RESUMEN
In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-beta. Crystallization studies by others have indicated that nuclear carriers such as importin-beta bind to phenylalanine-glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-beta identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-beta. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-beta binding to those repeats. In addition to these expected binding sequences for importin-beta, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-beta, indicating that additional Nup sequences may form binding sites for importin-beta.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Análisis por Matrices de Proteínas/métodos , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Sitios de Unión , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , beta Carioferinas/química , beta Carioferinas/genéticaRESUMEN
The nuclear import of classical nuclear localization signal-containing proteins depends on importin-alpha transport receptors. In budding yeast there is a single importin-alpha gene and in higher eukaryotes there are multiple importin-alpha-like genes, but in fission yeast there are two: the previously characterized cut15 and the more recently identified imp1. Like other importin-alpha family members, Imp1p supports nuclear protein import in vitro. In contrast to cut15, imp1 is not essential for viability, but imp1delta mutant cells exhibit a telophase delay and mild temperature-sensitive lethality. Differences in the cellular functions that depend on Imp1p and Cut15p indicate that they each have unique physiological roles. They also have common roles because the imp1delta and the cut15-85 temperature-sensitive mutations are synthetically lethal; overexpression of cut15 partially suppresses the temperature sensitivity, but not the mitotic delay in imp1delta cells; and overexpression of imp1 partially suppresses the mitotic defect in cut15-85 cells but not the loss of viability. Both Imp1p and Cut15p are required for the efficient nuclear import of both an SV40 nuclear localization signal-containing reporter protein and the Pap1p component of the stress response MAP kinase pathway. Imp1p and Cut15p are essential for efficient nuclear protein import in S. pombe.
Asunto(s)
Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , alfa Carioferinas/genética , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Letales/genética , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Estrés Oxidativo , Fenotipo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , alfa Carioferinas/metabolismoRESUMEN
Nuclear transport carriers interact with proteins of the nuclear pore complex (NPC) to transport their cargo across the nuclear envelope. One such carrier is nuclear transport factor 2 (NTF2), whose import cargo is the small GTPase Ran. A domain highly homologous to the small NTF2 protein (14kDa) is also found in a number of additional proteins, which together make up the NTF2 domain containing superfamily of proteins. Using structural, computational and biochemical analysis we have identified a functional site that is present throughout this superfamily, and our results indicate that this site functions as an NPC binding site in NTF2. Previously we showed that a D23A mutant of NTF2 exhibits increased affinity for the NPC. The mechanism of this mutation, however, was unknown as this region of NTF2 had not been implicated in binding to NPC proteins. Here we show that the D23A mutation in NTF2 does not result in gross structural changes affecting other known NPC binding sites. Instead, the D23 residue is located in an evolutionarily important region in the NTF2 domain containing superfamily, that in NTF2, is involved in binding to the NPC.
Asunto(s)
Núcleo Celular/metabolismo , Biología Computacional , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte Activo de Núcleo Celular , Alanina/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Escherichia coli/genética , Evolución Molecular , Células HeLa , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Proteínas de Transporte Nucleocitoplasmático/genética , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Proteína de Unión al GTP ran/metabolismoRESUMEN
ERK2 nuclear-cytoplasmic distribution is regulated in response to hormones and cellular state without the requirement for karyopherin-mediated nuclear import. One proposed mechanism for the movement of ERK2 into the nucleus is through a direct interaction between ERK2 and nucleoporins present in the nuclear pore complex. Previous reports have attributed regulation of ERK2 localization to proteins that activate or deactivate ERK2, such as the mitogen-activated protein (MAP) kinase kinase MEK1 and MAP kinase phosphatases. Recently, a small non-catalytic protein, PEA-15, has also been demonstrated to promote a cytoplasmic ERK2 localization. We found that the MAP kinase insert in ERK2 is required for its interaction with PEA-15. Consistent with its recognition of the MAP kinase insert, PEA-15 blocked activation of ERK2 by MEK1, which also requires the MAP kinase insert to interact productively with ERK2. To determine how PEA-15 influences the localization of ERK2, we used a permeabilized cell system to examine the effect of PEA-15 on the localization of ERK2 and mutants that have lost the ability to bind PEA-15. Wild type ERK2 was unable to enter the nucleus in the presence of an excess of PEA-15; however, ERK2 lacking the MAP kinase insert largely retained the ability to enter the nucleus. Binding assays demonstrated that PEA-15 interfered with the ability of ERK2 to bind to nucleoporins. These results suggest that PEA-15 sequesters ERK2 in the cytoplasm at least in part by interfering with its ability to interact with nucleoporins, presenting a potential paradigm for regulation of ERK2 localization.
