RESUMEN
Self-assembling systems of colloidal spheres are widely used as templates for the structured deposition of metals and semiconductors. Multilayer samples of ordered polystyrene spheres are prepared by a flow induced process. The subsequent surface activation by a dielectric barrier discharge in oxygen is followed by the fabrication of protecting polysiloxane layers. Electrochemical deposition of copper is used to test the stability of the pre-treated colloidal crystal. The arrangement of the spheres is preserved during the deposition process, due to the polysiloxane layer. The results of the consecutive preparation steps are investigated concerning topographical and chemical changes by atomic force microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy.
Asunto(s)
Coloides/síntesis química , Cristalización/métodos , Galvanoplastia/métodos , Nanopartículas/química , Nanopartículas/ultraestructura , Gases em Plasma/química , Poliestirenos/síntesis química , Ensayo de Materiales , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The growth suppressor protein p53 and the protein kinase CK2 are both implicated in cellular growth regulation. We previously found that p53 binds to protein kinase CK2 via its regulatory beta-subunit. In the present study, we analyzed the consequences of the binding of p53 to CK2 for the enzymatic activity of CK2 in vitro and in vivo. We found that the carboxy-terminus of p53 which is a potent transforming agent stimulated CK2 activity whereas full length wild-type p53 which is a growth suppressor inhibited the activity of protein kinase CK2. Inhibition of protein kinase CK2 by p53 was dose-dependent and was seen for various CK2 substrates. Experiments with heat-denatured p53 and the conformational mutant p53(R175H) revealed that an intact conformation of p53 seemed to be necessary. Transfection of wild-type and of mutant p53 into p53-/- cells showed that the inhibition of p53 on CK2 activity was also detectable in intact cells and specific for wild-type p53 indicating that the growth suppressing function of p53 might at least be partially achieved by down-regulation of protein kinase CK2.
Asunto(s)
Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Aminoácidos , Quinasa de la Caseína II , Línea Celular , Humanos , Fosforilación , Conformación Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Proteína p53 Supresora de Tumor/químicaRESUMEN
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
Muscarinic acetylcholine receptors (mAChRs) in exocrine tissue from the avian nasal salt gland are coupled to phospholipase C and generate inositol phosphate and Ca(2+) signals upon activation. An early effect of receptor activation in the secretory cells is a transient accumulation of c-Fos protein. In cooperation with constitutively expressed Jun, Fos presumably serves as a transcription factor altering gene expression during cell growth and differentiation processes in the gland associated with adaptation to osmotic stress in animals. Nothing is known, however, about the mAChR-dependent signaling pathways leading to Fos expression in these cells. By incubation of isolated nasal gland tissue in short-term culture with activators or inhibitors of signaling pathways and quantitative Western blot analysis of Fos abundance, we have now identified the sustained elevation of the intracellular Ca(2+) concentration and the activation of the p38 mitogen-activated protein (MAP) kinase as intermediate signaling elements for the regulation of c-Fos by muscarinic receptor activation. It is suggested that p38 MAP kinase, rather than exclusively mediating stress responses, is involved in the regulation of cellular growth and differentiation controlled by G protein-coupled receptors.
