RESUMEN
PURPOSE: Solid tumors involve an inflammatory microenvironment portrayed by immune cells playing role in cancer progression via inflammatory p38α mitogen-activated protein kinase (MAPK) molecule that produces pro-inflammatory cytokines-TNFα, IL1β and IL6. This study quantified and compared the expression of p38α in peripheral blood mononuclear cells (PBMCs) of HNSCC patients with the healthy subjects. METHODS: The PBMC were isolated from the 35 control and 83 HNSCC patients. The expression of p38α in PBMCs was assessed using surface plasmon resonance (SPR), ELISA and western blot analysis. RESULTS: p38α levels were found to be over-expressed in HNSCC patients 0.98 ng/μl (95 % CI 0.95-1.02) as compared to controls 0.46 ng/μl (95 % CI 0.42-0.50) (p < 0.0001). CONCLUSION: p38α is over-expressed in PBMCs of HNSCC patients and may play a role in the progression of cancer. This research may translate a protein marker for HNSCC to clinical oncologist for therapeutic intervention and use as a predictive marker (AU)
Asunto(s)
Humanos , Masculino , Femenino , Neoplasias/inducido químicamente , Neoplasias/metabolismo , Neoplasias/radioterapia , Neoplasias/diagnóstico , Protamina Quinasa/análisisRESUMEN
Our laboratory has previously described the presence of five tumor-specific low molecular weight isoforms of cyclin E in both tumor cell lines and breast cancer patient biopsies. We have also shown that one of these low forms arises from an alternate start site, whereas the other four appear as two sets of doublets following cleavage through an elastase-like enzyme. However, the origin of both sets of doublets was unknown. Here, we demonstrate that the larger isoform of each doublet is the result of phosphorylation at a key degradation site. Through site-directed mutagenesis of different phosphorylation sites within the cyclin E protein, we discovered that phosphorylation of threonine 395 is responsible for generating the larger isoform of each doublet. Because phosphorylation of threonine 395 has been linked to the proteasome-mediated degradation of full length cyclin E, we examined the stability of T395A phospho-mutants in both non-tumorigenic mammary epithelial cells and tumor cells. The results revealed that the low molecular weight isoforms appear to be stable in both a tumor cell line and a non-tumor forming cell line regardless of the presence of this critical phosphorylation site. The stability of low molecular weight cyclin E may have implications for both tumorigenesis and treatment of tumors expressing them.
Asunto(s)
Ciclina E/química , Ciclina E/metabolismo , Procesamiento Proteico-Postraduccional , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Neoplasias de la Mama/metabolismo , Línea Celular , Línea Celular Tumoral , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Riñón/citología , Modelos Biológicos , Datos de Secuencia Molecular , Peso Molecular , Fosforilación , Protamina Quinasa/análisis , Protamina Quinasa/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transfección , Quinasas p21 Activadas/metabolismoRESUMEN
Fertilisation in ascidian oocytes triggers a plasma membrane current, the release of intracellular calcium and the degradation of Maturation Promoting Factor (MPF) activity leading to the completion of meiosis and the initiation of embryo development. We have previously shown that the fertilisation current in ascidians is produced through the metabolism of nicotinamide nucleotide (NN) metabolites to ADP ribose. In this study we have used nicotinamide to test whether NN metabolism plays additional roles in fertilisation in ascidians. Nicotinamide treatment blocked calcium-induced calcium release (CICR) and arrested the cell cycle prior to the completion of meiosis I. Nicotinamide further prevented the abolition of MPF activity after fertilisation. Interestingly, nicotinamide treatment caused ascidian oocytes to form interphase-like pronuclei after fertilisation, despite the high MPF activity. The data demonstrate that NN metabolism is involved in calcium signalling through CICR and further suggest that a NN metabolite acts as a messenger connecting MPF activity to the formation of the meiotic apparatus.