Asunto(s)
Núcleo Celular/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fosfoproteínas/química , Transporte Activo de Núcleo Celular , Animales , Proteínas Reguladoras de la Apoptosis , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , MAP Quinasa Quinasa 1 , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Moleculares , Mutación , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/química , Técnicas del Sistema de Dos HíbridosRESUMEN
Regulator of chromosome condensation (RCC1) binding to chromatin is highly dynamic, as determined by fluorescence recovery after photobleaching analysis of GFP-RCC1 in stably transfected tsBN2 cells. Microinjection of wild-type or Q69L Ran markedly slowed the mobility of GFP-RCC1, whereas T24N Ran (defective in nucleotide loading) decreased it further still. We found significant alterations in the mobility of intranuclear GFP-RCC1 after treatment with agents that disrupt different Ran-dependent nuclear export pathways. Leptomycin B, which inhibits Crm1/RanGTP-dependent nuclear export, significantly increased the mobility of RCC1 as did high levels of actinomycin D (to inhibit RNA polymerases I, II, and III) or alpha-amanitin (to inhibit RNA polymerases II and III) as well as energy depletion. Inhibition of just mRNA transcription, however, had no affect on GFP-RCC1 mobility consistent with mRNA export being a Ran-independent process. In permeabilized cells, cytosol and GTP were required for the efficient release of GFP-RCC1 from chromatin. Recombinant Ran would not substitute for cytosol, and high levels of supplemental Ran inhibited the cytosol-stimulated release. Thus, RCC1 release from chromatin in vitro requires a factor(s) distinct from, or in addition to, Ran and seems linked in vivo to the availability of Ran-dependent transport cargo.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Proteínas de Ciclo Celular , Cromatina/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Nucleares , Proteína de Unión al GTP ran/metabolismo , Amanitinas/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Células Cultivadas , Cricetinae , Citosol/metabolismo , Dactinomicina/farmacología , Ácidos Grasos Insaturados/farmacología , Guanosina Trifosfato/metabolismo , Microinyecciones , Microscopía Fluorescente , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Simian virus 40 (SV40) is a DNA tumor virus known to induce cancers in laboratory animals. There are numerous reports of the detection of SV40 DNA and/or proteins in human malignancies of the same types as those induced by SV40 in animals, including brain cancers. However, known exposure to the virus has not yet been linked directly to cancer development in a specific individual. Here we describe the detection of SV40 sequences in the meningioma of a laboratory researcher who had a probable direct exposure to SV40 and subsequently developed a tumor positive for viral DNA sequences indistinguishable from those of the laboratory source. This case suggests a link between viral exposure and tumor development.
