RESUMEN
BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect of inactivation of the c-src gene. In response to adhesion on a fibronectin substrate, RPTPalpha-/- fibroblasts also exhibited characteristic deficiencies in integrin-mediated signalling responses, such as decreased tyrosine phosphorylation of the c-Src substrates Fak and p 130(cas), and reduced activation of extracellular signal regulated (Erk) MAP kinases. CONCLUSIONS: These observations demonstrate that RPTPalpha functions as a physiological upstream activator of Src-family kinases in fibroblasts and establish this tyrosine phosphatase as a newly identified regulator of integrin signalling.
Asunto(s)
Integrinas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Superficie Celular , Familia-src Quinasas/metabolismo , Animales , Adhesión Celular , Células Cultivadas , Activación Enzimática , Fibroblastos/citología , Fibroblastos/enzimología , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Genes src , Ratones , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Transducción de SeñalRESUMEN
Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does not require PTPase activity or posttranslational proteolytic cleavage of the R-PTP-kappa protein and is calcium independent. The results suggest that R-PTPases may provide a link between cell-cell contact and cellular signaling events involving tyrosine phosphorylation.
Asunto(s)
Proteínas Tirosina Fosfatasas/metabolismo , Animales , Adhesión Celular/fisiología , Agregación Celular/fisiología , Comunicación Celular/fisiología , Línea Celular , ADN Complementario/genética , Drosophila melanogaster , Espacio Extracelular/metabolismo , Expresión Génica , Humanos , Ratones , Conformación Proteica , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology, separated from the transmembrane segment by an uncharacteristically large juxta-membrane region. The extracellular domain of the R-PTP-kappa precursor protein contains an immunoglobulin-like domain and four fibronectin type III-like repeats, preceded by a signal peptide and a region of about 150 amino acids with similarity to the Xenopus A5 antigen, a putative neuronal recognition molecule (S. Takagi, T. Hsrata, K. Agata, M. Mochii, G. Eguchi, and H. Fujisawa, Neuron 7:295-307, 1991). Antibodies directed against the intra- and extracellular domains reveal that the R-PTP-kappa precursor protein undergoes proteolytic processing, following which both cleavage products remain associated. By site-directed mutagenesis, the likely cleavage site was shown to be a consensus sequence for cleavage by the processing endopeptidase furin, located in the fourth fibronectin type III-like repeat. In situ hybridization analysis indicates that expression of R-PTP-kappa in the central nervous system is developmentally regulated, with highest expression seen in actively developing areas and, in the adult, in areas capable of developmental plasticity such as the hippocampal formation and cerebral cortex. The mouse R-PTP-kappa gene maps to chromosome 10, at approximately 21 centimorgans from the centromere.
Asunto(s)
Envejecimiento/fisiología , Moléculas de Adhesión Celular/genética , Proteínas Tirosina Fosfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/enzimología , Moléculas de Adhesión Celular/metabolismo , Mapeo Cromosómico , Clonación Molecular/métodos , Embrión de Mamíferos , Embrión no Mamífero , Expresión Génica , Biblioteca de Genes , Ligamiento Genético , Marcadores Genéticos , Humanos , Sueros Inmunes , Hibridación in Situ , Antígenos Comunes de Leucocito/genética , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Especificidad de Órganos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , XenopusRESUMEN
The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , ADN/genética , Receptores ErbB/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Factores de Crecimiento TransformadoresRESUMEN
Tyrosine phosphorylation is controlled by a balance of tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Whereas the contribution of PTKs to breast tumorigenesis is the subject of intense scrutiny, the potential role of PTPs is poorly known. RPTPalpha is implicated in the activation of Src family kinases, and regulation of integrin signaling, cell adhesion, and growth factor responsiveness. To explore its potential contribution to human neoplasia, we surveyed RPTPalpha protein levels in primary human breast cancer. We found RPTPalpha levels to vary widely among tumors, with 29% of cases manifesting significant overexpression. High RPTPalpha protein levels correlated significantly with low tumor grade and positive estrogen receptor status. Expression of RPTPalpha in breast carcinoma cells led to growth inhibition, associated with increased accumulation in G0 and G1, and delayed tumor growth and metastasis. To our knowledge, this is the first example of a study correlating expression level of a specific bona fide PTP with neoplastic disease status in humans.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Proteínas Tirosina Fosfatasas/biosíntesis , Receptores de Superficie Celular , Animales , Neoplasias de la Mama/genética , División Celular/fisiología , Femenino , Amplificación de Genes , Humanos , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Células Tumorales CultivadasRESUMEN
To determine the capacity of the chicken c-erbA (cTR-alpha) gene product in regulating expression of known thyroid hormone-responsive genes, both the cTR-alpha and the viral v-erbA genes were expressed in FAO cells, a rat hepatoma cell line defective for functional thyroid hormone receptors. Upon nuclear expression of the cTR-alpha protein the cells become responsive to thyroid hormone, as detected by expression of a number of genes (malic enzyme, phosphoenolpyruvate carboxykinase, and Na+/K(+)-ATPase) reported to be indirectly induced by the hormone in vivo. In addition, our data show that the c-erbA product directly activates the Moloney murine leukemia virus promoter in a ligand-dependent manner. The data show that the chicken c-erbA-alpha protein can modulate the expression of rat genes under either direct or indirect control by thyroid hormone.
