RESUMO
Mitosis in early embryos often proceeds at a rapid pace, but how this pace is achieved is not understood. Here, we show that cyclin B3 is the dominant driver of rapid embryonic mitoses in the C. elegans embryo. Cyclins B1 and B2 support slow mitosis (NEBD to anaphase â¼600 s), but the presence of cyclin B3 dominantly drives the approximately threefold faster mitosis observed in wildtype. Multiple mitotic events are slowed down in cyclin B1 and B2-driven mitosis, and cyclin B3-associated Cdk1 H1 kinase activity is â¼25-fold more active than cyclin B1-associated Cdk1. Addition of cyclin B1 to fast cyclin B3-only mitosis introduces an â¼60-s delay between completion of chromosome alignment and anaphase onset; this delay, which is important for segregation fidelity, is dependent on inhibitory phosphorylation of the anaphase activator Cdc20. Thus, cyclin B3 dominance, coupled to a cyclin B1-dependent delay that acts via Cdc20 phosphorylation, sets the rapid pace and ensures mitotic fidelity in the early C. elegans embryo.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Ciclina B1 , Embrião não Mamífero , Mitose , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Ciclina B1/metabolismo , Ciclina B1/genética , Embrião não Mamífero/metabolismo , Fosforilação , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC2/genética , Ciclina B/metabolismo , Ciclina B/genética , Proteínas Cdc20/metabolismo , Proteínas Cdc20/genética , Ciclina B2/metabolismo , Ciclina B2/genéticaRESUMO
The study of the physiological and pathophysiological processes under extreme conditions facilitates a better understanding of the state of a healthy organism and can also shed light on the pathogenesis of diseases. In recent years, it has become evident that gravitational stress affects both the whole organism and individual cells. We have previously demonstrated that simulated microgravity inhibits proliferation, induces apoptosis, changes morphology, and alters the surface marker expression of megakaryoblast cell line MEG-01. In the present work, we investigate the expression of cell cycle cyclins in MEG-01 cells. We performed several experiments for 24 h, 72 h, 96 h and 168 h. Flow cytometry and Western blot analysis demonstrated that the main change in the levels of cyclins expression occurs under conditions of simulated microgravity after 96 h. Thus, the level of cyclin A expression showed an increase in the RPM group during the first 4 days, followed by a decrease, which, together with the peak of cyclin D, may indicate inhibition of the cell cycle in the G2 phase, before mitosis. In addition, based on the data obtained by PCR analysis, we were also able to see that both cyclin A and cyclin B expression showed a peak at 72 h, followed by a gradual decrease at 96 h. STED microscopy data also confirmed that the main change in cyclin expression of MEG-01 cells occurs at 96 h, under simulated microgravity conditions, compared to static control. These results suggested that the cell cycle disruption induced by RPM-simulated microgravity in MEG-01 cells may be associated with the altered expression of the main regulators of the cell cycle. Thus, these data implicate the development of cellular stress in MEG-01 cells, which may be important for proliferating human cells exposed to microgravity in real space.
