RESUMEN
Expression of the src homology 3 (SH3) domain-containing expressed in tumorigenic astrocytes (SETA) gene is associated with the tumorigenic state in astrocytes. SETA encodes a variety of adapter proteins containing either one or two SH3 domains, as suggested by the sequence heterogeneity of isolated cDNAs. Using both SH3 domains in a yeast two-hybrid screen of a glial progenitor cell cDNA library, we isolated the rat homolog of the ALG-2-interacting protein 1 or ALG-2-interacting protein X (AIP1/Alix). In vitro confrontation experiments showed that the SH3-N domain of SETA interacted with the proline-rich C terminus of AIP1. In co-immunoprecipitation experiments, SETA and AIP1 interacted and could form a complex with apoptosis-linked gene 2 protein. Endogenous SETA and AIP1 proteins showed similar patterns of staining in primary rat astrocytes. Misexpression of a variety of SETA protein isoforms in these astrocytes revealed that they localized to the actin cytoskeleton. Furthermore, SETA proteins containing the SH3-N domain were able to sensitize astrocytes to apoptosis induced by UV irradiation. Expression of the isolated SH3-N domain had the greatest effect in these experiments, indicating that interference in the interaction between endogenous SETA and AIP1 sensitizes astrocytes to apoptosis in response to DNA damage.
Asunto(s)
Apoptosis/fisiología , Astrocitos/citología , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Neoplasias/fisiología , Proteínas del Tejido Nervioso/fisiología , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Astrocitos/efectos de la radiación , ADN Complementario , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Unión Proteica , Ratas , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos , Rayos Ultravioleta , Xenopus , Dominios Homologos srcRESUMEN
Expression of the src homology 3 (SH3)-encoding, expressed in tumorigenic astrocytes (SETA) gene is associated with astrocyte transformation in culture and tumors in the adult brain. SETA binds to the apoptosis regulator apoptosis-linked gene 2 (ALG-2) interacting protein 1 (AIP1), and modulates apoptosis in astrocytes. The predicted protein structure of SETA revealed two SH3 domains, while related proteins were reported to have three. Here we report the identification of an additional SH3 domain N-terminal to the previously identified SETA sequence. Yeast two-hybrid screening of a p53(-/-) astrocyte cDNA library with this SH3 domain identified a novel gene, SETA binding protein 1 (SB1), with 55% amino acid identity to the renal tumor antigen, NY-REN-45. In vitro confrontation and co-immunoprecipitation experiments confirmed the binding of SB1 to SETA. Evidence that SETA binds to the CD2 protein, the proto-oncogene c-Cbl, and the signal transduction molecule Grb2, and can dimerize via its C-terminal coiled coil (CC) domain is also presented.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Antígenos de Neoplasias/química , Astrocitos/metabolismo , Antígenos CD2/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Células Cultivadas , Dimerización , Exones/genética , Proteína Adaptadora GRB2 , Eliminación de Gen , Biblioteca de Genes , Ratones , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , Ratas , Alineación de Secuencia , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
Differential display polymerase chain reaction analysis was used to compare five differentiation states of the O-2A progenitor-like cell line CG4: progenitor cells and cells at 12 h or 4 days after the induction of differentiation into oligodendrocytes or astrocytes. This led to the identification of 52 sequence tags that were expressed differentially with cellular phenotype. One sequence was upregulated during differentiation of CG4 cells and represented a novel gene that we named SETA (SH3 domain-containing gene expressed in tumorigenic astrocytes). This gene encodes an SH3 domain-containing adapter protein with sequence similarity to the CD2AP (CD2 adapter protein) and CMS (Cas ligand with multiple Src homology) genes. SETA mRNA was expressed at high levels in the developing rat brain but was barely detectable in the normal adult rat or human brain. However, SETA mRNA was found in approximately one half of the human gliomas tested, including astrocytomas grades II, III, and IV, as well as oligodendrogliomas, mixed oligoastrocytomas, and human glioma-derived cell lines. A rat glioma generated by treatment with the alkylating carcinogen ethylnitrosourea on postnatal day 1 and a derived cell line also expressed SETA mRNA. Furthermore, in an in vitro model of astrocytoma progression based on p53-/- astrocytes, expression of SETA was restricted to cells that are tumorigenic.
Asunto(s)
Astrocitos/fisiología , Neoplasias del Sistema Nervioso Central/etiología , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/metabolismo , Células Madre/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Línea Celular , ADN Complementario/genética , Presentación de Datos , Desarrollo Embrionario y Fetal , Glioma/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Neuroglía/citología , Neuroglía/metabolismo , Oligodendroglía/citología , Reacción en Cadena de la Polimerasa , Ratas , Células Madre/citologíaRESUMEN
Germline transmission of mutant p53 gene in cancer-prone families with Li-Fraumeni syndrome has revealed a new role for p53 in the genetic predisposition to cancer. The studies reported here focus on the analysis of the expression of normal and mutant p53 RNA and protein in germline configuration and demonstrate that normal skin fibroblasts derived from members of a family with Li-Fraumeni syndrome express mutant p53Gly----Asp(245) protein and RNA at levels similar to the wild-type p53. Thus, these fibroblasts represent a unique biological system in which endogenous promoters are utilized for the expression of both mutant and normal p53. We have further extended the earlier observations on the analysis of mutant p53 with a limited number of tumors derived from individuals with Li-Fraumeni syndrome. Tumors arising from two different germ layers in four individuals in a single family clearly exhibited the loss of the wild-type allele and the retention of the mutant allele observed in the normal skin fibroblasts derived from the same individuals. These observations further support the notion that germline p53 mutation plays a key role in the tumorigenesis of individuals with Li-Fraumeni syndrome.