RESUMEN
MicroRNAs (miRNAs) are ubiquitously expressed small, non-coding RNAs that negatively regulate gene expression at a post-transcriptional level. So far, over 1000 miRNAs have been identified in human cells and their diverse functions in normal cell homeostasis and many different diseases have been thoroughly investigated during the past decade. MiR-29, one of the most interesting miRNA families in humans to date, consists of three mature members miR-29a, miR-29b and miR-29c, which are encoded in two genetic clusters. Members of this family have been shown to be silenced or down-regulated in many different types of cancer and have subsequently been attributed predominantly tumor-suppressing properties, albeit exceptions have been described where miR-29s have tumor-promoting functions. MiR-29 targets expression of diverse proteins like collagens, transcription factors, methyltransferases and others, which may partake in abnormal migration, invasion or proliferation of cells and may favor development of cancer. Furthermore, members of the miR-29 family can be activated by interferon signaling, which suggests a role in the immune system and in host pathogen interactions, especially in response to viral infections. In this review, we summarize current knowledge on the genomic organization and regulation of the miR-29 family and we provide an overview of its implication in cancer suppression and promotion as well as in host immune responses. The numerous remarkable properties of these miRNAs and their often altered expression patterns might make the miR-29 family promising biomarkers and therapeutic targets for various diseases in future.
Asunto(s)
Genes Supresores de Tumor , Sistema Inmunológico/fisiología , MicroARNs/fisiología , Neoplasias/prevención & control , Humanos , Neoplasias/genéticaRESUMEN
Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS) proteins are potent feedback inhibitors of Jak-mediated signaling, we investigated their role in signal transduction through constitutively active Jak2 mutants. We selected two mutants, Jak2-V617F and Jak2-K539L, found in patients with MPDs and Jak2-T875N identified in acute megakaryoblastic leukemia. We found SOCS family members to be induced through Jak2-V617F in human leukemia cell lines expressing the mutant allele and in stable HEK transfectants inducibly expressing constitutively active Jak2 mutants. SOCS proteins were recruited to the membrane and bound to the constitutively active Jaks. In contrast to wild-type Jak2, the mutant proteins were constitutively ubiquitinated and degraded through the proteasome. Taken together, we show a SOCS-mediated downregulation of the constitutively active, disease-associated mutant Jak2 proteins. Furthermore, a threshold level of mutant Jak expression has to be overcome to allow full cytokine-independent constitutive activation of signaling proteins, which may explain progression to homozygocity in MPDs as well as gene amplification in severe phenotypes and leukemia.
Asunto(s)
Citocinas/fisiología , Janus Quinasa 2/fisiología , Transducción de Señal/fisiología , Proteínas Supresoras de la Señalización de Citocinas/fisiología , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Mutación , Complejo de la Endopetidasa Proteasomal/fisiología , Transporte de Proteínas , ARN Mensajero/análisis , Factor de Transcripción STAT5/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genéticaRESUMEN
IL-24, a member of the IL-10 family of cytokines, is produced by monocytes and Th2 cells. Interestingly, immune cells do not appear to express specific IL-24 receptor chains (IL-20R1/IL-20R2 and IL-22R/IL-20R2), it is therefore unlikely that IL-24 has classical immune-modulating properties. Skin, on the other hand, seems to represent a major target tissue for IL-24 and related cytokines such as IL-19, -20, and -22. However, the initial interest in IL-24 did not arise from its physiological signalling properties through its cognate receptors but rather because of its tentative ability to selectively kill different cancer cells. In an attempt to further investigate the signalling events underlying the IL-24-induced cancer cell death, we found that melanoma cell lines did not react in the expected and previously described way. Using several different forms and delivery modes of IL-24, we were unable to detect any apoptosis-inducing properties of this cytokine in melanoma cells. In the present 'Point of view' we will briefly summarize these findings and put them in context of published reports stating that IL-24 might be a long sought after treatment for several types of cancer.
