RESUMEN
The growth inhibitory functions of p53 are controlled in unstressed cells by rapid degradation of the p53 protein. One of the principal regulators of p53 stability is MDM2, a RING finger protein that functions as an E3 ligase to ubiquitinate p53. MDM2 promotes p53 nuclear export, and in this study, we show that ubiquitination of the C terminus of p53 by MDM2 contributes to the efficient export of p53 from the nucleus to the cytoplasm. In contrast, MDM2 did not promote nuclear export of the p53-related protein, p73. p53 nuclear export was enhanced by overexpression of the export receptor CRM1, although no significant relocalization of MDM2 was seen in response to CRM1. However, nuclear export driven by CRM1 overexpression did not result in the degradation of p53, and nuclear export was not essential for p53 degradation. These results indicate that MDM2 mediated ubiquitination of p53 contributes to both nuclear export and degradation of p53 but that these activities are not absolutely dependent on each other.
Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas Nucleares , Receptores Citoplasmáticos y Nucleares , Proteína p53 Supresora de Tumor/química , Western Blotting , División Celular , Línea Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Carioferinas/metabolismo , Lisina/química , Microscopía Fluorescente , Modelos Genéticos , Mutación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Transfección , Ubiquitina/metabolismo , Proteína Exportina 1RESUMEN
BACKGROUND: Bruton's tyrosine kinase (Btk) is essential for B cell development and function. Mutations of Btk elicit X-linked agammaglobulinemia in humans and X-linked immunodeficiency in the mouse. Btk has been proposed to participate in B cell antigen receptor-induced signaling events leading to activation of phospholipase C-gamma2 (PLCgamma2) and calcium mobilization. However it is unclear whether Btk activation is alone sufficient for these signaling events, and whether Btk can activate additional pathways that do not involve PLCgamma2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCgamma2-deficient DT40 B cell line. RESULTS: Activation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCgamma2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCgamma2, mobilization of intracellular calcium, activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, and apoptosis. In DT40 B cells deficient for PLCgamma2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis. CONCLUSIONS: These data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCgamma2 in B cells. While it is not known whether activated Btk:ER precisely mimics activated Btk, this conditional system will likely facilitate the dissection of the role of Btk and its family members in a variety of biological processes in many different cell types.
Asunto(s)
Linfocitos B/enzimología , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/fisiología , Agammaglobulinemia Tirosina Quinasa , Animales , Apoptosis , Linfocitos B/inmunología , Señalización del Calcio , Línea Celular , Sistema de Señalización de MAP Quinasas , Ratones , Mutación , Fosfolipasa C gamma , Proteínas Tirosina Quinasas/genética , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/metabolismo , Fosfolipasas de Tipo C/genéticaAsunto(s)
Proteínas Nucleares , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Replicación del ADN , Genes p53 , Humanos , Ratones , Modelos Biológicos , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
1. Parathyroid hormone-induced down-regulation was studied in the osteosarcoma cell line UMR-106. 2. A maximal priming does of bPTH (1-84) down-regulated PTH-responsiveness to 40% of its initial value; bPTH (1-41) was less effective than bPTH (1-84), whereas bPTH (42-84) had no effect, alone or in combination with bPTH (1-41). 3. A tentative model for the function of different domains of parathyroid hormone in down-regulation is suggested.
Asunto(s)
Adenilil Ciclasas/metabolismo , Osteoblastos/enzimología , Hormona Paratiroidea/farmacología , Animales , Bovinos , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Cinética , Osteoblastos/efectos de los fármacos , Osteosarcoma , Hormona Paratiroidea/administración & dosificación , Fragmentos de Péptidos/farmacología , Células Tumorales CultivadasRESUMEN
The protooncogene Ets-1 is a member of the c-Ets family of genes originally identified through their sequence homology to the v-ets gene of the avian erythroblastosis virus E26. Ets-like factors are characterised by a conserved 85 amino acid domain which appears to be essential for binding to purine rich DNA sequences. Sequences binding to Ets-1 were selected from a random oligonucleotide pool by immunoprecipitation and amplified using the Polymerase Chain Reaction. Oligonucleotides enriched by this procedure were cloned in plasmids and sequenced. Alignment of DNA sequences revealed GGAA and GGAT cores at about a 1.4:1 ratio. Preferred sequences were identified both 5' and 3' of the GGAW core, extending the binding site to ACMGGAWRTT. Analysis of the flanking sequences associated with GGAA and GGAT cores revealed differences which may have compensated for the generally lower affinity of binding sites containing a GGAT core. Lastly mutational analysis of one particular Ets-1 binding site was used to establish the relative importance for binding of some nucleotides within the core and to show that Ets-1 and the closely related Ets-2 proteins bind to similar sequences.