RESUMEN
Cloning by the transplantation of somatic nuclei into unfertilized eggs requires a dramatic remodeling of chromosomal architecture. Many proteins are specifically lost from nuclei, and others are taken up from the egg cytoplasm. Recreating this exchange in vitro, we identified the chromatin-remodeling nucleosomal adenosine triphosphatase (ATPase) ISWI as a key molecule in this process. ISWI actively erases the TATA binding protein from association with the nuclear matrix. Defining the biochemistry of global nuclear remodeling may facilitate the efficiency of cloning and other dedifferentiation events that establish new stem cell lineages.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Óvulo/fisiología , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción/metabolismo , Animales , Extractos Celulares , Citoplasma/fisiología , ADN/metabolismo , ADN Helicasas/metabolismo , Histonas/metabolismo , Microscopía Fluorescente , Matriz Nuclear/metabolismo , Nucleoplasminas , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIB , Xenopus , NucleolinaRESUMEN
Recent progress identifies targeted chromatin remodelling by co-repressor complexes as being an integral component of transcriptional silencing. Here we discuss how chromatin structure and the basal transcriptional machinery are manipulated by the co-repressor complex containing the Mi-2 nucleosomal ATPase, the histone-binding protein RbAp48 and histone deacetylase and by the co-repressor complex containing SIN3, RbAp48 and histone deacetylase. Remarkably, both of these complexes also contain methyl-CpG-binding proteins. This observation provides a molecular mechanism to integrate DNA methylation fully into gene control in vertebrates.
Asunto(s)
Cromatina/química , Cromatina/metabolismo , Proteínas Cromosómicas no Histona , Receptores de Esteroides/metabolismo , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Cromosomas/metabolismo , Islas de CpG/genética , Metilación de ADN , Silenciador del Gen , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Proteína 4 de Unión a Retinoblastoma , Factores de Transcripción/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
The nucleosomal ATPase ISWI is the catalytic subunit of several protein complexes that either organize or perturb chromatin structure in vitro. This work reports the cloning and biochemical characterization of a Xenopus ISWI homolog. Surprisingly, whereas we find four complex forms of ISWI in egg extracts, we find no functional homolog of NURF. One of these complexes, xACF, consists of ISWI, Acf1, and a previously uncharacterized protein of 175 kDa. Like both ACF and CHRAC, this complex organizes randomly deposited histones into a regularly spaced array. The remaining three forms include two novel ISWI complexes distinct from known ISWI complexes plus a histone-dependent ATPase complex. This comprehensive biochemical characterization of ISWI underscores the evolutionary conservation of the ACF/CHRAC family.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Drosophila , Factores de Transcripción/química , Proteínas de Xenopus , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Cromatina/química , Cromatina/metabolismo , Secuencia Conservada , ADN Complementario/metabolismo , Drosophila melanogaster , Electroforesis en Gel de Poliacrilamida , Evolución Molecular , Biblioteca de Genes , Heparina/metabolismo , Histonas/metabolismo , Immunoblotting , Datos de Secuencia Molecular , Isoformas de Proteínas , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismo , XenopusRESUMEN
The Mi-2 complex has been implicated in chromatin remodeling and transcriptional repression associated with histone deacetylation. Here, we use a purified Mi-2 complex containing six components, Mi-2, Mta 1-like, p66, RbAp48, RPD3, and MBD3, to investigate the capacity of this complex to destabilize histone-DNA interactions and deacetylate core histones. The Mi-2 complex has ATPase activity that is stimulated by nucleosomes but not by free histones or DNA. This nucleosomal ATPase is relatively inefficient, yet is essential to facilitate both translational movement of histone octamers relative to DNA and the efficient deacetylation of the core histones within a mononucleosome. Surprisingly, ATPase activity had no effect on deacetylation of nucleosomal arrays.
