RESUMEN
Prostate cancer is the most common malignant tumor in men and is normally associated with increased serum levels of prostate-specific antigen (PSA). Therefore, PSA is one potential target for a prostate cancer vaccine. In this study we analyzed the functionality of new bacterial PSA vaccines, expressed and secreted via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of Type I secretion systems (T1SS) using an attenuated Salmonella enterica serovar Typhimurium aroA strain as carrier. The data demonstrate that a bacterial live vaccine encompassing T1SS in combination with cholera toxin subunit B can be successfully used for delivery of PSA to induce cytotoxic CD8+ T-cell responses resulting in an efficient prevention of tumor growth in mice.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Toxina del Cólera/metabolismo , Inmunoterapia Activa , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/prevención & control , Vacunas contra la Salmonella/uso terapéutico , Salmonella typhimurium/genética , Vacunas Tifoides-Paratifoides/uso terapéutico , Animales , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Toxina del Cólera/genética , Toxina del Cólera/inmunología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Femenino , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos DBA , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/inmunología , Neoplasias de la Próstata/inmunología , Vacunas contra la Salmonella/genética , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/inmunología , Vacunas Tifoides-Paratifoides/genética , Vacunas Tifoides-Paratifoides/inmunología , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K(+) channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patch-clamp studies identified TPK1 as a voltage-independent and Ca(2+)-activated K(+) channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca(2+)- and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Vacuolas/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/aislamiento & purificación , Sitios de Unión , Calcio/metabolismo , Regulación hacia Abajo , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Fosforilación , Canales de Potasio de Dominio Poro en Tándem/química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.
Asunto(s)
Apoptosis/efectos de los fármacos , Interferón-alfa/farmacología , Neoplasias Pulmonares/patología , Proteínas Oncogénicas/metabolismo , Factor 1 de Elongación Peptídica/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Línea Celular Tumoral , Humanos , Inmunoprecipitación , Neoplasias Pulmonares/metabolismo , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Ubiquitina/metabolismoRESUMEN
The p150-Spir protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte. Spir proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spir encodes a zinc finger structure highly homologous to FYVE motifs. A region with high homology between the Spir family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as "Spir-box." The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A. Coexpression of p150-Spir and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spir protein colocalized specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome. The distinct Spir localization pattern was dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibited the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein was arrested in membrane structures, largely colocalizing with the TGN marker TGN46. Our findings that the Spir actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport.
Asunto(s)
Actinas/metabolismo , Proteínas de Drosophila , Proteínas de Microfilamentos/metabolismo , Células 3T3 , Actinas/química , Secuencia de Aminoácidos , Animales , Transporte Biológico , Drosophila , Ratones , Proteínas de Microfilamentos/química , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Despite the overall successful application of the tet-system to regulate gene expression in vitro and in vivo, nothing is known so far about the long-term stability of this system in transgenic mice. In this study, mice of generation F2, F3, F4, or F10 of two independent tTA(CMV) transgenic lines were bred with NZL-2 mice containing a tTA-responsive bidirectional promoter that allows the simultaneous expression of two reporter genes encoding luciferase and beta-galactosidase. Analysis of the expression of transgenes in double transgenic mice revealed a dramatic reduction of tTA transactivator mRNA over time. As a consequence, the expression of both reporter genes was gradually reduced from generation to generation in tissues of embryonic and adult NZL-2/tTA(CMV) mice. Luciferase activity in NZL-2/tTA(CMV)(F10) mice was reduced 8-10-fold compared to NZL-2/ tTA(CMV)(F2) mice, and beta-galactosidase expression was no longer detectable. In summary, we describe the long-term instability of the tet-system in our NZL-2/tTA(CMV) double transgenic mice. The molecular basis of this observation and experimental tools to overcome this limitation need to be addressed in future.