Asunto(s)
Citocinas/fisiología , Citocinas/uso terapéutico , Interleucinas/fisiología , Interleucinas/uso terapéutico , Neoplasias/fisiopatología , Neoplasias/terapia , Adenovirus Humanos/genética , Terapia Genética , Vectores Genéticos , Humanos , Interleucinas/genética , Modelos Biológicos , Transducción de SeñalRESUMEN
Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Células Mesangiales/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Anticuerpos Monoclonales/metabolismo , Becaplermina , Western Blotting , Línea Celular , Proliferación Celular/efectos de los fármacos , Colagenasas/metabolismo , Densitometría , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Metaloproteinasa 13 de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Factor de Crecimiento Derivado de Plaquetas/genética , Análisis por Matrices de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacología , Proteínas Proto-Oncogénicas c-sis , ARN Mensajero/metabolismo , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
A tripartite receptor comprising the external region of the erythropoietin (Epo) receptor, the transmembrane and JAK-binding domains of the gp130 subunit of the interleukin-6 (IL-6) receptor, and a seven amino acid STAT1 recruitment motif (Y440) from the interferon (IFN)-gamma receptor, efficiently mediates an IFN-gamma-like response. An analogous completely foreign chimeric receptor in which the Y440 motif is replaced with the Y905 motif from gp130 also mediates an IFN-gamma-like response, but less efficiently. The IFNGR1 signal-transducing subunit of the IFN-gamma receptor is tyrosine phosphorylated through the chimeric receptors and the endogenous IL-6 and OSM receptors. Cross phosphorylation of IFNGR1 is not, however, required for the IFN-gamma-like response through the chimeric receptors, nor does it mediate an IFN-gamma-like response to IL-6 or OSM. The data argue strongly for modular JAK/STAT signalling and against any rigid structural organization for the "pathways" involved. They emphasize the likely high degree of overlap between the signals generated from disparate JAK-receptor complexes and show that relatively minor changes in such complexes can profoundly affect the response.
Asunto(s)
Interferón gamma/metabolismo , Proteínas Nucleares , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Receptor de Interferón alfa y beta , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Receptores Inmunológicos/genética , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT1 , Transducción de Señal , Transactivadores/biosíntesis , Transactivadores/metabolismo , Receptor de Interferón gammaRESUMEN
The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin, Radixin, Moesin) homology domain comprising 19 conserved hydrophobic regions. To determine the role of this domain in governing recruitment of Jak1, but not Jak3, to the gp130 subunit of the interleukin-6 family of cytokine receptors, the interaction of three Jak1/Jak3 chimeras with gp130 was investigated. Chimeras 1, 2 and 3 (Jak1 FERM regions 1-19, 1-18 and 1-8/Jak3, respectively) were all enzymically active. Chimeras 1 and 2 interacted with the cytoplasmic domain of gp130, although less efficiently than Jak1. Only chimera 2, however, restored gp130 signalling in Jak1-negative cells. The data are consistent with recruitment of Jak1 to gp130 through the Jak1 FERM domain, but also emphasise the likely requirement for precise Jak/receptor orientation to sustain function.
Asunto(s)
Antígenos CD/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Receptor gp130 de Citocinas , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibrosarcoma , Humanos , Janus Quinasa 1 , Janus Quinasa 3 , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT1 , Transducción de Señal , Transactivadores/metabolismo , Células Tumorales CultivadasRESUMEN
Development of cytokine resistance is an important feature of melanoma cells during tumor progression. To study the mechanisms of interleukin-6 resistance, we examined an interleukin-6 sensitive (WM35) and an interleukin-6 unresponsive cell line (WM9). Interleukin-6 treatment resulted in rapid inhibition of cyclin-dependent kinase 2/cyclin E activity and accumulation of the hypophosphorylated retinoblastoma protein in WM35 but not in WM9 cells. In contrast to previous reports, no differences in the expression of the cyclin-dependent kinase 2 inhibitor p21Cip1/WAF1 upon interleukin-6 treatment were found in both cell lines. Interleukin-6-induced inhibition of cyclin-dependent kinase 2 was also not due to changes in protein expression of cyclin-dependent kinase 2, cyclin E, p27Kip1 and cdc25A, a phosphatase positively regulating cyclin-dependent kinase 2 activity. As it is established that interleukin-6 resistance of WM9 cells is not caused by differential interleukin-6 receptor expression, we studied whether this is due to defective interleukin-6 signaling in which activation of signal transducer and activator of transcription 3 is a critical step. WM9 cells showed reduced tyrosine phosphorylation, DNA binding, and delayed nuclear translocation of signal transducer and activator of transcription 3 as compared with WM35 cells. The kinase upstream of signal transducer and activator of transcription 3, Janus kinase 1, was constitutively tyrosine-phosphorylated in WM9 cells and did not respond to interleukin-6 with increased phosphorylation. As compared with WM35 cells, interleukin-6 treatment of WM9 cells was not paralleled by reduced activity of the mitogen-activated protein kinase kinase-1, which suppresses activation of signal transducer and activator of transcription 3. Our data suggest that resistance of advanced melanoma cells to interleukin-6 is associated with reduced inhibition of cyclin-dependent kinase 2, which appears to be a consequence of a complex alteration in interleukin-6 signal transduction.
