RESUMEN

Understanding how disruption of differentiation contributes to the cancer cell phenotype is required to identify alterations essential for malignant transformation and provide experimental basis for their correction. We investigated whether primary quail neuroretina cells, transformed by a conditional v-Src mutant (QNR/v-src(ts)), could revert to a normal phenotype, in response to the stable expression of constitutively active Notch1 intracellular domain (ICN). This model system was chosen because Notch signaling plays an instructive role in cell fate determination during NR development, and because the intrinsic capacity of QNR cultures to differentiate is blocked by v-Src. We report that stable ICN expression results in suppression of QNR/v-src(ts) cell transformation in the presence of an active oncoprotein. This phenotypic reversion coincides with a major switch in cell identity, as these undifferentiated cells acquire glial differentiation traits. Both changes appear to be mediated by CBF, a transcription factor that binds to ICN and activates target genes. Cells restored to a normal and differentiated phenotype have undergone changes in the functioning of signaling effectors, essentially regulating cell morphology and cytoskeleton organization. This dominant interference may be partially mediated by an autocrine/paracrine mechanism, as revertant cells secrete a factor(s), which inhibits transformation properties of QNR/v-src(ts) cells.

Asunto(s)

Transformación Celular Neoplásica , Neuronas/citología , Neuronas/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Animales , Biomarcadores , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Regulación hacia Abajo , Expresión Génica , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Proteína Oncogénica pp60(v-src)/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Codorniz , Transducción de Señal , Quinasas p21 Activadas , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

Tight regulation of the Src tyrosine kinase activity is essential for a variety of cellular processes, namely transitions of the cell cycle. The peaks of Src activity are dependent on its posttranslational modifications as well as on the regulation of gene expression. The 3'UTRs of mRNAs are often crucial for rapid changes of the protein level. The chicken c-src 3'UTR effects on gene expression have been explored. The c-src 3'UTR decreased the in vivo tumorigenic potential of the src-activated mutants in chickens. This corresponds with the finding that the c-src 3'UTR reduced the Src protein and src mRNA levels and luciferase activity in vitro. Our results suggest that the chicken c-src 3'UTR plays a role in the negative control of gene expression, either transcriptionally or posttranscriptionally.

Asunto(s)

Regiones no Traducidas 3'/genética , Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Regulación de la Expresión Génica/genética , Genes src/genética , Animales , Proteína Tirosina Quinasa CSK , División Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Embrión de Pollo , Regulación hacia Abajo/genética , Genes Reguladores/genética , Luciferasas/genética , Mutación/genética , Neuronas/citología , Neuronas/metabolismo , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Familia-src Quinasas

RESUMEN

We previously described the identification of quail MafA, a novel transcription factor of the Maf bZIP (basic region leucine zipper) family, expressed in the differentiating neuroretina (NR). In the present study, we provide the first evidence that MafA is phosphorylated and that its biological properties strongly rely upon phosphorylation of serines 14 and 65, two residues located in the transcriptional activating domain within a consensus for phosphorylation by mitogen-activated protein kinases and which are conserved among Maf proteins. These residues are phosphorylated by ERK2 but not by p38, JNK, and ERK5 in vitro. However, the contribution of the MEK/ERK pathway to MafA phosphorylation in vivo appears to be moderate, implicating another kinase. The integrity of serine 14 and serine 65 residues is required for transcriptional activity, since their mutation into alanine severely impairs MafA capacity to activate transcription. Furthermore, we show that the MafA S14A/S65A mutant displays reduced capacity to induce expression of QR1, an NR-specific target of Maf proteins. Likewise, the integrity of serines 14 and 65 is essential for the MafA ability to stimulate expression of crystallin genes in NR cells and to induce NR-to-lens transdifferentiation. Thus, the MafA capacity to induce differentiation programs is dependent on its phosphorylation.