Asunto(s)
Calcio/metabolismo , Fertilización/fisiología , Factor Promotor de Maduración/metabolismo , Niacinamida/farmacología , Oocitos/fisiología , Urocordados/fisiología , Animales , Técnicas In Vitro , Factor Promotor de Maduración/efectos de los fármacos , Meiosis/efectos de los fármacos , Meiosis/fisiología , Microscopía Confocal , Oocitos/citología , Protamina Quinasa/análisisAsunto(s)
Proteínas de Unión al Calcio/biosíntesis , Proteínas Portadoras , Proteínas de Ciclo Celular/biosíntesis , Ciclo Celular/fisiología , Embrión no Mamífero/fisiología , Proteínas Fúngicas/biosíntesis , Oocitos/citología , Oocitos/fisiología , Huso Acromático/fisiología , Huso Acromático/ultraestructura , Secuencia de Aminoácidos , Anafase , Animales , Evolución Biológica , Proteínas de Unión al Calcio/aislamiento & purificación , Proteínas de Ciclo Celular/aislamiento & purificación , Separación Celular/métodos , Sistema Libre de Células , Cromatografía de Afinidad , Secuencia Conservada , Embrión no Mamífero/citología , Femenino , Fertilización , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/aislamiento & purificación , Regulación del Desarrollo de la Expresión Génica , Proteínas Mad2 , Masculino , Metafase , Proteínas Nucleares , Protamina Quinasa/análisis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae , Xenopus laevisRESUMEN
Loss of adhesion of NRK fibroblasts to an appropriate surface leads to cell cycle arrest in late G1 and failure to produce cyclin A. Previously, we showed that adhesion-dependent expression of cyclin A is transcriptionally regulated. In an effort to identify elements of the adhesion-mediated signal transduction cascade upstream of cyclin A activation, we investigated the expression of cyclin E and its associated kinase activity in adherent and suspended NRK cells. Expression of cyclin E was found to be unaffected by suspension. However, cyclin E complexes immunoprecipitated from extracts prepared from NRK cells 12 h after release from G0 arrest were found to be catalytically inactive in suspended but not in adherent cells. This suspension-induced inhibition of cyclin E-associated kinase activity was not observed in NRK cells transformed by a c-Ha-ras oncogene containing a G12V mutation. When G0-synchronized NRK cells were transfected with a cyclin A promoter:luciferase reporter construct along with expression vectors for either wild-type cdk2 or a dominant-negative cdk2 mutant, transcriptional activation of cyclin A was found to be dependent on catalytically active cdk2. Inhibition of cyclin E/cdk2 complexes has frequently been attributed to association of the cdk inhibitors p21(Cip1) and p27(Kip1). However, no differences between adherent and suspended cells could be observed for either expression or cdk2 association of p21(Cip1) or p27(Kip1), nor were any proteins specifically associated with cdk2 or cyclin E in immunoprecipitates from metabolically labeled cell extracts. These results define a pathway through which an adhesion-generated signal controls cyclin A expression by modulating cyclin E/cdk2 activity.
Asunto(s)
Quinasas CDC2-CDC28 , Adhesión Celular/fisiología , Proteínas de Ciclo Celular , Ciclo Celular/fisiología , Transformación Celular Neoplásica , Quinasas Ciclina-Dependientes/biosíntesis , Ciclinas/biosíntesis , Genes ras , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Supresoras de Tumor , Animales , Línea Celular , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/análisis , Ciclinas/análisis , Ciclinas/metabolismo , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/metabolismo , Immunoblotting , Riñón , Luciferasas/biosíntesis , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/metabolismo , Protamina Quinasa/análisis , Protamina Quinasa/biosíntesis , Proteínas Serina-Treonina Quinasas/análisis , Ratas , Proteínas Recombinantes/biosíntesis , TransfecciónRESUMEN
A single-strand-specific endonuclease from mung bean sprouts is widely used in molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.
Asunto(s)
División Celular/fisiología , ADN de Plantas/metabolismo , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/metabolismo , Fabaceae/enzimología , Plantas Medicinales , Secuencia de Bases , Clonación Molecular , Reparación del ADN , Replicación del ADN , ADN de Plantas/biosíntesis , ADN Superhelicoidal/metabolismo , Datos de Secuencia Molecular , Protamina Quinasa/análisis , Análisis de Secuencia de ADN , Distribución TisularRESUMEN
Bovine oocytes were irradiated at germinal vesicle (GV) or metaphase II (MII) stage, after Hoechst staining, with chromosomally focused UV-C (254 nm) or UV-A ( > 330 nm). UV-C irradiation at GV stage did not inhibit germinal vesicle breakdown (GVBD) or chromosomal condensation; spindle formation was abolished and maturation promoting factor (MPF) levels failed to increase. UV-A irradiation at GV stage caused meiotic arrest at anaphase I; MPF levels were lower than control. UV-C irradiation at MII stage led to subsequent abnormal parthenogenetic activation when MPF levels failed to decrease. A normal male but no female pronucleus was formed at fertilization. UV-A irradiation at MII stage also caused abnormal activation; MPF levels declined normally. A normal male and abnormal female pronucleus formed at fertilization. UV-A irradiation results have implications for oocyte evaluation during development using Hoechst staining. UV-C irradiation is a potential means for oocyte enucleation in nuclear transfer.