Assuntos
Ciclo Celular , Ciclinas , Simulação de Ausência de Peso , Humanos , Linhagem Celular , Ciclinas/metabolismo , Ciclinas/genética , Células Progenitoras de Megacariócitos/metabolismo , Células Progenitoras de Megacariócitos/citologia , Ciclina A/metabolismo , Ciclina A/genética , Proliferação de Células , Ciclina B/metabolismo , Ciclina B/genéticaAssuntos
Proteínas Correpressoras , Ciclina B , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Sarcoma , Humanos , Masculino , Ciclina B/genética , Ciclina B/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sarcoma/genética , Sarcoma/patologia , Sarcoma/metabolismo , Hibridização in Situ Fluorescente , Diagnóstico Diferencial , Neoplasias de Tecidos Moles/genética , Neoplasias de Tecidos Moles/patologia , Neoplasias de Tecidos Moles/metabolismo , Imuno-Histoquímica , Adulto , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismoRESUMO
Meiosis is a complex variant of the mitotic cell cycle, and as such relies on many of the same proteins involved in mitosis, but utilizes these in novel ways. As in mitosis, Cdk1 and its cyclin partners, Cyclin A, B, and B3 are required at multiple steps in meiosis. Here, we study the effect of stabilized forms of the three mitotic cyclins to study the consequences of failure to degrade the cyclins in meiosis. We find that stabilized Cyclin B3 promotes ectopic microtubule polymerization throughout the egg, dependent on APC/C activity and apparently due to the consequent destruction of Cyclin A and Cyclin B. We present data that suggests CycB, and possibly CycA, can also promote APC/C activity at specific stages of meiosis. We also present evidence that in meiosis APC/CCort and APC/CFzy are able to target Cyclin B via a novel degron. Overall, our findings highlight the distinct functions of the three mitotic Cdk-cyclin complexes in meiosis.
Assuntos
Ciclina B , Ciclinas , Proteínas de Drosophila , Meiose , Mitose , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Ciclina B/metabolismo , Ciclina B/genética , Ciclinas/metabolismo , Ciclinas/genética , Ciclina A/metabolismo , Drosophila/metabolismo , Drosophila/genética , Microtúbulos/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genéticaRESUMO
Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.
Assuntos
Proteína Quinase CDC2 , Ciclina B , Proteína Quinase CDC2/metabolismo , Fosforilação , Ciclina B/genética , Ciclina B/metabolismo , Proteínas/metabolismo , MitoseRESUMO
Entry into mitosis has been classically attributed to the activation of a cyclin B/Cdk1 amplification loop via a partial pool of this kinase becoming active at the end of G2 phase. However, how this initial pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, depletion of the orthologue of FAM122A in C. elegans prevents entry into mitosis in germline stem cells. Moreover, data from Xenopus egg extracts strongly suggest that FAM122A-dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. Inhibition of this phosphatase allows subsequent phosphorylation of early mitotic substrates by cyclin A/Cdk, resulting in full cyclin B/Cdk1 and Greatwall (Gwl) kinase activation. Subsequent to Greatwall activation, Arpp19/ENSA become phosphorylated and now compete with FAM122A, promoting its dissociation from PP2A-B55 and taking over its phosphatase inhibition role until the end of mitosis.
Assuntos
Caenorhabditis elegans , Proteínas Serina-Treonina Quinases , Animais , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Mitose , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismoRESUMO
The cyclin-dependent kinase 1 (Cdk1)-cyclin B (CycB) complex plays critical roles in cell-cycle regulation. Before Drosophila male meiosis, CycB is exported from the nucleus to the cytoplasm via the nuclear porin 62kD (Nup62) subcomplex of the nuclear pore complex. When this export is inhibited, Cdk1 is not activated, and meiosis does not initiate. We investigated the mechanism that controls the cellular localization and activation of Cdk1. Cdk1-CycB continuously shuttled into and out of the nucleus before meiosis. Overexpression of CycB, but not that of CycB with nuclear localization signal sequences, rescued reduced cytoplasmic CycB and inhibition of meiosis in Nup62-silenced cells. Full-scale Cdk1 activation occurred in the nucleus shortly after its rapid nuclear entry. Cdk1-dependent centrosome separation did not occur in Nup62-silenced cells, whereas Cdk1 interacted with Cdk-activating kinase and Twine/Cdc25C in the nuclei of Nup62-silenced cells, suggesting the involvement of another suppression mechanism. Silencing of roughex rescued Cdk1 inhibition and initiated meiosis. Nuclear export of Cdk1 ensured its escape from inhibition by a cyclin-dependent kinase inhibitor. The complex re-entered the nucleus via importin ß at the onset of meiosis. We propose a model regarding the dynamics and activation mechanism of Cdk1-CycB to initiate male meiosis.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Masculino , Drosophila/metabolismo , Transporte Ativo do Núcleo Celular , Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Meiose , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ciclina B/metabolismoRESUMO
During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5â days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here, we show that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8â h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.
