RESUMEN
Phospholipase D (PLD)1 is quiescent in vitro and in vivo until stimulated by classical protein kinase C (PKC) isoforms, ADP-ribosylation factor or Rho family members. By contrast, PLD2 has high basal activity, and the mechanisms involved in agonist-induced activation of PLD2 are poorly understood. Using transiently transfected human embryonic kidney (HEK)-293 cells as a model system, we report in the present study that PLD2 overexpressed in HEK-293 cells exhibits regulatory properties similar to PLD1 when stimulated in response to insulin and phorbol ester. Co-expression of PLD1 or PLD2 with PKC alpha results in constitutive activation of both PLD isoforms, which cannot be further stimulated by insulin. Co-expression of PLD1 with phospholipase C (PLC)gamma has the same effect, while co-expression of PLD2 with PLC gamma allows PLD2 activity to be stimulated in an insulin-dependent manner. The PKC-specific inhibitors bisindolylmaleimide and Gö 6976 abolish insulin-induced PLD2 activation in HEK-293 cells co-expressing the insulin receptor, PLC gamma and PLD2, confirming that not only PLD1, but PLD2 as well, is regulated in a PKC-dependent manner. Finally, we provide evidence that PKC alpha is constitutively associated with PLD2. In summary, we demonstrate that insulin treatment results in activation of both PLD1 and PLD2 in appropriate cell types when the appropriate upstream intermediate signalling components, i.e. PKC alpha and PLC gamma, are expressed at sufficient levels.
Asunto(s)
Insulina/farmacología , Isoenzimas/metabolismo , Riñón/enzimología , Fosfolipasa D/biosíntesis , Proteína Quinasa C/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/metabolismo , Línea Celular , Humanos , Riñón/citología , Fosfolipasa C gamma , Fosfolipasa D/metabolismo , Unión Proteica , Proteína Quinasa C-alfaRESUMEN
The signaling routes linking G-protein-coupled receptors to mitogen-activated protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-kinase gamma (PI3Kgamma), and protein kinase C (PKC). To characterize the mitogenic pathway of bradykinin (BK), COS-7 cells were transiently cotransfected with the human bradykinin B(2) receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK is mediated via the alpha subunits of a G(q/11) protein. Both activation of Raf-1 and activation of MAPK in response to BK were blocked by inhibitors of PKC as well as of the epidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 cells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-Kgamma or Src kinase failed to diminish MAPK activation by BK. BK-induced translocation and overexpression of PKC isoforms as well as coexpression of inactive or constitutively active mutants of different PKC isozymes provided evidence for a role of the diacylglycerol-sensitive PKCs alpha and epsilon in BK signaling toward MAPK. In addition to PKC activation, BK also induced tyrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. Inhibition of PKC did not alter BK-induced transactivation, and blockade of EGF receptor did not affect BK-stimulated phosphatidylinositol turnover or BK-induced PKC translocation, suggesting that PKC acts neither upstream nor downstream of the EGF receptor. Comparison of the kinetics of PKC activation and EGF receptor transactivation in response to BK also suggests simultaneous rather than consecutive signaling. We conclude that in COS-7 cells, BK activates MAPK via a permanent dual signaling pathway involving the independent activation of the PKC isoforms alpha and epsilon and transactivation of the EGF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.
