RESUMEN
STAT transcription factors are induced by a number of growth factors and cytokines. Within minutes of induction, the STAT proteins are phosphorylated on tyrosine and serine residues and translocated to the nucleus, where they bind to their DNA targets. The leukemia inhibitory factor (LIF) mediates pleiotropic and sometimes opposite effects both in vivo and in cultured cells. It is known, for example, to prevent differentiation of embryonic stem (ES) cells in vitro. To get insights into LIF-regulated signaling in ES cells, we have analyzed protein-binding and transcriptional properties of STAT recognition sites in ES cells cultivated in the presence and in the absence of LIF. We have detected a specific LIF-regulated DNA-binding activity implicating the STAT3 protein. We show that STAT3 phosphorylation is essential for this LIF-dependent DNA-binding activity. The possibility that ERK2 or a closely related protein kinase, whose activity is modulated in a LIF-dependent manner, contributes to this phosphorylation is discussed. Finally, we show that the multimerized STAT3-binding DNA element confers LIF responsiveness to a minimal thymidine kinase promoter. This, together with our observation that overexpression of STAT3 dominant-negative mutants abrogates this LIF responsiveness, clearly indicates that STAT3 is involved in LIF-regulated transcriptional events in ES cells. Finally, stable expression of such a dominant negative mutant of STAT3 induces morphological differentiation of ES cells despite continuous LIF supply. Our results suggest that STAT3 is a critical target of the LIF signaling pathway, which maintains pluripotent cell proliferation.
Asunto(s)
Inhibidores de Crecimiento/farmacología , Interleucina-6 , Linfocinas/farmacología , Células Madre/citología , Transcripción Genética/efectos de los fármacos , Proteínas de Fase Aguda/metabolismo , Animales , Especificidad de Anticuerpos , Sitios de Unión/fisiología , Blastocisto/citología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/genética , Factor Inhibidor de Leucemia , Linfocinas/química , Linfocinas/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Mutagénesis/fisiología , Fosfoproteínas/inmunología , Fosforilación , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Recombinantes de Fusión/genética , Factor de Transcripción STAT3 , Células Madre/química , Células Madre/enzimología , Transactivadores/inmunología , Transactivadores/metabolismo , Tirosina/metabolismoRESUMEN
We recently isolated three cDNA clones encoding closely related proteins (ATFa1, ATFa2, and ATFa3) that belong to the activating transcription factor-cyclic AMP-responsive element family of cellular transcription factors. Using cotransfection experiments, we showed that these proteins mediate the transcriptional activation induced by the adenovirus E1a 13S mRNA gene product and that the zinc-binding domains present in both E1a conserved region 3 and the most N-terminal portion of the ATFa proteins play crucial roles in this activity. Reciprocal coimmunoprecipitation experiments demonstrated direct interactions between these proteins. Neither the conserved region 3 domain of E1a nor the N-terminal metal-binding element of ATFa is essential for these interactions. The simultaneous alteration of both the N-terminal and the C-terminal domains of ATFa abolished E1a binding, while either mutation alone failed to impair these interactions.