Assuntos
Proteínas de Drosophila , Meiose , Animais , Masculino , Meiose/genética , Espermatogênese/fisiologia , Prófase , Mitose , Espermatócitos/metabolismo , Drosophila/genética , Ciclina B/genética , Ciclina B/metabolismo , Proteínas de Drosophila/metabolismoRESUMO
Cell cycle progression during development is meticulously coordinated with differentiation. This is particularly evident in the Drosophila 3rd instar eye imaginal disc, where the cell cycle is synchronized and arrests at the G1 phase in the non-proliferative region (NPR), setting the stage for photoreceptor cell differentiation. Here, we identify the transcription factor Nuclear Factor-YC (NF-YC) as a crucial player in this finely tuned progression, elucidating its specific role in the synchronized movement of the morphogenetic furrow. Depletion of NF-YC leads to extended expression of Cyclin A (CycA) and Cyclin B (CycB) from the FMW to the NPR. Notably, NF-YC knockdown resulted in decreased expression of Eyes absent (Eya) but did not affect Decapentaplegic (Dpp) and Hedgehog (Hh). Our findings highlight the role of NF-YC in restricting the expression of CycA and CycB in the NPR, thereby facilitating cell-cycle synchronization. Moreover, we identify the transcriptional cofactor Eya as a downstream target of NF-YC, revealing a new regulatory pathway in Drosophila eye development. This study expands our understanding of NF-YC's role from cell cycle control to encompass developmental processes.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Discos Imaginais/metabolismo , Proteínas Hedgehog/metabolismo , Olho/metabolismo , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Ciclina B/metabolismoRESUMO
Adult stem cells maintain homeostatic self-renewal through the strategy of either population or single-cell asymmetry, and the former type of stem cells are thought to take passive while the latter ones take active competition for niche occupancy. Although the division ability of stem cells is known to be crucial for their passive competition, whether it is also crucial for active competition is still elusive. Drosophila female germline stem cells are thought to take active competition, and bam mutant germ cells are more competitive than wild-type germline stem cells for niche occupancy. Here we report that either cycB, cycE, cdk2, or rheb null mutation drastically attenuates the division ability and niche occupancy capacity of bam mutant germ cells. Conversely, accelerating their cell cycle by mutating hpo has an enhanced effect. Last but not least, we also determine that E-cadherin, which was proposed to be crucial previously, just plays a mild role in bam mutant germline niche occupancy. Together with previous studies, we propose that division ability plays a unified crucial role in either active or passive competition among stem cells for niche occupancy.
Assuntos
Células-Tronco Adultas , Proteínas de Drosophila , Animais , Feminino , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Transdução de Sinais/genética , Diferenciação Celular/genética , Ovário/metabolismo , Drosophila/genética , Células-Tronco Adultas/metabolismo , Células Germinativas/metabolismo , Nicho de Células-Tronco/genética , Drosophila melanogaster/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ciclina B/metabolismoAssuntos
Fertilização , Meiose , Feminino , Humanos , Meiose/genética , Oócitos , Ciclina B/metabolismoRESUMO
Cyclin B (CycB) plays essential roles in cell proliferation and promotes gonad development in many crustaceans. The goal of this study was to investigate the regulatory roles of this gene in the reproductive development of male oriental river prawns (Macrobrachium nipponense). A phylo-genetic tree analysis revealed that the protein sequence of Mn-CycB was most closely related to those of freshwater prawns, whereas the evolutionary distance from crabs was much longer. A quantitative PCR analysis showed that the expression of Mn-CycB was highest in the gonad of both male and female prawns compared to that in other tissues (p < 0.05), indicating that this gene may play essential roles in the regulation of both testis and ovary development in M. nipponense. In males, Mn-CycB expression in the testis and androgenic gland was higher during the reproductive season than during the non-reproductive season (p < 0.05), implying that CycB plays essential roles in the reproductive development of male M. nipponense. An RNA interference analysis revealed that the Mn-insulin-like androgenic gland hormone expression decreased as the Mn-CycB expression decreased, and that few sperm were detected 14 days after the dsCycB treatment, indicating that CycB positively affects testis development in M. nipponense. The results of this study highlight the functions of CycB in M. nipponense, and they can be applied to studies of male reproductive development in other crustacean species.
