RESUMEN
Efficient uptake of Yersinia pseudotuberculosis into cultured mammalian cells is the result of high-affinity binding of invasin to beta1 chain integrins. We demonstrate here that uptake requires Rac1 and Arp 2/3 function. Bacterial uptake was stimulated by GTPgammaS, but was inhibited in mammalian cells transfected with the interfering Rac1-N17 derivative. Rac1 was found to be activated in response to integrin engagement by invasin, whereas Rac1 and Arp 2/3 were found to be intensely localized around phagosomes bearing bacteria, indicating a specific role for Rac1 signalling from the nascent phagosome to downstream effectors. To determine whether the Arp 2/3 complex was a component of this proposed pathway, cells overproducing various derivatives of Scar1/WAVE1, an Arp 2/3-binding protein, were analysed. Sequestration of Arp 2/3 away from the phagocytic cup as a result of Scar1/WAVE1 overproduction dramatically inhibited uptake. To determine whether signalling from Rac1 to Arp 2/3 occurred via N-WASP, uptake was analysed in a cell line lacking expression of WASP and N-WASP. Uptake was unaffected by the absence of these proteins, indicating that beta1 integrin signalling from Rac1 to Arp 2/3 can occur in the absence of N-WASP function.
Asunto(s)
Adhesinas Bacterianas , Proteínas del Citoesqueleto , Integrina beta1/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Yersinia pseudotuberculosis/patogenicidad , Proteína de Unión al GTP rac1/metabolismo , Células 3T3 , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Actinas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Células COS , Línea Celular , Permeabilidad de la Membrana Celular , Chlorocebus aethiops , Combinación de Medicamentos , Humanos , Ratones , Aceites , Fagosomas/microbiología , Fenoles , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Infecciones por Yersinia pseudotuberculosis/microbiologíaRESUMEN
Mice with monoallelic inactivation of the CBP gene develop highly penetrant, multilineage defects in hematopoietic differentiation and, with advancing age, an increased incidence of hematologic malignancies. The latter are characterized, at least in some cases, by loss of heterozygosity (LOH) at the CBP locus. No such pathology was observed in wild-type or p300 heterozygous null mice of the same age and genetic background. Thus, a full complement of CBP, but not p300, is required for normal hematopoietic differentiation. These results also provide the first experimental evidence for the hypothesis that CBP has tumor-suppressing activity.
Asunto(s)
Genes Supresores de Tumor/genética , Neoplasias Hematológicas/genética , Hematopoyesis/genética , Proteínas Nucleares/genética , Transactivadores/genética , Animales , Southern Blotting , Western Blotting , Trasplante de Médula Ósea , Proteína de Unión a CREB , Trasplante de Células , Proteína p300 Asociada a E1A , Neoplasias Hematológicas/patología , Heterocigoto , Pérdida de Heterocigocidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/metabolismo , Fenotipo , Bazo/citología , Transactivadores/metabolismoRESUMEN
The transcriptional coactivator and integrator p300 and its closely related family member CBP mediate multiple, signal-dependent transcriptional events. We have generated mice lacking a functional p300 gene. Animals nullizygous for p300 died between days 9 and 11.5 of gestation, exhibiting defects in neurulation, cell proliferation, and heart development. Cells derived from p300-deficient embryos displayed specific transcriptional defects and proliferated poorly. Surprisingly, p300 heterozygotes also manifested considerable embryonic lethality. Moreover, double heterozygosity for p300 and cbp was invariably associated with embryonic death. Thus, mouse development is exquisitely sensitive to the overall gene dosage of p300 and cbp. Our results provide genetic evidence that a coactivator endowed with histone acetyltransferase activity is essential for mammalian cell proliferation and development.