Asunto(s)
Expresión Génica/efectos de los fármacos , Genes/efectos de los fármacos , Neoplasias Hepáticas Experimentales/ultraestructura , Proteínas Proto-Oncogénicas/farmacología , Receptores de Hormona Tiroidea/metabolismo , Hormonas Tiroideas/farmacología , Animales , Secuencia de Bases , Pollos , Cicloheximida/farmacología , Biblioteca Genómica , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Malato Deshidrogenasa/genética , Datos de Secuencia Molecular , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Ratas , Receptores de Hormona Tiroidea/deficiencia , Infecciones por Retroviridae/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Transcripción Genética/efectos de los fármacos , Triyodotironina/metabolismo , Triyodotironina/farmacologíaRESUMEN
Helicobacter pylori (H. pylori) produces vacuolating cytotoxin (VacA), a potent protein toxin, which is associated with gastric inflammation and ulceration. Recent studies demonstrated that connexins (Cxs), which are responsible for intracellular communication at gap junctions (GJs) as well as cell homeostasis, participate in VacA-induced cell death. We now demonstrate in AZ-521 cells that VacA increased cytoplasmic Cx43, accompanied by LC3-II generation in a time- and dose-dependent manner without induction of Cx43 mRNA expression. Inhibition of VacA-induced Rac1 activity prevented ERK phosphorylation and the increase in Cx43. Suppression of ERK activity and addition of N-acetyl-cysteine inhibited VacA-dependent increase in Cx43 and LC3-II. DIDS, an anion-selective inhibitor, suppressed VacA-dependent increase in Cx43, suggesting that VacA channel activity was involved in this pathway. By confocal microscopy, Cx43 increased by VacA was predominately localized in cholesterol-rich, detergent-resistant membranes including GJs, and a fraction of Cx43 was incorporated in endocytotic vesicles and autophagolysosomes. Accumulation of Cx43 was also observed in gastric mucosa from H. pylori-infected patients compared with healthy controls, suggesting that the pathogen caused a similar effect in vivo. Our findings show that VacA-mediated effects on autophagy inhibits turnover of Cx43, resulting in increased levels in the cytoplasm, leading eventually to apoptotic cell death.
RESUMEN
Receptor protein tyrosine phosphatases (RPTPs) comprise a family of proteins that feature intracellular phosphatase domains and an ectodomain with putative ligand-binding motifs. Several RPTPs are expressed in the brain, including RPTP-kappa which participates in homophilic cell-cell interactions in vitro [Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, J. Sap, Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region, Mol. Cell. Biol. 13 (1993) 2942-2951; J. Sap, Y.-P. Jiang, D. Friedlander, M. Grumet, J. Schlessinger, Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding, Mol. Cell. Biol. 14 (1994) 1-9]. The homology of RPTP-kappa's ectodomain to neural cell adhesion molecules indicates potential roles in developmental processes such as axonal growth and target recognition, as has been demonstrated for certain Drosophila RPTPs. The brain distribution of RPTP-kappa-expressing cells has not been determined, however. In a gene-trap mouse model with a beta-gal+neo (beta-geo) insertion in the endogenous RPTP-kappa gene, the consequent loss of RPTP-kappa's enzymatic activity does not produce any obvious phenotypic defects [W.C. Skarnes, J.E. Moss, S.M. Hurtley, R.S.P. Beddington, Capturing genes encoding membrane and secreted proteins important for mouse development, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6592-6596]. Nevertheless, since the transgene's expression is driven by the endogenous RPTP-kappa promoter, distribution of the truncated RPTP-kappa/beta-geo fusion protein should reflect the regional and cellular expression of wild-type RPTP-kappa, and thus may identify sites where RPTP-kappa is important. Towards that goal, we have used this mouse model to map the distribution of the truncated RPTP-kappa/beta-geo fusion protein in the adult mouse brain using beta-galactosidase as a marker enzyme. Visualization of the beta-galactosidase activity revealed a non-random pattern of expression, and identified cells throughout the CNS that display RPTP-kappa promoter activity. Several neural systems highly expressed the transgene-most notably cortical, olfactory, hippocampal, hypothalamic, amygdaloid and visual structures. These well-characterized brain regions may provide a basis for future studies of RPTP-kappa function.
