RESUMO
Cyclin-dependent kinases (Cdks) are key regulators of the eukaryotic cell division cycle. Cdk1 (Cdc2) and Cdk2 should be bound to regulatory subunits named cyclins as well as phosphorylated on a conserved Thr located in the T-loop for full enzymatic activity. Cdc2- and Cdk2-cyclin complexes can be inactivated by phosphorylation on the catalytic cleft-located Thr-14 and Tyr-15 residues or by association with inhibitory subunits such as p21(Cip1). We have recently identified a novel Cdc2 regulator named RINGO that plays an important role in the meiotic cell cycle of Xenopus oocytes. RINGO can bind and activate Cdc2 but has no sequence homology to cyclins. Here we report that, in contrast with Cdc2- cyclin complexes, the phosphorylation of Thr-161 is not required for full activation of Cdc2 by RINGO. We also show that RINGO can directly stimulate the kinase activity of Cdk2 independently of Thr-160 phosphorylation. Moreover, RINGO-bound Cdc2 and Cdk2 are both less susceptible to inhibition by p21(Cip1), whereas the Thr-14/Tyr-15 kinase Myt1 can negatively regulate the activity of Cdc2-RINGO with reduced efficiency. Our results indicate that Cdk-RINGO complexes may be active under conditions in which cyclin-bound Cdks are inhibited and can therefore play different regulatory roles.
Assuntos
Proteína Quinase CDC2/genética , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/genética , Ciclinas/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Animais , Quinase 2 Dependente de Ciclina , Humanos , Proteínas Recombinantes/genética , Xenopus , Proteínas de XenopusRESUMO
Xenopus oocytes arrested in prophase I resume meiotic division in response to progesterone and arrest at metaphase II. Entry into meiosis I depends on the activation of Cdc2 kinase [M-phase promoting factor (MPF)]. To better understand the role of Cdc2, MPF activity was specifically inhibited by injection of the CDK inhibitor, Cip1. When Cip1 is injected at germinal vesicle breakdown (GVBD) time, Cdc25 and Plx1 are both dephosphorylated and Cdc2 is rephosphorylated on tyrosine. The autoamplification loop characterizing MPF is therefore not only required for MPF generation before GVBD, but also for its stability during the GVBD period. The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), responsible for cyclin degradation, is also under the control of Cdc2; therefore, Cdc2 activity itself induces its own inactivation through cyclin degradation, allowing the exit from the first meiotic division. In contrast, cyclin accumulation, responsible for Cdc2 activity increase allowing entry into metaphase II, is independent of Cdc2. The c-Mos/mitogen-activated protein kinase (MAPK) pathway remains active when Cdc2 activity is inhibited at GVBD time. This pathway could be responsible for the sustained cyclin neosynthesis. In contrast, during the metaphase II block, the c-Mos/MAPK pathway depends on Cdc2. Therefore, the metaphase II block depends on a dynamic interplay between MPF and CSF, the c-Mos/MAPK pathway stabilizing cyclin B, whereas in turn, MPF prevents c-Mos degradation.
Assuntos
Proteína Quinase CDC2/fisiologia , Metáfase , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Oócitos/fisiologia , Proteínas Proto-Oncogênicas c-mos/fisiologia , Proteínas de Xenopus , Animais , Proteínas de Ciclo Celular , Ciclina B/metabolismo , Ciclina B1 , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/farmacologia , Feminino , Meiose , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Xenopus , Fosfatases cdc25/fisiologiaRESUMO
The protozoan parasite Theileria (spp. parva and annulata) infects bovine leukocytes and provokes a leukaemia-like disease in vivo. In this study, we have detected a type 1 serine/threonine phosphatase activity with phosphorylase a as a substrate, in protein extracts of parasites purified from infected B lymphocytes. In contrast to this type 1 activity, dose response experiments with okadaic acid (OA), a well characterised inhibitor of type 1 and 2A protein phosphatases, indicated that type 2A is the predominant activity detected in host B cells. Furthermore, consistent with polycation-specific activation of the type 2A phosphatase, protamine failed to activate the parasite-associated phosphorylase a phosphatase activity. Moreover, inhibition of phosphorylase a dephosphorylation by phospho-DARPP-32, a specific type 1 inhibitor, clearly demonstrated that a type 1 phosphatase is specifically associated with the parasite, while the type 2A is predominantly expressed in the host lymphocyte. Since an antibody against bovine catalytic protein phosphatase 1 (PP1) subunit only recognised the PP1 in B cells, but not in parasite extracts, we conclude that in parasites the PP1 activity is of parasitic origin. Intriguingly, since type 1 OA-sensitive phosphatase activity has been recently described in Plasmodium falciparum, we can conclude that these medically important parasites produce their one PP1.