Asunto(s)
Regulación de la Expresión Génica , Luciferasas/genética , Regiones Promotoras Genéticas/genética , Tetraciclina/metabolismo , Transgenes/genética , beta-Galactosidasa/genética , Envejecimiento/genética , Animales , Southern Blotting , Cruzamientos Genéticos , Embrión de Mamíferos/metabolismo , Femenino , Perfilación de la Expresión Génica , Genes Reporteros/genética , Luciferasas/análisis , Luciferasas/metabolismo , Masculino , Ratones , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , beta-Galactosidasa/análisis , beta-Galactosidasa/metabolismoRESUMEN
We have characterized the effects of different short-chain fatty acids (SCFAs) on cell growth and differentiation as well as the phosphorylation state of ERK1 and 2 in the human colon adenocarcinoma cell line HT-29. Of the five SCFAs tested, only butyrate and propionate impaired cellular proliferation. Moreover, butyrate and propionate specifically resulted in a decrease in ERK1 and 2 phosphorylation at 3 and 6 hours post-treatment, suggesting a correlation between the ability of these SCFAs to inhibit cellular proliferation and decrease ERK phosphorylation. Notably, the decrease in ERK phosphorylation was observed prior to the induction of the differentiation markers alkaline phosphatase (AP) and carcinoembryonic antigen (CEA) by butyrate and propionate from days 6 to 18 post-treatment. In the case of butyrate- and propionate-induced differentiation, ERK phosphorylation is a marker and may play a role in the proliferation and/or differentiation states of this cell line.
Asunto(s)
Butiratos/farmacología , Diferenciación Celular , Células HT29/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Propionatos/farmacología , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/metabolismo , Antígeno Carcinoembrionario/análisis , Antígeno Carcinoembrionario/metabolismo , Regulación hacia Abajo , Humanos , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Transducción de SeñalRESUMEN
The inhibitor of apoptosis proteins (IAPs) have been shown to interact with a growing number of intracellular proteins and pathways to fulfil their anti-apoptotic role. In the search for novel IAP-interacting proteins we identified the neurotrophin receptor-interacting MAGE homologue (NRAGE) as being able to bind to the avian IAP homologue ITA. This interaction requires the RING domain of ITA. NRAGE additionally coimmunoprecipitates with XIAP. When overexpressed in 32D cells NRAGE augments interleukin-3 withdrawal induced apoptosis, possibly through binding endogenous XIAP. Moreover, NRAGE is able to overcome the anti-apoptotic effect of Bcl-2.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Neoplasias , Proteínas/metabolismo , Animales , Sitios de Unión , Aves , Dimerización , Regulación de la Expresión Génica , Proteínas Inhibidoras de la Apoptosis , Proteínas de Insectos , Interleucina-3/deficiencia , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células Tumorales Cultivadas , Proteína Inhibidora de la Apoptosis Ligada a XRESUMEN
Bcl-2 family proteins play a critical role in the regulation of cell survival by controlling the activation of the cell death executing caspase machinery. Recent work demonstrated that they also provide a link between growth factor signaling and cell survival control. Raf-1 has been identified initially as an essential component of the mitogenic Ras-Raf-MEK-ERK cascade. However, expression of oncogenic Raf-1 also efficiently suppresses apoptotic cell death. This process requires mitochondrial translocation of Raf-1 which can be achieved either by co-expression of the anti-apoptotic protein Bcl-2 or by fusion with the transmembrane domain of the yeast outer mitochondrial membrane protein Mas 70p. It is currently unclear how mitochondrial Raf-1 prevents apoptosis. One possible mechanism involves the phosphorylation of the pro-apoptotic protein Bad resulting in the restoration of Bcl-2 function. Alternatively, the role of Bcl-2 could be limited to the mitochondrial translocation of Raf-1 and survival signaling by Raf-1 is Bcl-2 independent. To test for the mutual requirement of Raf-1 and Bcl-2 in apoptosis suppression the individual proteins were singly tested for survival activity in a genetic background which precludes the expression of the other. The results obtained in these studies demonstrate that ablation of Raf-1 or Bcl-2 expression in fibroblast cells significantly increases the sensitivity towards doxorubicin induced cell death. Reversion of the mutant phenotype could be achieved in either case by introducing a functional bcl-2 gene or a mitochondria targeted version of oncogenic Raf-1, demonstrating that each protein by itself is sufficient to confer protection. Our data thus suggest the existence of two separate pathways of survival signaling at the mitochondria controlled either by Bcl-2 or by Raf-1.