Asunto(s)
Química Encefálica/fisiología , Proteínas Tirosina Fosfatasas/genética , Factores de Edad , Animales , Northern Blotting , Regulación Enzimológica de la Expresión Génica/fisiología , Operón Lac , Mesencéfalo/química , Mesencéfalo/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas/fisiología , Prosencéfalo/química , Prosencéfalo/enzimología , ARN Mensajero/análisis , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Retina/química , Retina/enzimología , Rombencéfalo/química , Rombencéfalo/enzimología , Médula Espinal/química , Médula Espinal/enzimología , Transgenes/fisiología , beta-Galactosidasa/genéticaRESUMEN
Analysis of the localization of receptor-type protein tyrosine phosphatase-beta (RPTP-beta) by in situ hybridization and immunocytochemistry indicates that it is predominantly expressed in the developing central nervous system (CNS). RPTP-beta is highly expressed in radial glia and other forms of glial cells that play an important role during development. The immunoreactivity localizes to the radial processes of these cells, which act as guides during neuronal migration and axonal elongation. The pattern of RPTP-beta expression changes with the progression of glial cell differentiation. In the adult, high levels of RPTP-beta are seen in regions of the brain where there is continued neurogenesis and neurite outgrowth. The spatial and temporal patterns of RPTP-beta expression suggest that this receptor phosphatase plays a role in morphogenesis and plasticity of the nervous system.
Asunto(s)
Sistema Nervioso/crecimiento & desarrollo , Plasticidad Neuronal/fisiología , Proteínas Tirosina Fosfatasas/biosíntesis , Animales , Animales Recién Nacidos/fisiología , Axones/fisiología , Femenino , Inmunohistoquímica , Hibridación in Situ , Sistema Nervioso/citología , Sistema Nervioso/enzimología , Neuritas/fisiología , Neuroglía/fisiología , Embarazo , Proteínas Tirosina Fosfatasas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signaling pathways involving SRC family kinases, which result from their ability to control phosphorylation of both activating and inhibitory sites in these kinases and possibly also their substrates. Similarly, integrin signaling illustrates how phosphorylation of a single protein, or the activity of a pathway, can be controlled by multiple tyrosine phosphatases, attesting to the intricate integration of these enzymes in cellular regulation. Lastly, we are starting to appreciate the roles of intracellular topology, tyrosine phosphorylation and oligomerization among the many mechanisms regulating tyrosine phosphatase activity.
Asunto(s)
Comunicación Celular/fisiología , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Animales , HumanosRESUMEN
Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine-phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Antígenos Comunes de Leucocito/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Tirosina/análogos & derivados , Animales , Línea Celular , Proteína Adaptadora GRB2 , Humanos , Sustancias Macromoleculares , Fosforilación , Fosfotirosina , Ratas , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Transducción de Señal , Tirosina/metabolismoRESUMEN
The rat growth hormone (RGH) gene constitutes a well-documented model system for the direct regulation of transcription by thyroid hormones. In order to analyse its interaction with sequences in the RGH gene, we have overproduced the thyroid hormone receptor-alpha (c-erbA) protein using a vaccinia virus expression system. The expressed protein bound T3 and DNA-cellulose with expected affinities, and the major binding site for the receptor protein was found to be located in the third intron of the RGH gene. This site displayed significantly higher affinity for the receptor protein than a previously described thyroid hormone response element (TRE) in the promoter of this gene, and also conferred stronger hormone responsiveness in vivo to a heterologous promoter. The data suggest that this novel TRE plays a major role in the regulation of rat growth hormone gene expression by thyroid hormones.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Genes , Hormona del Crecimiento/genética , Intrones , Regiones Promotoras Genéticas , Transcripción Genética/efectos de los fármacos , Triyodotironina/farmacología , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Clonación Molecular , Desoxirribonucleasa I , Células HeLa/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Plásmidos , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Receptores de Hormona Tiroidea , Mapeo Restrictivo , TransfecciónRESUMEN
We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid extracellular domain (including signal peptide) of R-PTP-alpha is marked by a high serine/threonine content (32%) as well as eight potential N-glycosylation sites but displays no similarity to known proteins. Genetic mapping assigns the gene for R-PTP-alpha to mouse chromosome 2, closely linked to the Il-1a and Bmp-2a loci. The corresponding mRNA (3.0 kilobases) is expressed in most murine tissues and most abundantly expressed in brain and kidney. Antibodies against a synthetic peptide of R-PTP-alpha identified a 130-kDa protein in cells transfected with the R-PTP-alpha cDNA.