Asunto(s)

Leucina Zippers , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas del Ojo/genética , Glicoproteínas/genética , Células HeLa , Humanos , Lectinas Tipo C , Cristalino , Factores de Transcripción Maf de Gran Tamaño , Proteína Quinasa 3 Activada por Mitógenos , Proteína Quinasa 7 Activada por Mitógenos , Proteína Quinasa 8 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Conejos , Receptores Inmunológicos , Serina/genética , Serina/metabolismo , Transactivadores/genética , Transactivadores/fisiología , Transcripción Genética , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

The drm gene encodes a cystine knot-containing secreted and cell membrane-associated glycoprotein shown to be an antagonist of BMPs. Drm was recently reported to play a crucial role in limb bud development, by its capacity to bind BMPs. Here, we have studied the expression pattern of drm transcripts during chicken development, by using whole-mount in situ hybridization. We show that, from stage 22HH to stage 26HH, in addition to limb buds, drm is expressed in cephalic neural crest-derived branchial arches I, II and III, in the medio-dorsal lip of the myotome and in the superficial dermatome

Asunto(s)

Embrión no Mamífero/metabolismo , Cresta Neural/embriología , Biosíntesis de Proteínas , Proteínas , Animales , Proteínas Morfogenéticas Óseas , Embrión de Pollo , Citocinas , Embrión de Mamíferos/metabolismo , Hibridación in Situ , Factores de Tiempo

RESUMEN

The neuroretina is a functional unit of the central nervous system which arises through successive steps of division, growth arrest and differentiation of neuroectodermal precursors. Postmitotic quail neuroretina (QNR) cells are conditionally induced to divide upon infection with temperature sensitive mutants of Rous sarcoma virus (RSV), since QNR cell division can be arrested by either inactivating p60v-Src at the nonpermissive temperature (41 degrees C) or by serum deprivation at 37 degrees C. We are studying the transcriptional control of QR1, a neuroretina specific gene, whose expression is down-regulated in proliferating cells at 37 degrees C and is fully restored when these cells are made quiescent. We previously showed that this quiescence specific upregulation implicates a promoter region named A box, which binds Maf transcription factors. We report the identification of the C box, a second promoter sequence that activates QR1 transcription in non dividing cells. This sequence is able to form two DNA-protein complexes, one of which (C4) is predominantly detected in growth arrested NR cells. We identified the DNA binding site for C4 and described mutations that abolish both C4 binding and promoter activity in quiescent cells. Moreover, we show that a multimerized C box is able to stimulate a heterologous promoter in non dividing cells and constitutes, therefore, a novel quiescence responsive enhancer. Finally, we report that QR1 transcriptional response to cell quiescence requires cooperation between the C box and A box.

Asunto(s)

División Celular/genética , Proteínas del Ojo/genética , Proteína Oncogénica pp60(v-src)/fisiología , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Virus del Sarcoma Aviar/genética , Secuencia de Bases , Sitios de Unión , Coturnix/genética , Medio de Cultivo Libre de Suero/farmacología , ADN/genética , ADN/metabolismo , Regulación de la Expresión Génica/genética , Sustancias Macromoleculares , Proteínas Recombinantes de Fusión/biosíntesis , Retina/metabolismo , Temperatura , Transcripción Genética , Transfección

RESUMEN

Ras-induced cell transformation is mediated through distinct downstream signaling pathways, including Raf, Ral-GEFs-, and phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. In some cell types, strong activation of the Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) cascade leads to cell cycle arrest rather than cell division. We previously reported that constitutive activation of this pathway induces sustained proliferation of primary cultures of postmitotic chicken neuroretina (NR) cells. We used this model system to investigate the respective contributions of Ras downstream signaling pathways in Ras-induced cell proliferation. Three RasV12 mutants (S35, G37, and C40) which differ by their ability to bind to Ras effectors (Raf, Ral-GEFs, and the p110 subunit of PI 3-kinase, respectively) were able to induce sustained NR cell proliferation, although none of these mutants was reported to transform NIH 3T3 cells. Furthermore, they all repressed the promoter of QR1, a neuroretina growth arrest-specific gene. Overexpression of B-Raf or activated versions of Ras effectors Rlf-CAAX and p110-CAAX also induced NR cell division. The mitogenic effect of the RasC40-PI 3-kinase pathway appears to involve Rac and RhoA GTPases but not the antiapoptotic Akt (protein kinase B) signaling. Division induced by RasG37-Rlf appears to be independent of Ral GTPase activation and presumably requires an unidentified mechanism. Activation of either Ras downstream pathway resulted in ERK activation, and coexpression of a dominant negative MEK mutant or mKsr-1 kinase domain strongly inhibited proliferation induced by the three Ras mutants or by their effectors. Similar effects were observed with dominant negative mutants of Rac and Rho. Thus, both the Raf-MEK-ERK and Rac-Rho pathways are absolutely required for Ras-induced NR cell division. Activation of these two pathways by the three distinct Ras downstream effectors possibly relies on an autocrine or paracrine loop, implicating endogenous Ras, since the mitogenic effect of each Ras effector mutant was inhibited by RasN17.