Asunto(s)
Bradiquinina/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Receptores ErbB/fisiología , Proteínas Quinasas Activadas por Mitógenos , Proteína Quinasa C/fisiología , Receptores de Bradiquinina/fisiología , Transducción de Señal/fisiología , Activación Transcripcional , Androstadienos/farmacología , Animales , Células COS , Chlorocebus aethiops , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Proteínas de Unión al GTP/fisiología , Humanos , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/metabolismo , Receptor de Bradiquinina B2 , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Wortmanina , Familia-src Quinasas/fisiologíaRESUMEN
Mammalian phospholipase D (PLD) activity becomes up-regulated when cells are stimulated by a variety of hormones, growth factors, and other extracellular signals. Two distinct PLDs, PLD1 and PLD2, have been identified. The mechanism through which each PLD is activated, however, is poorly understood. Using transiently transfected human embryonic kidney fibroblasts (HEK293), we demonstrate here that PLD1 activity, and to a lesser extent PLD2 activity, is stimulated in response to epidermal growth factor (EGF). PLD2, but not PLD1, associates with the EGF receptor in a ligand-independent manner and becomes tyrosine-phosphorylated upon EGF receptor activation. Tyrosine 11 (Tyr-11) of PLD2 was identified as the specific phosphorylation site. Mutation of this residue to phenylalanine enhanced basal activity almost 2-fold, but did not alter the magnitude of the EGF-mediated increase in PLD2 activity. In conclusion, we show here for the first time agonist-stimulated activation of both PLD1 and PLD2 in vivo and provide evidence of a distinct type of interaction for each isoform with the EGF receptor. Moreover, our results suggest that agonist-induced tyrosine phosphorylation plays a role in PLD2 regulation.
Asunto(s)
Receptores ErbB/metabolismo , Fosfolipasa D/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Cartilla de ADN , Activación Enzimática , Humanos , Datos de Secuencia Molecular , Mutagénesis , FosforilaciónRESUMEN
The protein-tyrosine phosphatase SHP-1 binds to and dephosphorylates the epidermal growth factor receptor (EGFR), and both SH2 domains of SHP-1 are important for this interaction (Tenev, T., Keilhack, H., Tomic, S., Stoyanov, B., Stein-Gerlach, M., Lammers, R., Krivtsov, A. V., Ullrich, A., and Böhmer, F. D. (1997) J. Biol. Chem. 272, 5966-5973). We mapped the EGFR phosphotyrosine 1173 as the major binding site for SHP-1 by a combination of phosphopeptide activation, phosphopeptide competition, and receptor YF mutant analysis. Mutational conversion of the EGFR sequence 1171-1176 AEYLRV into the high affinity SHP-1 binding sequence LEYLYL of the erythropoietin receptor (EpoR) led to a highly elevated SHP-1 binding to the mutant EGFR (EGFR1171-1176EpoR) and in turn to an enhanced dephosphorylation of the receptor. SHP-1 expression interfered with EGF-dependent mitogen-activated protein kinase stimulation, and this effect was more pronounced in case of EGFR1171-1176EpoR. Reduced SHP-1 binding to the EGFR Y1173F mutant resulted in a reduced receptor dephosphorylation by coexpressed SHP-1 and less interference with EGF-dependent mitogen-activated protein kinase stimulation. The effects of receptor mutations on SHP-1 binding were, however, stronger than those on receptor dephosphorylation by SHP-1. Therefore, receptor dephosphorylation may be the result of the combined activity of receptor-bound SHP-1 and SHP-1 bound to an auxiliary docking protein.
Asunto(s)
Receptores ErbB/química , Receptores ErbB/fisiología , Fosfotirosina , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Clonación Molecular , Receptores ErbB/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosfopéptidos/metabolismo , Fosforilación , Mutación Puntual , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Tirosina Fosfatasas con Dominio SH2 , Transducción de Señal , Transfección , Dominios Homologos srcRESUMEN
Interleukin-1beta (IL-1beta) is cytotoxic to rat pancreatic beta-cells by inhibiting glucose oxidation, causing DNA damage and inducing apoptosis. Nitric oxide (NO) is a necessary but not sufficient mediator of these effects. IL-1beta induced kinase activity toward Elk-1, activation transcription factor 2, c-Jun, and heat shock protein 25 in rat islets. By Western blotting with phosphospecific antibodies and by immunocomplex kinase assay, IL-1beta was shown to activate extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38) in islets and rat insulinoma cells. Specific ERK1/2 and p38 inhibitors individually reduced but in combination blocked IL-1beta-mediated islet NO synthesis, and reverse transcription-polymerase chain reaction of inducible NO synthase mRNA showed that ERK1/2 and p38 controlled IL-1beta-induced islet inducible NO synthase expression at the transcriptional level. Hyperosmolarity caused phosphorylation of Elk-1, activation transcription factor 2, and heat shock protein 25 and activation of ERK1/2 and p38 in islets comparable to that induced by IL-1beta but did not lead to NO synthesis. Inhibition of p38 but not of ERK1/2 attenuated IL-1beta-mediated inhibition of glucose-stimulated insulin release. We conclude that ERK1/2 and p38 activation is necessary but not sufficient for IL-1beta-mediated beta-cell NO synthesis and that p38 is involved in signaling of NO-independent effects of IL-1beta in beta-cells.