Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.
- MeSH
- Anaphase MeSH
- Anaphase-Promoting Complex-Cyclosome metabolism MeSH
- cdc25 Phosphatases MeSH
- Meiosis * MeSH
- Metaphase MeSH
- Mice MeSH
- Oocytes * metabolism MeSH
- Mammals MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The role of hydrogen sulfide (H2S) is addressed in Xenopuslaevis oocytes. Three enzymes involved in H2S metabolism, cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, were detected in prophase I and metaphase II-arrested oocytes and drove an acceleration of oocyte meiosis resumption when inhibited. Moreover, meiosis resumption is associated with a significant decrease in endogenous H2S. On another hand, a dose-dependent inhibition was obtained using the H2S donor, NaHS (1 and 5 mM). NaHS impaired translation. NaHS did not induce the dissociation of the components of the M-phase promoting factor (MPF), cyclin B and Cdk1, nor directly impacted the MPF activity. However, the M-phase entry induced by microinjection of metaphase II MPF-containing cytoplasm was diminished, suggesting upstream components of the MPF auto-amplification loop were sensitive to H2S. Superoxide dismutase and catalase hindered the effects of NaHS, and this sensitivity was partially dependent on the production of reactive oxygen species (ROS). In contrast to other species, no apoptosis was promoted. These results suggest a contribution of H2S signaling in the timing of amphibian oocytes meiosis resumption.
- MeSH
- Apoptosis drug effects MeSH
- Cyclin B metabolism MeSH
- Cystathionine beta-Synthase antagonists & inhibitors metabolism MeSH
- Cystathionine gamma-Lyase antagonists & inhibitors metabolism MeSH
- Cytoplasm metabolism MeSH
- Maturation-Promoting Factor metabolism MeSH
- cdc25 Phosphatases metabolism MeSH
- Catalase metabolism MeSH
- Meiosis drug effects MeSH
- Metaphase drug effects MeSH
- Oocytes chemistry enzymology metabolism MeSH
- Meiotic Prophase I drug effects MeSH
- Protein Kinases metabolism MeSH
- Cell Cycle Proteins metabolism MeSH
- Xenopus Proteins metabolism MeSH
- Reactive Oxygen Species metabolism MeSH
- Signal Transduction drug effects MeSH
- Hydrogen Sulfide metabolism MeSH
- Sulfides metabolism pharmacology MeSH
- Sulfurtransferases antagonists & inhibitors metabolism MeSH
- Superoxide Dismutase metabolism MeSH
- Cell Survival drug effects MeSH
- Xenopus laevis MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Chromosome Aberrations MeSH
- Chromosome Deletion MeSH
- cdc25 Phosphatases genetics MeSH
- Immunomodulation MeSH
- Lenalidomide MeSH
- Humans MeSH
- Chromosomes, Human, Pair 5 * genetics MeSH
- Myelodysplastic Syndromes * drug therapy genetics pathology MeSH
- Tumor Suppressor Protein p53 MeSH
- Peptide Hydrolases genetics MeSH
- Protein Phosphatase 2 genetics MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Thalidomide * analogs & derivatives pharmacology therapeutic use MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified. SCOPE: Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco ('Samsun') cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied, although determination of endogenous cytokinin levels revealed a dramatic decrease in Spcdc25 transgenics. CONCLUSIONS: The data gained using the plants expressing yeast mitotic activator, Spcdc25, clearly argue for the existence and importance of activatory dephosphorylation at G₂/M transition and its interaction with cytokinin signalling in plants. The observed cytokinin-like effects of Spcdc25 expression are consistent with the concept of interaction between cell cycle regulators and phytohormones during plant development. The G₂/M control of the plant cell cycle, however, remains an elusive issue as doubts persist about the mode of activatory dephosphorylation, which in other eukaryotes is provided by Cdc25 phosphatase serving as a final all-or-nothing mitosis regulator.
- MeSH
- Cyclin-Dependent Kinases metabolism MeSH
- Cytokinins metabolism MeSH
- Eukaryotic Cells cytology MeSH
- cdc25 Phosphatases metabolism MeSH
- Phosphorylation MeSH
- G2 Phase MeSH
- Plants, Genetically Modified MeSH
- Mitosis MeSH
- Morphogenesis MeSH
- Schizosaccharomyces enzymology MeSH
- Nicotiana cytology embryology genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
Přeruš. str. : il., tab. ; 31 cm
Studium aktivace Chk1 a Chk2 kinázy v G1-fázi buněčného cyklu u myších embryonálních kmenových buněk. Charakterizace kináz zapojených do regulace kontrolního bodu v G1 fázi u myších embryonálních kmenových buněk. Studium vlivu aktivního onkoproteinu na průchod buněk buněčným cyklem u myších embryonálních kmenových buněk.; Study of activation of Chk1 and Chk2 kinases in G1 checkpoint in mouse embryonic stem cells. Characterization of kinases involved in regulation of G1 checkpoint in mouse embryonic stem cells. Study of influence of an active oncoprotein on the cell cycleprogression in mouse embryonic stem cells.
- MeSH
- Chromosome Breakpoints MeSH
- Cell Cycle MeSH
- Embryonic Stem Cells MeSH
- TNF Receptor-Associated Factor 2 MeSH
- cdc25 Phosphatases MeSH
- G1 Phase MeSH
- Hematopoietic Stem Cells physiology MeSH
- Methyltransferases MeSH
- Mice MeSH
- Tumor Suppressor Proteins analysis MeSH
- Tumor Suppressor Protein p53 analysis MeSH
- Myeloid-Lymphoid Leukemia Protein MeSH
- Ataxia Telangiectasia MeSH
- Check Tag
- Mice MeSH
- Conspectus
- Biochemie. Molekulární biologie. Biofyzika
- NML Fields
- biologie
- cytologie, klinická cytologie
- NML Publication type
- závěrečné zprávy o řešení grantu IGA MZ ČR
In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate-dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21(Cip1) accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.