Asunto(s)
Proteínas E1A de Adenovirus/fisiología , Adenovirus Humanos/genética , Proteínas Sanguíneas/fisiología , Regulación de la Expresión Génica , Factores de Transcripción/fisiología , Activación Transcripcional , Factores de Transcripción Activadores , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Relación Estructura-Actividad , Dedos de ZincRESUMEN
The human ATFa proteins belong to the CREB/ATF family of transcription factors. We have previously shown that the ATFa proteins may contribute to the modulation of the transcriptional activity of the Jun/Fos complexes (Chatton et al. (1994). Oncogene, 9, 375-385). We now show that a protein kinase activity is strongly associated with ATFa in vivo, as revealed by coimmunoprecipitation of ATFa/kinase complexes from whole cell extracts, with antibodies against ATFa. Two independent regions were found to be implicated in kinase binding: a major interaction site is located within the N-terminal 82 residues comprising an important metal-chelating element; a weaker binding site corresponds to the basic sequence element preceding the C-terminal leucine-zipper of ATFa. Induction experiments suggest that each of these ATFa domains may interact with different kinases. The major activity is associated with the ATFa N-terminal domain. Based on its response to various inducers, on both in vitro and in vivo binding assays, and on its immunological properties, this activity most likely corresponds to the 54/55 kDa JNK2 protein. Taken together, these observations suggest that the ATFa proteins, among other CREB/ATF proteins, may be important effectors of cell signalling pathways.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción Activadores , Animales , Sitios de Unión , Línea Celular , Humanos , MAP Quinasa Quinasa 4 , FosforilaciónRESUMEN
The ATFa proteins, which are members of the CREB/ATF family of transcription factors, display quite versatile properties. We have previously shown that they interact with the adenovirus E1a oncoprotein, mediating part of its transcriptional activity and heterodimerize with the Jun, Fos or related transcription factors, thereby modulating their DNA-binding specificity. In the present study, we report the sequence requirement of the N-terminal activation domain of ATFa and demonstrate the importance of specific threonine residues (Thr51 and Thr53) in addition to that of the metal-binding domain, in transcriptional activation processes. We also show that the N-terminal domain of ATFa which stably binds the Jun N-terminal kinase-2 (JNK2) (Bocco et al., 1996), is not a substrate for this kinase in vivo but, instead, serves as a JNK2-docking site for ATFa-associated partners like JunD, allowing them to be phosphorylated by the bound kinase.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Quinasas Activadas por Mitógenos , Fragmentos de Péptidos/fisiología , Proteínas Quinasas/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional/fisiología , Factor de Transcripción Activador 1 , Animales , Células COS , Línea Celular , Proteína Quinasa 9 Activada por Mitógenos , Fosforilación , Proteínas Proto-Oncogénicas c-jun/metabolismoRESUMEN
During the first half of 1993, bacteria that were isolated from clinical materials and found to have intermediate susceptibility by an agar dilution breakpoint method were collected in a large service laboratory in Germany. All of these isolates were gram-negative bacteria. They were re-tested employing full-scale agar dilution, broth microdilution, E-test and agar diffusion procedures. The results obtained indicated that 76.9% of the isolates were actually susceptible upon re-testing with a reference agar dilution technique. The reason for the discrepant results remained largely unclear. There was a high correlation between agar dilution and E-test results while the agreement with broth microdilution and agar diffusion was less satisfactory. It is suggested that the breakpoint between susceptible and intermediate categories currently recommended by DIN 58,940 (standard set by Deutsches Institut für Normung e.V.) be raised to reduce erroneous interpretations of minimum inhibitory concentrations.
Asunto(s)
Técnicas Bacteriológicas , Bacterias Gramnegativas/efectos de los fármacos , Imipenem/farmacología , Recuento de Colonia Microbiana , Medios de Cultivo , Bacterias Gramnegativas/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
The adenovirus EIa-inducible early EIIa (EIIaE) promoter is comprised of several sequence elements essential for constitutive and induced expression. We report here the purification of the host-cell factor that interacts with the major upstream element of this promoter, extending between positions -90 and -70 with respect to the main EIIaE cap site and exhibiting enhancer properties. The purified factor, which corresponds to a 40- to 43-kDa polypeptide, specifically binds to its recognition site and stimulates EIIaE promoter activity when added to an in vitro transcription system, reconstituted from purified factors and RNA polymerase. The implication of this factor in the control of the other adenovirus early genes is discussed.
Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Elementos de Facilitación Genéticos , Proteínas Oncogénicas Virales/genética , Regiones Promotoras Genéticas , Factores de Transcripción/aislamiento & purificación , Proteínas Precoces de Adenovirus , Adenovirus Humanos/genética , Cromatografía de Afinidad , Células HeLa , Humanos , Peso Molecular , Transcripción GenéticaRESUMEN
EIa-dependent transactivation of the adenovirus EIIa early (EIIaE) promoter is correlated with the activation of the cellular transcription factor E2F. In this study we identified a cellular protein, C alpha, that is distinct from E2F and that binds two sites in the EIIaE promoter, one of which overlaps with the proximal E2F binding site of the EIIaE promoter. The possible involvement of C alpha in the EIa responsiveness of this promoter is discussed.
Asunto(s)
Adenoviridae/genética , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Activación Transcripcional , Proteínas Precoces de Adenovirus , Secuencia de Bases , Unión Competitiva , Sondas de ADN , Células HeLa/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Oncogénicas Virales/aislamiento & purificación , Factores de Transcripción/aislamiento & purificaciónRESUMEN
As an approach to the identification of essential factors required for specific expression of the adenovirus type 2 EIIaE early (EIIaE) promoter, an in vitro system was established. Under appropriate conditions, using crude extracts of non-infected HeLa cells, efficient and accurate EIIaE expression has been reproduced. As in vivo, this transcription was strongly dependent upon the integrity of two non-consensus TATA-like elements, T1 and T2, corresponding to the major (EIIaE1) and minor (EIIaE2) start sites, respectively, as well as upon intact upstream elements (A, between -40 and -50 and B, between -70 and -90) common to both overlapping promoters. The implication of specific DNA-binding proteins in the transcriptional effects mediated by these elements was demonstrated by DNAse I footprinting analyses. Both crude nuclear extracts and partially purified fractions confer specific protection against DNAse I digestion to the T1 and B promoter elements defined above, and to a far upstream region (element C, between -110 and -150), which has previously been identified as a weaker promoter element by in vivo transcriptional studies. Separation of the T1 recognition factor from those which bind to the upstream elements B and C by chromatographic fractionation of the extracts has also been achieved.
Asunto(s)
Adenovirus Humanos/genética , Antígenos Virales de Tumores/genética , ADN Viral/genética , Genes Virales , Genes , Nucleoproteínas/metabolismo , Proteínas Oncogénicas Virales/genética , Regiones Promotoras Genéticas , Proteínas Precoces de Adenovirus , Adenovirus Humanos/metabolismo , Secuencia de Bases , ADN Viral/metabolismo , Células HeLa/metabolismo , Humanos , Nucleoproteínas/aislamiento & purificación , Unión Proteica , Transcripción GenéticaRESUMEN
In attempts to improve the post-translational modification and processing of recombinant factor IX (FIX) we have altered the cDNA sequence encoding pre-pro-FIX using site-directed mutagenesis and have expressed the variant cDNAs in BHK21 cells using a vaccinia-virus-derived vector. We find that substitution of the tyrosine residue at +1 for an alanine increases the biological activity of the recombinant molecules 2-fold. On the other hand, substitution of the proline at -3 for a valine results in no significant change to the specific activity of the protein. Other alterations to the N-terminus of the FIX proteins, in attempts to mimic other vitamin-K-dependent proteins, result in the failure to produce a secreted polypeptide. N-terminal sequence analysis of purified recombinant molecules reveals a correlation between specific activity and the efficiency of correct pro-sequence cleavage. gamma-Carboxylation analysis of purified recombinant proteins indicates that each molecule including unmutated FIX is completely gamma-carboxylated in this system. Thus the observed increase in biological activity of FIX variants containing an alanine at position +1 is not due to increased gamma-carboxylation but, at least in part, to more efficient pro-peptide cleavage.
Asunto(s)
Alanina/genética , Factor IX/genética , Variación Genética , Proteínas Recombinantes , Tirosina/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Deleción Cromosómica , Cricetinae , ADN/análisis , Factor IX/metabolismo , Expresión Génica , Datos de Secuencia MolecularRESUMEN
Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.