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Interleucina-1/farmacología , Islotes Pancreáticos/enzimología , Proteínas Quinasas Activadas por Mitógenos , Óxido Nítrico/biosíntesis , Páncreas/efectos de los fármacos , Animales , Activación Enzimática/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Insulina/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Concentración Osmolar , Fosforilación , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Endogámicas , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Growth factors are involved in a variety of cellular responses such as growth, differentiation, migration, metabolism, and transformation. Binding of the growth factor to its corresponding cell surface receptor results in activation of the receptor's intrinsic tyrosine kinase activity, and subsequently in activation of complex multistep signal transduction cascades. Activation of these interconnected signaling pathways eventually leads to a biological response, which involves changes in gene expression and protein synthesis. The biological response has been shown to be receptor-specific and also cell-type (tissue)-specific, indicating that various receptors activate distinct signal transduction pathways in one tissue and that one receptor activates different pathways in various tissues. What determines receptor specificity and tissue specificity? In this context, this article will focus on certain receptors with intrinsic tyrosine kinase activity, including receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, and nerve growth factor (NGF).
Asunto(s)
Sustancias de Crecimiento/fisiología , Transducción de Señal , Animales , Retroalimentación , Humanos , Membranas Intracelulares/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/fisiología , Especificidad por Sustrato , Factores de TiempoAsunto(s)
Diferenciación Celular , Neuronas/citología , Neuronas/fisiología , Receptores de Factores de Crecimiento/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Sitios de Unión , Receptores ErbB/metabolismo , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Neuritas/fisiología , Células PC12 , Fosfopéptidos/química , Ratas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factor de Crecimiento Nervioso/fisiología , Fosfolipasas de Tipo C/metabolismoAsunto(s)
Fosfotirosina/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Tirosina/metabolismo , Células 3T3 , Animales , Línea Celular , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/biosíntesis , Receptores ErbB/metabolismo , Homeostasis , Humanos , Ratones , Fosforilación , Proteínas Recombinantes de Fusión/metabolismo , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
Rapid and long term effects of protein kinase C alpha activation on receptor tyrosine kinase signaling parameters were investigated in human 293 embryonic fibroblasts and mouse NIH 3T3 cells. Within minutes of phorbol 12-myristate 13-acetate treatment, epidermal growth factor receptor and HER2 tyrosine phosphorylation was decreased, while platelet-derived growth factor receptor and insulin receptor autophosphorylation was upregulated. These effects are not mediated by protein kinase C-dependent receptor tyrosine kinase phosphorylation but apparently by activation or inactivation of receptor tyrosine kinase-specific phosphatases, as indicated by neutralization of these phenomena upon treatment of cells with sodium orthovanadate. In contrast to these short term effects, sustained activation of protein kinase C alpha by phorbol 12-myristate 13-acetate results in translocation of protein kinase C from the cytosol to the membrane fraction where it forms stable complexes with all receptor tyrosine kinases investigated. Ligand-induced receptor tyrosine kinase/protein kinase C association in NIH 3T3 fibroblasts is accompanied by a mobility shift of the receptor, indicating phosphorylation by activated protein kinase C. This phenomenon correlates with the disappearance of receptor tyrosine kinases from the cell surface, implying that this interaction plays a role in the process of receptor internalization and degradation. Interestingly, ligand-stimulated receptor down-regulation is also enhanced by overexpression of phospholipase C gamma, which strongly indicates a role for this common receptor tyrosine kinase substrate in negative regulation of growth factor signals.