Asunto(s)
Adenosina Trifosfato/farmacología , Autoantígenos/metabolismo , ADN Helicasas , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae , Acetilación , Acetiltransferasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Centrifugación por Gradiente de Densidad , Pollos , Cromatina/metabolismo , ADN/análisis , Electroforesis en Gel de Poliacrilamida , Eritrocitos/metabolismo , Histona Acetiltransferasas , Histona Desacetilasas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Nucleosomas/enzimología , Nucleosomas/metabolismo , Oocitos/metabolismo , XenopusRESUMEN
The nucleosome and chromatin fiber provide the common structural framework for transcriptional control in eukaryotes. The folding of DNA within these structures can both promote and impede transcription dependent on structural context. Importantly, neither the nucleosome nor the chromatin fiber is a static structure. Histone dissociation, histone modification, nucleosome mobility, and assorted allosteric transitions contribute to transcriptional control. Chromatin remodeling is associated with gene activation and repression. Energy-dependent processes mediate the assembly of both activating and repressive proteins into the nucleosomal infrastructure. Recent progress allows the structural consequences of these processes to be visualized at the chromosomal level. DNA and RNA polymerase, SWI/SNF complexes, histone deacetylases, and acetyltransferases are targeted by gene-specific regulators to mediate these structural transitions. The mistargeting of these enzymes contributes to human developmental abnormalities and tumorigenesis. These observations illuminate the roles of chromatin and chromosomal structural biology in human disease.
Asunto(s)
Cromatina/fisiología , Nucleosomas/fisiología , Transcripción Genética , Animales , Cromatina/genética , Cromatina/ultraestructura , ADN Polimerasa Dirigida por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Humanos , Nucleosomas/genética , Nucleosomas/ultraestructura , Activación TranscripcionalRESUMEN
Recent studies have shown that two nucleosome-remodeling complexes, NURF and CHRAC, open chromatin for transcription and replication by using their common catalytic subunit, the nucleosomal ATPase ISWI, to increase the mobility of nucleosomes relative to DNA sequence.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Cromatina/fisiología , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN , Proteínas de Drosophila , Proteínas de Insectos/fisiología , Chaperonas Moleculares , Proteínas Nucleares , Nucleosomas/fisiología , Pirofosfatasas , Factores de Transcripción/fisiología , Transcripción Genética , Adenosina Trifosfatasas/química , Animales , Histonas/fisiología , Proteínas de Insectos/química , Modelos Moleculares , Proteína 4 de Unión a Retinoblastoma , Factores de Transcripción/químicaRESUMEN
We have investigated the interactions of the methyl-CpG binding transcriptional repressor MeCP2 with nucleosomal DNA. We find that MeCP2 forms discrete complexes with nucleosomal DNA associating with methyl-CpGs exposed in the major groove via the methyl-CpG-binding domain (MBD). In addition to the MBD, the carboxyl-terminal segment of MeCP2 facilitates binding both to naked DNA and to the nucleosome core. These observations provide a molecular mechanism by which MeCP2 can gain access to chromatin in order to target corepressor complexes that further modify chromatin structure.
Asunto(s)
Proteínas Cromosómicas no Histona , Islas de CpG/genética , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Represoras/metabolismo , Animales , ADN/metabolismo , Huella de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Desoxirribonucleasa I , Fabaceae , Glutatión Transferasa/genética , Hidrólisis , Proteína 2 de Unión a Metil-CpG , Nucleasa Microcócica/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas de Plantas/metabolismo , Plantas Medicinales , Unión Proteica , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificación , Eliminación de Secuencia , Xenopus , Xenopus laevisRESUMEN
We describe histone-DNA cross-linking in a positioned nucleosome containing a thyroid hormone response element (TRE) from the Xenopus laevis thyroid hormone receptor betaA gene (TRbetaA). Histones H3 and H4 are cross-linked to DNA in the nucleosome core within 30 base pairs to either side of the dyad axis. Histone H2A cross-links to DNA in the core at the dyad axis, and histones H2A and H2B have extensive interactions with DNA 40-80 bp away from the dyad axis. Linker histone H5 and the globular domain of Xenopus H1(0) associate asymmetrically with DNA at one edge of the TRbetaA nucleosome. Nevertheless, the asymmetric association of H5 leads to a significant rearrangement of core histone-DNA contacts at the dyad axis of the nucleosome. In the presence of linker histone, cross-linkings of H4 within 15 bp to one side of the dyad axis, of histone H2A at the dyad axis, and of H2A and H2B 40-80 bp to one side of the dyad axis are all reduced. This reduction in cross-linking occurs preferentially on the side of the nucleosome to which H5 is bound. Our results indicate that core histone contacts within mononucleosomes are conformationally dynamic and that linker histone incorporation at the edge of the nucleosome can influence core histone-DNA interactions in an asymmetric way including contacts at the dyad axis.
