RESUMEN

To detect specific partners of the small Golgi-localized GTPase rab1b we generated rab1b mutants and used them as bait proteins in yeast two-hybrid screens. We isolated several specifically interacting clones. Two of them encode large protein fragments highly homologous to rat GM130 and to human Golgin95. The full-length human GM130 cDNA was cloned and its interaction with rab1b was characterized in detail by yeast two-hybrid and in vitro binding assays. Here we report for the first time that the rab1b protein interacts specifically with GM130 in a GTP-dependent manner and therefore needs the hypervariable regions of the N- and C-termini. We mapped the rab1b binding site of GM130 and provide evidence that it is different to the previously described p115 and Grasp65 binding sites of the GM130 protein.

Asunto(s)

Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab1 , Secuencia de Aminoácidos , Animales , Autoantígenos , Sitios de Unión , Proteínas Portadoras/metabolismo , Línea Celular , Clonación Molecular , ADN Complementario/metabolismo , Biblioteca de Genes , Proteínas de la Matriz de Golgi , Células HeLa , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Fluorescente , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Transformación Genética , Técnicas del Sistema de Dos Híbridos , beta-Galactosidasa/metabolismo , Proteínas de Unión al GTP rab/química

Asunto(s)

Mapeo Cromosómico , Cromosomas Humanos Par 8/genética , Proteínas del Citoesqueleto/genética , Proteínas del Tejido Nervioso/genética , Epilepsia Generalizada/genética , Dosificación de Gen , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular

RESUMEN

Idiopathic generalized epilepsy (IGE) comprises a heterogeneous group of disorders, in which a high genetic predisposition and a complex mode of inheritance have been suggested. However, genes, which confer liability to common IGE subtypes including juvenile myoclonic epilepsy (JME) and childhood absence epilepsy (CAE) have not been identified so far. Here, we tested the hypothesis that genetic variation in the human homolog of the <<<>>> (ARC) contributes to the etiology of common IGE disorders. The gene has recently been mapped to chromosome 8q24.3, a region which spans previously identified major IGE susceptibility loci. A systematic search for mutations was performed in 143 patients with a known family history of IGE. However, no evidence for functional variants was found in the ARC coding sequence. Nevertheless, we detected a novel common C489T single nucleotide polymorphism, which provides a useful marker in genetic linkage and association studies. By performing a population- and family-based study we however failed to show significant association between this novel single nucleotide polymorphism and IGE, a finding, which most likely rules out that genetic variation in or close to the ARC gene confers liability to common IGE subtypes.

Asunto(s)

Proteínas del Citoesqueleto/genética , Epilepsia Generalizada/genética , Mutación , Proteínas del Tejido Nervioso/genética , Adulto , Secuencia de Bases , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Cromosomas Humanos Par 8 , Femenino , Genes Inmediatos-Precoces , Predisposición Genética a la Enfermedad , Variación Genética , Heterocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia

RESUMEN

The rab1b GTPase is localized on the ER and cis-Golgi membranes and involved in early exocytotic membrane traffic processes. To analyze the influence of rab1b conformation on specific membrane targeting processes three point mutants simulating permanently inactive or active rab1b proteins were constructed and expressed in BHK cells. To increase the expression of the growth inhibiting rab1b S22N and rab1b N121I mutants we co-expressed the Mss4 protein and observed an elevated expression of the inactive rab1b mutants. Surprisingly, only the rab1b wild-type protein shows the correct intracellular localization, and a tight membrane association. We conclude that the targeting process of rab1b depends predominantly on GDP/GTP exchange.

Asunto(s)

Membranas Intracelulares/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab1 , Sustitución de Aminoácidos , Animales , Western Blotting , Línea Celular , Cricetinae , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , Epítopos/genética , Epítopos/metabolismo , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/metabolismo , Hemaglutininas Virales/genética , Hemaglutininas Virales/metabolismo , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismo , Transfección

