RESUMEN
The phosphatase of regenerating liver (PRL) is a group of protein tyrosine phosphatases that play a key role in cancer progression and metastasis. We previously showed that PRL-2 modulates intracellular Mg(2+) levels and sustains cancer phenotypes by binding to the Mg(2+) transporter CNNM3. However, the physiological functions of PRL-2 in animals remain largely unknown. To better understand which cell types are associated with PRL-2 function, we characterized its expression in mouse tissues using a PRL-2 ß-galactosidase reporter mouse model. Our results demonstrated that PRL-2 was ubiquitously expressed, with the highest expression levels observed in the hippocampal pyramidal neurons, ependymal cells, cone and rod photoreceptor cells, endocardium, vascular and bronchial smooth muscle, and collecting ducts in the kidney. On the other hand, PRL-2 expression was undetectable or very low in the parenchymal cells of the liver and pancreas. Our results also indicated that PRL-2 is involved in cell-type-specific Mg(2+) homeostasis and that PRL-2 expression is potentially inversely regulated by dietary Mg(2+) levels.
Asunto(s)
Suplementos Dietéticos , Proteínas Inmediatas-Precoces/análisis , Proteínas Inmediatas-Precoces/biosíntesis , Magnesio/farmacología , Proteínas Tirosina Fosfatasas/análisis , Proteínas Tirosina Fosfatasas/biosíntesis , Animales , Femenino , Homeostasis/efectos de los fármacos , Proteínas Inmediatas-Precoces/metabolismo , Magnesio/administración & dosificación , Magnesio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Tirosina Fosfatasas/metabolismoRESUMEN
Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor implicated in carcinogenesis. Here, the role of STAT3 pathway in the antitumor activity of an active ginseng saponin metabolite compound K (CK) was investigated in human multiple myeloma U266 cells. CK increased the cytotoxicity, accumulated the sub-G1 DNA population, cleaved poly (ADP-ribose) polymerase (PARP) and activated caspase-3 in U266 cells. Interestingly, CK inhibited phosphorylation of STAT3 and its upstream activators, the Janus activated kinase 1 (JAK1), but not JAK2. Furthermore, CK enhanced the expression of protein tyrosine phosphatase (PTP) SHP-1, but not PTEN. Additionally, CK down-regulated STAT3 target genes bcl-x(L), bcl-2, survivin, cyclin E and cyclin D1. Conversely, PTP inhibitor pervanadate reversed CK-mediated STAT3 inactivation and cleavages of caspase-3 and PARP. Overall, our findings demonstrate that JAK1/STAT3 signaling mediates CK-induced apoptosis in U266 cells and also suggest the chemopreventive potential of CK for treatment of multiple myeloma.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ginsenósidos/farmacología , Janus Quinasa 1/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Panax , Factor de Transcripción STAT3/antagonistas & inhibidores , Western Blotting , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/farmacología , Humanos , Janus Quinasa 1/metabolismo , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/biosíntesis , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Vanadatos/farmacologíaRESUMEN
With the exception of skin cancer, prostatic adenocarcinoma represents the most common cancer among men in the United States and the second most common cause of cancer mortality. Mortality is often associated with metastatic disease, which in the case of prostatic adenocarcinoma typically involves bones and only rarely affects the skin. Although clinical history and examination, laboratory tests and routine pathology can suggest the prostate as a source of metastatic disease, immunohistochemistry - specifically, for prostate-specific antigen (PSA) - is often used to help establish the diagnosis. We report a case of cutaneous metastatic prostatic adenocarcinoma presenting in the inguinal region of a 78-year-old man 5 years after his initial diagnosis. The case is unusual in that the clinical appearance mimicked a vascular proliferation and in that the metastatic prostatic adenocarcinoma failed to express PSA. Rather, expression of prostatic acid phosphatase was observed.