Asunto(s)
Quinasas CDC2-CDC28 , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/metabolismo , Interleucina-6/farmacología , Melanoma , Neoplasias Cutáneas , Transactivadores/metabolismo , Proteínas Supresoras de Tumor , Proteínas de Ciclo Celular/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/metabolismo , Proteínas de Unión al ADN/análisis , Fase G1/efectos de los fármacos , Fase G1/fisiología , Humanos , Janus Quinasa 1 , MAP Quinasa Quinasa 1 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Factor de Transcripción STAT3 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transactivadores/análisis , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimología , Tirosina/metabolismo , Fosfatasas cdc25/metabolismoRESUMEN
Hodgkin disease (HD) represents a malignant lymphoma in which the putative malignant Hodgkin and Reed-Sternberg cells are rare and surrounded by abundant reactive nonmalignant cells. It has been suggested that cytokines such as interleukin-6 (IL-6) are involved in the pathogenesis of the disease. The expression of the IL-6 receptor (IL-6R) complex and its link to the activation of signal transducers and activators of transcription (STAT) molecules in HD cell lines was investigated. Gel retardation and Western blot analyses revealed a high level of constitutively activated STAT3 in 5 of 7 HD cell lines, which could not be detected in Burkitt lymphoma cell lines. Different levels of IL-6R protein were measured in various HD cell lines: L428 and Dev cells were characterized by very low levels of gp80 and gp130, on KMH2 cells only gp130 but no gp80 was detected, whereas L540, L591, HDLM2, and L1236 were positive for both gp80 and gp130, suggesting a possible autocrine stimulation of STAT3. However, a further increase in STAT3 activation on IL-6 or IL-6/soluble IL-6R stimulation was not observed. Neutralizing monoclonal antibodies against IL-6, gp80, gp130, or both receptor subunits did not affect the proliferation or the constitutive activation of STAT molecules in HD cell lines. However, the tyrosine kinase inhibitor AG490 blocked the constitutive activation of STAT3 and inhibited spontaneous growth of HD tumor cells. The evidence suggests abnormal STAT signaling and growth regulation in Hodgkin cell lines. (Blood. 2001;98:762-770)
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Enfermedad de Hodgkin/patología , Transactivadores/metabolismo , Anticuerpos Monoclonales/farmacología , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos CD/farmacología , División Celular/efectos de los fármacos , Receptor gp130 de Citocinas , Proteínas de Unión al ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Enfermedad de Hodgkin/etiología , Enfermedad de Hodgkin/metabolismo , Humanos , Interleucina-6/inmunología , Interleucina-6/fisiología , Leucemia/metabolismo , Linfoma/metabolismo , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores de Interleucina-6/inmunología , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Transactivadores/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Janus kinase 1 (Jak1) is a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors. Here we show that the in vitro translated N-terminal domains of Jak1 are sufficient for binding to a biotinylated peptide comprising the membrane-proximal 73 amino acids of gp130, the signal-transducing receptor chain of interleukin-6-type cytokines. By the fold recognition approach amino acid residues 36-112 of Jak1 were predicted to adopt a beta-grasp fold, and a structural model was built using ubiquitin as a template. Substitution of Tyr(107) to alanine, a residue conserved among Jaks and involved in hydrophobic core interactions of the proposed beta-grasp domain, abrogated binding of full-length Jak1 to gp130 in COS-7 transfectants. By further mutagenesis we identified the loop 4 region of the Jak1 beta-grasp domain as essential for gp130 association and gp130-mediated signal transduction. In Jak1-deficient U4C cells reconstituted with the loop 4 Jak1 mutants L80A/Y81A and Delta(Tyr(81)-Ser(84)), the interferon-gamma, interferon-alpha, and interleukin-6 responses were similarly impaired. Thus, loop 4 of the beta-grasp domain plays a role in the association of Jak1 with both class I and II cytokine receptors. Taken together the structural model and the mutagenesis data provide further insight into the interaction of Janus kinases with cytokine receptors.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Receptores de Citocinas/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígenos CD/metabolismo , Sitios de Unión , Células COS , Receptor gp130 de Citocinas , Interferones/farmacología , Janus Quinasa 1 , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Homología de Secuencia de Aminoácido , Transducción de Señal , Tirosina/genética , Tirosina/metabolismoRESUMEN