Asunto(s)
Fosfoproteínas Fosfatasas/genética , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Encéfalo/enzimología , Mapeo Cromosómico , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Expresión Génica , Biblioteca de Genes , Genes , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Datos de Secuencia Molecular , Proteínas Tirosina Fosfatasas , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin to increase prolactin gene expression but potentiates the effects of epidermal growth factor and cAMP on prolactin promoter activity. RPTPalpha was the only protein-tyrosine phosphatase tested that did this. Thus, the effect of RPTPalpha on prolactin-chloramphenicol acetyltransferase (CAT) promoter activity is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent. Experiments with inhibitors of phosphatidylinositol 3-kinase suggest that insulin-increased prolactin-CAT expression is phosphatidylinositol 3-kinase-independent. These results suggest that RPTPalpha may be a physiological regulator of insulin action.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Insulina/farmacología , Prolactina/genética , Proteínas Tirosina Fosfatasas/fisiología , Receptores de Superficie Celular , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , AMP Cíclico/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Regiones Promotoras Genéticas , Proteínas Tirosina Fosfatasas/genética , Receptor de Insulina/metabolismo , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidoresRESUMEN
Receptor protein-tyrosine phosphatase RPTPalpha is found associated in vivo with the adaptor protein Grb2. Formation of this complex, which contains no detectable levels of Sos, is known to depend on a C-terminal phosphorylated tyrosine residue (Tyr798) in RPTPalpha and on the Src homology (SH) 2 domain in Grb2 (, ). We show here that association of Grb2 with RPTPalpha also involves a critical function for the C-terminal SH3 domain of Grb2. Furthermore, Grb2 SH3 binding peptides interfere with RPTPalpha-Grb2 association in vitro, and the RPTPalpha protein can dissociate the Grb2-Sos complex in vivo. These observations constitute a novel mode of Grb2 association and suggest a model in which association with a tyrosine-phosphorylated protein restricts the repertoire of SH3 binding proteins with which Grb2 can simultaneously interact. The function of the Tyr798 tyrosine phosphorylation/Grb2 binding site in RPTPalpha was studied further by expression of wild type or mutant RPTPalpha proteins in PC12 cells. In these cells, wild type RPTPalpha interferes with acidic fibroblast growth factor-induced neurite outgrowth; this effect requires both the catalytic activity and the Grb2 binding Tyr798 residue in RPTPalpha. In contrast, expression of catalytically active RPTPalpha containing a mutated tyrosine phosphorylation/Grb2 association site enhances neurite outgrowth. Our observations associate a functional effect with tyrosine phosphorylation of, and ensuing association of signaling proteins with, a receptor protein-tyrosine phosphatase and raise the possibility that RPTPalpha association may modulate Grb2 function and vice versa.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Unión Competitiva , Receptores ErbB/metabolismo , Humanos , Fosforilación , Conformación Proteica , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , TirosinaRESUMEN
CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein in which the extracellular and transmembrane domains of CD45 are replaced with those of the EGF receptor (EGFR) is able to restore TCR signaling in a CD45-deficient cell. Thus, the cytoplasmic domain of CD45 is necessary and sufficient for TCR signal transduction. Moreover, EGFR ligands functionally inactivate the EGFR-CD45 chimera in a manner that is dependent on dimerization of the chimeric protein. Inactivation of EGFR-CD45 chimera function results in the loss of TCR signaling, indicating that CD45 function is continuously required for TCR-mediated proximal signaling events. These results suggest that ligand-mediated regulation of receptor-PTPases may have mechanistic similarities with receptor tyrosine kinases.