Asunto(s)
Apoptosis , Mitocondrias/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Proteínas Proto-Oncogénicas c-raf/fisiología , Animales , Caspasas/fisiología , Supervivencia Celular , Células Cultivadas , Doxorrubicina/farmacologíaRESUMEN
Extracellular signal regulated kinase 5 (ERK5) is a novel member of the mitogen-activated protein kinase (MAPK) family with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle we examined a function of the cascade during muscle differentiation. We show that ERK5 is activated upon induction of differentiation in mouse myoblasts and that selective activation of the pathway results in promoter activation of differentiation-specific genes. Moreover, myogenic differentiation is completely blocked when ERK5 expression is inhibited by antisense RNA. Thus, we conclude that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculos/citología , Animales , Western Blotting , Diferenciación Celular , Línea Celular , Activación Enzimática , Genes Dominantes , Genes Reporteros , Humanos , MAP Quinasa Quinasa 5 , Sistema de Señalización de MAP Quinasas , Ratones , Proteína Quinasa 7 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Músculo Esquelético/citología , Oligonucleótidos Antisentido/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Transducción de Señal , Factores de Tiempo , Transducción Genética , TransfecciónRESUMEN
The ability to control gene expression in a temporal and spatial manner provides a new tool for the study of mammalian gene function particularly during development and oncogenesis. In this study the suitability of the tet-system for investigating embryogenesis was tested in detail. The tTACMV(M1) and rTACMV-3 (reverse Tc-controlled transactivator) transgenic mice were bred with NZL-2 bi-reporter mice containing the vector with a tTA/rTA responsive bidirectional promoter that allows simultaneous regulation of expression of two reporter genes encoding luciferase and beta-galactosidase. In both cases reporter genes were found to be expressed in a wide spectrum of tissues of double transgenic embryos and adult mice. The earliest expression was detected in tTACMV(M1)/NZL-2 embryos at embryonic day 10.5 (E10.5) and rTACMV-3/NZL-2 embryos at E13.5. Doxycycline abolished beta-gal expression in tTACMV(M1)/NZL-2 but induced it in rTACMV-3/NZL-2 embryos including late stages of embryo-genesis. The tTA and rtTA transactivators thus revealed a partially complementary mode of action during second half of embryonic development. These experiments demonstrated that both Tet regulatory systems function during embryonic development. We conclude that the Tet systems allows regulation of gene expression during embryonic development and that 'double reporter' animals like the NZL-2 mice are useful tools for the characterization of newly generated tet transactivator lines expressing tTA (or rtTA) in embryonic as well as in adult tissues.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Genes Reporteros/genética , Tetraciclina/metabolismo , Envejecimiento/metabolismo , Animales , Citomegalovirus/genética , Doxiciclina/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Luciferasas/análisis , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , beta-Galactosidasa/análisis , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
Influenza A virus infection of cells results in the induction of a variety of antiviral cytokines, including those that are regulated by transcription factors of the activating protein-1 (AP-1) family. Here we show that influenza virus infection induces AP-1-dependent gene expression in productively infected cells but not in cells that do not support viral replication. Among the AP-1 factors identified to bind to their cognate DNA element during viral infections of Madin-Darby canine kidney and U937 cells are those that are regulated via phosphorylation by JNKs. Accordingly, we observed that induction of AP-1-dependent gene expression correlates with a strong activation of JNK in permissive cells, which appears to be caused by viral RNA accumulation during replication. Blockade of JNK signaling at several levels of the cascade by transient expression of dominant negative kinase mutants and inhibitory proteins resulted in inhibition of virus-induced JNK activation, reduced AP-1 activity, and impaired transactivation of the IFN-beta promoter. Virus yields from transfected and infected cells in which JNK signaling was inhibited were higher compared with the levels from control cells. Therefore, we conclude that virus-induced activation of JNK and AP-1 is part of the innate antiviral response of the cell.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus de la Influenza A/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 4 , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Factor de Transcripción AP-1/fisiología , Factor de Transcripción Activador 2 , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Activación Enzimática , Células HeLa , Humanos , Interferón beta/genética , MAP Quinasa Quinasa 7 , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , ARN Viral/metabolismo , Especificidad de la Especie , Factores de Transcripción/metabolismo , Células U937 , Replicación ViralAsunto(s)
Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/metabolismo , Animales , División Celular , Membrana Celular/enzimología , Citosol/enzimología , Modelos Biológicos , Unión Proteica , Proteína Quinasa C/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Quinasas p21 ActivadasRESUMEN
Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.