The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated protein kinase (ERK)1 and ERK2, involved in regulating cell growth and differentiation, are constitutively active in A375 and WM239 human melanoma cells. Using PD098059, an inhibitor of MAPK kinase (MEK), we investigated the role of persistently activated ERK1/2 in cell growth. The inhibition of MAPK activity induced a dose-dependent growth arrest in G(0)/G(1) phase. Correspondingly, we observed the up-regulation of the cyclin-dependent kinase (Cdk) inhibitor p27/Kip1 and hypophosphorylation of the retinoblastoma protein. Further studies showed that PD098059 treatment significantly decreased Cdk2 kinase activity, most probably owing to an augmented level of p27/Kip1 associated with cyclin E-Cdk2 complexes. The accumulation of p27/Kip1 protein in A375 cells was attributed to its increased stability. Our findings suggest that constitutively active ERK1/2 kinases contribute to the growth of melanoma cells by negative regulation of the p27/Kip1 inhibitor.
Asunto(s)
Quinasas CDC2-CDC28 , Ciclo Celular/fisiología , División Celular/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Fase G1 , Genes Supresores de Tumor , Humanos , Melanoma , Proteína Quinasa 3 Activada por Mitógenos , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fase de Descanso del Ciclo Celular , Proteína de Retinoblastoma/metabolismo , Células Tumorales CultivadasRESUMEN
IL-5 is a major determinant in the survival, differentiation and effector-functions of eosinophils. It mediates its effect upon binding and activation of a membrane bound receptor (R), composed of a ligand-specific alpha-chain and a beta-chain, shared with the receptors for IL-3 and granulocyte-macrophage colony-stimulating factor. We have generated and mapped the epitopes of three monoclonal antibodies (mAb) directed against this cytokine: the strong neutralizing mAb 5A5 and 1E1, and the very weak neutralizing mAb H30. We found that H30 as well as 5A5 can increase proliferation above the level induced by human (h)IL-5 alone, in a JAK-2-dependent manner, and at every sub-optimal hIL-5 concentration analyzed. This effect is dependent on mAb-mediated cross-linking of IL-5R complexes, and is only observed on cell lines expressing a hybrid human/mouse IL-5Ralpha-chain. We discuss these findings in view of the stoichiometric and topological requirements for an activated IL-5R. Since humanized anti-IL-5 mAb are currently in clinical testing, our findings imply that such mAb should be carefully evaluated for their potentiating effects.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Interleucina-5/inmunología , Interleucina-5/farmacología , Proteínas Proto-Oncogénicas , Transducción de Señal/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta Inmunológica , Sinergismo Farmacológico , Mapeo Epitopo , Epítopos/inmunología , Humanos , Células Híbridas/efectos de los fármacos , Células Híbridas/metabolismo , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Interleucina-5/química , Janus Quinasa 2 , Ratones , Modelos Biológicos , Modelos Moleculares , Pruebas de Neutralización , Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Ratas , Agregación de Receptores/efectos de los fármacos , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-5 , Transfección , Células Tumorales CultivadasAsunto(s)
Citocinas/farmacología , Melanoma/tratamiento farmacológico , Antígenos CD/fisiología , Proteínas de Ciclo Celular/fisiología , División Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Receptor gp130 de Citocinas , Proteínas de Unión al ADN/fisiología , Humanos , Interleucina-6/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Melanoma/patología , Melanoma/fisiopatología , Glicoproteínas de Membrana/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Oncostatina M , Péptidos/farmacología , Transducción de Señal , Transactivadores/fisiología , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