Assuntos
Linfócitos B/parasitologia , Fosfoproteínas Fosfatases/metabolismo , Theileria parva/enzimologia , Animais , Bovinos , Células Cultivadas , Fosforilase a/metabolismo , Proteína Fosfatase 1RESUMO
Xenopus prophase oocytes reenter meiotic division in response to progesterone. The signaling pathway leading to Cdc2 activation depends on neosynthesized proteins and a decrease in PKA activity. We demonstrate that Eg2 protein, a Xenopus member of the Aurora/Ipl1 family of protein kinases, accumulates in response to progesterone and is degraded after parthenogenetic activation. The polyadenylation and cap ribose methylation of Eg2 mRNA are not needed for the protein accumulation. Eg2 protein accumulation is induced by progesterone through a decrease in PKA activity, upstream of Cdc2 activation. Eg2 kinase activity is undetectable in prophase and is raised in parallel with Cdc2 activation. In contrast to Eg2 protein accumulation, Eg2 kinase activation is under Cdc2 control. Furthermore, by using an anti-sense strategy, we show that Eg2 accumulation is not required in the transduction pathway leading to Cdc2 activation. Altogether, our results strongly suggest that Eg2 is not necessary for Cdc2 activation, though it could participate in the organization of the meiotic spindles, in agreement with the well-conserved roles of the members of the Aurora family, from yeast to man.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Oócitos/enzimologia , Progesterona/fisiologia , Proteínas Quinases/metabolismo , Animais , Aurora Quinases , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC2/fisiologia , Diferenciação Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Feminino , Meiose , Oócitos/metabolismo , Oócitos/fisiologia , Partenogênese , Fosforilação , Poli A/metabolismo , Proteínas Serina-Treonina Quinases , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Ribose/metabolismo , Proteínas de Xenopus , Xenopus laevisRESUMO
Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34(cdc2) could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34(cdc2), and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor-induced feedback. We report here that the cdk inhibitor p21(cip1), when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21(cip1), progesterone fails to induce the activation of MAPK or p34(cdc2), and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.
Assuntos
Ciclina B/metabolismo , Ciclinas/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Oócitos/metabolismo , Progesterona/farmacologia , Animais , Proteína Quinase CDC2/metabolismo , Ciclina B1 , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/farmacologia , Feminino , Fator Promotor de Maturação/metabolismo , Microinjeções , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitose , Ácido Okadáico/farmacologia , Oócitos/efeitos dos fármacos , Ligação Proteica , Proteínas Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , XenopusRESUMO
The auto-catalytic activation of the cyclin-dependent kinase Cdc2 or MPF (M-phase promoting factor) is an irreversible process responsible for the entry into M phase. In Xenopus oocyte, a positive feed-back loop between Cdc2 kinase and its activating phosphatase Cdc25 allows the abrupt activation of MPF and the entry into the first meiotic division. We have studied the Cdc2/Cdc25 feed-back loop using cell-free systems derived from Xenopus prophase-arrested oocyte. Our findings support the following two-step model for MPF amplification: during the first step, Cdc25 acquires a basal catalytic activity resulting in a linear activation of Cdc2 kinase. In turn Cdc2 partially phosphorylates Cdc25 but no amplification takes place; under this condition Plx1 kinase and its activating kinase, Plkk1 are activated. However, their activity is not required for the partial phosphorylation of Cdc25. This first step occurs independently of PP2A or Suc1/Cks-dependent Cdc25/Cdc2 association. On the contrary, the second step involves the full phosphorylation and activation of Cdc25 and the initiation of the amplification loop. It depends both on PP2A inhibition and Plx1 kinase activity. Suc1-dependent Cdc25/Cdc2 interaction is required for this process.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Meiose/fisiologia , Oócitos/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe , Tirosina 3-Mono-Oxigenase , Proteínas de Xenopus , Xenopus/fisiologia , Fosfatases cdc25/antagonistas & inibidores , Fosfatases cdc25/metabolismo , Proteínas 14-3-3 , Animais , Sistema Livre de Células , Criança , Proteínas Fúngicas/metabolismo , Humanos , Proteína Fosfatase 2 , Proteínas/metabolismoRESUMO