Asunto(s)

Sistema de Señalización de MAP Quinasas/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteínas Serina-Treonina Quinasas , Retina/citología , Proteínas ras/fisiología , Células 3T3 , Animales , División Celular , Células Cultivadas , Embrión de Pollo , Cloranfenicol O-Acetiltransferasa/biosíntesis , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Proteínas del Ojo/fisiología , Retroalimentación , Genes ras , Factores de Intercambio de Guanina Nucleótido , Sistema de Señalización de MAP Quinasas/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-raf/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Retina/metabolismo , Factores de Transcripción/fisiología , Transfección , Proteínas de Unión al GTP rac/fisiología , Proteínas de Unión al GTP ral/fisiología , Proteínas de Unión al GTP rho/fisiología

RESUMEN

The TrkC subfamily of primary high-affinity neurotrophin-3 receptors is composed of catalytic (kinase-containing; TrkC K) and noncatalytic (TrkC NC) isoforms generated by alternative splicing. We previously reported the presence of the mouse noncatalytic TrkC NC2 isoform in regions of neuronal differentiation [Menn, B., Timsit, S., Calothy, G. & Lamballe, F. (1998) J. Comp. Neurol., 401, 47-64]. In order to gain insight into specific roles for TrkC NC2 receptors during CNS neurogenesis, we compared its distribution with that of its catalytic counterparts and the p75NTR receptor in in vivo and in vitro model systems of early and late neuronal differentiation. We found that TrkC NC2 expression coincided with the exit of neuronal progenitors from the cell cycle and was maintained in differentiated cerebellar neurons. We also showed that, whilst TrkC K receptors were expressed both in mitotic and postmitotic cells, TrkC NC2 was present only in differentiating neural stem cell progeny, suggesting its involvement in neuronal and glial cell differentiation. During neuritogenesis of primary neocortical neurons, both TrkC isoforms as well as p75NTR were located in axonal and dendritic processes. However, whilst these various receptors were present in the same neuronal compartments, TrkC NC2 distribution was specifically restricted to distinct areas of extending neurites. Taken together, these findings suggest that spatiotemporal localization of the noncatalytic receptor could account for specific local effects of neurotrophin-3.

Asunto(s)

Neocórtex/química , Neocórtex/embriología , Receptor de Factor de Crecimiento Nervioso/genética , Receptor trkC/genética , Animales , Axones/química , Axones/enzimología , Dominio Catalítico/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Cerebelo/química , Cerebelo/embriología , Cerebelo/enzimología , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Conos de Crecimiento/química , Conos de Crecimiento/enzimología , Isomerismo , Ratones , Ratones Endogámicos C57BL , Mitosis/fisiología , Neocórtex/enzimología , Neuroglía/química , Neuroglía/citología , Neuroglía/enzimología , Neuronas/química , Neuronas/enzimología , Neuronas/ultraestructura , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptor de Factor de Crecimiento Nervioso/análisis , Receptor trkC/análisis , Receptor trkC/química , Células Madre/química , Células Madre/citología , Células Madre/enzimología

RESUMEN

Down-regulated by mos (Drm)/Gremlin is a highly conserved protein whose properties and expression pattern suggest a role in early development, tissue-specific differentiation, and cell transformation. We have investigated the biosynthesis and processing of Drm expressed endogenously in rat fibroblasts or overexpressed following transient or stable transfection. Analysis of metabolically labeled cells revealed that Drm exists in secreted and cell-associated forms that exhibit similar mobilities in SDS-polyacrylamide gel electrophoresis. Protein analysis indicated that Drm is present in two major species: a slow migrating glycosylated form and a nonglycosylated form. Both forms of Drm are able to undergo phosphorylation. Drm is released into the media within 30 min of synthesis and is detectable for up to 4-5 h, whereas the cell-associated form has a half-life of about 1 h. Confocal immunofluorescent microscopy indicates that Drm is present both on the external surface of expressing cells, as well as within the endoplasmic reticulum and the Golgi. Both glycosylated and nonglycosylated forms of Drm exhibit identical distributions and are able to antagonize bone morphogenetic protein signaling. Like the soluble form, the cell-associated forms are capable of binding (125)I-bone morphogenetic protein-4. These properties are consistent with a role for Drm in interfering with signaling and indicate that Drm may act at the cell surface during tissue development and transformation.

