RESUMEN
Fast excitatory transmission in the mammalian central nervous system is mediated by AMPA-type glutamate receptors. The tetrameric AMPA receptor complexes are composed of four subunits, GluR1-4. The GluR4 subunit is highly expressed in the cerebellum and the early postnatal hippocampus and is thought to be involved in synaptic plasticity and the development of functional neural circuitry through the recruitment of other AMPA receptor subunits. Previously, we reported an association of the human GluR4 gene (GRIA4) with schizophrenia. To examine the role of the GluR4 subunit in the higher brain function, we generated GluR4 knockout mice and conducted electrophysiological and behavioural analyses. The mutant mice showed normal long-term potentiation (LTP) in the CA1 region of the hippocampus. The GluR4 knockout mice showed mildly improved spatial working memory in the T-maze test. Although the retention of spatial reference memory was intact in the mutant mice, the acquisition of spatial reference memory was impaired in the Barnes circular maze test. The GluR4 knockout mice showed impaired prepulse inhibition. These results suggest the involvement of the GluR4 subunit in cognitive function.
Asunto(s)
Región CA1 Hipocampal/metabolismo , Potenciación a Largo Plazo/genética , Aprendizaje por Laberinto/fisiología , Inhibición Neural/genética , Receptores AMPA/genética , Estimulación Acústica , Animales , Región CA1 Hipocampal/efectos de los fármacos , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Clásico/fisiología , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Técnicas In Vitro , Inhibición Psicológica , Potenciación a Largo Plazo/fisiología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/genética , Tiempo de Reacción/fisiología , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismo , Reflejo de Sobresalto/efectos de los fármacos , Reflejo de Sobresalto/genética , Reflejo de Sobresalto/fisiología , Filtrado Sensorial/efectos de los fármacos , Filtrado Sensorial/genética , Filtrado Sensorial/fisiologíaRESUMEN
Chk1, a nuclear DNA damage/replication G2 checkpoint kinase, phosphorylates Cdc25 and causes its nuclear exclusion in yeast and mammalian cells, thereby arresting the cell at the G2 phase until DNA repair/replication is completed. Chk1 is also involved, at least in part, in the natural G2 arrest of immature Xenopus oocytes, but it is unknown how Chk1 inhibits Cdc25 function and undergoes regulation during oocyte maturation. By using enucleated oocytes, we show here that Chk1 inhibits Cdc25 function in the cytoplasm of G2-arrested oocytes and that Cdc25 is activated exclusively in the cytoplasm of maturing oocytes. Moreover, we show that Chk1 activity is not appreciably altered during maturation, being maintained at basal levels, and that C-terminal truncation mutants of Chk1 have very high kinase activities, strong abilities to inhibit maturation, and altered subcellular localization in oocytes. These results, together with other results, suggest that the Chk1/Cdc25 pathway is involved cytoplasmically in G2 arrest of Xenopus oocytes, but moderately and independent of the G2 checkpoint, and that the C-terminal region of Chk1 negatively regulates its kinase activity and also determines its subcellular localization. Based on these results, we discuss the possibility that Chk1 (with the basal activity) may function as an ordinary regulator of Cdc25 in oocytes (and in other cell types) and that Chk1 might be hyperactivated in response to the G2 checkpoint via its dramatic conformational change.
Asunto(s)
Citoplasma/metabolismo , Oocitos/fisiología , Proteínas Quinasas/metabolismo , Fosfatasas cdc25/metabolismo , Proteínas 14-3-3 , Animales , Compartimento Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Femenino , Fase G2/fisiología , Modelos Biológicos , Mutación , Fragmentos de Péptidos/metabolismo , Fosforilación , Proteínas Quinasas/genética , Tirosina 3-Monooxigenasa/metabolismo , Xenopus , Proteínas de XenopusRESUMEN
Nek2, a NIMA-related kinase, has been postulated to play a role in both the meiotic and mitotic cell cycles in vertebrates. Xenopus has two Nek2 splice variants, Nek2A and Nek2B, which are zygotic and maternal forms, respectively. Here we have examined the role of Nek2B in oocyte meiosis and early embryonic mitosis. Specific inhibition of Nek2B function does not interfere with the oscillation of Cdc2 activity in either the meiotic or mitotic cell cycles; however, it does cause abortive cleavage of early embryos, in which bipolar spindle formation is severely impaired due to fragmentation or dispersal of the centrosomes, to which endogenous Nek2B protein localizes. In contrast, inhibition of Nek2B function does not affect meiotic spindle formation in oocytes, in which functional centrosomes are absent. Thus, strikingly, Nek2B is specifically required for centrosome assembly and/or maintenance (and hence for normal bipolar spindle formation and cleavage) in early Xenopus embryos. Finally, (ectopic) Nek2A but not Nek2B is very labile in cleaving embryos, suggesting that Nek2A cannot replace the centrosomal function of Nek2B in early embryos.