RESUMEN

The organization of polymerized actin in the mammalian cell is regulated by several members of the rho family. Three rho proteins, cdc42, rac and rho act in a cascade to organize the intracellular actin cytoskeleton. Rho proteins are involved in the formation of actin stress fibers and adhesion plaques in fibroblasts. During transformation of mammalian cells by oncogenes the cytoskeleton is rearranged and stress fibers and adhesion plaques are disintegrated. In this paper we investigate the function of the rho protein in RR1022 rat fibroblasts transformed by the Rous sarcoma virus. Two activated mutants of the rho protein, rho G14V and rho Q63L, and a dominant negative mutant, rho N1171, were stably transfected into RR1022 cells. The resulting cell lines were analysed for the organization of polymerized actin and adhesion plaques. Cells expressing rho Q63L, but not rho wt, rho G14V or rho N1171, showed an altered morphology. These cells displayed a flat, fibroblast like shape when compared with the RR1022 ancestor cells. Immunofluorescence analyses revealed that actin stress fibers and adhesion plaques were rearranged in these cells. We conclude from these data that an active rho protein can restore elements of the actin cytoskeleton in transformed cells by overriding the tyrosine kinase phosphorylation induced by the pp60(v-src).

Asunto(s)

Transformación Celular Neoplásica/genética , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Genes src , Mutación , Actinas , Animales , Virus del Sarcoma Aviar/genética , Transporte Biológico , Adhesión Celular , Compartimento Celular , Tamaño de la Célula , Citoesqueleto , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Fenotipo , Prenilación de Proteína , Proteínas Tirosina Quinasas/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Transfección

RESUMEN

Overexpression of the crbB-2 gene-encoded p185(c-erbB-2) is correlated with early onset of metastasis in breast cancer patients. Furthermore, the detection of blood-borne epithelium-derived clustered cells expressing p185(c-erbB-2) was related to advanced stages in breast cancer. To further elucidate the receptor's function in the metastatic process of human breast cancers, we analyzed disaggregated cells and cell clusters from freshly dissected breast cancer tissues. We studied whether their capability of extravasation is correlated with their expression of c-erbB-2. A model for the venular wall was constructed by growing human umbilical vein endothelial cells (HUVECs) on porous membranes coated with basement membrane extracellular matrix. In four control breast cancer cell lines (SK-BR-3, MCF-7, MDA-MB-468, and MDA-MB-468, the latter transfected with a full-length c-erbB-2 cDNA vector) producing different levels of the c-erbB-2 receptor, the expression level correlated positively with the invasiveness of the cells. The invasive, predominantly clustered cells from 14 of 23 tumors were positively stained for p185(c-erbB-2) by immunocytochemistry. Furthermore, we show that the invasive cell populations express the metastasis-associated proteins matrix metalloproteinase MMP-2, CD44, and integrins alpha(v)beta3 and alpha6. In this first study on the behavior of cells and cell clusters from disaggregated operated cancers in an extravasation model, we could demonstrate the presence of c-erbB-2-expressing cell subpopulations within the individual breast cancers that are presumably of high metastatic potential.

Asunto(s)

Neoplasias de la Mama/metabolismo , Técnicas de Cultivo de Célula/métodos , Metástasis de la Neoplasia , Receptor ErbB-2/metabolismo , Western Blotting , Células Cultivadas , Técnicas de Cocultivo , Femenino , Gelatinasas/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Inmunohistoquímica , Integrinas/metabolismo , Queratinas/metabolismo , Metaloproteinasa 2 de la Matriz , Metaloendopeptidasas/metabolismo , Microscopía Electrónica , Invasividad Neoplásica , Receptor ErbB-2/genética , Transfección , Células Tumorales Cultivadas

RESUMEN

We have established three new cell lines deriving from malignant human gliomas. The cell lines were described in terms of both morphology and growth characteristics. Most cells in all three cell lines expressed the neuroepithelial marker protein GFAP. In terms of growth characteristics, the cells showed only slight differences. The cell lines showed no expression of the neural form of the c-src gene, pp60c-srcN, but did express the ubiquitous form, pp60c-src. The established glioma cell lines were also examined for expression of members of the neuropoietic cytokine family, CNTF and LIF, and their respective receptor components CNTFRalpha, LIFRbeta and gp130. With the exception of CNTFRalpha both the ligands and their receptor components were expressed in similar amounts in all three cell lines. The presence of ligand and receptor prompted us to study the effects of exogenously supplied factors on the growth of the glioma cell lines. Whereas LIF induced a high c-fos expression, only low c-fos induction was observed upon CNTF treatment. Accordingly, CNTF did not have any noticeable effects on glioma cell growth in culture, while LIF mediated an inhibiting effect on the growth of the three glioma cell lines in culture.