Asunto(s)
ADN/química , Histonas/química , Nucleosomas/química , Receptores de Hormona Tiroidea/química , Animales , Reactivos de Enlaces Cruzados , ADN/metabolismo , Histonas/metabolismo , Conformación de Ácido Nucleico , Nucleosomas/metabolismo , Unión Proteica , Conformación Proteica , Receptores de Hormona Tiroidea/metabolismo , Xenopus laevisRESUMEN
Histone deacetylase and chromatin assembly contribute to the control of transcription of the Xenopus TRbetaA gene promoter by the heterodimer of Xenopus thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). Addition of the histone deacetylase inhibitor Trichostatin A (TSA) relieves repression of transcription due to chromatin assembly following microinjection of templates into Xenopus oocyte nuclei, and eliminates regulation of transcription by TR-RXR. Expression of Xenopus RPD3p, the catalytic subunit of histone deacetylase, represses the TRbetaA promoter, but only after efficient assembly of the template into nucleosomes. In contrast, the unliganded TR-RXR represses templates only partially assembled into nucleosomes; addition of TSA also relieves this transcriptional repression. This result indicates the distinct requirements for chromatin assembly in mediating transcriptional repression by the deacetylase alone, compared with those needed in the presence of unliganded TR-RXR. In addition, whereas hormone-bound TR-RXR targets chromatin disruption as assayed through changes in minichromosome topology and loss of a regular nucleosomal ladder on micrococcal nuclease digestion, addition of TSA relieves transcriptional repression but does not disrupt chromatin. Thus, TR-RXR can facilitate transcriptional repression in the absence of hormone through mechanisms in addition to recruitment of deacetylase, and disrupts chromatin structure through mechanisms in addition to the inhibition or release of deacetylase.
Asunto(s)
Cromatina/metabolismo , Regulación de la Expresión Génica , Histona Desacetilasas/fisiología , Receptores de Hormona Tiroidea/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catálisis , Cromatina/genética , Cromatina/fisiología , Replicación del ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/biosíntesis , Histona Desacetilasas/genética , Ácidos Hidroxámicos/farmacología , Ligandos , Datos de Secuencia Molecular , Nucleosomas/genética , Nucleosomas/metabolismo , Receptores de Ácido Retinoico/fisiología , Receptores X Retinoide , Moldes Genéticos , Hormonas Tiroideas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , XenopusRESUMEN
A simple procedure for manufacturing microchips containing various gel-immobilized compounds is described. A gel photopolymerization technique is introduced to produce micromatrices of polyacrylamide gel pads (25 x 25 x 20 microm and larger) separated by a hydrophobic glass surface. A pin device for the manual application of a compound in solution onto the activated polyacrylamide gel pad for immobilization is described. Oligonucleotide, DNA, and protein microchips have been produced by this method and tested by hybridization and immunoanalysis monitored with a fluorescence microscope. The effect of the lengths of the immobilized oligonucleotides and the hybridized RNA and DNA on hybridization of the oligonucleotide microchips was evaluated. This method can also be used for manufacturing microchips containing a variety of other compounds.
Asunto(s)
Biotecnología/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia de ADN/instrumentación , Análisis de Secuencia de ARN/instrumentación , Anticuerpos , Especificidad de Anticuerpos , Cartilla de ADN , Técnica del Anticuerpo Fluorescente Indirecta , Microscopía Fluorescente , Oligonucleótidos/síntesis química , Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa/métodos , Proteínas/química , Proteínas/genéticaRESUMEN
The utility of parallel hybridization of environmental nucleic acids to many oligonucleotides immobilized in a matrix of polyacrylamide gel pads on a glass slide (oligonucleotide microchip) was evaluated. Oligonucleotides complementary to small-subunit rRNA sequences of selected microbial groups, encompassing key genera of nitrifying bacteria, were shown to selectively retain labeled target nucleic acid derived from either DNA or RNA forms of the target sequences. The utility of varying the probe concentration to normalize hybridization signals and the use of multicolor detection for simultaneous quantitation of multiple probe-target populations were demonstrated.
Asunto(s)
Técnicas Biosensibles , Microbiología Ambiental , Sondas de Oligonucleótidos/genética , Bacterias/genética , Secuencia de Bases , Estudios de Evaluación como Asunto , Hibridación de Ácido Nucleico , ARN Bacteriano/genética , ARN Ribosómico/genética , ARN Ribosómico 16S/genéticaRESUMEN
Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains.