Asunto(s)
Apoptosis/fisiología , Hipoxia/enzimología , MAP Quinasa Quinasa 4 , Melanoma/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Invasividad Neoplásica/fisiopatología , Neovascularización Patológica/enzimología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Hipoxia/fisiopatología , Inmunohistoquímica , Proteínas Quinasas JNK Activadas por Mitógenos , Melanoma/patología , Melanoma/fisiopatología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Necrosis , Neovascularización Patológica/fisiopatología , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética/fisiología , Transfección , Células Tumorales Cultivadas/enzimología , Células Tumorales Cultivadas/patología , Receptor fas/genéticaRESUMEN
Growth factor-induced signalling leads to activation of members of the Ras family and subsequent stimulation of different Raf isoforms. Within the mechanism of Raf activation, two isoforms of Raf, cRaf and BRaf, may cooperate. We investigated the relationship between cRaf and BRaf and found that active Ras induced heterodimerization of cRaf and BRaf, an effect that was dependent on the serine residue at position 621 of cRAF: Moreover, we also found that cRaf COOH-terminus constitutively associated with BRaf, whereas the NH(2) terminus did not, even in the presence of active RAS: These data suggest that Ras induces the cRaf-BRaf complex formation through the exposure of 14-3-3 binding sites in the COOH-terminus of cRAF: Thus, Ras-induced cRaf-Braf heterodimerization may explain the observed cooperativity of cRaf and BRaf in cells responding to growth factor signals.
Asunto(s)
Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas ras/fisiología , Proteínas 14-3-3 , Sitios de Unión , Dominio Catalítico , Línea Celular , Dimerización , Humanos , Fragmentos de Péptidos/metabolismo , Isoformas de Proteínas , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Influenza A viruses are important worldwide pathogens in humans and different animal species. The functions of most of the ten different viral proteins of this negative-strand RNA virus have been well elucidated. However, little is known about the virus-induced intracellular signalling events that support viral replication. The Raf/MEK/ERK cascade is the prototype of mitogen-activated protein (MAP) kinase cascades and has an important role in cell growth, differentiation and survival. Investigation of the function of this pathway has been facilitated by the identification of specific inhibitors such as U0126, which blocks the cascade at the level of MAPK/ERK kinase (MEK). Here we show that infection of cells with influenza A virus leads to biphasic activation of the Raf/MEK/ERK cascade. Inhibition of Raf signalling results in nuclear retention of viral ribonucleoprotein complexes (RNPs), impaired function of the nuclear-export protein (NEP/NS2) and concomitant inhibition of virus production. Thus, signalling through the mitogenic cascade seems to be essential for virus production and RNP export from the nucleus during the viral life cycle.
Asunto(s)
Butadienos/farmacología , Inhibidores Enzimáticos/farmacología , Virus de la Influenza A/fisiología , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitrilos/farmacología , Transporte Activo de Núcleo Celular , Animales , Western Blotting , Línea Celular , Genes Reporteros , Humanos , Inmunohistoquímica , Virus de la Influenza A/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Microscopía Confocal , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fosforilación , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/metabolismo , Transfección , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Two Ras effector pathways leading to the activation of Raf-1 and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting Raf-1 to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active Raf-1 is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic Raf-1. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.
Asunto(s)
Apoptosis/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-raf/genética , Transducción de Señal , Activación Enzimática , Humanos , MAP Quinasa Quinasa 1 , Plásmidos , Células Tumorales CultivadasRESUMEN
Embryonic sensory and motoneurons depend on neurotrophic factors for survival. Here we show that their survival requires B-Raf, which, in this function, cannot be substituted by C-Raf. Sensory and motoneurons from b-raf-deficient mice do not respond to neurotrophic factors for their survival. However, these primary neurons can be rescued by transfection of a b-raf expression plasmid. In contrast, c-raf-deficient neurons survive in response to neurotrophic factors, similarly to neurons from wild-type mice. This points to an essential and specific function of B-Raf in mediating survival of sensory and motoneurons during development.