Asunto(s)
Centrosoma/fisiología , Isoenzimas , Proteínas Serina-Treonina Quinasas/química , Proteínas de Xenopus , Xenopus/embriología , Animales , Western Blotting , Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Cicloheximida/farmacología , Embrión no Mamífero/fisiología , Activación Enzimática , Femenino , Meiosis , Microscopía Confocal , Mitosis , Oocitos/fisiología , Progesterona/metabolismo , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , Factores de Tiempo , Transcripción GenéticaRESUMEN
Meiotic cells undergo two successive divisions without an intervening S phase. However, the mechanism of S-phase omission between the two meiotic divisions is largely unknown. Here we show that Wee1, a universal mitotic inhibitor, is absent in immature (but not mature) Xenopus oocytes, being down-regulated specifically during oogenesis; this down-regulation is most likely due to a translational repression. Even the modest ectopic expression of Wee1 in immature (meiosis I) oocytes can induce interphase nucleus reformation and DNA replication just after meiosis I. Thus, the presence of Wee1 during meiosis I converts the meiotic cell cycle into a mitotic-like cell cycle having S phase. In contrast, Myt1, a Wee1-related kinase, is present and directly involved in G(2) arrest of immature oocytes, but its ectopic expression has little effect on the meiotic cell cycle. These results strongly indicate that the absence of Wee1 in meiosis I ensures the meiotic cell cycle in Xenopus oocytes. Based on these results and the data published previously in other organisms, we suggest that absence of Wee1 may be a well-conserved mechanism for omitting interphase or S phase between the two meiotic divisions.
Asunto(s)
Proteínas de Ciclo Celular , Meiosis/fisiología , Proteínas Nucleares , Oogénesis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas de Xenopus , Animales , Western Blotting , Replicación del ADN/fisiología , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica , Oocitos/citología , Profase/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S/fisiología , XenopusRESUMEN
Mos plays a crucial role in meiotic cell division in vertebrates. In Xenopus, Mos is involved in the initiation of oocyte maturation as an initiator and in the arrest at the metaphase II stage (MII) as a component of the cytostatic factor (CSF). The function of Mos is mediated by MAP kinase (MAPK). We investigated the function of the Mos/MAPK pathway during goldfish oocyte maturation induced by 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), a natural maturation-inducing hormone in fishes. Mos was absent in immature goldfish oocytes. It appeared before the onset of germinal vesicle breakdown (GVBD), increased to a maximum in mature oocytes arrested at MII and disappeared after fertilization. MAPK was activated after Mos synthesis but before maturation-promoting factor (MPF) activation, and its activity reached maximum at MII. Injection of either Xenopus or goldfish c-mos mRNA into one blastomere of 2-cell-stage Xenopus and goldfish embryos induced metaphase arrest, suggesting that goldfish Mos has a CSF activity. Injection of constitutively active Xenopus c-mos mRNA into immature goldfish oocytes induced MAPK activation, but neither MPF activation nor GVBD occurred. Conversely, the injection of goldfish c-mos antisense RNA inhibited both Mos synthesis and MAPK activation in the 17alpha,20beta-DP-treated oocytes, but these oocytes underwent GVBD. These results indicate that the Mos/MAPK pathway is not essential for initiating goldfish oocyte maturation despite its general function as a CSF. We discuss the general role of Mos/MAPK during oocyte maturation, with reference to the difference in contents of inactive MPF (pre-MPF) stored in immature oocytes.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Metafase , Proteínas Oncogénicas v-mos/fisiología , Oocitos/citología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Carpa Dorada , Datos de Secuencia Molecular , Proteínas Oncogénicas v-mos/química , Homología de Secuencia de Aminoácido , Especificidad de la Especie , XenopusRESUMEN
Nek2 kinase, a NIMA-related kinase, has been suggested to play both meiotic and mitotic roles in mammals, but its function(s) during development is poorly understood. We have isolated here cDNAs encoding a Xenopus homolog of mammalian Nek2 and have shown that Xenopus Nek2 has two structural variants, termed Nek2A and Nek2B. Nek2A, most likely a C-terminally spliced form, corresponds to the previously described human and mouse Nek2, while Nek2B is most probably a novel, C-terminally unspliced form of Nek2. As a consequence of this (probable) alternative splicing, Nek2B lacks the C-terminal 70-amino-acid sequence of Nek2A, which contains a PEST sequence (or a motif for rapid degradation). Western blot analysis reveals that Nek2A is expressed predominantly in the testis (presumably in spermatocytes) and very weakly in the stomach and, during development, only after the neurula stage. By contrast, Nek2B is expressed mainly in the ovary and in both primary and secondary oocytes and early embryos up to the neurula stage. These results suggest that Nek2A and Nek2B may play both meiotic and mitotic roles, but in a spatially and temporally complementary manner during Xenopus development, and that Nek2B, rather than Nek2A (or the conventional form of Nek2), may play an important role in early development. We discuss the possibility that a counterpart of Xenopus Nek2B might also exist and function in early mammalian development.