Asunto(s)

Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Citocinas , Glicosilación , Glicoproteínas de Membrana , Datos de Secuencia Molecular , Unión Proteica , Isoformas de Proteínas , Proteínas/genética , Ratas , Proteínas Recombinantes/metabolismo

RESUMEN

Members of the trk gene family encode neurotrophin receptors. The trkC locus encodes multiple neurotrophin-3 catalytic and noncatalytic receptor isoforms. We report the molecular cloning and characterization of mouse cDNAs encoding two noncatalytic TrkC receptors: novel isoforms designated as TrkC NC1 and TrkC NC2, the mouse homologue of the TrkC truncated form previously identified in rat (Tsoulfas et al. [1993] Neuron 10:975-990; Valenzuela et al. [1993] Neuron 10:963-974). We extensively analyzed the transcription pattern of these two noncatalytic isoforms and that of the catalytic isoforms by Northern blotting and in situ hybridization. We did not detect trkC NC1 transcripts in embryos, but we found that trkC NC1 expression is restricted to specific areas in adult brain. In contrast, trkC NC2 transcripts are readily detected early during embryogenesis and are expressed predominantly in adult brain and gonads. We also provide the first evidence for the existence of TrkC NC2 protein by using polyclonal antibodies that specifically recognize this isoform. By using in situ hybridization, we show for the first time that trkC NC2 transcripts are found in differentiating fields of maturing neurons and in mature neurons of laminar structures of adult brain. We also report a similarity of localization between trkC NC2 transcripts and markers of oligodendrocyte progenitors in the embryonic spinal cord. Furthermore, our results also show that trkC NC2 and trkC catalytic transcripts could be either codistributed (in the central and peripheral nervous system) or independently expressed, especially outside the nervous system. These results suggest that the TrkC NC2 isoform acts either independently or in association with its catalytic counterpart. Finally, we show that TrkC NC2 is expressed in dendrites of pyramidal neurons of hippocampus and cerebral cortex. We propose that this receptor is involved in proliferation of oligodendrocyte progenitors, neuronal differentiation, and synaptic plasticity and that it may also play a fundamental role in mediating neurotrophin-3 effects outside the nervous system.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica/fisiología , Isoformas de Proteínas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factor de Crecimiento Nervioso/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Encéfalo/metabolismo , Catálisis , Clonación Molecular , Desarrollo Embrionario y Fetal/fisiología , Código Genético , Inmunohistoquímica , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Receptor trkC

RESUMEN

Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.

Asunto(s)

Empalme Alternativo , Quinasa 1 de Quinasa de Quinasa MAP , Oncogenes , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , División Celular , Células Cultivadas , Embrión de Pollo , Activación Enzimática , Exones , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratones , Neuronas/citología , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-raf/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Retina , Transfección

RESUMEN

Transcription factors of the Maf proto-oncogene family have been shown to participate in the regulation of several differentiation specific genes. We previously reported that a member(s) of this family is involved in the regulation of the neuroretina specific gene, QR1, through a promoter region, designated the A box, that is closely related to the Maf recognition element (MARE). We undertook an identification of Maf family genes expressed in the quail neuroretina (QNR) and we report the isolation of mafA, a gene encoding a novel member of the large Maf proteins subgroup. Expression of this gene is developmentally regulated in the neuroretina. MafA is able to bind to MARE sequence and to heterodimerize with v-Maf, MafB, Jun and Fos, but not with the small MafF and MafK proteins. Accordingly, it is able to transactivate the QR1 promoter A box. We also show that increased expression of mafA induces sustained proliferation of postmitotic QNR cells.

