis by a signal transducing pathway in which the activation of the MEK–MAPK pathway is necessary, but in which Cdc25 phosphatase was not involved. Treatment with H-89, was able to overcome the inhibitory effect of PKA on GVBD; however, the inhibition of Cdc25 activity with NaVO3 was able to overcome the induction of GVBD by H-89. Although the connections between PKA and other signalling molecules that regulate oocytes maturation are still unclear, our results suggest that phosphatase Cdc25 may be the direct substrate of PKA. In Xenopus oocytes it was proposed that PP2A, a major Ser/Thr phosphatase present, is a negative regulator of Cdc2 activation. However, in Bufo arenarum oocytes, inhibition of Cdc25 with NaVO3 did not inhibit OA-induced maturation, suggesting that the target of PP2A was not the Cdc25 phosphatase. MAPK activation has been reported to be essential in Xenopus oocytes GVBD. In B. arenarum oocytes we demonstrated that the inhibition of MAPK by PD 98059 prevented the activation of MPF induced by OA, suggesting that the activation of the MAPK cascade produced an inhibition of Myt1 and, in consequence, the activation of MPF without participation of the Cdc25 phosphatase. Our results suggest that in incompetent oocytes of B. arenarum two signal transduction pathways may be involved in the control of MPF activation: (1) the inhibition of phosphatase 2A that through the MEK–MAPK pathway regulates the activity of the Myt1; and (2) the inhibition of AMPc–PKA, which affects the activity of the Cdc25 phosphatase.
Collapse
|
|
38
|
Jeong K, Jeong JY, Lee HO, Choi E, Lee H. Inhibition of Plk1 induces mitotic infidelity and embryonic growth defects in developing zebrafish embryos. Dev Biol 2010; 345:34-48. [PMID: 20553902 DOI: 10.1016/j.ydbio.2010.06.004] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2009] [Revised: 05/29/2010] [Accepted: 06/01/2010] [Indexed: 01/29/2023]
Abstract
Polo-like kinase 1 (Plk1) is central to cell division. Here, we report that Plk1 is critical for mitosis in the embryonic development of zebrafish. Using a combination of several cell biology tools, including single-cell live imaging applied to whole embryos, we show that Plk1 is essential for progression into mitosis during embryonic development. Plk1 morphant cells displayed mitotic infidelity, such as abnormal centrosomes, irregular spindle assembly, hypercondensed chromosomes, and a failure of chromosome arm separation. Consequently, depletion of Plk1 resulted in mitotic arrest and finally death by 6days post-fertilization. In comparison, Plk2 or Plk3 morphant embryos did not display any significant abnormalities. Treatment of embryos with the Plk1 inhibitor, BI 2536, caused a block in mitosis, which was more severe when used to treat plk1 morphants. Finally, using an assay to rescue the Plk1 morphant phenotype, we found that the kinase domain and PBD domains are both necessary for Plk1 function in zebrafish development. Our studies demonstrate that Plk1 is required for embryonic proliferation because its activity is crucial for mitotic integrity. Furthermore, our study suggests that zebrafish will be an efficient and economical in vivo system for the validation of anti-mitotic drugs.
Collapse
Affiliation(s)
- Kilhun Jeong
- Department of Biological Sciences and Institute of Molecular Biology and Genetics, College of Natural Sciences, Seoul National University, 599, Gwanak-Ro, Gwanak-Gu, Seoul 151-742, Korea
| | | | | | | | | |
Collapse
|
|
39
|
Canel N, Bevacqua R, Fernández-Martín R, Salamone DF. Activation with Ionomycin followed by Dehydroleucodine and Cytochalasin B for the Production of Parthenogenetic and Cloned Bovine Embryos. Cell Reprogram 2010; 12:491-9. [DOI: 10.1089/cell.2009.0109] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Natalia Canel
- Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires-CONICET, Buenos Aires, Argentina
| | - Romina Bevacqua
- Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires-CONICET, Buenos Aires, Argentina
| | - Rafael Fernández-Martín
- Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires-CONICET, Buenos Aires, Argentina
| | - Daniel F. Salamone
- Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires-CONICET, Buenos Aires, Argentina
| |
Collapse
|
|
40
|
Colnaghi R, Wheatley SP. Liaisons between survivin and Plk1 during cell division and cell death. J Biol Chem 2010; 285:22592-604. [PMID: 20427271 PMCID: PMC2903399 DOI: 10.1074/jbc.m109.065003] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2009] [Revised: 04/28/2010] [Indexed: 12/18/2022] Open
Abstract
Survivin and Plk1 kinase are important mediators of cell survival that are required for chromosome alignment, cytokinesis, and protection from apoptosis. Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis. Moreover, the expression of both survivin and Plk1 is deregulated in cancer. Given these similarities, we speculated that these two proteins may cooperate during mitosis and/or in cell death pathways. Here we report that survivin and Plk1 interact during mitosis and that Plk1 phosphorylates survivin at serine 20. Importantly, we find that overexpression of a non-phosphorylatable version, S20A, is unable to correct chromosomes connected to the spindle in a syntelic manner during prometaphase and allows cells harboring these maloriented chromosomes to enter anaphase, evading the spindle tension checkpoint. By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein. Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL. Together, these data suggest that phosphorylation of survivin at Ser(20) by Plk1 kinase is essential for accurate chromosome alignment and cell proliferation but is dispensable for its anti-apoptotic activity in cancer cells.
