RESUMEN
Human metapneumovirus (HMPV) encodes a small hydrophobic (SH) protein of unknown function. HMPV from which the SH open reading frame was deleted (HMPVΔSH) was viable and displayed similar replication kinetics, cytopathic effect and plaque size compared with wild type HMPV in several cell-lines. In addition, no differences were observed in infection efficiency or cell-to-cell spreading in human primary bronchial epithelial cells (HPBEC) cultured at an air-liquid interphase. Host gene expression was analyzed in A549 cells infected with HMPV or HMPVΔSH using microarrays and mass spectrometry (MS) based techniques at multiple time points post infection. Only minor differences were observed in mRNA or protein expression levels. A possible function of HMPV SH as apoptosis blocker, as proposed for several members of the family Paramyxoviridae, was rejected based on this analysis. So far, a clear phenotype of HMPV SH deletion mutants in vitro at the virus and host levels is absent.
Asunto(s)
Regulación de la Expresión Génica/genética , Metapneumovirus/genética , Metapneumovirus/fisiología , Proteínas Oncogénicas de Retroviridae/fisiología , Replicación Viral/genética , Western Blotting , Bronquios/citología , Línea Celular , Células Epiteliales/virología , Eliminación de Gen , Humanos , Espectrometría de Masas , Análisis por Micromatrices , Proteínas Oncogénicas de Retroviridae/deficienciaRESUMEN
Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.
Asunto(s)
Receptores ErbB/metabolismo , Lisosomas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Oncogénicas de Retroviridae/fisiología , Ubiquitina-Proteína Ligasas , Ubiquitina/metabolismo , Animales , Western Blotting , Células CHO , Línea Celular , Cricetinae , Regulación hacia Abajo , Embrión de Mamíferos , Endocitosis , Endosomas/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Fibroblastos/metabolismo , Expresión Génica , Humanos , Ratones , Mutación , Proteína Oncogénica v-cbl , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , ARN Mensajero/análisis , Proteínas Oncogénicas de Retroviridae/deficiencia , Proteínas Oncogénicas de Retroviridae/genética , Temperatura , TransfecciónRESUMEN
Cbl functions as an adaptor protein by interacting with other signalling molecules to form multimolecular complexes. Previous studies have proposed that Cbl is also a positive regulator of CrkL-C3G signalling, which leads to Rap1 activation. However, there is a lack of genetic evidence for a physiological function of Cbl in regulating this pathway. Here, we show that Cbl deficiency results in enhanced activation of Rap1. Cbl was shown to promote the ubiquitylation of CrkL without any apparent effect on its stability. Remarkably, the membrane translocation of C3G, its association with CrkL, and the guanine-nucleotide exchange activity of C3G were all increased in Cbl(-/-) thymocytes. Consistent with a function of Rap1 in integrin activation, enhanced integrin-mediated cell adhesion was also seen in Cbl(-/-) thymocytes. Thus, Cbl negatively regulates Rap1 activation, probably through a proteolysis-independent E3-ubiquitin-ligase activity of Cbl that modulates protein-protein interactions.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Ligasas/genética , Animales , Adhesión Celular , Línea Celular , Células Cultivadas , Regulación Enzimológica de la Expresión Génica , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Oncogénica v-cbl , Proteínas Recombinantes/metabolismo , Proteínas Oncogénicas de Retroviridae/deficiencia , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/metabolismo , Linfocitos T/inmunología , Linfocitos T/fisiología , Transfección , Ubiquitina-Proteína Ligasas , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Primary macrophages isolated from hck(-/-)fgr(-/-) mice display altered morphology and F-actin cytoskeletal structures and reduced migration. The ability of phorbol myristyl acetate (PMA), a protein kinase C activator that has been reported to increase macrophage spreading and carcinoma cell motility, to rescue these hck(-/-)fgr(-/-) defects was tested. Although PMA-treated wild-type and hck(-/-)fgr(-/-) macrophages exhibited a similar flattened, spread phenotype, PMA did not rescue the hck(-/-)fgr(-/-) macrophage migration defect. Instead, both PMA-treated wild type and hck(-/-)fgr(-/-) macrophages were defective in spontaneous and chemotactic migration and tyrosine phosphorylation of the Cbl protooncoprotein was decreased in both. Moreover, c-cbl(-/-) macrophages displayed the same impairment of motility as hck(-/-)fgr(-/-) macrophages and a similar morphology with less polarization and more dorsal ruffling than wild-type macrophages. As Hck and Fgr expression and activity were not decreased in c-cbl(-/-) macrophages, these results suggest that Cbl is likely to be an important downstream mediator of the Src family kinase-regulated macrophage motility pathway.
Asunto(s)
Actinas/metabolismo , Quimiotaxis de Leucocito/fisiología , Macrófagos/metabolismo , Proteínas Oncogénicas de Retroviridae/deficiencia , Actinas/efectos de los fármacos , Animales , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Células Cultivadas , Quimiotaxis de Leucocito/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Proteína Oncogénica v-cbl , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-hck , Proteínas Oncogénicas de Retroviridae/efectos de los fármacos , Proteínas Oncogénicas de Retroviridae/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacología , Tirosina/metabolismo , Familia-src Quinasas/efectos de los fármacos , Familia-src Quinasas/metabolismoRESUMEN
Responses to extracellular stimuli are often transduced from cell-surface receptors to protein tyrosine kinases which, when activated, initiate the formation of protein complexes that transmit signals throughout the cell. A prominent component of these complexes is the product of the proto-oncogene c-Cbl, which specifically targets activated protein tyrosine kinases and regulates their signalling. How, then, does this multidomain protein shape the responses generated by these signalling complexes?