Asunto(s)

Proteínas Aviares , Regulación de la Expresión Génica , Neuronas/citología , Proteínas Proto-Oncogénicas/metabolismo , Codorniz/genética , Retina/citología , Transactivadores/metabolismo , Factores de Transcripción , Proteínas Virales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Dimerización , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Mitógenos/genética , Datos de Secuencia Molecular , Proteína Oncogénica v-maf , Proteínas Oncogénicas/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores/genética , Activación Transcripcional

RESUMEN

BACKGROUND: Ksr (kinase supressor of Ras) was identified as a regulator of the Ras-MAP kinase (mitogen-activated protein kinase) pathway by genetic screens in Drosophila and Caenorhabditis elegans. Ksr is a kinase with similarities to the three conserved regions of Raf kinases, especially within the kinase domain. To investigate whether these structural similarities correlated with common functional properties, we examined the ability of mKsr-1, the murine homolog of Ksr, to interact with components of the vertebrate MAP kinase pathway. RESULTS: In the yeast two-hybrid interaction assay, mKsr-1 did not bind to either Ras, B-Raf or Raf-1, but interacted strongly with both MEK-1 and MEK-2, activators of MAP kinase. The Ksr-MEK interaction was confirmed by co-immunoprecipitation experiments. Ectopically expressed mKsr-1 co-precipitated with endogenous MEK-1 in COS-1 cells, and endogenous Ksr and MEK co-precipitated from PC12 cells. Phosphorylation of MEK by mKsr-1 was not detected, however. In contrast, the MEK subpopulation complexed with mKsr-1 in COS-1 cells or PC12 cells did not display kinase activity. This ability of Ksr to block MEK in an inactive form correlated with a biological response: mKsr-1 did not transform NIH3T3 cells, and, furthermore, mKsr-1 reduced Ras-induced transformation. Similarly, mKsr-1 inhibited the proliferation of embryonic neuroretina cells induced by Ras and B-Raf but not that induced by MEK. CONCLUSIONS: Our results suggest a novel mechanism for Ksr in regulating the MAP kinase pathway, at least in part through an ability to interact with MEK.

Asunto(s)

Transformación Celular Neoplásica/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Proteínas ras/antagonistas & inhibidores , Células 3T3 , Animales , Células COS , División Celular/efectos de los fármacos , Embrión de Pollo , Factor de Crecimiento Epidérmico/farmacología , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Ratones , Factores de Crecimiento Nervioso/farmacología , Células PC12 , Proteínas Proto-Oncogénicas c-raf/metabolismo , Ratas , Retina/efectos de los fármacos

RESUMEN

Retroviruses act as insertional mutagens and can also capture cellular sequences through a mechanism which initially requires the generation of RNA transcripts which fail to cleave and polyadenylate correctly. The correct termination of retroviral transcripts at the 3' LTR R/U5 junction is primarily dependent on the canonical AAUAAA polyadenylation signal, so we have analyzed the effect of mutating the polyadenylation signal sequences on the properties of a selectable murine retroviral vector. Mutation of consensus polyadenylation signal sequences in the 5' and/or 3' proviral LTRs demonstrated that a UA to GG change generated larger sized virus-specific RNA, consistent with loss of normal polyadenylation. Cell clones infected with viruses generated by proviral constructs containing this mutation in the 5' LTR express either normal-length or elongated viral RNA. Fused transcripts contained the mutant polyadenylation signal, while sequence analysis was consistent with the hypothesis that premature 5' to 3' primer strand transfer was responsible for the high frequency (80%) of wild-type polyadenylation. Cells infected by viruses from constructs mutated in both 5' and 3' proviral LTRs expressed poly(A)+ viral RNA between 0.3 and 3 kb larger than normal virus in 100% of infected clones, and sequence analysis of clones derived from either infected rodent or human cells confirmed that these transcripts contained both viral and adjacent cellular sequences. While mutant virus exhibits no increased ability to alter cell phenotypes, the read-through transcripts contain both unique and repetitive cell-derived sequences and can easily be recovered using PCR techniques, suggesting that these viruses may serve as effective tools for rapidly cloning cellular sequences and generating random genomic markers for gene mapping.

Asunto(s)

Virus de la Leucemia Murina de Moloney/genética , Poli A/metabolismo , Integración Viral , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis , Poli A/genética , Provirus/genética , ARN/metabolismo , ARN Viral/metabolismo , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Ribonucleasas/metabolismo