Asunto(s)
Calcio/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores Muscarínicos/metabolismo , Glándula de Sal/metabolismo , Animales , Técnicas de Cultivo , Patos , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de Superficie Celular/metabolismo , Glándula de Sal/citología , Glándula de Sal/efectos de los fármacos , Transducción de Señal , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The growth suppressor protein p53 plays a main part in cellular growth control. Two of its key functions are sequence specific DNA binding and transactivation. Functions of p53 in growth control are regulated at least in part by its interaction with protein kinases. p53 binds to protein kinase CK2, formerly known as casein kinase 2, and it is phosphorylated by this enzyme. CK2 is composed of two regulating beta-subunits and two catalytic alpha- or alpha'-subunits and the interaction with p53 is mediated by the regulatory beta-subunit of CK2. Recently we showed that the beta-subunit could inhibit the sequence specific DNA binding activity of p53 in vitro. Based on this finding, we asked if a coexpression of the beta-subunit of CK2 with p53 in mammalian cells could inhibit the DNA binding activity of p53 in a physiological context. We found that the coexpression of the beta-subunit showed the same inhibitory effect as in the previous assays with purified proteins. Then, we investigated the effects of the coexpression of the beta-subunit of CK2 on the transactivation and transrepression activity of p53. We found that transactivation of the mdm2, p21(WAF1/CIP1) and cyclin G promoter was inhibited in three different cell lines whereas transactivation of the bax promoter was not affected in COS1 cells but down-regulated in MCO1 and SaosS138V21 cells. p53 mediated transrepression of the fos promoter was not influenced by coexpression of the CK2 beta-subunit. Taken together we propose a cell type dependent fine regulation of the p53 transactivation function by the CK2 beta-subunit in vivo, which does not affect p53 mediated transrepression.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Células COS , Quinasa de la Caseína II , Línea Celular , ADN/genética , ADN/metabolismo , Expresión Génica , Genes fos , Genes p53 , Humanos , Ratones , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
p53 is one of the most powerful negative regulators of growth. To manage this in an efficient way it has to interact with a set of different cellular proteins. Most contacts with the cellular environment occur in the N- or the C-terminal domain of the protein. Since we previously found that p53 binds to the regulatory beta-subunit of CK2 we now analyzed N- and C-terminal domains of p53 separately for the binding of protein kinase CK2, an enzyme which seems to have a certain importance for proliferation processes. With different overlay assays we could map the binding domain of protein kinase CK2 to a sequence between amino acids 325-344, a region which coincides with the interaction domain of some other p53 binding proteins. We also found that the regulatory beta-subunit of protein kinase CK2 binds independent of the catalytic alpha-subunit to this C-terminal domain of p53.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Quinasa de la Caseína II , Dominio Catalítico , Humanos , Datos de Secuencia Molecular , Mapeo Peptídico , Unión Proteica , Proteína p53 Supresora de Tumor/químicaRESUMEN
Some of the numerous functions of the growth suppressor protein p53 are regulated by its interaction with viral and cellular proteins. C-terminal sequences of p53 are implicated in binding to the regulatory beta-subunit of protein kinase CK2. Using a p53-specific DNA binding element we found that the beta-subunit of CK2 inhibited the DNA binding of p53 whereas the alpha-subunit had no influence. The CK2 holoenzyme consisting of two alpha- and two beta-subunits led to a supershift in DNA binding of p53 similar to the p53-specific monoclonal antibody PAb421 as well as the C-terminus of p53. Thus, our results showed an individual role of the free beta-subunit of CK2 on the DNA binding activity of p53.
Asunto(s)
ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Quinasa de la Caseína II , Línea Celular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Fosforilación , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Recombinantes/metabolismo , Spodoptera , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
Quiescent non-permissive cells re-enter the cell cycle upon infection with the DNA tumor virus SV40. Before the expression of virus specific proteins and other cellular reactions there is an induced expression of the growth suppressor protein p53. p53 is known to be a substrate for protein kinase CK2 and in addition it is tightly associated with CK2 and both proteins are implicated in cell cycle regulation. No phosphorylation of p53 was observed in vivo until late in G(1)- or early S-phase. Immunopurified p53 from the early G(1)-phase of the cell cycle was not phosphorylated by an associated protein kinase activity. Furthermore, protein kinase CK2 could not phosphorylate p53 from the early G(1)-phase of the cell cycle and also immunopurified p53 from late G(1)- and S-phase which were dephosphorylated by alkaline and acid phosphatases. p53 from cells in S-phase is an efficient substrate. Moreover, in the presence of okadaic acid, a potent inhibitor of protein phosphatase PP2a, phosphorylation of p53 is detectable early in G(1)-phase of the cell cycle.
RESUMEN
Originally identified as multicopy suppressor of a lethal growth arrest caused by expression of a tumour mutant cDNA of p53 in fission yeast the tms1 gene product was found to form stable complexes with p53 in yeast. By using purified recombinant proteins multimeric complexes of tms1 and p53 could be demonstrated and recently the p53 binding site on the tms1 protein was established to the sequence YYITTEDFCT (aa 116-125) in the vicinity of a well conserved cell division motif. Here we report the precise mapping of the tms1 binding site on the p53 protein to the sequence LQIRGRERFE (aa 330-339) which defines a new functional domain on the p53 protein.