Assuntos
Decápodes , Palaemonidae , Animais , Feminino , Masculino , Palaemonidae/genética , Palaemonidae/metabolismo , Interferência de RNA , Ciclina B/genética , Ciclina B/metabolismo , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Sêmen/metabolismoRESUMO
The DNA repair scaffold SLX4 has multifaceted roles in genome stability, many of which depend on structure-selective endonucleases. SLX4 coordinates the cell cycle-regulated assembly of SLX1, MUS81-EME1, and XPF-ERCC1 into a tri-nuclease complex called SMX. Mechanistically, how the mitotic kinase CDK1 regulates the interaction between SLX4 and MUS81-EME1 remains unclear. Here, we show that CDK1-cyclin B phosphorylates SLX4 residues T1544, T1561, and T1571 in the MUS81-binding region (SLX4MBR). Phosphorylated SLX4MBR relaxes the substrate specificity of MUS81-EME1 and stimulates cleavage of replication and recombination structures, providing a biochemical explanation for the chromosome pulverization that occurs when SLX4 binds MUS81 in S-phase. Remarkably, phosphorylation of SLX4MBR drives folding of an SAP domain, which underpins the high-affinity interaction with MUS81. We also report the structure of phosphorylated SLX4MBR and identify the MUS81-binding interface. Our work provides mechanistic insights into how cell cycle-regulated phosphorylation of SLX4 drives the recruitment and activation of MUS81-EME1.
Assuntos
Endonucleases , Recombinases , Endonucleases/metabolismo , Fosforilação , Recombinases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Reparo do DNA , Ciclina B/metabolismo , Endodesoxirribonucleases/metabolismoRESUMO
To ensure successful offspring ploidy, vertebrate oocytes must halt the cell cycle in meiosis II until sperm entry. Emi2 is essential to keep oocytes arrested until fertilization. However, how this arrest is implemented exclusively in meiosis II and not prematurely in meiosis I has until now remained enigmatic. Using mouse and frog oocytes, we show here that cyclin B3, an understudied B-type cyclin, is essential to keep Emi2 levels low in meiosis I. Direct phosphorylation of Emi2 at an evolutionarily highly conserved site by Cdk1/cyclin B3 targets Emi2 for degradation. In contrast, Cdk1/cyclin B1 is inefficient in Emi2 phosphorylation, and this provides a molecular explanation for the requirement of different B-type cyclins for oocyte maturation. Cyclin B3 degradation at exit from meiosis I enables Emi2 accumulation and thus timely arrest in meiosis II. Our findings illuminate the evolutionarily conserved mechanisms that control oocyte arrest for fertilization at the correct cell-cycle stage, which is essential for embryo viability.