Asunto(s)
Proteínas de Ciclo Celular , Isoenzimas/genética , Meiosis/genética , Mitosis/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Xenopus , Xenopus/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Femenino , Masculino , Datos de Secuencia Molecular , Quinasa 1 Relacionada con NIMA , Quinasas Relacionadas con NIMA , Ovario/embriología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Caracteres Sexuales , Testículo/embriología , Distribución Tisular , Xenopus/embriologíaRESUMEN
Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. Here we have cloned a Xenopus homolog of Chk1, determined its developmental expression, and examined its possible role in prophase I arrest of oocytes. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. We discuss the possibility that Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes.
Asunto(s)
Oocitos/enzimología , Proteínas Quinasas/genética , Xenopus/embriología , Fosfatasas cdc25 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Clonación Molecular , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Meiosis/genética , Microinyecciones , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Progesterona/farmacología , Profase/genética , Proteínas Quinasas/metabolismo , ARN Mensajero/metabolismo , Alineación de Secuencia , Factores de Tiempo , Proteínas de XenopusAsunto(s)
Meiosis/fisiología , Oocitos/citología , Oogénesis/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Proteínas del Huevo/metabolismo , Femenino , Humanos , Factor Promotor de Maduración/fisiología , Profase , Proteínas Proto-Oncogénicas c-mos/fisiología , Factores de TiempoRESUMEN
In almost all animal species, immature oocytes are arrested naturally in the first meiotic prophase, with a large nucleus called the germinal vesicle. A number of previous studies showed that both activation of maturation/M phase-promoting factor (MPF) (assayed by semiquantitative cytological methods) and some other maturational events occur essentially normally in enucleated oocytes from many amphibian species and mice. Hence, for nearly three decades, it has generally been believed that nuclear material is dispensable for MPF activation and the meiotic cell cycle in vertebrate oocytes. Here, we have challenged this view by examining the histone H1 kinase activities and the molecular forms of MPF in experimentally manipulated Xenopus oocytes. We show that oocytes injected with nuclear material undergo much more rapid MPF activation and maturation than uninjected control oocytes. Conversely, enucleated oocytes, unlike nucleated counterparts, undergo only weak MPF activation in meiosis I and no detectable MPF reactivation in meiosis II, the latter accompanying inhibitory tyrosine phosphorylation of cdc2 kinase, the catalytic subunit of MPF. These results argue strongly that nuclear material is indispensable for the meiotic cell cycle, particularly MPF reactivation (or cdc2 tyrosine dephosphorylation) on entry into meiosis II, in Xenopus oocytes. The classical and general view may thus need reconsideration.
Asunto(s)
Ciclo Celular/fisiología , Núcleo Celular/fisiología , Factor Promotor de Maduración/biosíntesis , Oocitos/citología , Oocitos/fisiología , Animales , Proteína Quinasa CDC2/biosíntesis , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Femenino , Cinética , Meiosis , Mesotelina , Ratones , Mitosis , Modelos Biológicos , Oocitos/efectos de los fármacos , Progesterona/farmacología , Proteínas Quinasas/metabolismo , XenopusRESUMEN
Cyclin B, a regulatory subunit of maturation/M-phase promoting factor (MPF), has several subtypes in many vertebrate species. However, it is not known whether the different B-type cyclins have any different functions in vertebrate cells, although their subcellular localizations seem to differ largely from each other. To examine the roles of two major B-type cyclins, B1 and B2, in spindle formation in M phase, we overexpressed their N-termini in Xenopus oocytes; the N-termini of cyclins B1 and B2 contained a cytoplasmic retention signal (CRS), and hence their overexpressions were expected to competitively inhibit the subcellular localizations of the endogenous cyclins B1 and B2, respectively. Upon entry into meiosis I, oocytes overexpressing the cyclin B1 N-terminus formed an apparently normal bipolar spindle, but those oocytes overexpressing the cyclin B2 N-terminus formed a monopolar (or monoastral) spindle. This defect in bipolar spindle formation was observed only when the cyclin B2 N-terminus contained its own CRS sequence, and was able to be rescued by overexpression of full-length cyclin B2. These results suggest, for the first time, that the correct subcellular localization of cyclin B2, but not of cyclin B1, is essential for (the initiation of) bipolar spindle formation in Xenopus oocytes.