Asunto(s)
Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células 3T3 , Animales , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Humanos , Ratones , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND: Mitogen-activated protein (MAP) kinase is the central component of a signal transduction pathway that is activated by growth factors interacting with receptors that have protein tyrosine kinase activity. The stimulation of PC12 phaeochromocytoma cells with nerve growth factor leads to the sustained activation and nuclear translocation of the p42 and p44 isoforms of MAP kinase and induces the differentiation of these chromaffin cells to a sympathetic-neuron-like phenotype. In contrast, stimulation with epidermal growth factor induces a transient activation of p42 and p44 MAP kinases without pronounced nuclear translocation and does not trigger cell differentiation. We have examined whether the differential activation of MAP kinases forms the basis of the differential response of the cells to the two factors. RESULTS: By overexpressing either wild-type or mutant receptors for epidermal growth factor in PC12 cells, we found that p42 and p44 MAP kinase activity remains elevated for longer in cells that overexpress receptors than in untransfected cells. Epidermal growth factor promotes both a striking nuclear translocation of p42 MAP kinase and the differentiation of the overexpressing cells. CONCLUSIONS: Our results strongly suggest that the distinct effects of nerve growth factor and epidermal growth factor on PC12 cell differentiation can be explained by differences in the extent and duration of activation of p42 and p44 MAP kinases in response to the two factors, without invoking a signal transduction pathway specific to nerve growth factor.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/fisiología , Células PC12/efectos de los fármacos , Animales , Transporte Biológico , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/enzimología , Activación Enzimática , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Técnica del Anticuerpo Fluorescente , Expresión Génica , Factores de Crecimiento Nervioso/farmacología , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Transducción de Señal , TransfecciónRESUMEN
Src homology 3 (SH3) domains are found in a variety of proteins that are involved in signal transduction or represent components of the cytoskeleton. These domains are thought to serve as modules that mediate specific protein-protein interactions that include proline-rich sequences on the target protein. We have identified proteins of 110, 80, 65, and 43 kDa in human embryonic fibroblasts that bind specifically to the SH3 domain of phospholipase C gamma, a primary substrate of receptor tyrosine kinases, and characterized the 110-kDa band as the microtubule-activated GTPase dynamin. In addition, dynamin binds the son of sevenless adaptor protein GRB-2 with even higher affinity. This interaction does not require the dynamin GTPase function and involves a proline-rich target sequence between residues 812 and 820 of dynamin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , GTP Fosfohidrolasas/metabolismo , Microtúbulos/enzimología , Proteínas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Secuencia de Aminoácidos , Dinaminas , Proteína Adaptadora GRB2 , Genes src , Glutatión Transferasa/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión , Transducción de SeñalRESUMEN
Differentiation and survival of neuronal cell types requires the action of neurotrophic polypeptides such as nerve growth factor (NGF). In the central and peripheral nervous system and the phaeochromocytoma cell model PC12, NGF exerts its effects through the activation of the signalling capacity of Trk, a receptor tyrosine kinase (RTK) which upon interaction with NGF becomes phosphorylated on tyrosines and thereby acquires the potential to interact with signal-transducing proteins such as phospholipase C-gamma (PLC gamma), phosphatidylinositol-3'-kinase (PI3'-K) and SHC. Mutagenesis of the specific binding sites for these src homology 2 (SH2) domain-containing substrates within the Trk cytoplasmic domain suggests a non-essential function of PI3'-K and reveals a major role for the signal controlled by the SHC binding site at tyrosine 490 and a co-operative function of the PLC gamma-mediated pathway for neuronal differentiation of PC12 cells.
Asunto(s)
Células PC12/fisiología , Proteínas Tirosina Quinasas/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal/fisiología , Animales , Secuencia de Bases , Diferenciación Celular/fisiología , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/farmacología , Neuronas/fisiología , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Recombinantes/metabolismo , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
Rat-1 fibroblasts stably overexpressing high levels of human insulin receptor were used as a model system to study the effects of hyperglycemia on insulin receptor tyrosine kinase (IRK) activity and protein kinase C (PKC) translocation in parallel in the intact cell. Glucose (10-25 mM) induced a significant reduction of IRK activity (tyrosine phosphorylation of IR-beta-subunit and IR-substrate-1) within 10 min. This effect was paralleled by a rapid translocation of several PKC isoforms (cPKC alpha, nPKC delta, nPKC epsilon, nPKC zeta) to the plasma membrane within 1 min. Kinetics of IRK inhibition and PKC translocation are consistent with the idea that the glucose effect on IRK is mediated by PKC activation. This hypothesis is supported by further observations. Addition of the protein kinase C inhibitor H-7 can prevent the effect of glucose on IRK. Inhibition of IRK is also observed after stimulation of the cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which can substitute for a physiological activator of PKC. Glucose (25 mM) increases the 32P incorporation in serine residues of the beta-subunit of IRK. We conclude that high levels of glucose induce inhibition of IRK in vivo. There is indirect evidence that this effect is mediated by a glucose-induced PKC translocation/activation and serine phosphorylation of the insulin receptor.