The activation of Cdc2 kinase induces the entry into M-phase of all eukaryotic cells. We have developed a cell-free system prepared from prophase-arrested Xenopus oocytes to analyze the mechanism initiating the all-or-none activation of Cdc2 kinase. Inhibition of phosphatase 2A, the major okadaic acid-sensitive Ser/Thr phosphatase, in these extracts, provokes Cdc2 kinase amplification and concomitant hyperphosphorylation of Cdc25 phosphatase, with a lag of about 1 h. Polo-like kinase (Plx1 kinase) is activated slightly after Cdc2. All these events are totally inhibited by the cdk inhibitor p21(Cip1), demonstrating that Plx1 kinase activation depends on Cdc2 kinase activity. Addition of a threshold level of recombinant Cdc25 induces a linear activation of Cdc2 and Plx1 kinases and a partial phosphorylation of Cdc25. We propose that the Cdc2 positive feedback loop involves two successive phosphorylation steps of Cdc25 phosphatase: the first one is catalyzed by Cdc2 kinase and/or Plx1 kinase but it does not modify Cdc25 enzymatic activity, the second one requires a new kinase counteracted by phosphatase 2A. Furthermore we demonstrate that, under our conditions, Cdc2 amplification and MAP kinase activation are two independent events.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fator Promotor de Maturação/biossíntese , Oócitos/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citosol/enzimologia , Ativação Enzimática/efeitos dos fármacos , Retroalimentação/efeitos dos fármacos , Fator Promotor de Maturação/metabolismo , Ácido Okadáico/farmacologia , Oócitos/citologia , Prófase , Proteína Fosfatase 2 , Proteínas Tirosina Fosfatases/farmacologia , Xenopus , Fosfatases cdc25RESUMO
Oogonia undergo numerous mitotic cell cycles before completing the last DNA replication and entering the meiotic prophase I. After chromosome pairing and chromatid exchanges between paired chromosomes, the oocyte I remains arrested at the diplotene stage of the first meiotic prophase. Oocyte growth then occurs independently of cell division; indeed, during this growth period, oocytes (4n DNA) are prevented from completing the meiotic divisions. How is the prophase arrest regulated? One of the players of the prophase block is the high level of intracellular cAMP, maintained by an active adenylate cyclase. By using lethal toxin from Clostridium sordellii (LT), a glucosyltransferase that glucosylates and inactivates small G proteins of the Ras subfamily, we have shown that inhibition of either Ras or Rap or both proteins is sufficient to release the prophase block of Xenopus oocytes in a cAMP-dependent manner. The implications of Ras family proteins as new players involved in the prophase arrest of Xenopus oocytes will be discussed here.
Assuntos
Oócitos/química , Oócitos/citologia , Prófase/fisiologia , Proteínas ras/fisiologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Oócitos/enzimologia , XenopusRESUMO
The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that modifies and inhibits small G proteins of the Ras family, Ras and Rap, as well as Rac proteins. LT induces cdc2 kinase activation and germinal vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. Toxin B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2 activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xenopus oocyte. In oocyte extracts, LT catalyzes the incorporation of [14C]glucose into a group of proteins of 23 kDa and into one protein of 27 kDa. The 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras antibodies and probably corresponds to K-Ras. Microinjection of LT into oocytes together with UDP-[14C]glucose results in a glucosylation pattern similar to the in vitro glucosylation, indicating that the 23- and 27-kDa proteins are in vivo substrates of LT. In vivo time-course analysis reveals that the 27-kDa protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the 23-kDa proteins are partially glucosylated at GVBD. This observation suggests that the 27-kDa Ras protein could be the in vivo target of LT allowing cdc2 kinase activation. Interestingly, inactivation of Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that occurs normally around GVBD.