The common use of the cytokine receptor gp130 has served as an explanation for the extremely redundant biological activities exerted by interleukin (IL)-6-type cytokines. Indeed, hardly any differences in signal transduction initiated by these cytokines are known. In the present study, we demonstrate that oncostatin M (OSM), but not IL-6 or leukemia inhibitory factor, induces tyrosine phosphorylation of the Shc isoforms p52 and p66 and their association with Grb2. Concomitantly, OSM turns out to be a stronger activator of ERK1/2 MAPKs. Shc is recruited to the OSM receptor (OSMR), but not to gp130. Binding involves Tyr(861) of the OSMR, located within a consensus binding sequence for the Shc PTB domain. Moreover, Tyr(861) is essential for activation of ERK1/2 and for full activation of the alpha(2)-macroglobulin promoter, but not for an exclusively STAT-responsive promoter. This study therefore provides evidence for qualitative differential signaling mechanisms exerted by IL-6-type cytokines.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Interleucina-6/farmacología , Proteínas/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal , Animales , Células COS , Dimerización , Proteína Adaptadora GRB2 , Inhibidores de Crecimiento/farmacología , Factor Inhibidor de Leucemia , Linfocinas/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oncostatina M , Péptidos/farmacología , Regiones Promotoras Genéticas , Ratas , Receptores de Citocinas/química , Receptores de Oncostatina M , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Tirosina/fisiología , alfa-Macroglobulinas/genéticaAsunto(s)
Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Fibrosis/enzimología , Fibrosis/metabolismo , Gelatinasas/metabolismo , Humanos , Metaloproteinasa 9 de la Matriz/genética , Datos de Secuencia Molecular , Mutación , Ratas , Células Tumorales CultivadasRESUMEN
Prior activation of mitogen-activated protein kinases by phorbol 13-myristate 12-acetate (PMA) results in an inhibition of interleukin (IL)-6-induced activation of the Janus kinase/signal transducer and activator of transcription (STAT) signaling pathway which is most likely mediated by the induction of suppressor of cytokine signaling-3 and requires the specific SHP2 binding site Y759 of the IL-6 signal transducer gp130. In this study, we demonstrate that PMA inhibits STAT activation by IL-6 and the related cytokine leukemia inhibitory factor (LIF) but not by oncostatin M (OSM). Since the LIF receptor also contains an SHP2 recruitment site whereas the OSM receptor lacks such a module, we propose that two SHP2 binding modules within a homo- or heterodimeric receptor are necessary to mediate the PMA inhibitory effect.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interleucina-6/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Represoras , Acetato de Tetradecanoilforbol/farmacología , Transactivadores/metabolismo , Factores de Transcripción , Secuencias de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Sitios de Unión , Línea Celular , Receptor gp130 de Citocinas , Dimerización , Activación Enzimática/efectos de los fármacos , Inhibidores de Crecimiento/antagonistas & inhibidores , Inhibidores de Crecimiento/farmacología , Humanos , Interleucina-5/farmacología , Interleucina-6/farmacología , Péptidos y Proteínas de Señalización Intracelular , Janus Quinasa 1 , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Linfocinas/antagonistas & inhibidores , Linfocinas/farmacología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oncostatina M , Péptidos/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Receptores de Citocinas/química , Receptores de Citocinas/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-5 , Receptores OSM-LIF , Receptores de Oncostatina M , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , TransfecciónRESUMEN
Stimulation of the IL-6R complex leads to Src homology domain containing tyrosine phosphatase 2 (SHP2) recruitment to the receptor subunit gp130 and its subsequent tyrosine phosphorylation. SHP2 is a two-SH2 domain-containing protein tyrosine phosphatase that is activated by many cytokines and growth factors. SHP2 counteracts the activation of transcription factors of the STAT family and the induction of IL-6-responsive genes. Tyrosine 759 of gp130, the signal transducing subunit of the IL-6R complex, is essential for the phosphorylation of SHP2. Mutation of tyrosine 759 to phenylalanine leads to an enhanced inducibility of IL-6-dependent genes. Here we demonstrate that no further tyrosines in the cytoplasmic part of gp130 are required for the phosphorylation of SHP2. We also tested whether the tyrosine 759 motifs in both subunits of the gp130 dimer are required for SHP2 association and tyrosine phosphorylation. Interestingly, one SHP2-recruiting phosphotyrosine motif in a single chain of the gp130 dimer is sufficient to mediate SHP2 association to the gp130 receptor subunit and its tyrosine phosphorylation as well as to attenuate IL-6-dependent gene induction. Furthermore, we show that repression of gene induction via Y759 does not require the presence of the SHP2 and STAT recruitment sites within the same receptor subunit, but within the same receptor complex. The Y759 motif in gp130 also attenuates gene induction mediated by the oncostatin M and leukemia inhibitory factor receptor complexes, which both contain gp130 as the shared subunit.