Collapse
Affiliation(s)
- Rita Colnaghi
- From the Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom and
| | - Sally P. Wheatley
- the School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2UH, United Kingdom
| |
Collapse
|
|
41
|
Van Horn RD, Chu S, Fan L, Yin T, Du J, Beckmann R, Mader M, Zhu G, Toth J, Blanchard K, Ye XS. Cdk1 activity is required for mitotic activation of aurora A during G2/M transition of human cells. J Biol Chem 2010; 285:21849-57. [PMID: 20444701 PMCID: PMC2898447 DOI: 10.1074/jbc.m110.141010] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2009] [Revised: 05/04/2010] [Indexed: 11/06/2022] Open
Abstract
In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G(2)/M transition. We find that activation of Aurora A first occurs at centrosomes at late G(2) and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.
Collapse
Affiliation(s)
- Robert D. Van Horn
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| | - Shaoyou Chu
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| | - Li Fan
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| | - Tinggui Yin
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| | - Jian Du
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| | - Richard Beckmann
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| | - Mary Mader
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| | - Guoxin Zhu
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| | - John Toth
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| | - Kerry Blanchard
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| | - Xiang S. Ye
- From Lilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, Indiana 46285
| |
Collapse
|
|
42
|
Beria I, Ballinari D, Bertrand JA, Borghi D, Bossi RT, Brasca MG, Cappella P, Caruso M, Ceccarelli W, Ciavolella A, Cristiani C, Croci V, De Ponti A, Fachin G, Ferguson RD, Lansen J, Moll JK, Pesenti E, Posteri H, Perego R, Rocchetti M, Storici P, Volpi D, Valsasina B. Identification of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as a new class of orally and selective Polo-like kinase 1 inhibitors. J Med Chem 2010; 53:3532-51. [PMID: 20397705 DOI: 10.1021/jm901713n] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Polo-like kinase 1 (Plk1) is a fundamental regulator of mitotic progression whose overexpression is often associated with oncogenesis and therefore is recognized as an attractive therapeutic target in the treatment of proliferative diseases. Here we discuss the structure-activity relationship of the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline class of compounds that emerged from a high throughput screening (HTS) campaign as potent inhibitors of Plk1 kinase. Furthermore, we describe the discovery of 49, 8-{[2-methoxy-5-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide, as a highly potent and specific ATP mimetic inhibitor of Plk1 (IC(50) = 0.007 microM) as well as its crystal structure in complex with the methylated Plk1(36-345) construct. Compound 49 was active in cell proliferation against different tumor cell lines with IC(50) values in the submicromolar range and active in vivo in the HCT116 xenograft model where it showed 82% tumor growth inhibition after repeated oral administration.
Collapse
Affiliation(s)
- Italo Beria
- Nerviano Medical Sciences Srl, Oncology, Viale Pasteur 10, 20014 Nerviano, (Mi), Italy.