Asunto(s)
Neoplasias Encefálicas/virología , Laboratorios de Hospital , Personal de Laboratorio Clínico , Meningioma/virología , Infecciones por Polyomavirus , Virus 40 de los Simios/inmunología , Infecciones Tumorales por Virus , Adulto , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/inmunología , Neoplasias Encefálicas/líquido cefalorraquídeo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/etiología , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/cirugía , Línea Celular Tumoral , Femenino , Gadolinio , Humanos , Imagen por Resonancia Magnética , Meningioma/líquido cefalorraquídeo , Meningioma/diagnóstico , Meningioma/etiología , Meningioma/patología , Meningioma/cirugía , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Factores de Riesgo , Análisis de Secuencia de ADN , Texas , Resultado del TratamientoRESUMEN
Until very recently, the vertebrate protein Npap60/Nup50 was thought merely to be a component of the nuclear pore complex (NPC). This conclusion was based on the observations that Npap60/Nup50 localizes at the NPC by immunofluorescence and electron microscopy and also contains FG (Phe-Gly) repeats, a motif commonly found in nucleoporins but not in proteins located elsewhere. However, far from being a fixed structural component of the NPC, it now appears as though Npap60 can shuttle from one side of the NPC to the other. Most significantly, a recent paper shows that Npap60 enhances the nuclear import of a cargo possessing a basic nuclear localization sequence by associating directly with the import cargo-carrier complex and (presumably) moving through the NPC with it. Several NPC proteins have now been shown to be mobile in the NPC, and this new report might indicate that these 'mobile' nucleoporins play a more active role in the nuclear transport of cargo than was previously appreciated.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Células Eucariotas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos/fisiología , Animales , HumanosRESUMEN
Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activated smooth muscle gene targets. CRP2 was found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocked proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converted pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibited the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation.
Asunto(s)
Proteínas Aviares , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas Portadoras/metabolismo , Diferenciación Celular , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteína beta Potenciadora de Unión a CCAAT/química , Proteína beta Potenciadora de Unión a CCAAT/genética , Sistema Cardiovascular/citología , Sistema Cardiovascular/embriología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Embrión de Pollo , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Factores de Unión al ADN Específico de las Células Eritroides , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Proteínas con Dominio LIM , Sustancias Macromoleculares , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Respuesta Sérica/metabolismo , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Rran-dependent nuclear transport requires a nuclear pool of RanGTP both for the assembly of export complexes and the disassembly of import complexes. Accordingly, in order for these processes to proceed, Ran-dependent nuclear import and export assays in vitro require the addition of GTP to produce RanGTP. Notably, no ATP requirement can be detected for these transport processes in vitro. But in vivo, when cells are depleted of ATP by the addition of sodium azide and 2-deoxyglucose to block ATP production by oxidative phosphorylation and glycolysis, respectively, Ran-dependent nuclear import and export are rapidly inhibited. This raised the question of whether there is an ATP requirement for these nuclear transport pathways in an intact cell that has remained undetected in vitro. Here we report that the free (but not total) GTP concentration rapidly drops to an undetectable level upon ATP depletion as does the availability of RanGTP. Our conclusion is that the inhibition of Ran-dependent nuclear transport observed upon ATP depletion in vivo results from a shortage of RanGTP rather than the inhibition of some ATP-dependent process.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Nucleares/metabolismo , Proteína de Unión al GTP ran/antagonistas & inhibidores , Adenosina Trifosfato/análisis , Antimetabolitos/farmacología , Transporte Biológico/efectos de los fármacos , Núcleo Celular , Inhibidores Enzimáticos/farmacología , Femenino , Células HeLa , Humanos , Cinética , Ribavirina/farmacología , Células Tumorales CultivadasRESUMEN
In stimulated cells, the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) concentrates in the nucleus. Evidence exists for CRM1-dependent, mitogen-activated protein kinase kinase-mediated nuclear export of ERK2, but its mechanism of nuclear entry is not understood. To determine requirements for nuclear transport, we tagged ERK2 with green fluorescent protein (GFP) and examined its nuclear uptake by using an in vitro import assay. GFP-ERK2 entered the nucleus in a saturable, time- and temperature-dependent manner. Entry of GFP-ERK2, like that of ERK2, required neither energy nor transport factors and was visible within minutes. The nuclear uptake of GFP-ERK2 was inhibited by wheat germ agglutinin, which blocks nuclear entry by binding to carbohydrate moieties on nuclear pore complex proteins. The nuclear uptake of GFP-ERK2 also was reduced by excess amounts of recombinant transport factors. These findings suggest that ERK2 competes with transport factors for binding to nucleoporins, which mediate the entry and exit of transport factors. In support of this hypothesis, we showed that ERK2 binds directly to a purified nucleoporin. Our data suggest that GFP-ERK2 enters the nucleus by a saturable, facilitated mechanism, distinct from a carrier- and energy-dependent import mechanism and involves a direct interaction with nuclear pore complex proteins.