Asunto(s)
Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/fisiología , Interleucina-6/química , Interleucina-6/fisiología , Linfocinas/química , Linfocinas/fisiología , Péptidos/química , Péptidos/fisiología , Transducción de Señal/inmunología , Proteínas de Fase Aguda/antagonistas & inhibidores , Proteínas de Fase Aguda/biosíntesis , Secuencias de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/fisiología , Receptor gp130 de Citocinas , Dimerización , Activación Enzimática/genética , Activación Enzimática/inmunología , Regulación de la Expresión Génica/inmunología , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Linfocinas/genética , Linfocinas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiología , Ratones , Mutagénesis Sitio-Dirigida , Oncostatina M , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Citocinas/antagonistas & inhibidores , Receptores de Citocinas/metabolismo , Receptores de Citocinas/fisiología , Receptores OSM-LIF , Receptores de Oncostatina M , Proteínas Tirosina Fosfatasas con Dominio SH2 , Transducción de Señal/genética , Activación Transcripcional , Tirosina/química , Tirosina/genética , Dominios Homologos src/inmunologíaRESUMEN
gp130 is the common signal-transducing receptor chain of interleukin (IL)-6-type cytokines. Here we describe, for the first time, a single amino acid substitution (Trp(666)-->Ala) in the membrane-proximal interbox1/2 region that abrogates activation of STAT (signal transducer and activator of transcription) transcription factors and the proliferative response of pro-B-cell transfectants. Moreover, association of the Janus kinase JAK1 is prevented. No signalling of heterodimeric IL-5 receptor (IL-5R)/gp130 chimaeras occurs in COS-7 cells, even when only a single cytoplasmic chain of a gp130 dimer contains the Trp(666)Ala mutation, indicating that it acts dominantly.
Asunto(s)
Alanina/química , Antígenos CD/química , Antígenos CD/metabolismo , Interleucina-6/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Triptófano/química , Animales , Linfocitos B/metabolismo , Células COS , División Celular , Receptor gp130 de Citocinas , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Janus Quinasa 1 , Ratones , Mutagénesis Sitio-Dirigida , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Interleucina/metabolismo , Receptores de Interleucina-5 , Transducción de Señal , Activación Transcripcional , TransfecciónRESUMEN
The interleukin-6 (IL-6) receptor complex comprises the IL-6 receptor (IL-6R, gp80) and the signal transducer gp130. Binding of IL-6 to its receptor results in dimerization of gp130, activation of the Jak/STAT pathway, and in a down-regulation of IL-6 binding sites by endocytosis. The STAT activation after stimulation is transient, being maximal after 15-30 min and disappearing after 60-90 min. The mechanism which leads to the termination of the signal is still unknown. In this paper we have studied whether the down-modulation of the STAT signal requires the endocytosis of the receptor complex. Our results suggest that the desensitization of the IL-6 signal is not due to internalization of the receptor complex but requires de novo protein synthesis.
Asunto(s)
Antígenos CD/metabolismo , Proteínas de Unión al ADN/metabolismo , Endocitosis , Interleucina-6/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina-6/metabolismo , Transactivadores/metabolismo , Animales , Antígenos CD/genética , Línea Celular , Receptor gp130 de Citocinas , Eritropoyetina/metabolismo , Humanos , Interleucina-6/farmacología , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/genética , Ratones , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Células Tumorales CultivadasRESUMEN
The commonly accepted model of STAT factor activation at the cytoplasmic part of the receptor assumes that signal transducers and activators of transcription (STATs) are recruited from a cytoplasmic pool of monomeric STAT proteins. Based on a previous observation that non-phosphorylated STAT3-Src homology 2 domains dimerize in vitro, we investigated whether the observed dimerization is of physiological relevance within the cellular context. We show that STAT1 and STAT3 are pre-associated in non-stimulated cells. Apparently, these complexes are not able to translocate into the nucleus. We provide evidence that the event of STAT activation is more complex than previously assumed.