Asunto(s)
Cromosomas/efectos de la radiación , ADN/efectos de la radiación , Factor Promotor de Maduración/biosíntesis , Meiosis/efectos de la radiación , Oocitos/efectos de la radiación , Rayos Ultravioleta , Animales , Bencimidazoles/toxicidad , Bovinos , Daño del ADN , Femenino , Colorantes Fluorescentes/toxicidad , Masculino , Protamina Quinasa/análisis , Interacciones Espermatozoide-Óvulo , Rayos Ultravioleta/efectos adversosRESUMEN
To better understand the relationship between the proliferation of human lymphoid cells and the expression of cdk1, a catalytic subunit of the histone H1 kinase (H1K), we examined its mRNA and protein content in 3 B-cell lines: Ramos, Reh-6 and IARC 963. Cells were elutriated according to their position in the cell cycle. Cell fractions were analyzed for cdk1 mRNA and protein cellular content by Northern blot and immunoblot, respectively, as well as for H1K activity. Both mRNA and protein amounts and H1K activity varied according to cell cycle phase, the lowest values being observed in G1-enriched fractions. For comparison, elutriated fractions were also tested for the expression of cdk2 and cdk4 proteins. Both showed some variations among fractions, but they were less clear than those of cdk1. We also tested 29 samples of lymphoid neoplastic and non-neoplastic tissues for proliferative activity (percentage of S and G2/M cells estimated by flow cytometry) and expression of cdk1, cdk2 and cdk4 proteins. We found a significant correlation between the percentage of cells in S or S + G2/M phases and cdk1 protein content but not cdk2 or cdk4 content. We conclude that cdk1 expression in human lymphoid cells varies during the cell cycle at both mRNA and protein levels.
Asunto(s)
Proteína Quinasa CDC2/biosíntesis , Quinasas CDC2-CDC28 , Ciclo Celular , División Celular , Expresión Génica , Leucemia Linfocítica Crónica de Células B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Biomarcadores , Linfoma de Burkitt , Proteína Quinasa CDC2/análisis , Línea Celular , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/análisis , Quinasas Ciclina-Dependientes/biosíntesis , ADN de Neoplasias/análisis , ADN de Neoplasias/metabolismo , Citometría de Flujo , Humanos , Cinética , Leucemia de Células B , Leucemia Linfocítica Crónica de Células B/metabolismo , Linfocitos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Protamina Quinasa/análisis , Protamina Quinasa/biosíntesis , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/biosíntesis , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Células Tumorales CultivadasRESUMEN
The mitotic state is associated with a generalized repression of transcription. We show that mitotic repression of RNA polymerase III transcription can be reproduced by using extracts of synchronized HeLa cells. We have used this system to investigate the molecular basis of transcriptional repression during mitosis. We find a specific decrease in the activity of the TATA-binding-protein (TBP)-containing complex TFIIIB. TBP itself is hyperphosphorylated at mitosis, but this does not appear to account for the loss of TFIIIB activity. Instead, one or more TBP-associated components appear to be regulated. The data suggest that changes in the activity of TBP-associated components contribute to the coordinate repression of gene expression that occurs at mitosis.
