RESUMEN
In Xenopus oocyte, the formation of complexes between neosynthesized cyclins and Cdc2 contributes to Cdc2 kinase activation that triggers meiotic divisions. It has been proposed that cytoplasmic membranes could be involved in this process. To investigate this possibility, we have injected in the oocyte two undegradable human cyclin A2 mutants anchored to the endoplasmic reticulum (ER) membrane. They encode fusion proteins between the truncated cyclin A2-Delta152 and a viral or cellular ER-targeting domain. We show that both mutants are fully functional as mitotic cyclins when expressed in Xenopus oocytes, bind Cdc2 and activate M-phase promoting factor.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclina A/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Oocitos/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Membrana Celular/metabolismo , Ciclina A2 , Quinasa 2 Dependiente de la Ciclina , Activación Enzimática , Proteínas de Xenopus , Xenopus laevisRESUMEN
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.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Metafase/fisiología , Oocitos/citología , Animales , Proteínas de Ciclo Celular/metabolismo , Femenino , Ligasas/metabolismo , Meiosis , Ubiquitina-Proteína Ligasas , Xenopus laevisRESUMEN
We have cloned and sequenced a Xenopus cDNA referred to as EF-1 delta 2. The cDNA is homologous to EF-1 delta 1 encoding for EF-1 delta a protein of the guanine-nucleotide exchange complex of elongation factor-1 (EF-1). The protein sequence deduced from the cDNA, contains the two characteristic features of EF-1 delta protein, the leucine-zipper domain and the guanine-nucleotide exchange domain. In vitro and in vivo translation leads to the production of a 36-kDa protein from EF-1 delta and a 34-kDa protein from EF-1 delta 1. The clone EF-1 delta 2 therefore encodes for authentic p36 protein of EF-1 beta gamma delta complex, while EF-1 delta 1 encodes for a newly characterised p34 protein of the leucine zipper family. Both EF-1 delta proteins are simultaneously present in oocytes extracts, at a molecular ratio around 1:10 for p34 versus p36 proteins. Both are associated in a macromolecular structure that is greater than 750 kDa upon gel filtration. The two proteins are targets for Cdc2 kinase in meiotic maturation.
Asunto(s)
Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Clonación Molecular , ADN Complementario/genética , Femenino , Nucleótidos de Guanina/metabolismo , Técnicas In Vitro , Leucina Zippers/genética , Sustancias Macromoleculares , Meiosis , Datos de Secuencia Molecular , Peso Molecular , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Factor 1 de Elongación Peptídica , Factores de Elongación de Péptidos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Xenopus laevisRESUMEN
Brefeldin A, a fungal metabolite which disrupts protein traffic, provokes indirect activation of cdc2 protein kinase in Xenopus oocytes. Cdc2 protein kinase activation was judged by MPF (M-phase factor) transfer activity, histone H1 kinase activity, and phosphorylation in vivo of the guanine-nucleotide exchange complex EF-1 beta gamma delta. Oocytes resumed complete meiosis upon brefeldin A treatment. Cdc2 protein kinase, MAP kinase, cyclin B, MPF, and protein synthesis changes were all comparable in brefeldin A-treated oocytes and in progesterone-induced oocytes. ED50 for brefeldin A was 0.6 microM. Brefeldin A activation of cdc2 protein kinase occurs with a long time course. Simultaneous treatment of the oocytes at a subthreshold concentration of 1 nM progesterone and 30 microM brefeldin A considerably shortened the kinetics of maturation. Brefeldin A induction of maturation was sensitive to drugs that act on cAMP metabolism. ID50 for IBMX was 0.1 mM, compared to 1 mM for progesterone-treated oocytes. Brefeldin A inhibited protein traffic in oocytes as determined from protein export experiments. ID50 was between 0.1 and 1 microM. Our results give new insights into the possible mechanism of induction of meiotic maturation and further demonstrate that brefeldin A acts on cell cycle regulatory elements.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclopentanos/farmacología , Factor Promotor de Maduración/metabolismo , Meiosis/efectos de los fármacos , Oocitos/efectos de los fármacos , 1-Metil-3-Isobutilxantina/farmacología , Animales , Brefeldino A , Ciclo Celular/efectos de los fármacos , Toxina del Cólera/farmacología , Colforsina/análogos & derivados , Colforsina/farmacología , AMP Cíclico/fisiología , Cicloheximida/farmacología , Activación Enzimática/efectos de los fármacos , Oocitos/enzimología , Oogénesis/efectos de los fármacos , Factor 1 de Elongación Peptídica , Factores de Elongación de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Progesterona/farmacología , Protamina Quinasa/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Xenopus laevisRESUMEN
Elongation factor-1 (EF-1) is a major substrate for cdc2 kinase in Xenopus oocytes. The guanine-nucleotide exchange factor EF-1 beta gamma delta, appears to have a highly complex macromolecular structure containing several GTP/GDP exchange proteins, valyl-tRNA synthetase, and a putative anchoring protein EF-1 gamma. During meiotic cell division, the factor becomes phosphorylated by cdc2 kinase, not only on EF-1 gamma, but also on two different phospho-acceptors on EF-1 delta. Phosphorylation is concomitant with changes in protein synthesis in vivo. Xenopus oocytes, and potentially all cells, contain a multitude of heteromeric forms of the complex which postulates that EF-1 beta gamma delta is not a "house keeping" factor but a sophisticated regulatory element.