Asunto(s)
Ciclina B/fisiología , Oocitos/fisiología , Señales de Clasificación de Proteína , Huso Acromático/fisiología , Secuencia de Aminoácidos , Animales , Ciclina B/biosíntesis , Ciclina B/genética , Ciclina B1 , Citoplasma , Expresión Génica , Meiosis/fisiología , Datos de Secuencia Molecular , Oocitos/metabolismo , Xenopus laevisAsunto(s)
Proteínas Proto-Oncogénicas c-mos , Animales , Transformación Celular Neoplásica/metabolismo , Ciclina B/metabolismo , Implantación del Embrión , Femenino , Meiosis , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-mos/metabolismo , Proteínas Proto-Oncogénicas c-mos/fisiologíaRESUMEN
In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/metabolismo , Meiosis , Oocitos/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Ciclo Celular , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Factor Promotor de Maduración/metabolismo , Metafase , Datos de Secuencia Molecular , Oocitos/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mos/metabolismo , Conejos , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal , Xenopus , Proteínas de XenopusRESUMEN
We have cloned a cDNA encoding a novel B regulatory subunit of protein phosphatase 2A (PP2A) from a Xenopus oocyte cDNA library. The novel B subunit, termed B beta', shows the strongest overall sequence similarity to, but a distinct N-terminal sequence from, the beta isoform of the human/rat B subunit. When expressed ectopically in Xenopus oocytes, the B beta' isoform can augment the endogenous PP2A activity and inhibit oocyte maturation induced by progesterone. These results suggest that the B beta' isoform can form a complex with other PP2A subunits to make a trimeric PP2A holoenzyme in Xenopus oocytes and may negatively control the initiation of oocyte maturation.
Asunto(s)
Oocitos/enzimología , Fosfoproteínas Fosfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Proteína Fosfatasa 2 , Ratas , Homología de Secuencia de Aminoácido , XenopusRESUMEN
Mos, a protein kinase, is specifically expressed and functions during meiotic maturation (or G2/M progression) of vertebrate oocytes. When expressed ectopically, however, it can also readily induce oncogenic transformation (or uncontrolled G1/S transitions) in somatic cells. In both of these cell types, Mos activates mitogen-activated protein kinase (MAPK), which seems largely to mediate its different functions in both oocyte maturation and cellular transformation. In oocyte maturation, the Mos-MAPK pathway probably serves to activate and stabilize M-phase promoting factor (MPF) (possibly by inhibiting some negative regulator(s) of this factor), while in cellular transformation, it seems to stabilize and activate the nuclear oncoprotein c-Fos as well as to induce transcription of its gene. Thus, the different functions of Mos in oocytes and somatic cells may arise chiefly from its different MAPK-mediated targets in the respective cell types. This review discusses the cellular basis that may enable Mos to act differently in oocytes and somatic cells.
Asunto(s)
Oocitos/fisiología , Proteínas Proto-Oncogénicas c-mos/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Ciclo Celular/fisiología , Transformación Celular Neoplásica , Femenino , Meiosis/fisiología , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oogénesis/fisiología , XenopusRESUMEN
From a cDNA library of Xenopus laevis (Xl) oocytes, we isolated a cDNA encoding a putative protein phosphatase homologous to mammalian Cdc25A. Sequence analysis predicts that the Xl cdc25A gene product (Xl Cdc25A) consists of 521 amino acid residues and shares overall 55% identity with human Cdc25A. When its mRNA is injected into Xl oocytes, Xl Cdc25A can act as a potent M phase inducer.