RESUMEN

Using differential display analysis, we compared the expression of RNA in v-mos-transformed cells and their flat revertant and isolated a novel gene, drm (down-regulated in mos-transformed cells), whose expression is down-regulated in parental v-mos-transformed cells but which is expressed at a high level in the revertant and normal rat fibroblasts (REF-1 cells). Analysis of different oncogene-transformed cells revealed that drm gene expression was also suppressed in REF-1 cells transformed by v-ras, v-src, v-raf, and v-fos. The drm cDNA contains a 184-amino-acid-protein-encoding open reading frame which shows no significant homologies to known genes in DNA databases. Polyclonal antibodies raised against drm peptide detect a protein with the predicted size of 20.7 kDa in normal cells and under nonpermissive conditions in cells conditionally transformed by v-mos but not in parental v-mos-transformed cells. Northern analysis of normal adult tissues shows that drm is expressed as a 4.4-kb message in a tissue-specific manner, with high expression in the brain, spleen, kidney, and testis and little or no expression in the heart, liver, and skeletal muscle. In situ hybridization analysis in adult rat tissue reveals good correlation with this pattern and indicates that drm mRNA is most highly expressed in nondividing and terminally differentiated cells, such as neurons, type 1 lung cells, and goblet cells. Transfection of a drug-selectable drm expression vector dramatically reduced the efficiency of colony formation in REF-1 and CHO cells, and the drm-transfected REF-1 survivors expressed low or nondetectable levels of exogenous drm mRNA. The toxic effects of drm can be overcome by cotransfection with constructs expressing oncogenic ras; furthermore, cells expressing high levels of drm and conditionally transformed with mos-expressing Moloney murine sarcoma virus rapidly undergo apoptosis when shifted to the nonpermissive temperature. Taken together, our data suggest that cells expressing high levels of drm undergo apoptotic death in the absence of oncogene-induced transformation and that drm represents a novel gene with potential roles in cell growth control or viability and tissue-specific differentiation.

Asunto(s)

Transformación Celular Neoplásica/genética , Fibroblastos/citología , Proteínas/genética , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , Proteínas Morfogenéticas Óseas , División Celular , Línea Celular , Línea Celular Transformada , Citocinas , ADN Complementario/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes mos/fisiología , Datos de Secuencia Molecular , Peso Molecular , Oncogenes , Especificidad de Órganos , Proteínas/análisis , Proteínas/química , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

We previously reported the isolation of PR2257, a novel avian sarcoma retrovirus which transduced the c-src protooncogene. The v-src gene of PR2257 differs from the c-src gene by a sequence change after amino acid 525, resulting in the replacement of tyrosine 527 by a valine, and an extension of the open reading frame into the non coding region of c-src. We investigated the respective roles of Tyr527 mutation and of the C-terminal extension in activating the oncogenic properties of c-src. Therefore we overexpressed the wild type c-src gene and c-src variants, carrying either a substitution of tyrosine 527 or an extension of the C-terminus or both modifications in combination, in chicken embryo fibroblasts and post mitotic neuroretina (NR) cells, using replication defective retroviruses. We also used in vivo inoculation of plasmid DNA to assess the tumorigenicity of the various c-src genes. We report that, in contrast to previous results, overexpression of c-src is sufficient to induce NR cell division. While mutation of tyrosine 527 alone significantly activates c-src transforming and tumorigenic properties, its combination with the C-terminal extension of PR2257 confers to this gene full oncogenic properties and increased metastatic potential as compared to the v-src of Rous sarcoma virus strains.

Asunto(s)

Diferenciación Celular/genética , División Celular/genética , Transformación Celular Neoplásica/genética , Genes src/genética , Familia-src Quinasas/genética , Animales , Pollos , Vectores Genéticos , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Fenotipo , Retroviridae/genética , Transfección , Familia-src Quinasas/metabolismo

RESUMEN

We reported previously that post-mitotic chicken embryonic neuroretina (NR) cells are induced to proliferate following in vitro infection with RAV-1, a retrovirus that does not carry an oncogene. NR cell multiplication results from the frequent activation and subsequent retroviral transduction of two related serine/threonine protein kinases, the c-mil/c-raf or c-Rmil/B-raf genes. We also showed that a very early event in the activation of these proto-oncogenes is the synthesis of chimeric mRNAs containing viral and cellular sequences joined by a splicing mechanism. In the current study, we have examined the ability of RAV-1 to induce proliferation of quail NR cells. By using the reverse transcription-polymerase chain reaction technique, we identified, in several proliferating quail NR cultures infected with RAV-1, a chimeric mRNA containing cellular sequences joined to the RAV-1 splice donor site. These cellular sequences are derived from a gene designated R10, which is expressed through a 1.9-kilobase (kb) mRNA detected in several embryonic tissues. A second transcript of 2.3 kb is specifically expressed in the NR, where both transcripts are developmentally regulated. The R10 cDNA encodes a 251-amino acid polypeptide that contains a leucine zipper motif. It exhibits significant similarity with the putative D52/N8L protein, encoded by an mRNA reported previously to be overexpressed in human breast and lung carcinomas. By using polyclonal antibodies specific for its amino-terminal and leucine zipper-containing regions, we identified the R10 gene product as a cytoplasmic protein of 23 kDa in cultured avian fibroblasts. A second protein of 30 kDa is detected in post-mitotic NR cells that express the 2.3-kb transcript. We also show, by in vitro transcription/translation and immunoprecipitation, that the R10 protein can readily form homodimers, presumably through its leucine zipper motif.

