RESUMEN
Two human hSos1 isoforms (Isf I and Isf II; Rojas et al., Oncogene 12, 2291-2300, 1996) defined by the presence of a distinct 15 amino acid stretch in one of them, were compared biologically and biochemically using representative NIH3T3 transfectants overexpressing either one. We showed that hSos1-Isf II is significantly more effective than hSos1-Isf I to induce proliferation or malignant transformation of rodent fibroblasts when transfected alone or in conjunction with normal H-Ras (Gly12). The hSos1-Isf II-Ras cotransfectants consistently exhibited higher saturation density, lower cell-doubling times, increased focus-forming activity and higher ability to grow on semisolid medium and at low serum concentration than their hSos1-Isf I-Ras counterparts. Furthermore, the ratio of GTP/GDP bound to cellular p21ras was consistently higher in the hSos1-Isf II-transfected clones, both under basal and stimulated conditions. However, no significant differences were detected in vivo between Isf I- and Isf II-transfected clones regarding the amount, stability and subcellular localization of Sos1-Grb2 complex, or the level of hSos1 phosphorylation upon cellular stimulation. Interestingly, direct Ras guanine nucleotide exchange activity assays in cellular lysates showed that Isf II transfectants consistently exhibited about threefold higher activity than Isf I transfectants under basal, unstimulated conditions. Microinjection into Xenopus oocytes of purified peptides corresponding to the C-terminal region of both isoforms (encompassing the 15 amino acid insertion area and the first Grb2-binding motif) showed that only the Isf II peptide, but not its corresponding Isf I peptide, was able to induce measurable rates of meiotic maturation, and synergyzed with insulin, but not progesterone, in induction of GVBD. Our results suggest that the increased biological potency displayed by hSos1-Isf II is due to higher intrinsic guanine nucleotide exchange activity conferred upon this isoform by the 15 a.a. insertion located in proximity to its Grb2 binding region.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteína Adaptadora GRB2 , Factores de Intercambio de Guanina Nucleótido , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Isoformas de Proteínas , Proteínas/genética , Transfección , Transformación Genética , Factores de Intercambio de Guanina Nucleótido ras , Proteínas ras/metabolismoRESUMEN
A neural specific protein, CRMP-2 (for Collapsin Response Mediator Protein-2), is considered to mediate collapsin-induced growth cone collapse during neural development. We have isolated the Xenopus homologue of the CRMP-2 (XCRMP-2) cDNA and studied the expression of XCRMP-2 mRNA and protein during neural induction. Induction of XCRMP-2 mRNA and protein expression, like N-CAM, occurred at the midgastrula stage and increased through early neural developmental stages. Whole mount in situ hybridization demonstrated that expression of XCRMP-2 mRNA was localized in neural tissues such as the neural plate and tube at early stages, while its expression in the brain, spinal cord, and eyes was observed at later stages. Immunostaining of Xenopus embryos with the antibody against CRMP-2 also showed that the protein was specifically expressed in the neural tissues at early stages. XCRMP-2 expression was induced by neural inducers such as noggin and chordin which antagonize a neural inhibitor, BMP4. A dominant negative BMP receptor also induced XCRMP-2 expression, suggesting that transcription of XCRMP-2 gene was negatively regulated by the BMP4 signaling. These results indicate that expression of XCRMP-2 is an early response marking neural commitment, and that transcriptional control of XCRMP-2 gene, is one of the targets of BMP4 signaling.