Assuntos
Proteínas F-Box , Animais , Ciclina B/metabolismo , Ciclina B1 , Ciclinas/metabolismo , Proteínas F-Box/genética , Fertilização , Masculino , Meiose , Camundongos , Oócitos/metabolismo , Sêmen/metabolismo , Vertebrados/metabolismoRESUMO
The term M-phase supershift denotes the phosphorylation-dependent substantial increase in the apparent molecular weight of numerous proteins of varied biological functions during M-phase induction. Although the M-phase supershift of multiple key mitotic regulators has been attributed to the multisite phosphorylation catalyzed by the Cdk1/cyclin B/Cks complex, this view is challenged by multiple lines of paradoxical observations. To solve this problem, we reconstituted the M-phase supershift of Xenopus Cdc25C, Myt1, Wee1A, APC3, and Greatwall in Xenopus egg extracts and characterized the supershift-producing phosphorylations. Our results demonstrate that their M-phase supershifts are each due to simultaneous phosphorylation of a considerable portion of S/T/Y residues in a long intrinsically disordered region that is enriched in both S/T residues and S/TP motifs. Although the major mitotic kinases in Xenopus egg extracts, Cdk1, MAPK, Plx1, and RSK2, are able to phosphorylate the five mitotic regulators, they are neither sufficient nor required to produce the M-phase supershift. Accordingly, inhibition of the four major mitotic kinase activities in Xenopus oocytes did not inhibit the M-phase supershift in okadaic acid-induced oocyte maturation. These findings indicate that the M-phase supershift is produced by a previously unrecognized category of mitotic phosphorylation that likely plays important roles in M-phase induction.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Xenopus , Animais , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Mitose , Ácido Okadáico/metabolismo , Oócitos/metabolismo , Fosforilação , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ('CDK') CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1, confirming and extending previous results. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle, but is degraded after all divisions have ceased. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1 signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by 'comets' of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC modulate microtubule function and assembly while regulating mitotic progression. Genetic results suggest an independent additional role for the APC in regulating sister chromatid cohesion; this role is likely conserved across eukaryotes.
Assuntos
Chlamydomonas , Ciclina B , Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chlamydomonas/genética , Chlamydomonas/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/genética , Mitose/genética , Fuso Acromático/metabolismoRESUMO
RNA stability control contributes to the proper expression of gene products. Messenger RNAs (mRNAs) in eukaryotic cells possess a 5' cap structure and the 3' poly(A) tail which are important for mRNA stability and efficient translation. The Ccr4-Not complex is a major cytoplasmic deadenylase and functions in mRNA degradation. The CLB1-6 genes in Saccharomyces cerevisiae encode B-type cyclins which are involved in the cell cycle progression together with the cyclin-dependent kinase Cdc28. The CLB genes consist of CLB1/2, CLB3/4, and CLB5/6 whose gene products accumulate at the G2-M, S-G2, and late G1 phase, respectively. These Clb protein levels are thought to be mainly regulated by the transcriptional control and the protein stability control. Here we investigated regulation of CLB1-6 expression by Ccr4. Our results show that all CLB1-6 mRNA levels were significantly increased in the ccr4Δ mutant compared to those in wild-type cells. Clb1, Clb4, and Clb6 protein levels were slightly increased in the ccr4Δ mutant, but the Clb2, Clb3, and Clb5 protein levels were similar to those in wild-type cells. Since both CLB6 mRNA and Clb6 protein levels were most significantly increased in the ccr4Δ mutant, we further analyzed the cis-elements for the Ccr4-mediated regulation within CLB6 mRNA. We found that there were destabilizing sequences in both coding sequence and 3' untranslated region (3' UTR). The destabilizing sequences in the coding region were found to be both within and outside the sequences corresponding the cyclin domain. The CLB6 3' UTR was sufficient for mRNA destabilization and decrease of the reporter GFP gene and this destabilization involved Ccr4. Our results suggest that CLB6 expression is regulated by Ccr4 through the coding sequence and 3' UTR of CLB6 mRNA.
