RESUMEN
RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA in vitro. These activities were inhibited by reducing agents, but no disulphide bonds were detected between RalA and ERp57. Mutation of all four of ERp57's active site cysteine residues blocked sensitivity to reducing agents, suggesting that redox-dependent conformational changes in ERp57 affect binding to RalA. Mutations in the switch II region of the GTPase domain of RalA specifically reduced or abolished binding to ERp57, but did not block GTP-specific binding to known RalA effectors, the exocyst and RalBP1. Oxidative treatment of A431 cells with H(2)O(2) inhibited cellular RalA activity, and the effect was exacerbated by expression of recombinant ERp57. The oxidative treatment significantly increased the amount of RalA localised to the cytosol. These findings suggest that ERp57 regulates RalA signalling by acting as a redox-sensitive guanine-nucleotide dissociation inhibitor (RalGDI).
Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Guanosina Difosfato/metabolismo , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Estrés Oxidativo , Péptidos/química , Unión Proteica , Proteína Disulfuro Isomerasas/química , Ratas , Fracciones Subcelulares/metabolismoRESUMEN
We have examined the role of endogenous 70-kDa S6 kinase (p70(S6K)) in actin cytoskeletal organization and cell migration in Swiss 3T3 fibroblasts. Association of p70(S6K) with the actin cytoskeleton was demonstrated by cosedimentation of p70(S6K) with F-actin and by subcellular fractionation in which p70(S6K) activity was measured in the F-actin cytoskeletal fraction. Immunocytochemical studies showed that p70(S6K), Akt1, PDK1, and p85 phosphoinositide 3-kinase (PI 3-kinase) were localized to the actin arc, a caveolin-enriched cytoskeletal structure located at the leading edge of migrating cells. Using a phospho-specific antibody to mammalian target of rapamycin (mTOR), we find that activated mTOR is enriched at the actin arc, suggesting that activation of the p70(S6K) signaling pathway is important to cell migration. Using the actin arc to assess migration, epidermal growth factor (EGF) stimulation was found to induce actin arc formation, an effect that was blocked by rapamycin treatment. We show further that actin stress fibers may function to down-regulate p70(S6K). Fibronectin stimulated stress fiber formation in the absence of growth factors and caused an inactivation of p70(S6K). Conversely, cytochalasin D and the Rho kinase inhibitor Y-27632, both of which cause stress fiber disruption, increased p70(S6K) activity. These studies provide evidence that the p70(S6K) pathway is important for signaling at two F-actin microdomains in cells and regulates cell migration.
Asunto(s)
Movimiento Celular , Citoesqueleto/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/fisiología , Actinas/metabolismo , Animales , Citoesqueleto/ultraestructura , Regulación hacia Abajo , Factor de Crecimiento Epidérmico/farmacología , Sustancias Macromoleculares , Ratones , Unión Proteica , Proteínas Quinasas S6 Ribosómicas 70-kDa/biosíntesis , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Fibras de Estrés/fisiología , Células 3T3 SwissAsunto(s)
Bioquímica/métodos , Histonas/química , Animales , Blastocisto/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Clonación Molecular , ADN/química , Histonas/aislamiento & purificación , Humanos , Ratones , Microscopía Fluorescente , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/metabolismo , Xenopus laevisRESUMEN
Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Endocitosis/fisiología , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismo , Secuencias de Aminoácidos , Animales , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/genética , Dinamina I/genética , Dinamina I/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Péptidos/química , Péptidos/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Proteína Quinasa C-alfa , Purinas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Roscovitina , Serina/metabolismo , Ovinos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sinaptosomas/química , Sinaptosomas/metabolismo , Sinaptosomas/ultraestructuraRESUMEN