Assuntos
Toxinas Bacterianas/toxicidade , Proteína Quinase CDC2/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Oócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Clostridium , Ativação Enzimática/efeitos dos fármacos , Feminino , Oócitos/efeitos dos fármacos , XenopusRESUMO
Metaphase II arrest of Xenopus oocyte is characterized by the presence of M-phase-promoting factor (MPF) and of a microtubular spindle, both of which are stable in the presence of protein synthesis inhibitors. We studied in vivo this equilibrium state that is settled during meiotic maturation. At time of germinal vesicle breakdown (GVBD), cdc2 kinase and MAP kinase activities are stimulated. A component of the cyclin ubiquitin ligase, CDC27, is phosphorylated at the same time and remains phosphorylated until fertilization, indicating that an important component of the ligase complex is modified as early as GVBD. During a first period extending from GVBD until the cortical anchorage of the metaphase II spindle, homogeneous pools of cdc2 kinase and mitogen-activated protein (MAP) kinase activities are present in oocyte and are strictly dependent on protein turnover, since protein synthesis inhibition induces their total inactivation and drives oocytes into interphase. The metaphase II spindle, once anchored into the cortex, is no more sensitive to protein synthesis inhibition, likewise MAP kinase activity. During this cellular arrest, cdc2 kinase is divided into two distinctly regulated pools. The first one contains cyclin B that actively turns over and is subjected to a microtubular checkpoint. The second one is stable. Alteration of intracellular compartmentation of metaphase II oocytes either by gentle centrifugation or by cold shock inactivates MAP kinase and targets all cyclin B molecules for full destruction. We therefore suggest that MAP kinase participates to the cytostatic activity by preventing part of cyclin B molecules from entering the ubiquitination/degradation machinery which is still turned on in metaphase II oocytes.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Metáfase/fisiologia , Oócitos/citologia , Animais , Proteínas de Ciclo Celular/metabolismo , Feminino , Ligases/metabolismo , Meiose , Ubiquitina-Proteína Ligases , Xenopus laevisRESUMO
The effects of HIV-1 encoded proteins NCp7, vpr and NCp7:vpr complex on the activity of protein phosphatase-2A0 have been tested. We report that NCp7 is an activator of protein phosphatase-2A0 and that vpr activated protein phosphatase-2A0 only slightly. We also report that NCp7 and vpr form a tight complex which becomes a more potent activator of protein phosphatase-2A0 than NCp7 alone. The ability of NCp7 to activate protein phosphatase-2A0 is regulated by vpr. The C-terminal portion of vpr prevents NCp7 from activating protein phosphatase-2A0 while the N-terminal portion of vpr potentiates the effect of NCp7 on the activity of protein phosphatase-2A0. Our findings indicate that vpr may be acting as a targeting subunit which directs NCp7 to activate protein phosphatase-2A0. In view of the fact that protein phosphatase-2A functions as an inhibitor of G0 to M transition of the cell cycle and is involved in other key cellular processes such as the control of RNA transcription, the results presented in this report may explain how HIV-1 causes cell cycle arrest which may lead to CD4+ T cell depletion and also how it disturbs normal cellular processes of its host cell.
Assuntos
Proteínas do Capsídeo , Capsídeo/metabolismo , Produtos do Gene gag/metabolismo , Produtos do Gene vpr/metabolismo , HIV-1/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Virais , Animais , Capsídeo/genética , Ativação Enzimática , Produtos do Gene gag/genética , Produtos do Gene vpr/genética , Proteína Fosfatase 2 , Suínos , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
In Xenopus prophase-blocked oocytes, it is assumed that progesterone interacts with the plasma membrane to initiate a signalling cascade that ultimately leads to MPF activation. Progesterone regulates negatively the cAMP pathway through an inhibition of adenylate cyclase. However, the mechanisms linking the initial action of the hormone with adenylate cyclase activity remain to be elucidated. Here, we demonstrate that PDMP, an inhibitor of glucosphingolipid synthesis, triggers oocyte meiotic maturation in a cAMP- and cycloheximide-dependent manner, whereas exogenous ceramide is unefficient. We propose that sphingolipid metabolism and targeting represent an important regulatory process of oocyte meiosis.