Asunto(s)

Neoplasias Encefálicas/patología , Glioma/patología , Inhibidores de Crecimiento/farmacología , Interleucina-6 , Linfocinas/farmacología , Aneuploidia , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Neoplasias Encefálicas/química , División Celular , Aberraciones Cromosómicas , Factor Neurotrófico Ciliar , Receptor gp130 de Citocinas , Regulación Neoplásica de la Expresión Génica , Genes fos , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteína Ácida Fibrilar de la Glía/genética , Glioma/genética , Glioma/metabolismo , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/genética , Humanos , Cariotipificación , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Linfocinas/biosíntesis , Linfocinas/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Desnudos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas pp60(c-src)/biosíntesis , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Receptor de Factor Neurotrófico Ciliar , Receptores de Citocinas/biosíntesis , Receptores de Citocinas/genética , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Receptores de Factor de Crecimiento Nervioso/genética , Receptores OSM-LIF , Células Tumorales Cultivadas

RESUMEN

We examined the recently synthesized and characterized polyoxometalate compound (NH4)10[Co2Sb2W20 O70(H2O)6] (POM1). The inhibitory potency of POM1 was studied in tissue culture experiments with uninfected and Rous sarcoma virus (RSV)-infected chicken embryo fibroblasts (CEF) in vivo. We measured a considerable decrease in total cellular phosphotyrosine content in treated infected cells in vivo. POM1 treatment of SR-RSV-A infected CEF in vivo resulted in decreased pp60v-src activity, possibly due to a reduced rate of v-src translation caused by the inhibitory effect of POM1 on the RSV encoded reverse transcriptase activity (RT) activity. In further studies we were able to demonstrate the inhibitory effect of this complex on the RT activity of RSV in vitro and in vivo.

Asunto(s)

Virus del Sarcoma Aviar/fisiología , Transformación Celular Viral/efectos de los fármacos , Fibroblastos/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Compuestos de Tungsteno/farmacología , Animales , Embrión de Pollo , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/virología , Citometría de Flujo , Proteína Oncogénica pp60(v-src)/metabolismo , Fosforilación , Transfección , Proteínas Virales/análisis

RESUMEN

Increases in the average gene copy number (AGCN) of the erbB oncogenes, especially the erbB-2 gene, have been found in a variety of human cancers. Such information is useful with respect to prognosis of the disease. Poor reproducibility and DNA damage complicate quantitative polymerase chain reaction (PCR)-based methods. Therefore, we describe a quantitative PCR method for the estimation of AGCN in the oncogenes erbB-1 (egfr), erbB-2, and erbB-3. The method comprises a competitive and differential PCR in a one-tube reaction (competitive-differential PCR, or cdPCR). Genomic sequences of the oncogene and the human beta-globin (HBB) reference gene and two competitors for the oncogene and reference gene were amplified with two primer pairs simultaneously. The competitors were chosen to be amplified with the same efficiency as the genomic sequences. Using this method, we confirmed egfr and erbB-2 amplification in cancer cell lines and tumor tissue, and we also detected erbB-3 amplifications. Furthermore, gene dosage decreases were detectable, e.g., an erbB-2 hemizygosity in MCF-7 cells. Thus, cdPCR facilitates the detection of both increases and decreases in AGCN with high reproducibility, sensitivity, and accuracy. This method is therefore suitable for clinical studies on erbB oncogene dosage deviations.

Asunto(s)

Dosificación de Gen , Genes erbB/genética , Neoplasias/genética , Reacción en Cadena de la Polimerasa/métodos , Receptores ErbB/genética , Genes erbB-1/genética , Genes erbB-2/genética , Humanos , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-3

RESUMEN

Applying different hybridization techniques we could confirm existence and transcription of sequences related to the proto-oncogene c-src in freshwater and marine species of sponges, the most primitive metazoan animals. By using the in situ hybridization technique, we were able to demonstrate for the first time that the cell-type-specific expression, characteristic for c-src related genes in higher vertebrates, is even realized in a freshwater sponge (Spongilla lacustris). The highest amount of c-src related transcripts was found in the omnipotent stem cells, the archaeocytes, and in the cells forming the inner epithelia, the endopinacocytes, which probably are involved in signal transduction.