Nitric oxide (NO) inhibits both actively induced and transferred autoimmune encephalomyelitis. To explore potential mechanisms, we examined the ability of NO to inhibit migration of T lymphoblasts through both collagen matrices and monolayers of rat brain endothelial cells. The NO donor 1-hydroxy-2-oxo-3, 3-bis (2-aminoethyl)-1-triazene (HOBAT) inhibited migration in a concentration-dependent manner. NO pretreatment of T cells inhibited migration through untreated endothelial cells, but NO pretreatment of endothelial cells had no inhibitory effect on untreated T cells. Therefore NO's migration inhibitory action was mediated through its effect on T cells and not endothelial cells. HOBAT did not inhibit migration by inducing T-cell death but rather by polarizing the T cells, resulting in a morphology suggestive of migrating cells. P70S6 kinase, shown to have a role in NO-induced migration inhibition in fibroblasts, had no role in the inhibitory effect of NO on T-cell migration. Thus, HOBAT did not alter p70S6K activity nor did rapamycin, a specific inhibitor of p70S6K, inhibit HOBAT-induced T-cell morphological changes or T-cell migration. We suggest that NO-induced morphological changes result in T cells with predefined migratory directionality, thus limiting the ability of these cells to respond to other migratory signals.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Actinas/análisis , Movimiento Celular , Encefalomielitis Autoinmune Experimental/inmunología , Óxido Nítrico/fisiología , Linfocitos T/inmunología , Animales , Encéfalo/citología , Moléculas de Adhesión Celular/metabolismo , Muerte Celular , Línea Celular , Movimiento Celular/efectos de los fármacos , Polaridad Celular , Células Cultivadas , Perros , Endotelio/fisiología , Activación de Linfocitos , Modelos Inmunológicos , Donantes de Óxido Nítrico/farmacología , Ratas , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Fibras de Estrés/ultraestructura , Linfocitos T/citología , Linfocitos T/ultraestructura , Uniones Estrechas/ultraestructura , Triazenos/farmacologíaRESUMEN
Determining how chromatin is remodelled during early development, when totipotent cells begin to differentiate into specific cell types, is essential to understand how epigenetic states are established. An important mechanism by which chromatin can be remodelled is the replacement of major histones with specific histone variants. During early mammalian development H2A.Z plays an essential, but unknown, function(s). We show here that undifferentiated mouse cells of the inner cell mass lack H2A.Z, but upon differentiation H2A.Z expression is switched on. Strikingly, H2A.Z is first targeted to pericentric hetero chromatin and then to other regions of the nucleus, but is excluded from the inactive X chromosome and the nucleolus. This targeted incorporation of H2A.Z could provide a critical signal to distinguish constitutive from facultative heterochromatin. In support of this model, we demonstrate that H2A.Z can directly interact with the pericentric heterochromatin binding protein INCENP. We propose that H2A.Z functions to establish a specialized pericentric domain by assembling an architecturally distinct chromatin structure and by recruiting specific nuclear proteins.
Asunto(s)
Desarrollo Embrionario y Fetal , Heterocromatina/metabolismo , Histonas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Compensación de Dosificación (Genética) , Femenino , Técnica del Anticuerpo Fluorescente , Histonas/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos Híbridos , Cromosoma XRESUMEN
The Drosophila melanogaster flightless I gene is required for normal cellularization of the syncytial blastoderm. Highly conserved homologues of flightless I are present in Caenorhabditis elegans, mouse, and human. We have disrupted the mouse homologue Fliih by homologous recombination in embryonic stem cells. Heterozygous Fliih mutant mice develop normally, although the level of Fliih protein is reduced. Cultured homozygous Fliih mutant blastocysts hatch, attach, and form an outgrowing trophoblast cell layer, but egg cylinder formation fails and the embryos degenerate. Similarly, Fliih mutant embryos initiate implantation in vivo but then rapidly degenerate. We have constructed a transgenic mouse carrying the complete human FLII gene and shown that the FLII transgene is capable of rescuing the embryonic lethality of the homozygous targeted Fliih mutation. These results confirm the specific inactivation of the Fliih gene and establish that the human FLII gene and its gene product are functional in the mouse. The Fliih mouse mutant phenotype is much more severe than in the case of the related gelsolin family members gelsolin, villin, and CapG, where the homozygous mutant mice are viable and fertile but display alterations in cytoskeletal actin regulation.