Assuntos
Proteína Quinase CDC2/metabolismo , Inibidores Enzimáticos/farmacologia , Glicoesfingolipídeos/biossíntese , Morfolinas/farmacologia , Oócitos/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Toxina da Cólera/farmacologia , Cicloeximida/farmacologia , Ativação Enzimática , Feminino , Glicoesfingolipídeos/antagonistas & inibidores , Técnicas In Vitro , Cinética , Oócitos/citologia , Oócitos/efeitos dos fármacos , Progesterona/farmacologia , Prófase , Transdução de Sinais/efeitos dos fármacos , Xenopus laevisRESUMO
Growing stage IV Xenopus oocytes are unresponsive to progesterone treatment. They contain a store of preMPF composed of tyrosine phosphorylated p34cdc2 and cyclin B2. The endogenous store of preMPF cannot be recruited by cdc25 protein phosphatase or cyclin protein microinjections. This is in contrast with full-grown stage VI oocytes where microinjections of these proteins are known to activate the autoamplification of MPF. When cyclins are microinjected into stage IV oocytes, they associate with endogenous free p34cdc2 and the illegitimate complexes undergo phosphorylation on tyrosine 15. High doses of human cyclin A allow, however, part of the neoformed complexes to be activated as an histone-H1 kinase; this partial activation of p34cdc2 is sufficient to induce germinal vesicle breakdown in these small oocytes. Co-injections of cyclin A or cyclin B together with okadaic acid (10 microM in the microinjection solution), an inhibitor of protein phosphatase 2A (PP2A), lead to the full activation of neoformed p34cdc2/cyclin complexes. These results indicate that small oocytes possess an active tyrosine kinase that inactivates new p34cdc2/cyclin complexes. Inhibition of PP2A by okadaic acid prevents this inactivation reaction and conversely allows the illegitimate complex to be activated. Neither the activating phosphorylation on threonine 161 nor the inactivating phosphorylation on tyrosine 15 take place in stage IV enucleated oocytes. Altogether, our results show that the accumulation of inactive p34cdc2/cyclin B2 during the long-lasting prophase of the oocyte is positively controlled by PP2A through the tyrosine phosphorylation of p34cdc2.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclinas/metabolismo , Ciclinas/farmacologia , Oócitos/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Animais , Proteína Quinase CDC2/isolamento & purificação , Ciclinas/isolamento & purificação , Ativação Enzimática , Éteres Cíclicos/farmacologia , Feminino , Homeostase , Humanos , Microinjeções , Ácido Okadáico , Oócitos/citologia , Oócitos/efeitos dos fármacos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfotirosina , Protamina Quinase/metabolismo , Ligação Proteica , Proteína Fosfatase 2 , Tirosina/análogos & derivados , Tirosina/metabolismo , Xenopus laevisRESUMO
cDNA clones encoding the 65-kDa (PR65) and 55-kDa (PR55) regulatory subunits of protein phosphatase 2A from Xenopus laevis were isolated by homology screening with the corresponding human cDNAs, and used to analyze the developmental expression patterns of these genes. The PR65 subunit was found to be encoded by two genes, termed XPR65 alpha and XPR65 beta. The open reading frames of the alpha and beta cDNAs both span 1767 bp, and predict proteins of 64.4 kDa and 65.3 kDa, respectively, that are 87% identical. The predicted amino acid sequence of XPR65 alpha showed 95% and 84% identity with human PR65 alpha and PR65 beta proteins, respectively, whereas the identity of XPR65 beta with the same proteins was 87% and 86.5%, respectively. Only one type of Xenopus PR55 (XPR55) was isolated that showed 93% and 84% similarity to human PR55 alpha and PR55 beta, respectively. Analysis of the N-terminal region of XPR55 with the same regions of human PR55 alpha and PR55 beta, indicates that the XPR55 is the Xenopus homolog of the human PR55 alpha isoform. Despite the overall similarity with PR55 from other species, XPR55 has an N-terminal extention of at least 24 amino acids. In the ovary, a transcript of 2.8 kb, encoding the XPR65 beta, was predominantly expressed and these XPR65 beta mRNAs are present at a constant level during oogenesis until late embryogenesis. Expression of the 2.4-kb XPR65 alpha was low until the larval stage, then dramatically increased. In all adult tissues except ovary, the 2.4-kb alpha-specific mRNA was more abundant than the 2.8-kb beta transcript. Two transcripts of 2.4 kb and 2.5 kb, encoding the XPR55 subunit, were detected at a constant level throughout Xenopus oogenesis and during embryogenesis. Both transcripts were also expressed at similar levels in all adult tissues, but in a tissue-specific manner. Analysis of the XPR55 and XPR65 proteins using antibodies to recombinant proteins revealed that the overall levels of the two proteins were constant, in good agreement with mRNA data.