Asunto(s)
Interferón-alfa/farmacología , Interferón gamma/farmacología , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Antígenos CD/genética , Antígenos CD/metabolismo , Secuencia de Bases , Línea Celular , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Mutación del Sistema de Lectura , Humanos , Interleucina-6/farmacología , Janus Quinasa 1 , Janus Quinasa 2 , Datos de Secuencia Molecular , Mutación , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Proteínas Recombinantes de Fusión , Proteínas Recombinantes , Factor de Transcripción STAT1 , Transactivadores/metabolismo , Receptor de Interferón gammaRESUMEN
We present a further development in the technology of sequencing by hybridization to oligonucleotide microchips (SHOM) and its application to diagnostics for genetic diseases. A robot has been constructed to manufacture sequencing "microchips." The microchip is an array of oligonucleotides immobilized into gel elements fixed on a glass plate. Hybridization of the microchip with fluorescently labeled DNA was monitored in real time simultaneously for all microchip elements with a two-wavelength fluorescent microscope equipped with a charge-coupled device camera. SHOM has been used to detect beta-thalassemia mutations in patients by hybridizing PCR-amplified DNA with the microchips. A contiguous stacking hybridization technique has been applied for the detection of mutations; it can simplify medical diagnostics and enhance its reliability. The use of multicolor monitoring of contiguous stacking hybridization is suggested for large-scale diagnostics and gene polymorphism studies. Other applications of the SHOM technology are discussed.
Asunto(s)
Análisis Mutacional de ADN/métodos , Enfermedades Genéticas Congénitas/diagnóstico , Técnicas Genéticas , Oligonucleótidos/genética , Secuencia de Bases , Biotecnología , ADN/genética , Análisis Mutacional de ADN/instrumentación , Cartilla de ADN/genética , Estudios de Evaluación como Asunto , Enfermedades Genéticas Congénitas/genética , Técnicas Genéticas/instrumentación , Globinas/genética , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Robótica/instrumentación , Talasemia beta/diagnóstico , Talasemia beta/genéticaRESUMEN
The isolation and complementation of mutant human cell lines has established an essential role for the JAK (Janus kinase) family of protein tyrosine kinases and STAT (signal transduction and transcription) factors in the Interferon response pathways. Activation of STATs by JAKs occurs in receptor complexes at the cell membrane. Activated STATs form homo- or heterodimers and, with or without additional factors, migrate to the nucleus to initiate transcription. Different STAT combinations interact differentially with related DNA response elements. Signalling pathways of this novel type are likely utilized by a wide variety of polypeptide ligands. Data from the IL2, IL6 and IFN systems indicate a major role for the tyrosine phosphorylated receptor/JAK complexes (rather than substrate specificity of the JAKs per se) in STAT selection. The mutant cell lines lacking individual JAKs and STATs are being used together with kinase-negative JAK mutants which differentially affect the IFN-gamma, and IFN-alpha beta and IL-6 pathways in the further analysis of these and additional systems.
Asunto(s)
Interferones/fisiología , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas , Transducción de Señal/fisiología , Factores de Transcripción/fisiología , Citocinas/fisiología , Humanos , Janus Quinasa 1 , Janus Quinasa 2RESUMEN
The activation of Janus protein tyrosine kinases (Jak) and STAT (signal transducer and activator of transcription) proteins has recently been linked to the signal transduction mechanism of several cytokines. IL-7 was observed to induce a rapid and dose-dependent tyrosine phosphorylation of Jak 1 and Jak 3 and concomitantly, the tyrosine phosphorylation and DNA binding activity of multiple STAT proteins. The STAT proteins utilized by IL-7 were identical to those induced by IL-2 and could be identified as various STAT 5 isoforms. Moreover, the induction of both Jak 1 and 3, and STAT 5 activity strongly correlated with the growth-promoting effects of IL-7, suggesting that this signal transduction mechanism may play a key role in IL-7-induced proliferation.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Interleucina-7/farmacología , Proteínas de la Leche , Proteínas Tirosina Quinasas/biosíntesis , Linfocitos T/efectos de los fármacos , Transactivadores/biosíntesis , Animales , Secuencia de Bases , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Humanos , Interleucina-2/farmacología , Janus Quinasa 1 , Janus Quinasa 3 , Ratones , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT5 , Linfocitos T/metabolismoRESUMEN