- MeSH
- Autoantigens genetics metabolism MeSH
- Cell Cycle genetics MeSH
- Genes, cdc MeSH
- Chromatin * genetics metabolism MeSH
- Chromosomes metabolism MeSH
- DNA genetics metabolism MeSH
- DNA Breaks, Double-Stranded MeSH
- cdc25 Phosphatases genetics metabolism MeSH
- Radiation, Ionizing MeSH
- Mi-2 Nucleosome Remodeling and Deacetylase Complex genetics metabolism MeSH
- Humans MeSH
- RNA, Small Interfering metabolism pharmacology MeSH
- Cell Line, Tumor MeSH
- DNA Repair * MeSH
- DNA Damage * physiology MeSH
- RNA Interference MeSH
- Signal Transduction * genetics MeSH
- Ubiquitin genetics metabolism MeSH
- Ubiquitination MeSH
- Check Tag
- Humans MeSH
Cyclin-dependent kinase two (Cdk2) is the major regulator of the G1/S transition and the target of an activated G1 checkpoint in somatic cells. In the presence of DNA damage, Cdk2 kinase activity is abrogated by a deficiency of Cdc25A phosphatase, which is marked by Chk1/Chk2 for proteasomal degradation. Embryonic stem cells (ESCs) lack a G1 checkpoint response. In this study, we analyzed the G1 checkpoint pathways in mouse ESCs (mESCs) in the presence of DNA double-strand breaks evoked by ionizing radiation (IR). We show that checkpoint pathways, which operate during G1 phase in somatic cells, are activated in mESCs after IR; however, Cdk2 activity is not abolished. We demonstrate that Cdc25A is degraded in mESCs, but this degradation is not regulated by Chk1 and Chk2 kinases because they are sequestered to the centrosome. Instead, Cdc25A degradation is governed by glycogen synthase kinase-3beta kinase. We hypothesize that Cdc25A degradation does not inhibit Cdk2 activity because a considerable proportion of Cdk2 molecules localize to the cytoplasm and centrosomes in mESCs, where they may be sheltered from regulation by nuclear Cdc25A. Finally, we show that a high Cdk2 activity, which is irresponsive to DNA damage, is the driving force of the rapid escape of mESCs from G1 phase after DNA damage.
- MeSH
- Enzyme Activation genetics MeSH
- Cell Line MeSH
- Cell Cycle genetics MeSH
- Genes, cdc physiology MeSH
- Centrosome enzymology MeSH
- Cyclin-Dependent Kinase 2 genetics MeSH
- Cytoplasm enzymology MeSH
- DNA genetics radiation effects MeSH
- Embryonic Stem Cells cytology enzymology MeSH
- cdc25 Phosphatases genetics MeSH
- G1 Phase genetics MeSH
- Radiation, Ionizing MeSH
- Glycogen Synthase Kinase 3 metabolism MeSH
- Mice MeSH
- DNA Repair MeSH
- DNA Damage genetics MeSH
- Protein Serine-Threonine Kinases genetics MeSH
- Protein Kinases genetics MeSH
- Signal Transduction genetics MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Embryonic stem cells progress very rapidly through the cell cycle, allowing limited time for cell cycle regulatory circuits that typically function in somatic cells. Mechanisms that inhibit cell cycle progression upon DNA damage are of particular importance, as their malfunction may contribute to the genetic instability observed in human embryonic stem cells (hESCs). In this study, we exposed undifferentiated hESCs to DNA-damaging ultraviolet radiation-C range (UVC) light and examined their progression through the G1/S transition. We show that hESCs irradiated in G1 phase undergo cell cycle arrest before DNA synthesis and exhibit decreased cyclin-dependent kinase two (CDK2) activity. We also show that the phosphatase Cdc25A, which directly activates CDK2, is downregulated in irradiated hESCs through the action of the checkpoint kinases Chk1 and/or Chk2. Importantly, the classical effector of the p53-mediated pathway, protein p21, is not a regulator of G1/S progression in hESCs. Taken together, our data demonstrate that cultured undifferentiated hESCs are capable of preventing entry into S-phase by activating the G1/S checkpoint upon damage to their genetic complement.
- MeSH
- Cell Differentiation MeSH
- Cell Line MeSH
- Cyclin-Dependent Kinase 2 metabolism MeSH
- cdc25 Phosphatases metabolism MeSH
- G1 Phase radiation effects MeSH
- Stem Cells cytology metabolism radiation effects MeSH
- Humans MeSH
- DNA Damage MeSH
- Protein Serine-Threonine Kinases metabolism MeSH
- Protein Kinases metabolism MeSH
- S Phase radiation effects MeSH
- Signal Transduction MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life. Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling. In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B-Cdk1.
- MeSH
- Enzyme Activation MeSH
- Antigens * genetics metabolism MeSH
- Cell Line MeSH
- Centrosome metabolism MeSH
- Cyclin B genetics metabolism MeSH
- cdc25 Phosphatases genetics metabolism MeSH
- Humans MeSH
- Microcephaly genetics physiopathology MeSH
- Mitosis * physiology MeSH
- Protein Kinases * genetics metabolism MeSH
- Nerve Tissue Proteins * genetics metabolism MeSH
- RNA Interference MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a -/- mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.