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fosfoproteínas Fosfatases/genética , Proteínas de Plantas/genética , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Ciclo Celular , Clonagem Molecular , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/imunologia , Proteínas de Plantas/imunologia , Proteína Fosfatase 2 , RNA Mensageiro/análiseRESUMO
Activation of the cyclin B-cdc2 kinase mitotic inducer involves dephosphorylation of two inhibitory residues, tyrosine 15 and threonine 14, cdc25 is the specific phosphatase that directly dephosphorylates and activates the cdc2 kinase, cdc25 activity is regulated by phosphorylation. Both phosphatases 1 and 2A could act as cdc25-specific inhibitory phosphatases. Although the cyclin B-cdc2 complex plays a role in activating cdc25, it is highly probable that a distinct protein kinase is involved as a trigger in cdc25 activation. The implication of raf kinase as a cdc25-specific activating kinase in human cells and Xenopus oocytes is discussed.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Meiose , Mitose , Fosfoproteínas Fosfatases/fisiologia , Animais , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Ativação Enzimática , Humanos , Modelos Biológicos , Fosforilação , Xenopus , Fosfatases cdc25RESUMO
Microinjection of bacterially expressed human cdc25A protein into Xenopus prophase oocytes provokes the activation of p34cdc2 kinase and the tyrosine dephosphorylation of p34cdc2 in the presence or absence of protein synthesis. The level of p34cdc2 kinase activity then drops in parallel with the degradation of cyclin B2 and finally increases again to stabilize at a high level. Cdc25 microinjection induces the assembly of a metaphase I spindle which is abnormally located in the deep cytoplasm. Moreover, oocytes arrest at the metaphase I stage and do not reach metaphase II even 10 h after cdc25 microinjection. The extended metaphase I period observed in cdc25-injected oocytes results from an equilibrium between degradation of cyclins and synthesis of new cyclins. This is in contrast with progesterone-stimulated oocytes where cyclin degradation is turned off when oocytes enter metaphase II. During metaphase I, the reactivation of MPF activity can be disrupted in two different ways: 1) cycloheximide, an inhibitor of protein synthesis, by preventing the synthesis of new cyclins, provokes the disappearance of MPF kinase activity and the reformation of a nucleus; 2) when the cAMP level is increased during the metaphase I period in cdc25-injected oocytes, MPF kinase activity drops following a rephosphorylation of tyrosine 15 of p34cdc2, while the cyclin turn-over remains unaffected. Moreover, increasing the cAMP level in prophase oocytes totally prevents the action of cdc25. Our results indicate that in Xenopus oocytes, the PKA pathway negatively regulates the activation of MPF and the activity of p34cdc2/cyclin B complex through tyrosine phosphorylation of p34cdc2 during metaphase I.
Assuntos
Proteínas de Ciclo Celular/administração & dosagem , Proteínas de Ciclo Celular/farmacologia , Fator Promotor de Maturação/metabolismo , Meiose/fisiologia , Metáfase/fisiologia , Oócitos/citologia , Fosfoproteínas Fosfatases/administração & dosagem , Fosfoproteínas Fosfatases/farmacologia , Fosfoproteínas Fosfatases/fisiologia , Xenopus laevis/genética , Animais , Proteína Quinase CDC2/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclinas/metabolismo , Cicloeximida/farmacologia , Feminino , Microinjeções , Fosforilação , Prófase/efeitos dos fármacos , Precursores de Proteínas/metabolismo , Inibidores da Síntese de Proteínas/metabolismo , Fuso Acromático/genética , Fatores de Tempo , Fosfatases cdc25RESUMO
Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase II oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase I and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s).