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
43
|
Kim JA, Lee J, Margolis RL, Fotedar R. SP600125 suppresses Cdk1 and induces endoreplication directly from G2 phase, independent of JNK inhibition. Oncogene 2010; 29:1702-16. [PMID: 20062077 PMCID: PMC3145494 DOI: 10.1038/onc.2009.464] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2009] [Revised: 10/14/2009] [Accepted: 10/22/2009] [Indexed: 12/20/2022]
Abstract
Cell cycle controls ensure that DNA replication (S phase) follows mitosis resulting in two precise copies of the genome. A failure of the control mechanisms can result in multiple rounds of DNA replication without cell division. In endoreplication, cells with replicated genomes bypass mitosis, then replicate their DNA again, resulting in polyploidy. Endoreplication from G2 phase lacks all hallmarks of mitosis. Using synchronized cells, we show that the c-Jun N-terminal kinase (JNK) inhibitor, SP600125, prevents the entry of cells into mitosis and leads to endoreplication of DNA from G2 phase. We show that cells proceed from G2 phase to replicate their DNA in the absence of mitosis. This effect of SP600125 is independent of its suppression of JNK activity. Instead, the inhibitory effect of SP600125 on mitotic entry predominantly occurs upstream of Aurora A kinase and Polo-like kinase 1, resulting in a failure to remove the inhibitory phosphorylation of Cdk1. Importantly, our results directly show that the inhibition of Cdk1 activity and the persistence of Cdk2 activity in G2 cells induces endoreplication without mitosis. Furthermore, endoreplication from G2 phase is independent of p53 control.
Collapse
Affiliation(s)
- JA Kim
- Sidney Kimmel Cancer Center, San Diego, CA, USA
| | - J Lee
- Sidney Kimmel Cancer Center, San Diego, CA, USA
| | - RL Margolis
- Sidney Kimmel Cancer Center, San Diego, CA, USA
- Burnham Institute for Medical Research, La Jolla, CA, USA
| | - R Fotedar
- Sidney Kimmel Cancer Center, San Diego, CA, USA
- Burnham Institute for Medical Research, La Jolla, CA, USA
| |
Collapse
|
|
44
|
Zhao C, Gong L, Li W, Chen L. Overexpression of Plk1 promotes malignant progress in human esophageal squamous cell carcinoma. J Cancer Res Clin Oncol 2010; 136:9-16. [PMID: 19572149 DOI: 10.1007/s00432-009-0630-4] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2008] [Accepted: 06/12/2009] [Indexed: 11/28/2022]
Abstract
PURPOSE Plk1, belonging to a family of serine/threonine kinases, is involved in spindle formation and chromosome segregation during mitosis and therefore, in the regulation of cell cycle. Plk1 was found to be overexpressed in various human tumors. In the present work, we investigated the expression of human esophageal squamous cell carcinoma (ESCC) to determine whether Plk1 has a role in malignant progress. METHODS Immunohistochemistry and Western blotting were performed to define the expressions of Plk1 in ESCC tissues and normal adjacent tissues. Transfection of cells with small interference RNA, growth suppression assay, and Transwell assay were used to determine the potential role of Plk1 in ESCC malignant progress. RESULTS Plk1 was overexpressed in 69.6% of the ESCC tissues. In addition, the extent of Plk1 expression was closely correlated with differentiation grades and invasiveness in ESCC. We also found that the downregulation of endogenous Plk1 in human ESSC cell lines Eca-109 and EC9706 significantly decreased cells proliferation and migrating ability. CONCLUSIONS Our results show that Plk1 expression is elevated in ESCC tissues and is associated with differentiation grades and invasiveness in ESCC, indicating that overexpression of Plk1 may play an important role in carcinogenesis and malignant progress of ESCC.
Collapse
Affiliation(s)
- Chunling Zhao
- Basic Medicine Department, Weifang Medical University, 261053 Weifang, Shandong, People's Republic of China.
| | | | | | | |
Collapse
|
|
45
|
Abstract
The core machinery that drives the eukaryotic cell cycle has been thoroughly investigated over the course of the past three decades. It is only more recently, however, that light has been shed on the mechanisms by which elements of this core machinery are modulated to alter cell cycle progression during development. It has also become increasingly clear that, conversely, core cell cycle regulators can play a crucial role in developmental processes. Here, focusing on findings from Drosophila melanogaster and Caenorhabditis elegans, we review the importance of modulating the cell cycle during development and discuss how core cell cycle regulators participate in determining cell fates.