RESUMEN

We developed a rapid method for quantification of tyrosine phosphorylation in immunophenotypically defined cell populations in specimens of whole blood and unprocessed bone marrow. Samples were formaldehyde-fixed and cells were permeabilized. Phosphotyrosine residues and surface antigens were simultaneously stained by monoclonal antibodies and visualized by flow cytometry. The accuracy of the method was confirmed by demonstration of an increase of phosphotyrosine levels in pp60v-src transformed fibroblasts. In blood of healthy donors, monocytes and granulocytes showed higher levels of phosphotyrosine than lymphocytes. CD34+ peripheral blood stem cells showed slightly increased tyrosine phosphorylation compared to autologous lymphocytes. Significantly elevated levels of phosphotyrosine were demonstrated in leukaemic blasts compared to lymphocytes (P = 0.01).

Asunto(s)

Médula Ósea/metabolismo , Pruebas Hematológicas/métodos , Fosfotirosina/metabolismo , Enfermedad Aguda , Citometría de Flujo , Humanos , Leucemia Mieloide/diagnóstico , Fosforilación , Fosfotirosina/sangre

RESUMEN

Small GTP binding proteins of the rab/YPT family are essential regulators of vectorial transport in the eukaryotic cell. Members of the rab/YPT1 family are found on the cytoplasmic surface of distinct intracellular membrane compartments. Membrane attachment is facilitated by a C-terminal geranylgeranyl moiety. In this report we investigated posttranslational modification and membrane binding of the rab6 protein, a member of the rab/YPT family located on the Golgi apparatus. A set of point mutations, which simulate the GDP or GTP bound conformation, was introduced into the rab6 cDNA. The mutated cDNAs were expressed in insect cells and the ability of the protein products to undergo geranylgeranyl modification and membrane association was assessed by Triton X-114 partition and cell fractionation. We report here that the modification of rab6 in insect cells depends on protein conformation. Only the GDP bound form, but not the GTP bound form is isoprenylated and subsequently membrane bound.

Asunto(s)

Proteínas Portadoras/metabolismo , Nucleótidos/farmacología , Prenilación de Proteína/genética , Spodoptera/metabolismo , Proteínas de Unión al GTP rab , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Western Blotting , Células Cultivadas , GTP Fosfohidrolasas/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Datos de Secuencia Molecular , Mutagénesis/genética , Mutación Puntual/genética , Unión Proteica/genética , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo

RESUMEN

We investigated the transforming domain of a recently isolated feline sarcoma virus (TP1-FeSV) which encodes a fgr-related tyrosine kinase expressed as a gag-fgr fusion protein. The gag portion was removed and replication-competent expression vectors (RCAS) with inserted v-fgr sequences were established. Chicken embryo fibroblasts (CEF) were transfected and monitored for replication, integration and transcription of the proviral constructs. We demonstrated that transfected cells display morphological changes and are able to form colonies in soft-agar. This suggests that the gag portion of the fusion protein from TP1-FeSV is not necessary for the transformation of fibroblasts.

Asunto(s)

Transformación Celular Viral , Proteínas Tirosina Quinasas/genética , Proteínas Oncogénicas de Retroviridae/genética , Virus del Sarcoma Felino/genética , Animales , Sitios de Unión , Células Cultivadas , Embrión de Pollo , Clonación Molecular , ADN Viral/análisis , Fibroblastos , Genes Virales , Genes gag , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/fisiología , ARN Viral/análisis , Proteínas Oncogénicas de Retroviridae/química , Proteínas Oncogénicas de Retroviridae/fisiología , Virus del Sarcoma Felino/enzimología , Virus del Sarcoma Felino/fisiología , Transfección

RESUMEN

We investigated the expression of the tyrosine kinase encoding proto-oncogenes c-src and fyn during in vitro differentiation of the human promyelocytic leukemia cell line HL-60 and an HL-60 derived cell line (HL-GOT) with an altered T-cell receptor gene. In HL-60 cells, the fyn and c-src encoded tyrosine kinases are activated in a differentiation-dependent manner, whereas in HL-60T cells high levels of pp59(fyn) and pp60(c-src) specific kinase activities are already present in the untreated cells. From the data obtained we conclude that untreated HL-60T cells represent a more differentiated type of cells compared to the untreated promyelocytic HL-60 cells.