Asunto(s)
Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/genética , Sistema Nervioso/embriología , Secuencia de Aminoácidos , Animales , Proteínas Morfogenéticas Óseas/biosíntesis , Embrión no Mamífero/metabolismo , Gástrula/metabolismo , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Sistema Nervioso/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Semaforina-3A , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Transcripción Genética , XenopusRESUMEN
We have cloned the cDNA encoding bovine CRMP-2 from bovine brains. A full length cDNA encoding bovine CRMP-2 was isolated and sequenced. The deduced amino acid sequence reveals that the gene encodes a protein of 572 amino acids and is highly homologous to Caenorhabditis elegans unc-33, which controls the guidance and outgrowth of neuronal axons. The CRMP-2 transcript was present in bovine brains but not non-neural tissues, and its protein product existed in both soluble and membrane-bound forms. The expression of CRMP-2 protein and mRNA was upregulated during neuronal differentiation of rat PC12 cells. Immunoprecipitation of PC12 cell extracts shows that CRMP-2 was co-immunoprecipitated with a 190 kDa protein (p190). Both CRMP-2 and p190 were phosphorylated on serine residues in vivo and in vitro in a kinase assay of CRMP-2 immune complexes. Immunocytochemistry shows that CRMP-2 was exclusively localized in both the central and peripheral nervous systems in mouse embryos and detectable in the adult brain although the level of CRMP-2 decreased. The protein was expressed in the axon, dendrite, and cytoplasm of postmitotic neurons and in the cytoplasm of oligodendrocytes and astrocytes. The CRMP-2 gene maps to the region of mouse chromosome 14 syntenic with human chromosome 8p21. Taken together, these data suggest multi-functional roles for CRMP-2 in developing and adult nervous systems, and the biological activity of CRMP-2 could be regulated by phosphorylation reaction.
Asunto(s)
Axones/fisiología , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , ADN Complementario/aislamiento & purificación , Genes de Helminto , Proteínas del Helminto/genética , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Desarrollo Embrionario y Fetal/fisiología , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/análisis , Células PC12 , Ratas , Semaforina-3A , Homología de Secuencia de Ácido NucleicoRESUMEN
mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutation and with a Src homology 2 deletion mutation resulted in a stimulation and suppression of the mSOS activity, respectively. The complex formations of Fyn-Shc, Shc-Grb2, and Grb2-mSOS were detected in the activated Fyn-transformed cells, whereas the SH2 deletion mutant of Fyn failed to form a complex with mSOS. Moreover, tyrosine phosphorylation of Shc was induced by the overexpression of the activated Fyn. These findings support the idea that Fyn activates the activity of mSOS bound to Grb2 through tyrosine phosphorylation of Shc. Unlike the current prevailing model, Fyn-induced activation of Ras might involve the stimulation of the catalytic guanine nucleotide exchange activity of mSOS.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Activación Enzimática , Factor 2 Eucariótico de Iniciación/metabolismo , Proteína Adaptadora GRB2 , Expresión Génica , Factores de Intercambio de Guanina Nucleótido , Cinética , Ratones , Mutagénesis Sitio-Dirigida , Mutación Puntual , Unión Proteica , Proteínas/aislamiento & purificación , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas c-fyn , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Proteínas Recombinantes , Eliminación de Secuencia , Linfocitos T Colaboradores-Inductores/inmunología , Transfección , Factores de Intercambio de Guanina Nucleótido ras , Dominios Homologos srcRESUMEN
We report biochemical evidence that epidermal growth factor and platelet-derived growth factor stimulate the Ras guanine nucleotide exchange factor activity in quiescent NIH 3T3 cells. Moreover, the exchange activity is constitutively enhanced in NIH 3T3 cells transformed by Src and ErbB2 oncogenic tyrosine protein kinases (TPKs), whereas transformation by oncogenic Mos and Raf does not alter the activity. GTPase-activating protein activity was not affected under these conditions. Overexpression of pp60c-Src mutants containing activated and suppressor TPK mutations resulted in stimulation and inhibition of the exchange factor activity, respectively. A TPK inhibitor, genistein, prevented the activation of the exchange factor in epidermal growth factor/platelet-derived growth factor-treated cells and src-transformed cells. Furthermore, the exchange factor activity bound to an anti-phosphotyrosine antibody immunoaffinity column. These findings suggest that the guanine nucleotide exchange factor, but not GTPase-activating protein, plays a major role in the Ras activation in cell proliferation initiated by growth factor receptor TPKs and malignant transformation by oncogenic TPKs and that tyrosine phosphorylation of either the exchange factor or a tightly bound protein(s) may mediate the activation of the exchange factor by these TPKs.