Collapse
Affiliation(s)
- Yemima Budirahardja
- Swiss Institute for Experimental Cancer Research (ISREC Sciences, Swiss Federal Institute of Technology), Lausanne, Switzerland
| | | |
Collapse
|
|
46
|
Pomerening JR. Positive-feedback loops in cell cycle progression. FEBS Lett 2009; 583:3388-96. [PMID: 19818353 DOI: 10.1016/j.febslet.2009.10.001] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2009] [Revised: 09/28/2009] [Accepted: 10/01/2009] [Indexed: 01/19/2023]
Abstract
A positive-feedback loop is a simple motif that is ubiquitous to the modules and networks that comprise cellular signaling systems. Signaling behaviors that are synonymous with positive feedback include amplification and rapid switching, maintenance, and the coherence of outputs. Recent advances have been made towards understanding how positive-feedback loops function, as well as their mechanistic basis in controlling eukaryotic cell cycle progression. Some of these advances will be reviewed here, including: how cyclin controls passage through Start and maintains coherence of G1/S regulon expression in yeast; how Polo-like kinase 1 activation is driven by Bora and Aurora A, and its expression is stimulated by Forkhead Box M1 in mammalian cells; and how some of the various dynamic behaviors of spindle assembly and anaphase onset can be produced.
Collapse
Affiliation(s)
- Joseph R Pomerening
- Department of Biology, Interdisciplinary Program in Biochemistry, Indiana University, 212 S. Hawthorne Drive Simon Hall Room 043F, Bloomington, IN 47405-7003, United States.
| |
Collapse
|
|
47
|
Involvement of the dehydroleucodine alpha-methylene-gamma-lactone function in GVBD inhibition inBufo arenarumoocytes. ZYGOTE 2009; 18:41-9. [DOI: 10.1017/s0967199409990086] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
SummaryDehydroleucodine (DhL), a sesquiterpenic lactone, was isolated and purified from aerial parts ofArtemisia douglasianaBesser, a medicinal herb used in Argentina. DhL is an alpha-methylene butyro-gamma-lactone ring connected to a seven-membered ring fused to an exocyclic alpha,beta-unsaturated cyclopentenone ringIt has been previously shown that DhL selectively induces a dose-dependent transient arrest in G2of both meristematic cells and vascular smooth muscle cells. Treatment with DhL induces an inhibition of spontaneous and progesterone-induced maturation in a dose-dependent manner inBufo arenarumfully grown oocytes arrested at G2, at the beginning of meiosis I. However, the nature of the mechanisms involved in the process is still unknown.The aim of this work was to analyse whether DhL's alpha-methylene-gamma-lactone function is responsible for the inhibition effect on meiosis reinitiation ofBufo arenarumoocytes as well as some of the transduction pathways that could be involved in this effect using a derivative of DhL inactivated for alpha-methylenelactone, the 11,13-dihydro-dehydroleucodine (2H-DhL).The use of 2H-DhL in the maturation promoting factor (MPF) amplification experiments by injection of both cytoplasm with active MPF and of germinal vesicle content showed results similar to the ones obtained with DhL, suggesting that the hydrogenated derivative would act in a similar way to DhL.Pretreatment with DhL or 2H-DhL did not affect the percentage of germinal vesicle breakdown (GVBD) induced by H89, a protein kinase A (PKA) inhibitor, which suggests that these lactones would act on another step of the signalling pathway that induces MPF activation. The fact that both DhL and 2H-Dhl inhibit GVBD induced by okadaic acid microinjection suggests that they could act on the activity of the Myt1 kinase. This idea is supported by the experiments of injection of GV contents in which an inhibitory effect of these lactones on GVBD was also observed.Our results indicate that the inhibitory effect on meiosis progression of DhL does not depend only on the activity of the alpha-methylenelactone function, as its hydrogenated derivative, 2H-DhL, in which this function has been inactivated, causes similar effects on amphibian oocytes. However, 2H-DhL was less active than DhL as higher doses were required to obtain a significant inhibition. On the other hand, the analysis of the participation of certain mediators in some of the signalling pathways leading to MPF activation suggests that the Myt1 kinase could be a target of these lactones, while cdc25 phosphatase would not be affected. Besides, the PKA inhibition assays indicate that these lactones would act earlier in the signalling pathways.