Assuntos
Ciclina B , Ribonucleases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regiões 3' não Traduzidas/genética , Ciclina B/metabolismo , Ciclinas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Fase S , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Regulation of the cell cycle is an understudied response to oxygen deprivation among crustaceans. The virile crayfish, Orconectes virilis, is a freshwater crustacean that when challenged by environmental oxygen limitation undergoes metabolic rate depression (to ~30% of normal levels) and switches to anaerobic metabolism to generate energy. To understand how crayfish regulate the cell cycle in response to anoxia, key proteins involved in cell cycle control were analyzed in muscle and hepatopancreas. At the G1/S barrier, an overall upregulation of positive regulators of cell cycle progression was indicated by the responses of G1 cyclins (cyclin D and cyclin E) and Cyclin dependent kinases (CDK4, CDK6 and CDK2) under anoxia. Although the levels of Cyclin kinase inhibitors (CKIs) at this juncture were also upregulated (P15/16 and P21 (T145) in muscle and P16 (S152) in hepatopancreas), levels of a major regulator of this phase and driver to S-phase, E2F1, were significantly higher in both tissues in conjunction with deactivation of its inhibitor, Retinoblastoma (Rb) protein. At the G2/M barrier, expression profiles of the G2 cyclin B suggested cell cycle progression despite overall trend of higher activities of checkpoint kinases, (Chk1 (S317) and Chk2 (S19)), that also negatively regulate the cyclin B-CDK1 complex via CdC25C (cell division cycle 25) whose levels remained unchanged. Overall, the present study suggests continued cell cycle progression, albeit with potential deceleration, as indicated by checkpoint kinases and kinase inhibitor profiles that might play a role in protecting tissues from apoptotic damage under chronic anoxic stress.
Assuntos
Astacoidea , Proteínas de Ciclo Celular , Animais , Astacoidea/metabolismo , Ciclo Celular/fisiologia , Ciclina B/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Água Doce , Hepatopâncreas/metabolismo , Hipóxia/metabolismo , Músculos/metabolismo , Oxigênio/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases , Proteína do Retinoblastoma/metabolismo , CaudaRESUMO
We describe a method for rapid identification of protein kinase substrates within the nuclear envelope. Open mitosis in higher eukaryotes is characterized by nuclear envelope breakdown (NEBD) concerted with disassembly of the nuclear lamina and dissociation of nuclear pore complexes (NPCs) into individual subcomplexes. Evidence indicates that reversible phosphorylation events largely drive this mitotic NEBD. These posttranslational modifications likely disrupt structurally significant interactions among nucleoporins (Nups), lamina and membrane proteins of the nuclear envelope (NE). It is therefore critical to determine when and where these substrates are phosphorylated. One likely regulator is the mitotic kinase: Cdk1-Cyclin B. We employed an "analog-sensitive" Cdk1 to bio-orthogonally and uniquely label its substrates in the NE with a phosphate analog tag. Subsequently, peptides covalently modified with the phosphate analogs are rapidly purified by a tag-specific covalent capture and release methodology. In this manner, we were able to confirm the identity of known Cdk1 targets in the NE and discover additional candidates for regulation by mitotic phosphorylation.
Assuntos
Membrana Nuclear , Fosfopeptídeos , Animais , Ciclina B/química , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Células HeLa , Humanos , Mitose , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosfatos/metabolismo , Fosfopeptídeos/isolamento & purificação , Fosfopeptídeos/metabolismo , RatosRESUMO
BACKGROUND: Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for the maturation of cardiopulmonary function. Preterm and low-birth-weight infants often experience liver dysfunction after birth because their livers are immature. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration on the maturation of liver hepatocytes in preterm rats. METHODS AND RESULTS: Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational days 17 and 19 before cesarean section. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the mRNA levels of albumin, hepatocyte nuclear factor-4 alpha (HNF4α), hepatocyte growth factor (HGF), thymus cell antigen 1 (Thy-1), cyclin B, and Cyclin-dependent kinase 1 (CDK1) in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay were performed to examine protein production. The hepatocytes enlarged because of growth and prenatal DEX administration. Albumin, HNF4α, and HGF levels increased secondary to growth and prenatal DEX administration. The levels of the cell cycle markers cyclin B and CDK1 gradually decreased during growth and with DEX administration. CONCLUSIONS: The results suggest that prenatal GC administration leads to hepatocyte maturation via expression of HNF4α and HGF in preterm fetuses.