Asunto(s)

Proteínas Portadoras/genética , Proteínas del Ojo/genética , Proteínas de Neoplasias/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Compartimento Celular , Coturnix , Citoplasma/química , ADN Complementario/genética , Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Humanos , Leucina Zippers , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Mapeo Restrictivo , Retina/química , Alineación de Secuencia

RESUMEN

A rat brain synaptosomal protein of 110,000 M(r) present in a fraction highly enriched in adenylyl cyclase activity was microsequenced (Castets, F., G. Baillat, S. Mirzoeva, K. Mabrouk, J. Garin, J. d'Alayer, and A. Monneron. 1994. Biochemistry. 33:5063-5069). Peptide sequences were used to clone a cDNA encoding a novel, 780-amino acid protein named striatin. Striatin is a member of the WD-repeat family (Neer, E.J., C.J. Schmidt, R. Nambudripad, and T.F. Smith. 1994. Nature (Lond.). 371:297-300), the first one known to bind calmodulin (CaM) in the presence of Ca++. Subcellular fractionation shows that striatin is a membrane-associated, Lubrol-soluble protein. As analyzed by Northern blots, in situ hybridization, and immunocytochemistry, striatin is localized in the central nervous system, where it is confined to a subset of neurons, many of which are associated with the motor system. In particular, striatin is conspicuous in the dorsal part of the striatum, as well as in motoneurons. Furthermore, striatin is essentially found in dendrites, but not in axons, and is most abundant in dendritic spines. We propose that striatin interacts, through its WD-repeat domain and in a CaM/Ca(++)-dependent manner, with one or several members of a surrounding cluster of molecules engaged in a Ca(++)-signaling pathway specific to excitatory synapses.

Asunto(s)

Adenilil Ciclasas/análisis , Proteínas de Unión a Calmodulina/análisis , Sistema Nervioso Central/química , Dendritas/química , Adenilil Ciclasas/química , Adenilil Ciclasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión a Calmodulina/química , Proteínas de Unión a Calmodulina/genética , Fraccionamiento Celular , Clonación Molecular , Cuerpo Estriado/química , AMP Cíclico/biosíntesis , ADN Complementario/genética , Masculino , Datos de Secuencia Molecular , Peso Molecular , Neuronas Motoras/química , Péptidos/química , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Análisis de Secuencia , Análisis de Secuencia de ADN , Solubilidad

RESUMEN

Recent studies suggested the existence of Ras/B-Raf/ MEK-1 complexes and a critical role for B-Raf in regulating the MAP kinase/ERKs signalling pathway. We report, here, that both Ras and MEK-1 proteins interact physically with B-Raf proteins in the yeast two-hybrid system. In addition, by screening a mouse brain cDNA library, we isolated additional B-Raf interacting proteins. These include three members of the 14-3-3 proteins family (eta, theta and zeta) and the MEK-2 protein. We also show that c-Raf-1, previously reported to interact with beta and zeta 14-3-3 proteins, also interacts with eta and theta 14-3-3 proteins in the two-hybrid system. By using different portions of the B-Raf protein, we mapped the regions of the protein involved in these interactions. Specifically, we have characterized B-Raf specific sequences required for an efficient interaction with MEK proteins. We show that, consequently, B-Raf interacts with MEK-1 and MEK-2 with a better affinity than does c-Raf-1, thus strengthening the notion that B-Raf is a stronger MEK activator than c-Raf-l. Our results also suggest that a MEK specific sequence, not present in MAP kinase kinases which are not activated by members of the Raf family, is required for the interaction with Raf proteins.