Collapse
|
|
48
|
Kishi K, van Vugt MATM, Okamoto KI, Hayashi Y, Yaffe MB. Functional dynamics of Polo-like kinase 1 at the centrosome. Mol Cell Biol 2009; 29:3134-50. [PMID: 19307309 PMCID: PMC2682011 DOI: 10.1128/mcb.01663-08] [Citation(s) in RCA: 74] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2008] [Revised: 11/20/2008] [Accepted: 03/12/2009] [Indexed: 11/20/2022] Open
Abstract
Polo-like kinase 1 (Plk1) functions as a key regulator of mitotic events by phosphorylating substrate proteins on centrosomes, kinetochores, the mitotic spindle, and the midbody. Through mechanisms that are incompletely understood, Plk1 is released from and relocalizes to different mitotic structures as cells proceed through mitosis. We used fluorescence recovery after photobleaching to examine the kinetics of this process in more detail. We observed that Plk1 displayed a range of different recovery rates that differ at each mitotic substructure and depend on both the Polo-box domain and a functional kinase domain. Upon mitotic entry, centrosomal Plk1 becomes more dynamic, a process that is directly enhanced by Plk1 kinase activity. In contrast, Plk1 displays little dynamic exchange at the midbody, a process that again is modulated by the kinase activity of Plk1. Our findings suggest that the intrinsic kinase activity of Plk1 triggers its release from early mitotic structures and its relocalization to late mitotic structures. To assess the importance of Plk1 dynamic relocalization, Plk1 was persistently tethered to the centrosome. This resulted in a G(2) delay, followed by a prominent prometaphase arrest, as a consequence of defective spindle formation and activation of the spindle checkpoint. The dynamic release of Plk1 from early mitotic structures is thus crucial for mid- to late-stage mitotic events and demonstrates the importance of a fully dynamic Plk1 at the centrosome for proper cell cycle progression. This dependence on dynamic Plk1 was further observed during the mitotic reentry of cells after a DNA damage G(2) checkpoint, as this process was significantly delayed upon centrosomal tethering of Plk1. These results indicate that mitotic progression and control of mitotic reentry after DNA damage resides, at least in part, on the dynamic behavior of Plk1.
Collapse
Affiliation(s)
- Kazuhiro Kishi
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | | | | | | | | |
Collapse
|
|
49
|
Zhang L, Hou SY, Wang D, Wu K, Xia L. Effects of thioglycolic acid on progesterone-induced maturation of Xenopus oocytes. JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH. PART A 2009; 72:1123-1131. [PMID: 20077179 DOI: 10.1080/15287390902953519] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
In order to examine the effects of thioglycolic acid (TGA) on reproduction, Xenopus oocytes were treated with different concentrations of TGA. During culture, frequencies of germinal vesicle breakdown (GVBD) and MI-MII transition were determined. Samples collected at indicated times were subjected to immunoblotting. Data indicated that TGA accelerated the frequency of GVBD, but inhibited polar body extrusion and formation of MII-arrested eggs in a concentration-dependent manner. At 4 h after progesterone addition, phosphorylation of extracellular signal-regulated kinase (ERK) and p90 ribosomal S6 kinase, two members of the mitogen-activated protein kinase (MAPK) pathway, was upregulated in TGA-treated oocytes. The regulatory subunit of M-phase promoting factor (MPF)-cyclin B was also upregulated by TGA, while phospho-Cdc2 was downregulated. At 8 h, Cdc2 dephosphorylation and cyclin B1 were downregulated by TGA treatment. However, TGA exerted no effect on Mos, an MAPKKK (MAPK kinase kinase). In conclusion, TGA has the potential to inhibit in vitro maturation of Xenopus oocyte with increased GVBD frequency accompanied by alterations in protein expression and phosphorylation involved in MPF and MAPK pathways. Since egg formation is essential to maintain appropriate reproductive capacity, our findings may have certain toxicological implications.
Collapse
Affiliation(s)
- Ling Zhang
- Department of Nutrition and Food Hygiene, Harbin Medical University, Heilongjiang, China
| | | | | | | | | |
Collapse
|
|
50
|
The spindle pole body plays a key role in controlling mitotic commitment in the fission yeast Schizosaccharomyces pombe. Biochem Soc Trans 2008; 36:1097-101. [PMID: 18793196 DOI: 10.1042/bst0361097] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Commitment to mitosis is regulated by a conserved protein kinase complex called MPF (mitosis-promoting factor). MPF activation triggers a positive-feedback loop that further promotes the activity of its activating phosphatase Cdc25 and is assumed to down-regulate the MPF-inhibitory kinase Wee1. Four protein kinases contribute to this amplification loop: MPF itself, Polo kinase, MAPK (mitogen-activated protein kinase) and Greatwall kinase. The fission yeast SPB (spindle pole body) component Cut12 plays a critical role in modulating mitotic commitment. In this review, I discuss the relationship between Cut12 and the fission yeast Polo kinase Plo1 in mitotic control. These results indicate that commitment to mitosis is co-ordinated by control networks on the spindle pole. I then describe how the Cut12/Plo1 control network links growth control signalling from TOR (target of rapamycin) and MAPK networks to the activation of MPF to regulate the timing of cell division.
Collapse
|