RESUMEN

Through the development of a new colorimetric and non-radioactive method it is possible to measure PTPase activity in cell extracts. Applying this method we were able to demonstrate that intracellular PTPase activity in chicken embryo fibroblasts increases significantly after RSV induced transformation of these cells. PTPase activity in normal and transformed cells is inhibited by microM concentrations of vanadate, molybdate and zinc ion, but is not affected by mM concentrations of calcium, magnesium and sodium fluoride. The transformation specific activation of the PTPase seems to represent an early parameter during the events of cellular transformation.

Asunto(s)

Virus del Sarcoma Aviar/genética , Transformación Celular Viral/fisiología , Proteínas Tirosina Fosfatasas/análisis , 4-Nitrofenilfosfatasa/metabolismo , Animales , Extractos Celulares/análisis , Células Cultivadas , Embrión de Pollo , Fibroblastos/enzimología , Calor , Inmunoglobulina G/metabolismo , Mutación , Proteína Oncogénica pp60(v-src)/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores

RESUMEN

In one of the simplest metazoan organisms, the sponge Spongilla lacustris, at least four different src-related kinase genes (srk1-4) are expressed, all of which show a high degree of similarity to the c-src genes of vertebrates. Whereas srk2 and srk3 are clearly unrelated at the nucleic acid level, srk1 and srk4 share identical sequences in the 5' parts of their cDNAs. The cloning of several primer extension clones and genomic polymerase chain reaction experiments confirmed the hypothesis of an alternative splicing of tandemly arranged carboxy-terminal parts of srk1 and srk4. The genomic sequence encoding both proteins was found to be interrupted at the splice point by an intron which is located in the same position as one of the introns in the chicken src gene, which is the only gene conserved in invertebrates and vertebrates. All four srk genes are expressed in adult sponges as mRNA transcripts of about 2.2 kb. Tyrosine kinase activity of a src-related kinase could be detected in adult sponges but not in their resting form (gemmulae), and may reflect the activity of the srk protein products. Spongilla lacustris is the simplest organism from which a protein tyrosine kinase gene has been isolated. The presence of at least four such genes in the evolutionary ancient and primitive phylum Porifera suggests that tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms and that their activity may be involved in aggregation and cell-cell recognition.

Asunto(s)

Genes src/genética , Familia de Multigenes/genética , Poríferos/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Proteína Tirosina Quinasa CSK , Clonación Molecular , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/genética , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Quinasas/química , Proteínas Proto-Oncogénicas pp60(c-src)/química , Mapeo Restrictivo , Familia-src Quinasas

RESUMEN

Cell transformation in vivo seems to be a multistep process. In in vitro studies certain combinations of two oncogenes, a cytoplasmic gene product together with a nuclear gene product, are sufficient to transform primary rodent cells. Polyoma virus large T antigen can immortalize and, in cooperation with polyoma virus middle T antigen, transform primary cells. On the other hand mutant mouse p53 can also immortalize and, in cooperation with an activated Ha-ras oncogene, transform primary cells. In the present study we analyzed whether mutant p53 can replace polyoma virus large T antigen in a cell transformation assay with polyoma virus middle T antigen. Transfection of mutant p53 alone resulted in a cell line which had retained the actin cable network, grew poorly in medium with low concentration of serum, and failed to grow in semisolid agar. Cotransfection of mutant p53 together with polyoma virus middle T led to cells which grew in medium containing low serum concentration, grew well in semisolid agar, and displayed an altered morphology with the tendency to overgrow the normal monolayer. By these criteria these cells were considered fully transformed. The rate of p53 synthesis was similar in both cell lines. However, only p53 from the transformed cell line turned out to be stable. Cells transformed by mutant p53 and polyoma virus middle T expressed nearly the same amount of the c-src-encoded pp60c-src protein as cells transformed by the same p53 and cotransfected activated Ha-ras oncogene. However, only the polyoma virus middle T/p53-transformed cells exhibited an elevated level of pp60c-src-specific tyrosine kinase activity. Thus, despite different mechanisms leading to cell transformation, mutant p53 can replace polyoma virus large T antigen and polyoma virus middle T can replace the activated Ha-ras oncogene in cell transformation.