Asunto(s)
Glucosa/farmacología , Insulina/farmacología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Receptor de Insulina/antagonistas & inhibidores , Receptor de Insulina/metabolismo , 3-O-Metilglucosa , Animales , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Cinética , Manitol/farmacología , Metilglucósidos/farmacología , Fosfatos/metabolismo , Fosforilación , Fosfoserina/análisis , Fosfotreonina/análisis , Fosfotirosina , Ratas , Receptor de Insulina/biosíntesis , Receptor de Insulina/aislamiento & purificación , Acetato de Tetradecanoilforbol/farmacología , Transfección , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
Chimeric receptors composed of the human epidermal growth factor receptor (EGF-R) extracellular domain fused to wild-type and truncated platelet-derived growth factor receptor (PDGF-R) intracellular sequences were stably expressed in NIH 3T3 cells devoid of endogenous EGF-Rs. This experimental system allowed us to investigate the biological activity of PDGF-R cytoplasmic-domain mutants in PDGF-R-responsive NIH 3T3 cells by activating PDGF-specific signaling pathways with EGF. Deletion of 74 carboxy-terminal amino acids severely impaired the ability of the PDGF-R cytoplasmic domain to associate with cellular substrates in vitro. This deletion also inhibited receptor and substrate phosphorylation, reduced the receptor's mitogenic activity, and completely abolished its oncogenic signaling potential. Surprisingly, removal of only six additional amino acids, including Tyr-989, restored substantial receptor and substrate phosphorylation capacity as well as transforming potential and yielded a receptor with wild-type levels of ligand-induced mitogenic activity. However, the ability of this chimera to bind phospholipase C gamma was severely impaired in comparison with the ability of the wild-type receptor, while the association with other cellular proteins was not affected. Further deletion of 35 residues, including Tyr-977, nearly abolished all PDGF-R cytoplasmic-domain biological signaling activities. None of the three C-terminal truncations completely abolished the mitogenic potential of the receptors or had any influence on ligand binding or receptor down regulation. Together, these data implicate the 80 C-terminal-most residues of the PDGF-R, and possibly Tyr-989, in phospholipase C gamma binding, while receptor sequences upstream from Asp-988 appear to be essential for specific interactions with other cellular polypeptides such as ras GTPase-activating protein and phosphatidylinositol 3-kinase. Thus, the mutants described here allow the separation of distinct PDGF-activated signaling pathways and demonstrate that phospholipase C gamma phosphorylation is not required for mitogenesis and transformation.
Asunto(s)
Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Células 3T3 , Animales , División Celular , Clonación Molecular , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Ratones , Mutagénesis , Fosforilación , Receptores del Factor de Crecimiento Derivado de Plaquetas/química , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
A chimeric receptor consisting of an epidermal growth factor (EGF) receptor ligand-binding domain and platelet-derived growth factor (PDGF) receptor transmembrane and cytoplasmic signalling domains has been constructed and shown to be fully functional in phosphorylation, mitogenesis, transformation, Ca2+ release, and pH change assays. Expression of this receptor in EGF receptor-deficient, PDGF-responsive NIH 3T3 cells allows the activation of PDGF signalling pathways by EGF. This system was used to examine the function of kinase insertion sequences (KIS). While a mutant with a KIS deletion of 83 amino acids displayed a significant but reduced ability to induce mitogenic, transforming, and Ca2+ release responses in transfected cells, deletion of 20 additional amino acids resulted in abolishment of such activities. This differential loss of signalling potential correlated with the reduced or abolished potential of these receptor mutants to phosphorylate cellular substrates such as PLC gamma. Our results suggest an integral role for KIS in PDGF receptor cytoplasmic domain conformation and an involvement in substrate interaction, but provide no evidence for an exclusive role of KIS in the mediation of biological signals.