Assuntos
Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Ativação Enzimática , Éteres Cíclicos/farmacologia , Feminino , Técnicas In Vitro , Fator Promotor de Maturação/farmacologia , Proteína Quinase 1 Ativada por Mitógeno , Ácido Okadáico , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Fosforilação , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Tirosina Quinases/isolamento & purificação , Tirosina/metabolismo , Xenopus laevisRESUMO
A cytological analysis was performed in order to determine how the formation of the metaphase I- and metaphase II-spindles is dependent upon p34cdc2 kinase activity and protein synthesis during the meiotic maturation of Xenopus oocytes. The p34cdc2 kinase activity increases abruptly during the prophase-prometaphase I transition, then drops to a minimum level at the metaphase I/anaphase I transition and further increases again until reaching a maximum stable level at metaphase II. The injection of an indestructible cyclin B into oocytes arrests the maturation process at the onset of anaphase I and prevents the re-increase of p34cdc2 activity which accompanies normal entry into metaphase II. Inhibition of protein synthesis between the germinal vesicle breakdown and the onset of metaphase I spindle induces exit from M-phase and leads to an 'interphase-like' state characterized by the organization of nuclear-like structures. In contrast, inhibition of protein synthesis at metaphase II stage does not affect the metaphase II spindle nor p34cdc2 activity, indicating that metaphase I- and metaphase II-spindles are not regulated by the same effectors. When protein synthesis is inhibited before induction of M-phase by MPF transfer, it prevents the formation of the metaphase I spindle, despite a transient elevated level of p34cdc2 activity. To dissociate the role of protein synthesis and of p34cdc2 kinase activity, the indestructible cyclin B was microinjected in the absence of protein synthesis. This allows the in vivo maintenance of a stable p34cdc2 activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ciclinas/farmacologia , Proteínas do Ovo/biossíntese , Metáfase/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Anáfase/fisiologia , Animais , Proteína Quinase CDC2/metabolismo , Senescência Celular/fisiologia , Feminino , Interfase/fisiologia , Fator Promotor de Maturação/metabolismo , Meiose/fisiologia , Oócitos/citologia , Fuso Acromático/fisiologia , Xenopus laevisRESUMO
Microinjection of a bacterially expressed stable delta 90 sea urchin cyclin B into Xenopus prophase oocytes, in absence or presence of cycloheximide, provokes the activation of histone H1 kinase and the tyrosine dephosphorylation of p34cdc2. Unexpectedly, when prophase oocytes are submitted to a treatment known to elevate the intracellular cAMP level (3-isobutyl-1-methylxanthine and cholera toxin), delta 90 cyclin has no effect and the oocytes remain blocked in prophase. This inhibition is reverted by the microinjection of the inhibitor of cAMP-dependent protein kinase. When delta 90 cyclin is microinjected into oocytes depleted of endogenous cyclins (cycloheximide-treated metaphase I) and in the presence of a high intracellular concentration of cAMP, p34cdc2 kinase is tyrosine rephosphorylated. Altogether, our results indicate that in Xenopus oocyte, cAMP-dependent protein kinase (A-kinase) controls the formation of the cyclin B/p34cdc2 complex which remains inactive and tyrosine phosphorylated.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclinas/farmacologia , Oócitos/metabolismo , Proteínas Quinases/fisiologia , Animais , Ciclinas/antagonistas & inibidores , Cicloeximida/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Microinjeções , Fosforilação/efeitos dos fármacos , Prófase , Inibidores de Proteínas Quinases , Tirosina/metabolismo , XenopusRESUMO
Xenopus oocytes are blocked in prophase of the first meiotic division. During the G2/M transition drastic changes occur both in the cytoskeletal organization and in the capacity of tubulin to polymerize. Posttranslational modification of tubulin isoforms might be one of the factors that control the dynamic properties of microtubules. We have therefore analysed, by two-dimensional polyacrylamide gel electrophoresis, the isotubulins purified from Xenopus oocytes, and we show that tubulin is resolved into at least four alpha-isoforms and four beta-isoforms. We have identified a basic alpha (alpha b)-tubulin isoform which is specific to prophase arrested oocyte and that progressively disappears during meiotic maturation; its decrease is initiated when the nuclear envelope breaks down and is controlled by the nucleus. Using 35S methionine labelled oocytes we demonstrate that the disappearance of the alpha b isotubulin results from both an arrest of its biosynthesis after maturation, and from posttranslational modification which induces a shift of this alpha-isoform to a more acidic pI. Moreover, in vitro experiments using 35S prelabelled tubulin purified from prophase oocytes show that metaphase extracts containing MPF activity are able to induce the acidification of the alpha b-isoform, suggesting that the observed posttranslational modification might be regulated by p34cdc2. However, the nature of this modification remains to be elucidated.