Asunto(s)

Antígenos Transformadores de Poliomavirus/genética , Transformación Celular Neoplásica , Transformación Celular Viral , Proteína p53 Supresora de Tumor/genética , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Western Blotting , Células Cultivadas , Microscopía Fluorescente , Proteína Oncogénica pp60(v-src)/genética , Proteína Oncogénica pp60(v-src)/metabolismo , Ratas , Transfección/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

To date the src gene family consists of at least 9 closely related protein tyrosine kinases belonging to the non-receptor type of kinases: c-src, c-yes, c-fgr, fyn, lyn, lck, hck, tkl and bkl. We have intensively studied the expression of the c-src gene during evolution and with respect to its possible functions in the processes of cellular differentiation and proliferation. From our results we conclude, that the c-src encoded tyrosine kinase could play a role in the development and/or maintenance of the multicellular organisation of primitive organisms like sponges or coelenterates. With respect to its tissue-specific expression pattern with neuronal cells always displaying elevated levels of pp60c-src and the observation that synaptophysin, the major constituent of the synaptic vesicle membrane protein, is phosphorylated by the c-src encoded tyrosinekinase in vitro and in intact synaptic vesicles, we suggest an essential role for pp60c-src in signal transduction pathways and/or axonal transport mechanisms in neurons.

Asunto(s)

Genes src , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Animales , Virus del Sarcoma Aviar/genética , Línea Celular

RESUMEN

Expression of pp60c-src, the first well defined proto-oncogene product, is developmentally regulated and tissue-specific, with neuronal tissues displaying high amounts of the c-src encoded pp60c-src kinase activity. In the central nervous system pp60c-src is preferentially expressed in regions characterized by a high content of grey matter and elevated density of nerve terminals. In this study we show for the first time a direct interaction between pp60c-src and synaptophysin as a physiological target protein in neurons by demonstrating that endogenous pp60c-src is able to phosphorylate synaptophysin (p38). p38 is a major constituent of the synaptic vesicle membrane protein and is thought to play a key role in the exocytosis of small synaptic vesicles and possibly small clear vesicles in neuroendocrine cells.

Asunto(s)

Proteínas de la Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Anticuerpos Monoclonales , Western Blotting , Fraccionamiento Celular , Técnicas In Vitro , Peso Molecular , Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/análisis , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas pp60(c-src) , Ratas , Sinaptofisina

RESUMEN

Enhanced protein phosphorylation seems to be characteristic for cell transformation. Viral or cellular oncogene products which are functionally implicated in cell transformation sometimes activate protein kinases, or they are protein kinases themselves. In the present paper we have shown that a protein kinase activity is tightly associated with immunopurified oncoprotein p53, either uncomplexed or in complex with SV40 large T antigen. Furthermore, we could demonstrate that the protein kinase associated with immunopurified p53 was independent of SV40 large T antigen. p53 in the immunocomplexes served as a substrate for this protein kinase. Phosphoamino acid analysis of in vitro phosphorylated p53 revealed a phosphorylation predominantly on serine residues similar to p53 phosphorylated in vivo. The use of different monoclonal antibodies did not reveal a total inhibition of the protein kinase activity. However, p53 precipitated with monoclonal antibodies which recognize a C-terminal domain, was phosphorylated in vitro to a lesser extent than p53 which was precipitated with monoclonal antibodies that recognize an N-terminal epitope. All subclasses of immunopurified p53 separable by sucrose density gradients or by sequential immunoprecipitation exhibited a protein kinase activity and served as substrates for this protein kinase. Moreover, a protein kinase activity was found to be associated with baculovirus expressed p53 which allows us to attribute this enzymatic activity more directly to p53.

Asunto(s)

Proteínas Oncogénicas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Aminoácidos/análisis , Animales , Antígenos de Diferenciación de Linfocitos T/metabolismo , Línea Celular , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Oncogénicas/inmunología , Proteínas Oncogénicas/aislamiento & purificación , Fosfoproteínas/análisis , Fosfoproteínas/inmunología , Fosfoproteínas/aislamiento & purificación , Fosforilación , Pruebas de Precipitina , Ratas , Proteína p53 Supresora de Tumor