Asunto(s)
ADN Polimerasa III/metabolismo , Regulación de la Expresión Génica , Mitosis/genética , TATA Box , Transcripción Genética , Secuencia de Bases , Western Blotting , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosforilación , Protamina Quinasa/análisis , Fracciones Subcelulares/metabolismo , Factor de Transcripción TFIIIB , Factores de Transcripción/metabolismoRESUMEN
To study the role of the metaphase spindle during the period of oocyte activation, mouse oocytes were fertilised or activated parthenogenetically in the presence or absence of the microtubule inhibitor nocodazole. In both cases, nocodazole caused the disappearance of the spindle and prevented the passage of the oocytes into interphase. However, the calcium spiking responses of the oocytes were not affected by nocodazole, being repetitive after fertilisation and a single spike after activation. If, after their activation or fertilisation in nocodazole, oocytes were later removed from the drug, only those that had been fertilised progressed into interphase. This progress was associated with continuing calcium spiking. Moreover, both the spiking and the progress to interphase could be blocked or reduced in incidence by removal of external calcium or addition of 5,5'-dimethyl BAPTA-AM. Oocytes that had been activated by ethanol in the presence of nocodazole and then removed from it, to allow re-formation of the spindle, only progressed into interphase if given a second exposure to ethanol, thereby eliciting a second calcium transient. These results show that exit from meiotic M-phase requires the simultaneous presence of a fully intact spindle during the release of calcium and that those factors leading to the degradation of cyclin B are only activated transiently. Since cyclin is being degraded continuously in the metaphase-II-arrested mouse oocyte and since this degradation is microtubule-dependent, these data suggest that the superimposition of a high concentration of intracellular calcium is required to tilt the equilibrium further in favour of cyclin degradation if exit from M-phase is to occur.
Asunto(s)
Calcio/metabolismo , Ciclo Celular , Oocitos/citología , Huso Acromático/fisiología , Animales , Cromosomas/fisiología , Cromosomas/ultraestructura , Etanol/farmacología , Femenino , Fertilización , Fura-2 , Inmunohistoquímica , Meiosis , Ratones , Microscopía Fluorescente , Microtúbulos/efectos de los fármacos , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Mitosis , Nocodazol/farmacología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Protamina Quinasa/análisis , Huso Acromático/ultraestructura , Factores de TiempoRESUMEN
Two different fractions of cdk2 and cdc2 have been found in the nucleus of HeLa cells. One, which can be extracted by nuclease treatment, possibly associated with DNA- or RNA-containing structures and another one, which is bound to the nuclear matrix. Nuclear cdk2 forms high molecular weight complexes which migrate at the same position as DNA polymerase alpha and proliferating cell nuclear antigen in sucrose gradient centrifugation experiments. These results suggest that nuclear cdk2 complexes could be associated with the replication factories. Immunoprecipitation experiments reveal that nuclear cdk2 complexes display histone H1-kinase activity and phosphorylate a protein of 18 kDa which is present in these complexes.
Asunto(s)
Quinasas CDC2-CDC28 , Núcleo Celular/metabolismo , Quinasas Ciclina-Dependientes , Ciclinas/metabolismo , Protamina Quinasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Western Blotting , Fraccionamiento Celular , Núcleo Celular/química , Centrifugación por Gradiente de Densidad , Quinasa 2 Dependiente de la Ciclina , Ciclinas/análisis , Células HeLa , Humanos , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Protamina Quinasa/análisis , Proteínas Serina-Treonina Quinasas/análisisRESUMEN
The E6 and E7 proteins of human papillomavirus (HPV) types 16 and 18 are expressed in cell lines derived from cervical cancers and can immortalize primary human keratinocytes. Since expression of E6/E7 has been shown to induce mitotic defects and karyotype instability in primary human cells, we investigated the effect of these viral oncoproteins on the expression and activity of mitotic regulatory proteins. Primary human keratinocytes immortalized by the entire genome or by only the E6/E7 genes of HPV types 16 and 18 displayed 5- to 20-fold increases in the abundance of p34cdc2, cyclin B and cyclin A when compared with normal parental cells. Results obtained from normal and immortalized cells that were derived from identical single donors were similar to those from mixed donor cultures. Increased protein levels were achieved without corresponding increases in mRNA, indicating alterations in translational and/or post-translational control. The histone H1 kinase activities associated with these regulatory proteins were also elevated, but to a lesser extent than the protein levels. Because p34cdc2, cyclin B and cyclin A regulate the entry into and exit from mitosis, increased expression and activity of these proteins could contribute to the mitotic defects and chromosomal aberrations associated with HPV-induced immortalization.
