RESUMEN
Complex neuronal functions rely upon the precise sorting, targeting, and restriction of receptors to specific synaptic microdomains. Little is known, however, of the molecular signals responsible for mediating these selective distributions. Here we report that metabotropic glutamate receptor subtype 7a (mGluR7a) is polarized at the basolateral surface when expressed in Madin-Darby canine kidney (MDCK) epithelial cells but is not polarized when expressed in cultured hippocampal neurons. Truncation of the mGluR7 cytoplasmic tail produces a protein that is restricted to a perinuclear intracellular compartment in both neurons and MDCK cells, where this protein colocalizes with a trans-Golgi network antigen. The mGluR7 cytoplasmic domain appended to the transmembrane portion of the vesicular stomatitis virus G protein and the ectodomain of human placental alkaline phosphatase is distributed over the entire cell surface in cultured neurons. When expressed in MDCK cells, this construct remains in an intracellular compartment distinct from endosomes or lysosomes. Thus, the cytoplasmic tail domain of mGluR7 is necessary but not sufficient for polarized targeting in MDCK monolayers, whereas in neurons the cytoplasmic tail is sufficient for cell surface expression but not polarization. Additional mechanisms are likely required to mediate mGluR7 neuronal polarization and synaptic clustering.
Asunto(s)
Epitelio/metabolismo , Neuronas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Citoplasma/metabolismo , Perros , Endocitosis , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Receptores de Glutamato Metabotrópico/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Cadenas Pesadas de Miosina , Miosina Tipo II , Miosina Tipo V , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Proteínas de Schizosaccharomyces pombe , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/efectos de los fármacos , Actinas/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteínas Portadoras/genética , División Celular , Polaridad Celular , Proteínas Fúngicas/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía por Video/métodos , Mutación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/ultraestructura , Tiazoles/farmacología , TiazolidinasRESUMEN
Sec2p is required for the polarized transport of secretory vesicles in S. cerevisiae. The Sec2p NH(2) terminus encodes an exchange factor for the Rab protein Sec4p. Sec2p associates with vesicles and in Sec2p COOH-terminal mutants Sec4p and vesicles no longer accumulate at bud tips. Thus, the Sec2p COOH terminus functions in targeting vesicles, however, the mechanism of function is unknown. We found comparable exchange activity for truncated and full-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate. Full-length Sec2-GFP, similar to Sec4p, concentrates at bud tips. A COOH-terminal 58-amino acid domain is necessary but not sufficient for localization. Sec2p localization depends on actin, Myo2p and Sec1p, Sec6p, and Sec9p function. Full-length, but not COOH-terminally truncated Sec2 proteins are enriched on membranes. Membrane association of full-length Sec2p is reduced in sec6-4 and sec9-4 backgrounds at 37 degrees C but unaffected at 25 degrees C. Taken together, these data correlate loss of localization of Sec2 proteins with reduced membrane association. In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the notion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4p.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Actinas/metabolismo , Transporte Biológico , División Celular , Membrana Celular/química , Membrana Celular/metabolismo , Polaridad Celular , Citoesqueleto/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP/genética , Genes Fúngicos/genética , Genes Fúngicos/fisiología , Factores de Intercambio de Guanina Nucleótido , Cinética , Fosforilación , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Eliminación de Secuencia/genética , Temperatura , Vacuolas/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The DNA binding activity of wild type p53 is central to its activity. The "central" part of the molecule, where most mutations appear in primary human tumors, is the actual DNA binding domain. The C-terminal part was shown to exert a negative effect on the DNA binding activity. In the present study we show that while anti-p53 antibodies recognizing the C terminus of the wild type p53 facilitate DNA binding activity, blocking of the wild type specific epitope by specific anti-p53 antibodies, inhibited the DNA binding activity of the wild type p53 protein. An alternatively spliced p53 protein exhibits an augmented DNA binding activity. The fact that most p53 mutants have lost the wild type p53 conformation specific epitope, coupled with the observation that blocking of this site by binding specific antibodies, prevents the interaction of wild type p53 with DNA, suggests that maintaining the correct structural conformation of this site is central for DNA binding activity. Still, the internal structure of the p53 target and particularly the length of the sequence between the two tandem inverted repeats, is critical for protein-DNA interaction behavior.