The protein tyrosine kinases JAK1, JAK2 and Tyk2 and STATs (signal transducers and activators of transcription) 1 and 3 are activated in response to interleukin-6 (IL-6) in human fibrosarcoma cells. In mutant cells lacking JAK1, JAK2 or Tyk2, the absence of one kinase does not prevent activation of the others; activation does not, therefore, involve a sequential three-kinase cascade. In the absence of JAK1, the phosphorylation of the gp130 subunit of the IL-6 receptor and the activation of STATs 1 and 3 are greatly reduced. JAK1 is also necessary for the induction of IRF1 mRNA, thus establishing a requirement for the JAK/STAT pathway in the IL-6 response. JAK2 and Tyk2 although activated cannot, in the absence of JAK1, efficiently mediate activation of STATs 1 and 3. A kinase-negative mutant of JAK2 can, however, inhibit such activation, and ancillary roles for JAK2 and Tyk2 are not excluded. A major role for JAK1 and the nonequivalence of JAK1 and JAK2 in the IL-6 response pathway are, nevertheless, clearly established for these cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interleucina-6/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Transducción de Señal , Transactivadores/metabolismo , Adolescente , Secuencia de Bases , Western Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Fibrosarcoma , Humanos , Janus Quinasa 1 , Janus Quinasa 2 , Datos de Secuencia Molecular , Mutagénesis , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosfotirosina , Proteínas/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transducción de Señal/efectos de los fármacos , TYK2 Quinasa , Transfección , Células Tumorales Cultivadas , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
The activation of Janus protein tyrosine kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins by interleukin (IL)-2, the T cell antigen receptor (TCR) and interferon (IFN) alpha was explored in human peripheral blood-derived T cells and the leukemic T cell line Kit225. An IL-2-induced increase in JAK1 and JAK3, but not JAK2 or Tyk2, tyrosine phosphorylation was observed. In contrast, no induction of tyrosine phosphorylation of JAKs was detected upon stimulation of the TCR. IFN alpha induced the tyrosine phosphorylation of JAK1 and Tyk2, but not JAK2 or JAK3. IFN alpha activated STAT1, STAT2 and STAT3 in T cells, but no detectable activation of these STATs was induced by IL-2. However, IL-2 regulates the DNA binding and tyrosine phosphorylation of two STAT-like protein complexes which do not include STAT1, STAT2 or STAT3. STAT4 is not activated by IL-2. The activation of STAT5 cannot be excluded, so the IL-2-activated complexes most probably include at least one novel STAT. No STAT activity was detected in TCR-stimulated lymphocytes, indicating that the JAK/STAT pathway defined in this study constitutes an IL-2R-mediated signaling event which is not shared by the TCR. Finally, in other cell types the correlation between JAK1 activation and the induction of STAT1 has suggested that JAK1 may activate STAT1. The observation that IL-2 and IFN alpha activate JAK1 to a comparable degree, but only IFN alpha activates STAT1, indicates that JAK1 activation is not the only determining factor for STAT1 activation. Moreover, the data show that JAK1 stimulation is also not sufficient for STAT3 activation.
Asunto(s)
Interferón-alfa/farmacología , Interleucina-2/farmacología , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/enzimología , Transactivadores/metabolismo , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Janus Quinasa 1 , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Fosforilación , Receptores de IgG/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Tirosina/metabolismoRESUMEN
The recently identified JAK-STAT signal transduction pathway plays a major role in signalling the arrival of cytokines at the cell surface and can, in part, account for the pleiotropic and redundant effects of cytokines.
Asunto(s)
Citocinas/fisiología , Transducción de Señal/fisiología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Janus Quinasa 3 , Modelos Biológicos , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/fisiología , Receptores de Citocinas/genética , Receptores de Citocinas/fisiología , Transactivadores/genética , Transactivadores/fisiologíaRESUMEN
We have produced a cell line which lacks the protein tyrosine kinase JAK1 and is completely defective in interferon response. Complementation of this mutant with JAK1 restored the response, establishing the requirement for JAK1 in both the interferon-alpha/beta and -gamma signal transduction pathways. The reciprocal interdependence between JAK1 and Tyk2 activities in the interferon-alpha pathway, and between JAK1 and JAK2 in the interferon-gamma pathway, may reflect a requirement for these kinases in the correct assembly of interferon receptor complexes.