Asunto(s)
Proteína Quinasa CDC2/fisiología , Factores de Elongación de Péptidos/metabolismo , Animales , Meiosis , Oocitos , Factor 1 de Elongación Peptídica , Fosforilación , XenopusRESUMEN
A new physiological target for Cdc2 protein kinase has been identified. It corresponds to a protein EF-1 delta, a constituent of the nucleotide exchange factor EF-1 beta gamma delta, involved in the elongation step of protein synthesis. EF-1 delta is phosphorylated by Cdc2 kinase on threonine and serine residues. Threonine has been identified as Thr122 in the sequence VQVTPAAK. During oocyte maturation, Thr122 is phosphorylated at metaphase, when p34cdc2 is active. Phosphorylation studies revealed the presence of two post-translational regulated forms of EF-1 delta protein. Identification of two isoforms of the delta protein, together with the presence of two guanine-nucleotide exchange proteins (beta and delta) and physiologically regulated phosphorylation sites by Cdc2 kinase on gamma and delta proteins, implicate that EF-1 beta gamma delta exists in the cell under a multitude of macromolecular forms which suggests that EF-1 beta gamma delta is a sophisticated regulatory factor rather than a "housekeeping" element of the cell.
Asunto(s)
Factor Promotor de Maduración/metabolismo , Oocitos/metabolismo , Factores de Elongación de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteína Quinasa CDC2/metabolismo , Metafase , Datos de Secuencia Molecular , Oocitos/citología , Oogénesis , Factor 1 de Elongación Peptídica , Fosforilación , Especificidad por Sustrato , Xenopus laevisRESUMEN
EF-1 delta is a physiological substrate for cdc2 protein kinase in Xenopus oocytes. The protein is part of the nucleotide exchange factor EF-1 beta gamma delta, involved in the elongation step of protein synthesis. We show that EF-1 delta exists under four isoforms in the prophase oocyte, all phosphorylable by casein kinase II. Each of the prophase isoforms was further separated into a 36 and a 38 kDa form upon phosphorylation by cdc2 kinase which therefore reveals the existence of eight different isoforms. Phosphorylation by cdc2 kinase can be monitored as the electrophoretic mobility dedoublement 36/38 kDa. Developmental regulation of EF-1 delta was analyzed. The cdc2 kinase-induced change occures at meiotic division, after complete oogenesis and perdures during early development. It is therefore a phosphorylation memory signal for early development.
Asunto(s)
Oocitos/crecimiento & desarrollo , Factores de Elongación de Péptidos/metabolismo , Animales , Proteína Quinasa CDC2/metabolismo , Meiosis , Factor 1 de Elongación Peptídica , Fosforilación , Xenopus laevisAsunto(s)
Factores de Elongación de Péptidos/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Factores de Intercambio de Guanina Nucleótido , Datos de Secuencia Molecular , Factor 1 de Elongación Peptídica , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , XenopusRESUMEN
The cdc2 protein kinase phosphorylates elongation factor-1 gamma (EF-1 gamma) during meiotic maturation of Xenopus oocytes. A synthetic peptide P2: PKKETPKKEKPA matching the cDNA-deduced sequence of EF-1 gamma was an in vitro substrate for cdc2 protein kinase and inhibited phosphorylation of EF-1 gamma. Tryptic hydrolysis of EF-1 gamma and the P2 peptide, both phosphorylated by cdc2 protein kinase, resulted in multiple partial digestion products generated by the presence of barely hydrolysable bonds. The two peptides obtained from the hydrolysis of EF-1 gamma comigrated exactly in two-dimensional separation with two of the P2 peptide hydrolysates. EF-1 gamma therefore contains one unique phosphoacceptor for cdc2 protein kinase, identified as threonine-230.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Factores de Elongación de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Datos de Secuencia Molecular , Factor 1 de Elongación Peptídica , Fosforilación , Treonina/metabolismo , XenopusAsunto(s)
Factores de Elongación de Péptidos/genética , Ribonucleoproteínas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Biblioteca de Genes , Datos de Secuencia Molecular , Factor 1 de Elongación Peptídica , Factores de Elongación de Péptidos/química , Ribonucleoproteínas/química , Xenopus/genéticaRESUMEN
A polyclonal antibody was raised against bacterially produced catalytic alpha subunit of protein phosphatase 2A (PP2AC) cloned from Xenopus ovarian library. The amount of PP2AC in Xenopus oocytes determined by Western blot analysis was 1 ng/microgram of cytosolic protein. The antibody depleted PP2AC from oocyte extracts in association with 6 components (40, 62, 65, 80, 85 and 90 kDa). Prophase- and metaphase-arrested oocytes contained identical amounts of PP2AC. Metaphase oocytes showed one specific change in the 62 kDa protein associated with PP2AC.
