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1.
PLoS One ; 8(3): e58395, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23516471

RESUMO

Functional and mechanistic studies of Wnt signaling have been severely hindered by the inaccessibility of bioactive proteins. To overcome this long-standing barrier, we engineered and characterized a panel of Chinese hamster ovary (CHO) cell lines with inducible transgenes encoding tagged and un-tagged human WNT1, WNT3A, WNT5A, WNT7A, WNT11, WNT16 or the soluble Wnt antagonist Fzd8CRD, all integrated into an identical genomic locus. Using a quantitative real-time bioluminescence assay, we show that cells expressing WNT1, 3A and 7A stimulate Wnt/beta-catenin reporter activity, while the other WNT expressing cell lines interfere with this activation. Additionally, in contrast to WNT3A, WNT1 only exhibits activity when cell-associated, and thus only signals to neighboring cells. The reporter assay also revealed a rapid decline of Wnt activity at 37°C, indicating that Wnt activity is highly labile. These engineered cell lines will reduce the cost of making and purifying Wnt proteins and serve as a continuous, reliable and regulatable source of Wnts to research laboratories around the world.


Assuntos
Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Células CHO , Cricetinae , Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Wnt/isolamento & purificação
2.
Cell Signal ; 22(3): 564-71, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19932175

RESUMO

Post-translational modifications play important roles during the stabilisation and activation of p53 by various genotoxic and non-genotoxic stresses. Ser392 has been reported to be a major UV-stimulated phosphorylation site that is modified through the p38 MAPK pathway in a manner that may involve recruitment of CK2. Here we show that phosphorylation of Ser392 is an integral event that occurs not only in response to UV, but also during the induction of p53 by a range of stimuli including treatment of cells with the MDM2 inhibitor, Nutlin 3a. Strikingly, phosphorylation of Ser392 and Ser33 was also observed following induction of the p53 pathway by ARF which has previously been thought to induce p53 in a phosphorylation-independent manner. The induction of Ser392 phosphorylation by diverse stimuli can be explained by a common mechanism in which its phosphorylation at a low rate, coupled with the rapid turnover of p53, limits the accumulation of phosphorylated molecules until a stimulus stabilises p53 and allows the Ser392-phosphorylated p53 to accumulate. We also provide biological evidence that Ser392 phosphorylation is not mediated by a UV-associated route involving p38 MAPK, either directly or indirectly via CK2. These data suggest that, physiologically, Ser392 may be phosphorylated by an, as yet, unidentified protein kinase.


Assuntos
Serina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Caseína Quinase II/metabolismo , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Fosforilação , Piperazinas/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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