Asunto(s)
Proteína Quinasa CDC2/análisis , Proteína Quinasa CDC2/fisiología , Ciclinas/análisis , Ciclinas/fisiología , Proteínas de Unión al ADN , Queratinocitos/química , Queratinocitos/citología , Mitosis/fisiología , Papillomaviridae/fisiología , Proteínas Represoras , Northern Blotting , Proteína Quinasa CDC2/genética , Línea Celular/virología , Ciclinas/genética , Femenino , Citometría de Flujo , Humanos , Queratinocitos/efectos de los fármacos , Masculino , Proteínas Oncogénicas Virales/farmacología , Proteínas E7 de Papillomavirus , Pruebas de Precipitina , Protamina Quinasa/análisis , Protamina Quinasa/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Células Tumorales Cultivadas/virologíaRESUMEN
beta gamma subunits of G proteins were purified from starfish oocytes, and their role in the induction of oocyte maturation by 1-methyladenine was investigated. When injected into starfish oocytes, the purified beta gamma subunit of the starfish G protein induced germinal vesicle breakdown (GVBD) faster than that of bovine brain G protein. Injection of the starfish beta gamma into cytoplasm near the germinal vesicle (GV) induced GVBD earlier than when injected into the GV or the cytoplasm near the plasma membrane. Fluorescent-labeled beta gamma was retained in the injected area even after GVBD. Injected beta gamma also induced the formation of maturation-promoting factor as well as an increase of histone H1 kinase activity. These results suggest that beta gamma dissociates from alpha-subunit by the stimulation of 1-methyladenine and interacts with a cytoplasmic effector, which results in formation of active cdc2 kinase.
Asunto(s)
Citoplasma/metabolismo , Proteínas de Unión al GTP/fisiología , Oocitos/fisiología , Estrellas de Mar/fisiología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Química Encefálica , Bovinos , Proteínas de Unión al GTP/química , Hormonas de Invertebrados/farmacología , Factor Promotor de Maduración/análisis , Factor Promotor de Maduración/fisiología , Microinyecciones , Membrana Nuclear/metabolismo , Oocitos/citología , Oocitos/efectos de los fármacos , Protamina Quinasa/análisis , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Rat vascular smooth muscle cells were synchronized to the quiescent state (G0) by serum deprivation and then stimulated to enter the cell cycle by serum refeeding. At various times of the cell cycle, cells were analyzed for the expression of p34cdc2 and mitogen-activated protein kinase homologues by immunoblotting and for kinase activity toward histone H1, myelin basic protein, and caldesmon. A small amount of p34cdc2 was expressed in the G0/G1 phase (0 to 8 hours). At the G1/S transition (12 hours), the level of p34cdc2 started to accumulate and increased by 60-fold at G2/M (18 hours), accompanied by a more slowly migrating band. Histone H1 kinase activity was undetectable in anti-p34cdc2 immunoprecipitates in the G0/G1 cells but appeared around the G1/S boundary and peaked at G2/M (18 hours). The caldesmon kinase activity exhibited two distinct phases: the first appeared at G0/G1 (0 to 8 hours), and the second appeared at G1/S and continued through G2/M. Two mitogen-activated protein kinase isoforms were expressed throughout the cell cycle. Anti-mitogen-activated protein kinase immunoprecipitates possessed kinase activities toward myelin basic protein and caldesmon, which were activated within 15 minutes after serum stimulation and declined within a few hours. These findings suggest that p34cdc2 and mitogen-activated protein kinase homologues may play significant roles in regulating the progression of the cell cycle of smooth muscle cells, the former at the G2/M transition and the latter at the G0/G1 transition.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular , Proteínas Quinasas Activadas por Mitógenos , Músculo Liso Vascular/citología , Proteínas de Schizosaccharomyces pombe , Animales , Proteína Quinasa CDC2/análisis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular , Citometría de Flujo , Proteínas Fúngicas/metabolismo , Fase G1 , Masculino , Proteína Quinasa 3 Activada por Mitógenos , Músculo Liso Vascular/metabolismo , Protamina Quinasa/análisis , Proteínas Quinasas/análisis , Ratas , Ratas Wistar , Fase de Descanso del Ciclo Celular , Fase SRESUMEN
Phosphorylation events are major regulatory mechanisms of signal transduction pathways that regulate gene expression and cell growth. To study the potential involvement of serine-threonine specific phosphatases in these processes we used okadaic acid (OA), an inhibitor of type 1 and type 2A protein phosphatases. Here we present evidence that OA arrests cells at defined points in the cell cycle. Concomitantly, expression and associated histone H1 kinase activity of cdc2 and cyclin A, two cell cycle regulatory proteins, are repressed by this agent. Furthermore, phosphorylation of the tumor suppressor protein retinoblastoma, an event thought to be necessary in order to permit cells to proliferate, is inhibited when OA is present. These effects are fully reversible since removal of OA restores cdc2 and cyclin A expression as well as histone H1 kinase activity, and the cells resume growth. Since cdc2 and cyclin A have previously been shown to be absolutely required for cell cycle progression it is likely that blockage of synthesis of these components contributes to the cytostatic effects of OA. Furthermore, our results suggest a positive role for OA sensitive protein phosphatases in the regulation of expression of these cell cycle regulatory proteins.