Asunto(s)
ADN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Anticuerpos Monoclonales , Sitios de Unión , Clonación Molecular , ADN Complementario , Epítopos , Humanos , Pruebas de Precipitina , Unión ProteicaRESUMEN
p53 was shown to play a central role in the maintenance of genomic integrity. The present experiments suggest that p53 is involved in the control of cell ploidity. Using a p53 non-producer cell line, M1/2, that was reconstituted to express either wild type or mutant p53 protein, by infection with the temperature sensitive (Ts) p53Val135 virus, it was found that both loss of wild type p53 or overexpression of mutant p53, may be associated with the generation of cell polyploidity. Overexpression of mutant p53 protein enhanced the appearance of giant cells that further accumulated following gamma-irradiation. Expression of wild type p53 reduced the level of giant cells which accumulated in the parental M1/2 p53 non-producer cells following gamma-irradiation. This activity of the wild type p53 seems to be mediated by either the reduction in the rate of giant cell generation, as observed in M1/2 derived cell lines expressing low levels of wild type p53 protein or by facilitating their apoptosis, as observed in wild type p53 high-producer cells. The latter conclusion is further supported by the observation that isolated giant cells are directly induced to undergo apoptosis following wild type p53 expression.
Asunto(s)
Leucemia Mielomonocítica Aguda/genética , Ploidias , Proteína p53 Supresora de Tumor/fisiología , Animales , Células Clonales , ADN de Neoplasias/metabolismo , Rayos gamma , Leucemia Mielomonocítica Aguda/patología , Ratones , Mutación , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Nuclear Localization Signals (NLS) have been found to mediate the import of proteins into the nucleus. Proteins interacting directly with NLS control the subcellular localization of nucleophilic proteins. The p53 protein is spatially regulated throughout the cell cycle and this regulation has been shown to be dependent on the presence of its NLS sequences. We identified three novel cDNA clones that were isolated from an expression library because they encode polypeptides that bind a synthetic peptide containing the major NLS of p53 (NLS I). These clones were found to share a common domain encoded by p(CA)n repeats; a simple sequence length polymorphism (SSLP). THis is the first report where p(CA)n repeats were found to encode protein. One cDNA clone encodes a full length, 16 kDa protein, designated spot-1, that is represented in cells predominantly as oligomers. spot-1 interacts with the NLS I of p53 through its p(CA)n repeat. Cell fractionation and immunofluorescence analysis demonstrated that spot-1 is a nuclear protein which, in fibroblasts, co-localizes with p53.
Asunto(s)
Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Fosfoproteínas/genética , Fosfoproteínas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
DNA-binding activity of the wild-type p53 is central to its function in vivo. However, recombinant or in vitro translated wild-type p53 proteins, unless modified, are poor DNA binders. The fact that the in vitro produced protein gains DNA-binding activity upon modification at the C terminus raises the possibility that similar mechanisms may exist in the cell. Data presented here show that a C-terminal alternatively spliced wild-type p53 (ASp53) mRNA expressed by bacteria or transcribed in vitro codes for a p53 protein that efficiently binds DNA. Our results support the conclusion that the augmented DNA binding activity of an ASp53 protein is probably due to attenuation of the negative effect residing at the C terminus of the wild-type p53 protein encoded by the regularly spliced mRNA (RSp53) rather than acquisition of additional functionality by the alternatively spliced C' terminus. In addition, we found that ASp53 forms a complex with the non-DNA-binding RSp53, which in turn blocks the DNA-binding activity of ASp53. Interaction between these two wild-type p53 proteins may underline a mechanism that controls the activity of the wild-type p53 protein in the cell.