Asunto(s)
Receptores ErbB/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Western Blotting , Calcio/metabolismo , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Receptores ErbB/genética , Humanos , Cinética , Leucemia , Antígenos Comunes de Leucocito/genética , Ligandos , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/inmunología , Factores de Tiempo , Transfección , Células Tumorales CultivadasRESUMEN
To identify and characterize the hormone-binding domain of the thyroid hormone receptor, we analyzed the ligand-binding capacities of proteins representing chimeras between the normal receptor and P75gag-v-erbA, the retrovirus-encoded form deficient in binding ligand. Our results show that several mutations present in the carboxy-terminal half of P75gag-v-erbA co-operate in abolishing hormone binding, and that the ligand-binding domain resides in a position analogous to that of steroid receptors. Furthermore, a point mutation that is located between the putative DNA and ligand-binding domains of P75gag-v-erbA and that renders it biologically inactive fails to affect hormone binding by the c-erbA protein. These results suggest that the mutation changed the ability of P75gag-v-erbA to affect transcription since it also had no effect on DNA binding. Our data also suggest that hormone-independent activity of P75gag-v-erbA provided a selective advantage to the avian erythroblastosis virus during the original selection for a highly oncogenic strain of the virus.
Asunto(s)
Genes , Proto-Oncogenes , Receptores de Hormona Tiroidea/genética , Animales , Pollos , Quimera , Clonación Molecular , Cinética , Mutación , Plásmidos , Biosíntesis de Proteínas , Receptores de Hormona Tiroidea/metabolismo , Proteínas Recombinantes/metabolismo , Hormonas Tiroideas/metabolismo , Transcripción GenéticaRESUMEN
Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site-directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane-proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45-deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR-mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand-induced negative regulation of PTPase function. We also demonstrate that the phosphorylation of the C-terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45-deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C-terminal phosphorylated Lck molecules.
Asunto(s)
Antígenos Comunes de Leucocito/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Catálisis , Análisis Mutacional de ADN , Receptores ErbB/genética , Receptores ErbB/metabolismo , Antígenos Comunes de Leucocito/genética , Ligandos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Datos de Secuencia Molecular , Fosforilación , Mutación Puntual , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Tirosina/metabolismoRESUMEN
Several recent observations, such as the identification of the cellular homologue of the v-erb-A oncogene as a thyroid-hormone receptor, have strongly implicated nuclear oncogenes in transcriptional control mechanisms. The v-erb-A oncogene blocks the differentiation of erythroid cells, and changes the growth requirements of fibroblasts and erythroblasts. Mutations in v-erb-A protein have led to the loss of its affinity for thyroid hormones but do not affect its DNA-binding ability, a property required for biological activity. We report here the identification of a novel thyroid-hormone response element (TRE) in the long terminal repeat of Moloney murine leukaemia virus that binds the c-erb-A-alpha protein. The v-erb-A protein abolishes the responsiveness of this TRE to thyroid hormone, although it has a lower affinity than the normal receptor for the TRE. The data indicate that overexpressed v-erb-A protein negatively interferes with normal transcriptional-control mechanisms, and that amino-acid substitutions have altered its DNA-binding properties.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Oncogenes , Proteínas de los Retroviridae/genética , Hormonas Tiroideas/farmacología , Transcripción Genética/efectos de los fármacos , Secuencia de Bases , Técnicas de Inmunoadsorción , Virus de la Leucemia Murina de Moloney/genética , Proteínas Oncogénicas v-erbA , Plásmidos , Regiones Promotoras Genéticas , Receptores de Hormona Tiroidea/fisiología , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas de los Retroviridae/farmacología , Homología de Secuencia de Ácido Nucleico , Simplexvirus/genética , Transfección , Triyodotironina/farmacologíaRESUMEN
The v-erbA oncogene, one of the two oncogenes of the avian erythroblastosis virus, efficiently blocks erythroid differentiation and suppresses erythrocyte-specific gene transcription. Here we show that the overexpressed thyroid hormone receptor c-erbA effectively modulates erythroid differentiation and erythrocyte-specific gene expression in a T3-dependent fashion, when introduced into erythroid cells via a retrovirus. In contrast, the endogenous thyroid hormone receptor does not detectably affect erythroid differentiation. The analysis of a series of chimeric v-/c-erbA proteins suggests that the v-erbA oncoprotein has lost one type of thyroid hormone receptor function (regulating erythrocyte gene transcription in response to T3), but constitutively displays another function: it represses transcription in the absence of T3. The region responsible for the loss of hormone-dependent regulator activity of v-erbA has been mapped to the very C-terminus of c-erbA, encompassing a cluster of highly conserved amino acid residues with the potential to form an amphipathic alpha-helix.