Asunto(s)
Éteres Cíclicos/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Protamina Quinasa/antagonistas & inhibidores , Proteína de Retinoblastoma/metabolismo , Células 3T3 , Animales , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Regulación hacia Abajo , Ratones , Ácido Ocadaico , Fosforilación , Protamina Quinasa/análisisRESUMEN
Microinjection of transforming p21ras into Xenopus oocytes caused a time-dependent increase in the level of total cell protein phosphorylation that culminated with germinal vesicle breakdown (GVBD). The same pattern of phosphorylation was observed in oocytes matured by either progesterone or insulin. Treatment with cycloheximide (CHX) completely blocked both GVBD and the associated de novo phosphorylations induced by the hormones, but did not abolish p21ras-induced maturation nor the occurrence of associated maturation promoting factor (MPF)-dependent and -independent phosphorylations. Thus, induction of GVBD by p21ras in the absence of protein synthesis correlated with the activation of cytosolic MPF-associated kinase activity similar in specificity on exogenous (histone H1) and endogenous (47 kDa and a 42 kDa proteins) substrates to the MPF activity of hormonally-matured oocytes. The injection of p21ras in the presence of CHX caused also activation of other kinase(s) proceeding MPF activation which were responsible for the phosphorylation of endogenous substrates including a 41 kDa protein and a 92 kDa protein kinase that comigrated, respectively, with bands recognized specifically by antibodies to MAP2 kinase and S6 kinase. The phosphorylation of those bands correlated also with the activation of cytosolic kinases acting specifically on myelin basic protein (MBP) and a S6-derived peptide as substrates. These results indicate that, in the absence of protein synthesis, p21ras is able to activate phosphorylation events leading to GVBD and suggest that this oncoprotein can participate in at least two separate pathways of MPF activation. We propose that the activation of MAP/MBP kinases and S6 kinases is an early effect of p21ras oncoproteins.
Asunto(s)
Factor Promotor de Maduración/metabolismo , Meiosis/efectos de los fármacos , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Tirosina Quinasas/análisis , Proteínas Proto-Oncogénicas p21(ras)/farmacología , Animales , Cicloheximida/farmacología , Activación Enzimática , Femenino , Microinyecciones , Proteína Quinasa 1 Activada por Mitógenos , Oocitos/fisiología , Fosforilación , Protamina Quinasa/análisis , Proteínas Quinasas S6 Ribosómicas , Xenopus laevisRESUMEN
The c-mos gene product (c-Mos) encodes a serine/threonine kinase required for activation of pre-MPF (maturation-promoting factor) to MPF in oocytes undergoing meiosis and also for stabilization of MPF leading to metaphase arrest in unfertilized eggs. In order to determine whether the v-mos gene product (v-Mos) causes neoplastic transformation via interaction with cell cycle control elements, we have searched for proteins that interact with v-Mos. Extracts of NIH3T3 cells transformed by v-Mos encoded by Moloney murine sarcoma virus (Mo-MuSV) were examined by gel filtration, by immunoprecipitation with antibodies to a conserved region of p34cdc2, and by binding to beads that contain cross-linked p13suc1, a protein known to bind p34cdc2. Gel filtration detected a 500-kDa complex that contained v-Mos and a p34cdc2 isoform, termed p35cdk. The 500-kDa macromolecular complex also exhibited histone H1 phosphorylation activity, consistent with the presence of a cdc2 isoform. The identity of p35cdk is based on its recognition by anti-cdc2 PSTAIR but not by anti-cdc2 C-terminal antibodies, which detect authentic p34cdc2. Structures containing v-Mos and p35cdk were also detected by experiments involving co-immunoprecipitation of v-Mos with anti-cdc2 PSTAIR antibodies. Furthermore, both v-Mos and the p35cdk co-precipitated with p13suc1-Sepharose beads. Our findings raise the possibility of a v-Mos-p35cdk regulatory interaction in cells transformed by Mo-MuSV.
