RESUMEN
Fibroblast growth factor-2 (FGF-2) is a powerful mitogen and angiogenic factor whose expression is strongly regulated at the translational level. The constitutive upregulation of FGF-2 isoforms in transformed cells prompted us to investigate the post-transcriptional effects of a tumour suppressor, p53, on FGF-2 expression. We show here in human primary skin fibroblasts that the cell density-dependent variation of FGF-2 mRNA translatability was inversely correlated with endogenous p53 expression. Transient cell transfection revealed an inhibitory effect of wild-type p53 on the expression of chimeric FGF--CAT proteins. RNAse mapping experiments ruled out any effect of p53 on FGF--CAT mRNA accumulation, suggesting a translational inhibition. This inhibition was mediated by the FGF-2 mRNA leader, but not by vascular endothelial growth factor or platelet derived growth factor mRNA leaders. Neither p53-like protein p73, nor p21/waf had any inhibitory activity. Furthermore a set of hot spot mutants of p53 bearing mutations in the DNA binding domain had no post-transcriptional inhibitory effect. In contrast a p53 mutant of the transactivating domain was still able to block FGF--CAT expression, indicating that the post-transcriptional activity of p53 described here was independent of the trans-activation of target genes. Such data reveal a novel mechanism by which p53 efficiently blocks the expression of a major proliferating, anti-apoptotic and angiogenic gene.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Genes p53/fisiología , Apoptosis , Factores de Crecimiento Endotelial/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/fisiología , Humanos , Linfocinas/genética , Neoplasias/etiología , Factor de Crecimiento Derivado de Plaquetas/genética , ARN Mensajero/análisis , Transactivadores/fisiología , Activación Transcripcional , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Numerous evidence indicates that some of the activities of fibroblast growth factor 2 (FGF-2) depend on an intracrine mode of action. Recently, we showed that three high molecular mass (HMM) nuclear forms of FGF-2 are part of a 320-kDa protein complex while the cytoplasmic AUG-initiated form is included in a 130-kDa complex. Consequently, the characterization of FGF endogenous targets has become crucial to allow the elucidation of their endogenous activities. Through the screening of GAL4-based yeast two-hybrid expression libraries, we have isolated a gene encoding a nuclear protein of 55 kDa, FIF (FGF-2-interacting-factor), which interacts specifically with FGF-2 but not with FGF-1, FGF-3, or FGF-6. In this system, FIF interacts equally well with the NH2-extended 24-kDa FGF form as with the 18-kDa form, indicating that the FIF-binding motif is located in the last 155 amino acids of FGF-2. Nevertheless, coimmunoprecipitation experiments showed an exclusive association with HMM FGF-2. The predicted protein contains a canonical leucine zipper domain and three overlapping hydrophobic heptad repeats. The region spanning these repeats is, together with a region located in the N-terminal part of the FIF protein, implicated in the binding to FGF-2. In contrast to the full-length FIF protein, several deletion constructs were able to transactivate a lac-Z reporter gene. Furthermore, the COOH-terminal part, but not the full-length FIF protein, has previously been shown to exhibit antiapoptotic properties. Thus we discuss the possibility that these activities could reflect a physiological function of FIF through its interaction with FGF-2.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Línea Celular , Clonación Molecular , Regulación de la Expresión Génica , Humanos , Leucina Zippers , Mamíferos , Datos de Secuencia Molecular , Señales de Localización Nuclear , Pruebas de Precipitina , Isoformas de Proteínas , Secuencias Repetitivas de Aminoácido , Activación Transcripcional , Técnicas del Sistema de Dos HíbridosRESUMEN
Dimerization is a prerequisite for many growth factors in their receptor activation leading to cellular response. FGF-1 and FGF-2, members of the Fibroblast Growth Factor (FGF) family, were shown to form non-covalent dimers and oligomers in vitro. Using the two-hybrid system as an in vivo binding assay we show here that of three representative members of the FGF family, only FGF-2 is able to homodimerize. Moreover the FGF-2 isoforms could heterodimerize. Two single-point mutants (T121F and W123R), defective in their dimerization capability, were isolated through random mutagenesis and were used to study the role of FGF-2 dimerization with regard to its biological activity. Remarkably, these mutant proteins were still able to induce cell differentiation, but were strongly affected in their capacity to promote cell proliferation. This study thus highlights the uncoupling between proliferation and differentiation FGF-2 signaling pathways and the crucial role of FGF-2 dimerization in the mitogenic activity of this factor.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/fisiología , Animales , Células CHO , Bovinos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Células Cultivadas , Cricetinae , Dimerización , Factor 2 de Crecimiento de Fibroblastos/genética , Heparina/metabolismo , Mutagénesis , Células PC12 , Mutación Puntual , Unión Proteica , Conformación Proteica , Ratas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/fisiologíaRESUMEN
Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus.
Asunto(s)
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Línea Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Células L , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Fosfoproteínas/química , Ingeniería de Proteínas , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , NucleolinaRESUMEN
Benzylphenoxyethanamine derivatives are known to display antiproliferative activities on tumor cell lines consistently with their binding affinity to the microsomal antiestrogen binding site. In the present study we show that pyrrolidinobenzylphenoxyethanamine, a new efficient compound of this series, exhibits reversible effects on exponentially growing adult bovine aortic endothelial cells inducing (1) lamellated cytoplasmic inclusions, (2) cell proliferation inhibition, (3) dose-dependent transition delay of cells in the G0-G1 phase of the cell cycle. Complete reversal of these effects is achieved only by withdrawing the drug from the medium. The ultrastructural cellular modifications disappeared, and flow cytometry and thymidine incorporation analysis showed the effect on degree of synchronization of this one-step methodology.