RESUMEN
Human p53 protein was found to be functional in fission yeast in terms of growth repression and checkpoint control. Expression of wild-type p53 or the hot spot mutant p53His273 results in dramatic morphological changes and loss of viability of recipient yeast cells. Overexpression of cdc25C phosphatase, the mitotic activator of cdc2, results in suppression of a p53-induced growth arrest. In order to understand the interplay between p53 and cdc25C in mammalian cells we isolated and sequenced cdc25C cDNA from the epidermoid carcinoma cell line A431, which is known to carry the p53His273 mutation. Two different transcripts of the human cdc25C gene were detected by RT-PCR analysis - one full-length transcript and a shortened version (cdc25Cdm) that carries two deletions in the 5'-region of the gene. In normal human skin fibroblasts only one full-length cdc25C transcript was detected. The two different transcripts code for proteins with a molecular weight of 55 kDa and 46 kDa, respectively. Both cdc25C cDNAs from A431 cells were found to complement a conditional lethal cdc25.22 mutant strain as well as a cdc25 deletion strain of Schizosaccharomyces pombe indicating that functional proteins were translated. Expression of cdc25Cdm variant leads to a stronger uncoupling of DNA replication from mitosis than expression of cdc25C suggesting that the deletion within the amino-terminus of cdc25C leads to a protein which might contribute some potential for oncogenic transformation. As with cdc25C, uncoupling of the DNA synthesis checkpoint by cdc25Cdm was reversed by coexpression of wild-type p53.
Asunto(s)
Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Laríngeas/patología , Proteínas de Neoplasias/genética , Transcripción Genética , Fosfatasas cdc25/genética , Empalme Alternativo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/genética , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/fisiología , Transformación Celular Neoplásica/genética , Replicación del ADN , ADN Complementario/genética , Inducción Enzimática , Proteínas Fúngicas/genética , Genes p53 , Prueba de Complementación Genética , Humanos , Neoplasias Laríngeas/enzimología , Neoplasias Laríngeas/genética , Mitosis/genética , Mitosis/fisiología , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Neoplasias/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S/genética , Fase S/fisiología , Schizosaccharomyces/genética , Eliminación de Secuencia , Proteína p53 Supresora de Tumor/biosíntesis , Fosfatasas cdc25/biosíntesis , Fosfatasas cdc25/fisiología , ras-GRF1/genéticaRESUMEN
Human p53 is a growth suppressor which not only functions in mammalian cells but also in fission yeast. It was previously shown that the cell cycle regulating phosphatase cdc25C suppresses the p53 induced growth arrest in fission yeast. In the present study we analysed the mechanism of this suppression. We found that cdc25C directly interacts with p53. By using different deletion mutants the binding region was narrowed down on the polypeptide chain of p53 to amino acids 287-340. To test the functional significance we analysed the effect of this interaction on the DNA binding activity of p53. As shown by band shift experiments binding of cdc25C to p53 does not modify the DNA binding activity of p53. Our data suggest that the observed suppression of the p53 induced growth arrest by cdc25C might be achieved by direct binding of cdc25C to the C-terminus of p53.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Fosfatasas cdc25/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Ciclo Celular/química , Línea Celular , Células Clonales , Secuencia de Consenso , ADN/química , ADN/metabolismo , Humanos , Mutagénesis , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Spodoptera , Transfección , Proteína p53 Supresora de Tumor/química , Fosfatasas cdc25/químicaRESUMEN
We produced a rabbit monoclonal antibody (MAb) against human CDC25C phosphatase. The antibody reacts with a minimal epitope between amino acids 291-295 in the highly conserved C-terminal region of CDC25C. The antibody recognizes denatured CDC25C of recombinant and mammalian origin in Western blot analysis. The corresponding rabbit polyclonal serum is able to immunoprecipitate the native protein, but this ability has been lost during the selection procedure. Although the production of the rabbit MAb requires more effort and patience than the mouse MAb technology, it offers a true alternative in case of antigens that are not immunogenic in mice.