RESUMO
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.
Assuntos
Adesinas Bacterianas , Proteínas do Citoesqueleto , Integrina beta1/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Yersinia pseudotuberculosis/patogenicidade , Proteínas rac1 de Ligação ao GTP/metabolismo , Células 3T3 , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Actinas/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Células COS , Linhagem Celular , Permeabilidade da Membrana Celular , Chlorocebus aethiops , Combinação de Medicamentos , Humanos , Camundongos , Óleos , Fagossomos/microbiologia , Fenóis , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Infecções por Yersinia pseudotuberculosis/microbiologiaRESUMO
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.
Assuntos
Genes Supressores de Tumor/genética , Neoplasias Hematológicas/genética , Hematopoese/genética , Proteínas Nucleares/genética , Transativadores/genética , Animais , Southern Blotting , Western Blotting , Transplante de Medula Óssea , Proteína de Ligação a CREB , Transplante de Células , Proteína p300 Associada a E1A , Neoplasias Hematológicas/patologia , Heterozigoto , Perda de Heterozigosidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , Fenótipo , Baço/citologia , Transativadores/metabolismoRESUMO
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.