Asunto(s)

Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Serina-Treonina Quinasas/metabolismo , Señales de Clasificación de Proteína/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiología , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cricetinae , Humanos , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Ratones , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-raf , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Proteínas ras/genética , Proteínas ras/metabolismo

RESUMEN

Neural retina development results from growth arrest of neuroectodermal precursors and differentiation of postmitotic cells. The QRI gene is specifically expressed in Müller retinal glial cells. Its expression coincides with the stage of withdrawal from the cell cycle and establishment of differentiation and is repressed upon induction of retinal cell proliferation by the v-src gene product. In this report, we show that the QR1 gene encodes several glycosylated proteins that are secreted and can either associate with the extracellular matrix or remain diffusible in the medium. By using pulse-chase experiments, the 100-103 kDa forms seem to appear first and are specifically incorporated into the extracellular matrix, whereas the 108 and 60 kDa polypeptides appear later and are detected as soluble forms in the culture medium. We also report that expression of the QR1 gene is developmentally regulated in the chicken. Its mRNA is first detectable at embryonic day 10, reaches a maximal level at embryonic day 15 and is no longer detected at embryonic day 18. Immunolocalization of the QR1 protein in chicken retina sections during development shows that expression of the protein parallels the differentiation pattern of post-miotic cells (in particular Müller cells and rods), corresponding to the two differentiation gradients in the retina: from the ganglion cell layer to the inner nuclear layer and outer nuclear layer, and from the optic nerve to the iris. At embryonic day 10, expression of the QR1 protein(s) is restricted to the optic nerve region and the inner nuclear layer, colocalizing with Müller cell bodies. As development proceeds, QR1 protein localization spreads towards the iris and towards the outer nuclear layer, following Müller cell elongations towards the photoreceptors. Between embryonic days 16 and 18, the QR1 protein is no longer detectable in the optic nerve region and is concentrated around the basal segment of the photoreceptors in the peripheral retina. Our results suggest a role for the QR1 gene product in the process of growth arrest and establishment of photoreceptor differentiation.

Asunto(s)

Coturnix/embriología , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Retina/embriología , Animales , Diferenciación Celular/genética , División Celular/genética , Retina/citología , Retina/metabolismo

RESUMEN

Four different transformed cell lines were isolated as a result of independent infection of primary hamster fibroblasts by Rous sarcoma virus (RSV SR-D stocks). These lines differ by the level of their spontaneous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70-200 metastatic nodules in the lung and/or lymph nodes of inoculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, LM). All cell lines contained one copy of integrated and expressed intact RSV provirus. The difference in the amount of v-src protein in cell lines was not correlated with their metastatic potential in vivo. Complete v-srcHM and v-srcLM genes were cloned from corresponding gene libraries and sequenced. In the unique region of both v-src isoforms a GC-rich insert of 60 nucleotides (20 a.a.) was found. The presence of this insert explains the unusual apparent molecular weight of protein encoded by v-srcHM and vsrcLM: 62 kDA. Both genes had 10 identical amino acid changes when compared to the known RSV SR-D v-src sequence. v-srcHM and v-srcLM differ by several amino acid changes. Most of them are localized in the unique domain and the extreme carboxy-terminal region of the of the oncoprotein. Both v-src variants and chimeric v-src with mutually substituted parts were subcloned in a retroviral vector and introduced into avian neuroretina cells. Significant differences in the morphology of transformed neuroretinal cells were associated with the mutations in the carboxy-terminal region of the v-src oncogene. Low metastatic HET-SR cells transfected with v-srcHM and the chimeric gene v-src-LH remarkably increased their metastatic potential. In contrast, this effect was not observed when the same cells were transfected with v-srcLM and the chimeric v-srcHL gene. Specific changes in the distribution of fibronectin matrix typical for high metastatic cells were found in the lines transfected with v-srcHM.

Asunto(s)

Virus del Sarcoma Aviar/genética , Transformación Celular Neoplásica , Transformación Celular Viral , Genes src , Mutación , Metástasis de la Neoplasia/genética , Neoplasias Experimentales/virología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Transformada , Pollos , Clonación Molecular , Cricetinae , ADN Viral/genética , Fibronectinas/metabolismo , Genes Virales , Mesocricetus , Datos de Secuencia Molecular , Neoplasias Experimentales/genética , Plásmidos , Proteínas Recombinantes de Fusión/genética , Retina/patología