Asunto(s)
Ganglios Espinales/embriología , Neuronas Motoras/fisiología , Neuronas Aferentes/fisiología , Proteínas Proto-Oncogénicas c-raf/fisiología , Médula Espinal/embriología , Animales , Supervivencia Celular/fisiología , Células Cultivadas , Embrión de Mamíferos/fisiología , Desarrollo Embrionario y Fetal/fisiología , Ganglios Espinales/citología , Ratones , Médula Espinal/citologíaRESUMEN
Influenza A virus infection of cells results in the induction of a variety of antiviral cytokines, including those that are regulated by transcription factors of the activating protein-1 (AP-1) family. Here we show that influenza virus infection induces AP-1-dependent gene expression in productively infected cells but not in cells that do not support viral replication. Among the AP-1 factors identified to bind to their cognate DNA element during viral infections of Madin-Darby canine kidney and U937 cells are those that are regulated via phosphorylation by JNKs. Accordingly, we observed that induction of AP-1-dependent gene expression correlates with a strong activation of JNK in permissive cells, which appears to be caused by viral RNA accumulation during replication. Blockade of JNK signaling at several levels of the cascade by transient expression of dominant negative kinase mutants and inhibitory proteins resulted in inhibition of virus-induced JNK activation, reduced AP-1 activity, and impaired transactivation of the IFN-beta promoter. Virus yields from transfected and infected cells in which JNK signaling was inhibited were higher compared with the levels from control cells. Therefore, we conclude that virus-induced activation of JNK and AP-1 is part of the innate antiviral response of the cell.
Asunto(s)
Gripe Humana/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Orthomyxoviridae/fisiología , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Animales , Línea Celular , Regulación Viral de la Expresión Génica , Humanos , Gripe Humana/genética , Gripe Humana/virología , MAP Quinasa Quinasa 4 , Replicación ViralRESUMEN
The cytokine-induced C-C chemokine monocyte chemoattractant protein-1 (MCP-1) is an important regulator of leukocyte recruitment to sites of inflammatory challenge. Here, it is demonstrated that the widely distributed contact hapten NiCl(2), like tumor necrosis factor alpha (TNFalpha), induces monocyte-chemoattractant activity in primary human endothelial cells via induction of MCP-1. NiCl(2) rapidly activated mitogen-activated protein (MAP) kinase p38, and inhibition of p38 partially blocked NiCl(2)-induced MCP-1 messenger RNA and protein expression. Both NiCl(2)- and TNFalpha-induced MCP-1 synthesis was sensitive to D609, an inhibitor of phosphatidylcholine-dependent phospholipase C (PC-PLC). NiCl(2)-induced MCP-1 synthesis required activation of NF-kappaB since mutation of NF-kappaB-binding sites in the promoter resulted in complete loss of inducible promoter activity. Consistent with that finding, stimulation with NiCl(2) or TNFalpha activated IkappaB kinase-beta (IKKbeta), and transient transfection of dominant-negative IKKbeta strongly inhibited NiCl(2)- and TNFalpha-induced MCP-1 expression. However, D609 and the specific p38 inhibitor SB202190 did not affect NiCl(2)- and TNFalpha-induced IKKbeta activation, NF-kappaB DNA-binding activity, or transcriptional activity of a Gal4p65 fusion protein. This indicates that p38- and PC-PLC-dependent pathways directly regulate the transcriptional activity of NF-kappaB factors in the transcriptional complex. Consistent with that, inhibition of p38 blocked enhanced transcriptional activity induced by the transcriptional coactivator p300. Thus, it was concluded that at least 3 independent pathways regulate MCP-1 expression in endothelial cells. Its induction requires activation of the IKKbeta/IkappaBalpha/NF-kappaB signaling pathway, resulting in nuclear accumulation of p65 and subsequent recruitment of cofactors. Proper assembly and activity of this transcriptional complex is further modulated by the p38 MAP kinase cascade and a PC-PLC-dependent pathway.