Asunto(s)
Quimera , Receptores de Superficie Celular/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Calcio/metabolismo , Línea Celular , ADN/genética , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/fisiología , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Datos de Secuencia Molecular , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Pruebas de Precipitina , Receptores de Superficie Celular/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas , Timidina/metabolismo , Transfección , Tirosina/metabolismoRESUMEN
By Western blot technique, 519 samples of human sera were tested for the presence of antibodies to the human papillomavirus (HPV) type 16 proteins E4 and E7 that had been expressed in Escherichia coli as fusion proteins. Sera were obtained from patients attending the University hospitals for reasons unrelated to HPV infections (controls), from patients with HPV-associated lesions, as well as from patients suffering from cervical cancer. Within the control population, 18.1% of them had antibodies that reacted with the E4 protein, and 3.9% of them had antibodies that reacted with the E7 protein. No sex-specific difference in the antibody prevalence was observed. The highest proportion of anti-E4 antibody-positive individuals (40.7%) was observed in the age group between 11 and 20 years. The frequency of anti-E4-positive sera was threefold higher in patients with HPV-associated genital lesions than that in age-matched controls. Antibodies against the HPV16 E7 protein were found 14 times more frequently in patients with cervical cancer, compared with age- and sex-matched controls (P less than .00001). From these data, we concluded that anti-E4 antibodies may be correlated with virus replication and that anti-E7 antibodies may represent a marker for cervical cancer development.
Asunto(s)
Anticuerpos Antivirales/análisis , Proteínas Bacterianas/sangre , Papillomaviridae/inmunología , Transfección/inmunología , Infecciones Tumorales por Virus/inmunología , Neoplasias del Cuello Uterino/inmunología , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Especificidad de Anticuerpos , Western Blotting/métodos , Niño , Preescolar , Femenino , Alemania Occidental/epidemiología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Papillomaviridae/genética , Plásmidos/genética , Prevalencia , Infecciones Tumorales por Virus/sangre , Infecciones Tumorales por Virus/epidemiología , Neoplasias del Cuello Uterino/sangreRESUMEN
The upstream regulatory region of the human papilloma virus-16 (HPV-16) genomic DNA contains a sequence element with a large degree of homology to the partially palindromic sequence GGTACANNNTGTTCT, which is the consensus sequence of the glucocorticoid responsive elements of known genes regulated by this steroid hormone. DNase I and dimethylsulfate protection experiments reveal the binding of this sequence by rat glucocorticoid receptor protein. A 400-bp DNA segment centrally containing this sequence confers strong inducibility by dexamethasone to the promoter p97 of HPV-16 and to the Herpes simplex virus thymidine kinase promoter, as judged by chloramphenicol acetyltransferase activity and RNase protection assays. The same DNA segment, that does not contain the consensus sequences of all papilloma viruses relevant for E2 protein-mediated transcription enhancement, functions in an enhancer-like fashion in addition to its glucocorticoid responsive action. This hormone-independent transcription enhancement is absent in human MCF7 cells, but is strong in human HeLa cells where the combined activity of the constitutive and the steroid hormone-dependent enhancer elements stimulate transcription by a factor of 500. This cell type specificity of the HPV-16 enhancer may be responsible for the tissue tropism of the virus. These observations and the presence of numerous homologies to known enhancers of cellular and viral genes suggest a complex pattern of activation of the human papilloma virus-16 promoters.