Asunto(s)
Meiosis , Oocitos/enzimología , Fosfoproteínas Fosfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , División Celular , ADN/genética , Femenino , Biblioteca de Genes , Datos de Secuencia Molecular , Oocitos/citología , Ovario/enzimología , Fosfoproteínas Fosfatasas/análisis , Fosfoproteínas Fosfatasas/aislamiento & purificación , Proteína Fosfatasa 2 , Mapeo Restrictivo , XenopusRESUMEN
The M-phase-specific cdc2 (cell division control) protein kinase (a component of the M-phase-promoting factor) was found to activate casein kinase II in vitro. The increase in casein kinase II activity ranged over 1.5-5-fold. Increase in activity was prevented if ATP was replaced during the activation reaction by a non-hydrolysable analogue. Alkaline phosphatase treatment of the activated enzyme decreased the activity to the basal level. The beta subunit of casein kinase II was phosphorylated by cdc2 protein kinase at site(s) different from the autophosphorylation sites of the enzyme. Phosphoamino acid analysis showed that the beta subunit was phosphorylated by cdc2 protein kinase at threonine residues while autophosphorylation involved serine residues. Casein kinase II may be part of the cascade which leads to increased phosphorylation of many proteins at M-phase and therefore be involved in the pleiotropic effects of M-phase-promoting factor.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Autorradiografía , Caseína Quinasas , Ciclo Celular , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Femenino , Técnicas In Vitro , Cinética , Factor Promotor de Maduración/metabolismo , Mitosis , Fosforilación , Estrellas de Mar , Xenopus laevisRESUMEN
M-Phase specific protein kinase or cdc2 protein kinase is a component of MPF (M-Phase promoting factor). During meiotic maturation of Xenopus oocytes, cdc2 protein kinase is activated in correlation with MPF activity. A protein phosphorylation cascade takes place involving several protein kinases, among which casein kinase II, and different changes associated with meiosis occur such as germinal vesicle breakdown, chromosome condensation, cytoskeletal reorganization and increase in protein synthesis. Our results provide a biochemical link between cdc2 protein kinase and protein synthesis since they show that the kinase phosphorylates in vitro a p47 protein identified as elongation factor EF1 (gamma subunit) and that the in vitro site of p47 corresponds to the site phosphorylated in vivo. Immunofluorescence showed that the elongation factor (EF1-beta gamma) is localized in the oocyte cortex. Furthermore, they show that cdc2 kinase phosphorylates and activates casein kinase II in vitro, strongly supporting the view that casein kinase II is involved in the phosphorylation cascade originated by cdc2 kinase.
Asunto(s)
Meiosis , Oocitos/citología , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Animales , Proteína Quinasa CDC2 , Femenino , Modelos Biológicos , Oocitos/enzimología , Oocitos/fisiología , Fosfoproteínas/aislamiento & purificación , Fosforilación , XenopusRESUMEN
A high molecular mass complex isolated from Xenopus laevis oocytes contains three main proteins, respectively p30, p36 and p47. The p47 protein has been reported to be an in vivo substrate of the cell division control protein kinase p34cdc2. From polypeptide sequencing, we now show that the p30 and the p47 correspond to elongation factor EF-1 beta and EF-1 gamma. Furthermore, the p30 and p36 proteins were phosphorylated in vitro by casein kinase II.
Asunto(s)
Sustancias de Crecimiento/metabolismo , Oocitos/análisis , Factores de Elongación de Péptidos/aislamiento & purificación , Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Caseína Quinasas , Electroforesis en Gel de Poliacrilamida , Factor Promotor de Maduración , Datos de Secuencia Molecular , Factor 1 de Elongación Peptídica , Péptidos/aislamiento & purificación , Fosforilación , Proteínas Quinasas/metabolismo , Homología de Secuencia de Ácido Nucleico , XenopusRESUMEN
This paper describes the purification of a 47 kDa protein from Xenopus laevis oocytes that becomes phosphorylated when the oocytes undergo meiotic maturation. This protein (p47) is part of a high molecular mass complex containing at least two other proteins of molecular mass 30 and 36 kDa. This complex can be isolated from stage VI oocytes before maturation. We obtained a pattern for phosphopeptides in p47 phosphorylated in vivo very similar to that of the purified protein phosphorylated in vitro by p34cdc2 (a H1 kinase which is a component of the M-phase promoting factor) and [gamma-32P]ATP. Therefore, the purified p47, already described as a marker of MPF activity, is the first reported in vivo substrate for the cell division control kinase.