Asunto(s)
Transformación Celular Neoplásica , Proteínas Quinasas/análisis , Proteínas Oncogénicas de Retroviridae/análisis , Células 3T3 , Secuencia de Aminoácidos , Animales , Ciclo Celular , Factor Promotor de Maduración/metabolismo , Mesotelina , Ratones , Datos de Secuencia Molecular , Proteínas Oncogénicas v-mos , Pruebas de Precipitina , Protamina Quinasa/análisis , Proteínas Serina-Treonina Quinasas , Proteínas Oncogénicas de Retroviridae/toxicidadRESUMEN
Calcium/calmodulin dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase. We have created a calcium/calmodulin independent form of this enzyme by truncation. Expression of this enzyme fragment in a rabbit reticulocyte lysate yields a constitutive enzyme with specific activity similar to the activated native enzyme. We have established mammalian cell lines that transiently express this constitutive enzyme using the glucocorticoid-inducible mouse mammary tumor virus long terminal repeat. The transient increase in kinase activity results in a complete cessation of cell cycle progression. This block develops as a consequence of a specific arrest of the cell cycle in G2. During the block, increases in histone H1 kinase activity present in p13 beads or anti-cdc2 immunoprecipitates are seen in parallel with the accumulation of cells at G2, arguing that the arrest is not due to a failure to activate cdc2 as a histone H1 kinase. These results suggest that other changes in serine/threonine protein phosphorylation besides those involved in activation of cdc2 as a histone H1 kinase may be necessary for proper G2-M transition.
Asunto(s)
Ciclo Celular/fisiología , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Encéfalo/enzimología , Proteína Quinasa CDC2/análisis , Proteína Quinasa CDC2/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Ciclo Celular/efectos de los fármacos , Línea Celular , Deleción Cromosómica , Ciclinas/análisis , Ciclinas/metabolismo , Dexametasona/farmacología , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Virus del Tumor Mamario del Ratón/genética , Ratones , Datos de Secuencia Molecular , Plásmidos , Protamina Quinasa/análisis , Protamina Quinasa/metabolismo , Biosíntesis de Proteínas , Proteínas Quinasas/análisis , Proteínas Quinasas/genética , ARN/genética , ARN/aislamiento & purificación , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Protein kinases in the cytosol of whole bovine lactating mammary gland were separated by phosphocellulose chromatography. Five protein kinases (identified as histone kinase and casein kinases A, B, C, and D) were characterized and compared with kinases from other tissues. The histone kinase activity was identified as cyclic AMP-dependent protein kinase. The casein kinases differed in their activities toward a variety of proteins (alpha s1-casein, native and dephosphorylated beta-casein, and alpha-lactalbumin) and peptides. Based on substrate specificity studies and the inhibitory effects of heparin, 2,3-diphosphoglycerate, and guanosine triphosphate, kinases B, C, and D were tentatively identified as glycogen synthase kinase-3, casein kinase I, and casein kinase II, respectively.
Asunto(s)
Bovinos/metabolismo , Lactancia/metabolismo , Glándulas Mamarias Animales/enzimología , Proteínas Quinasas/metabolismo , Animales , Caseína Quinasas , Cromatografía , Femenino , Embarazo , Protamina Quinasa/análisis , Protamina Quinasa/metabolismo , Proteínas Quinasas/análisis , Proteínas Quinasas/fisiologíaRESUMEN
The cAMP level and the activity of cAMP-dependent and independent protein kinases were measured in adenomatous polyps, villous polyps (premalignant condition) and adenocarcinomas. The cAMP concentration and ratio of cAMP level versus cAMP-independent casein kinase activity were significantly lower in adenocarcinomas compared to adenomatous polyps, thus permitting differentiation between benign and malignant lesions. The cAMP versus cAMP-dependent histone kinase level ratio was used as a test for differentiation between malignancies and premalignant condition. It was markedly lower in adenocarcinomas than in villous polyps.