Asunto(s)
Proteínas de Unión al ADN , Elementos de Facilitación Genéticos , Genes Reguladores , Genes Virales , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Regiones Promotoras Genéticas , Neoplasias del Cuello Uterino/genética , Secuencia de Bases , Línea Celular , Dexametasona/farmacología , Dihidrotestosterona/farmacología , Elementos de Facilitación Genéticos/efectos de los fármacos , Femenino , Genes/efectos de los fármacos , Genes Reguladores/efectos de los fármacos , Genes Virales/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Papillomaviridae/efectos de los fármacos , Homología de Secuencia de Ácido Nucleico , Simplexvirus/genética , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
A number of human papillomavirus (HPV) type 16 proteins have recently been identified in human cervical carcinoma cell lines using polyclonal antisera against papillomavirus gene products expressed in Escherichia coli. E7 protein has been found to be the most abundant papillomavirus protein in these cells. Here we describe a panel of monoclonal antibodies recognizing a 15K Mr non-glycosylated cytoplasmic HPV-16 E7 protein. One of the antibodies cross-reacted with HPV-18 E7 protein.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Papillomaviridae/inmunología , Proteínas Virales/inmunología , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
We have constructed and functionally tested a cassette-vector-system for the transcription and translation of open reading frames (ORFs) in cells of higher eukaryotes. The vectors are derived from the plasmid pBR322 and can be selected and amplified in Escherichia coli. Alternative eukaryotic promoters can be inserted between the restriction sites SphI and KpnI, translation initiation motifs between KpnI and BglII, linkers for the adjustment of the translation reading frame and the insertion of genes or gene segments between BglII and HindIII, followed by a HindIII-EcoRI segment with splicing and polyadenylation signals derived from SV40. A prototype vector system, pORFEX11, 12 and 13, contains the strong cytomegalovirus immediately early promoter and a 10-bp motif of the SV40 T-antigen translation start. Polylinkers derived from pUC18 permit the insertion of ATG-less ORFs downstream from the ATG of the vector. Either of the three alternative polylinkers adjusts the appropriate translation frame. A similar construct contains the regulatable promoter of the Drosophila heat shock gene 70. We inserted genes or gene segments, that code for the bacterial chloramphenicol acetyltransferase, the bacterial gene conferring resistance against hygromycin, and the ORF E7 of the human papillomavirus type 18 into these vectors. After transfection of mouse L fibroblasts, all proteins and functions were expressed in accordance with the prediction. In transiently transfected L cells, the E7 protein expressed from pORFEX12 constitutes approximately 2.0% of total cell protein. This E7 protein could be localized by immunocytochemistry as a cytoplasmic component.
Asunto(s)
Proteínas de Unión al ADN , Genes Virales , Genes , Vectores Genéticos , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Biosíntesis de Proteínas , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Enzimas de Restricción del ADN , Escherichia coli/genética , Células L/enzimología , Ratones , Plásmidos , Timidina Quinasa/genética , TransfecciónRESUMEN
We have sequenced 1730 bp of human papilloma virus type 18 (HPV 18) DNA containing the open reading frames (ORF) E6, E7, the N-terminal part of E1 and, additionally, 120 bp of the N-terminal part of L1. Based on these sequencing data, together with the human papilloma virus type 16 (HPV 16) DNA sequence published recently, we identified and cloned the ORF E6, E7, E1 and L1 of HPV 18 and the ORF E6, E7, E1, E4, E5, L2 and L1 of HPV 16 into prokaryotic expression vectors. The expression system used provides fusions to the N-terminal part of the MS2 polymerase gene controlled by the heat-inducible lambda PL promoter. Using the purified fusion proteins as immunogens we raised antisera against the proteins encoded by the ORF E6, E7 and E1 of HPV 18 as well as those encoded by the ORF E6, E7, E4 and L1 of HPV 16. By Western blot analysis we could show that the E7 gene product is the most abundant protein in cell lines containing HPV 16 or HPV 18 DNA. It is a cytoplasmic protein of 15 kd in the SiHa and the CaSki cell lines which contain HPV 16 DNA, and 12 kd in the HeLa, the C4-1 and the SW756 cell lines which contain HPV 18 DNA. These results were confirmed by in vitro translation of hybrid-selected HPV 16 and HPV 18 specific poly(A)+ RNA from SiHa, CaSki and HeLa cells. Additionally, these experiments led to the identification of an 11-kd E6 and a 10-kd E4 protein in the CaSki cell line as well as a 70-kd E1 protein in HeLa cells.