Asunto(s)
Proteínas de Ciclo Celular/genética , Meiosis/genética , Oogénesis/genética , Proteínas de Xenopus , Fosfatasas cdc25 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , ADN Complementario , Humanos , Datos de Secuencia Molecular , Oocitos/fisiología , Fosfoproteínas Fosfatasas/genética , Homología de Secuencia de Aminoácido , Xenopus laevisAsunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Transformación Celular Neoplásica , Meiosis , Oocitos/citología , Proteínas Proto-Oncogénicas c-mos/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Proteínas Proto-Oncogénicas c-mos/fisiologíaRESUMEN
AML1 (also called PEBP2alphaB, CBFA2, or CBFalpha2) is one of the most frequently disrupted genes in chromosome abnormalities seen in human leukemias. It has been reported that AML1 plays several pivotal roles in myeloid hematopoietic differentiation and other biological phenomena, probably through the transcriptional regulation of various relevant genes. Here, we investigated the mechanism of regulation of AML1 functions through signal transduction pathways. The results showed that AML1 is phosphorylated in vivo on two serine residues within the proline-, serine-, and threonine-rich region, with dependence on the activation of extracellular signal-regulated kinase (ERK) and with interleukin-3 stimulation in a hematopoietic cell line. These in vivo phosphorylation sites of AML1 were phosphorylated directly in vitro by ERK. Although differences between wild-type AML1 and phosphorylation site mutants in DNA-binding affinity were not observed, we have shown that ERK-dependent phosphorylation potentiates the transactivation ability of AML1. Furthermore the phosphorylation site mutations reduced the transforming capacity of AML1 in fibroblast cells. These data indicate that AML1 functions are potentially regulated by ERK, which is activated by cytokine and growth factor stimuli. This study provides some important clues for clarifying unidentified facets of the regulatory mechanism of AML1 function.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al ADN , Leucemia Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Humanos , Leucemia Mieloide/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , TransfecciónRESUMEN
Metaphase arrest in meiosis I or II before fertilization is a common and unique feature of oogenesis in many animal species. How and why oocytes from many species are arrested at metaphase, rather than after the completion of meiosis, has long remained a mystery. This article reviews recent advances in our understanding of the mechanisms and biological significance of meiotic metaphase arrest in animal oocytes.
RESUMEN
c-Fos is associated with c-Jun to increase the transcription of a number of target genes and is a nuclear proto-oncoprotein with a very short half-life. This instability of c-Fos may be important in regulation of the normal cell cycle. Here we report a mechanism for degradation of c-Fos. Coexpression of c-Fos and c-Jun in HeLa cells caused marked increase in the instability of c-Fos, whereas v-Fos, the retroviral counterpart of c-Fos, was stable irrespective of the coexpression of c-Jun. Interestingly, deletion of the C-terminal PEST region of c-Fos, which is altered in v-Fos by a frameshift mutation, greatly enhanced its stability, with loss of the effect of c-Jun on its stability. c-Fos synthesized in vitro was degraded by the 26S proteasome in a ubiquitin-dependent fashion. Simple association with c-Jun had no effect on the degradation of c-Fos, but the additions of three protein kinases, mitogen-activated protein kinase, casein kinase II, and CDC2 kinase, resulted in marked acceleration of its degradation by the proteasome-ubiquitin system, though only in the presence of c-Jun. In contrast, v-Fos and c-Fos with a truncated PEST motif were not degraded, suggesting that they escaped from down-regulation by breakdown. These findings indicate a new oncogenic pathway induced by acquisition of intracellular stability of a cell cycle modulatory factor.
Asunto(s)
Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/fisiología , Secuencia de Aminoácidos , Mutación del Sistema de Lectura , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Oncogénicas v-fos/metabolismo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , Ubiquitinas/fisiologíaRESUMEN
The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate ERK1 and 2 mitogen-activated protein (MAP) kinases by direct phosphorylation of MAPK/ERK kinase (MEK). ERK activation is essential for oncogenic transformation of NIH 3T3 cells by Mos. In this study, we examined how mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway reaches the nucleus to activate downstream target genes. We show that c-Fos (the c-fos protooncogene product), which is an intrinsically unstable nuclear protein, is metabolically highly stabilized, and greatly enhances the transforming efficiency of NIH 3T3 cells, by Mos. This stabilization of c-Fos required Mos-induced phosphorylation of its C-terminal region on Ser362 and Ser374, and double replacements of these serines with acidic (Asp) residues markedly increased the stability and transforming efficiency of c-Fos even in the absence of Mos. Moreover, activation of the ERK pathway was necessary and sufficient for the c-Fos phosphorylation and stabilization by Mos. These results indicate that c-Fos undergoes stabilization, and mediates at least partly the oncogenic signalling, by the Mos/MEK/ERK pathway. The present findings also suggest that, in general, the ERK pathway may regulate the cell fate and function by affecting the metabolic stability of c-Fos.