Asunto(s)
Empalme Alternativo , Secuencia de Aminoácidos , ADN/metabolismo , Eliminación de Secuencia , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Unión Competitiva , Ratones , Datos de Secuencia Molecular , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Interaction of wild type p53 with specific DNA target sequences, which is dictated by several structural domains, can be modified by blocking the different antigenic epitopes of the protein. Comparison of p53 protein expressed by recombinant bacteria (wtp53-Bac) to that produced in an eukaryotic system by a vaccinia expression vector (wtp53-Vac), indicated that only the later exhibited spontaneous DNA-binding activity. Furthermore, DNA-binding patterns of these wild type p53 proteins were affected differently by their interactions with monoclonal anti-p53 antibodies recognizing individual antigenic epitopes of the molecule. While the vaccinia derived p53 that spontaneously bound DNA is supershifted by the N'-terminal specific antibodies PAb-248, the bacterial derived p53 protein that retains this antigenic epitope but does not bind DNA spontaneously, is not affected. The C'-terminal specific PAb-421 antibodies accelerated binding of the bacterial p53 protein and modified the pattern of the interaction of the vaccinia derived p53 DNA. DNA-binding patterns generated by PAb-421 and PAb-248, suggest that either interaction of wild type p53 is dependent on modification of the p53 protein or that it interacts with cellular factors which their activity can be mimicked by PAb-421. Saturation of both types of wild type p53 with several anti-p53 monoclonal antibodies directed against the wild type p53 specific epitope that maps to the N'-terminal border of the DNA-binding region, blocked specific DNA-binding. The fact that most p53 mutants have lost the wild type p53 conformation specific epitope coupled with the observation that blocking of this site by binding specific antibodies, prevents the interaction of wild type p53 with DNA, suggests that maintaining the correct structural conformation of this site is central for DNA-binding activity. The wild type specific epitope which maps to the N'-terminal border of the DNA-binding region is neighboring the first beta-strand detected by the recent crystallographic analysis.
Asunto(s)
ADN/metabolismo , Epítopos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Ratones , Datos de Secuencia Molecular , Unión Proteica/inmunología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
Inactivation of the p53 tumor suppressor gene plays a major role in malignant transformation. The central question in this issue is concerned with the understanding of the function of p53 in normal cells and its deregulation in cancer cells. Several in vitro and in vivo experimental models have indicated that induction of cells to undergo differentiation involve up-regulation in the expression of the p53. In the case of B cell differentiation, p53 was found to be involved in several steps of the differentiation pathway. The conclusion that p53 plays a role in normal development and differentiation in vivo is substantiated by the observation that p53 is expressed during embryonic development and is detected at low levels in a number of organs of adult mice. Accentuated levels of p53 in testes of adult mice, suggests that p53 plays a role in the meiotic process of spermatogenesis. B cell differentiation and spermatogenesis are biological pathways which normally involve DNA reshuffling and rearrangements. In accordance with the notion that p53 is associated with DNA repair it is tempting to speculate that at least in these physiological pathways p53 functions as a master gene that controls genome integrity.
Asunto(s)
Diferenciación Celular , Proteína p53 Supresora de Tumor/fisiología , Animales , Linfocitos B/fisiología , Reparación del ADN , Humanos , Masculino , EspermatogénesisRESUMEN
c-Myc and wild-type p53 have been shown to play important roles in the regulation of cellular proliferation and oncogenic transformation. We have previously shown that the p53 promoter contains a conserved consensus recognition sequence for the basic-helix-loop-helix-containing proteins, identical to the specific binding site for c-Myc/Max heterodimers. Here, we demonstrate that this element, which is required for full promoter activity, is bound by in vitro translated c-Myc/Max heterodimers. Furthermore, we found that in cotransfection assays, c-Myc trans-activates the p53 promoter as well as a hybrid herpes simplex virus-thymidine kinase promoter containing multiple copies of a synthetic p53-derived c-Myc binding site. The p53 promoter deleted of the basic-helix-loop-helix consensus recognition sequence is not trans-activated by c-Myc, thus suggesting that c-Myc trans-activates the p53 promoter through the basic-helix-loop-helix recognition motif. These findings raise the possibility that the p53 gene may be a potential target for trans-activation by c-Myc in vivo.