Asunto(s)
Adenocarcinoma/secundario , Neoplasias de la Próstata/patología , Neoplasias Cutáneas/secundario , Fosfatasa Ácida , Anciano , Humanos , Inmunohistoquímica , Masculino , Antígeno Prostático Específico/biosíntesis , Proteínas Tirosina Fosfatasas/biosíntesis , Resección Transuretral de la PróstataRESUMEN
The activation of signal transducers and activators of transcription 3 (STAT3) has been linked with carcinogenesis through survival, proliferation, and angiogenesis of tumor cells. Agents that can suppress STAT3 activation have potential not only for prevention but also for treatment of cancer. In the present report, we investigated whether 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), an analogue of vitamin K, and isolated from chitrak (Plumbago zeylanica), an Ayurvedic medicinal plant, can modulate the STAT3 pathway. We found that plumbagin inhibited both constitutive and interleukin 6-inducible STAT3 phosphorylation in multiple myeloma (MM) cells and this correlated with the inhibition of c-Src, Janus-activated kinase (JAK)1, and JAK2 activation. Vanadate, however, reversed the plumbagin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that plumbagin induced the expression of the protein tyrosine phosphatase, SHP-1, and silencing of the SHP-1 abolished the effect of plumbagin. This agent also downregulated the expression of STAT3-regulated cyclin D1, Bcl-xL, and vascular endothelial growth factor; activated caspase-3; induced poly (ADP ribose) polymerase cleavage; and increased the sub-G(1) population of MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the plumbagin-induced apoptosis. When compared with AG490, a rationally designed STAT3/JAK2 inhibitor, plumbagin was found more potent in suppressing the proliferation of cells. Plumbagin also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells. Overall, these results suggest that the plumbagin inhibits STAT3 activation pathway through the induction of SHP-1 and this may mediate the sensitization of STAT3 overexpressing cancers to chemotherapeutic agents.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Naftoquinonas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/biosíntesis , Factor de Transcripción STAT3/antagonistas & inhibidores , Vitamina K 3/análogos & derivados , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , ADN/metabolismo , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/genética , Inducción Enzimática/efectos de los fármacos , Humanos , Modelos Biológicos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Vitamina K 3/farmacologíaRESUMEN
BACKGROUND: Cementum is a key component of a functional periodontal organ. However, regenerating lost cementum is difficult and often incomplete. Identifying molecular mediators of cementoblast differentiation and function should lead to better targeted treatment for periodontitis. Prostaglandins increase mineralization of murine cementoblastic OCCM cells and alveolar bone formation, whereas the cytokine interleukin-1 (IL-1) inhibits alveolar bone formation. We hypothesized that differentially induced primary genes in OCCM cells may mediate anabolic and catabolic responses. Our objective was to identify primary genes differentially induced by the synthetic prostanoid fluprostenol and IL-1 in cementoblastic cells. METHODS: Confluent OCCM cells were pretreated with the protein synthesis inhibitor cycloheximide followed by fluprostenol or IL-1 for 1.5 hours. cDNA generated from each group was used for cDNA subtraction hybridization to identify differentially induced genes. Preferential gene induction was verified by Northern blot analysis. RESULTS: Thirteen fluprostenol- and seven IL-1-regulated genes were identified. Among the fluprostenol-induced genes was mitogen-activated protein (MAP) kinase phosphatase 1 (MKP1), a negative regulator of MAP kinase signaling. To verify the cDNA subtraction hybridization results, OCCM cells were treated with fluprostenol or prostaglandin F2 (PGF2), and MKP1 mRNA levels were determined. The 0.001 to 1 microM fluprostenol and 0.01 to 1 microM PGF2 significantly induced MKP1 mRNA levels, which peaked at 1 hour of treatment and returned to baseline at 2 hours. CONCLUSIONS: Fluprostenol enhanced, whereas IL-1 inhibited, OCCM mineralization. Using cDNA subtraction hybridization, we identified primary genes that correlate with the observed anabolic and catabolic responses. These findings further our understanding of cementoblast function and suggest that differentially induced genes may mediate cementum formation and resorption.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cemento Dental/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Interleucina-1/farmacología , Fosfoproteínas Fosfatasas/genética , Prostaglandinas F Sintéticas/farmacología , Proteínas Tirosina Fosfatasas/genética , Calcificación de Dientes/genética , Animales , Northern Blotting , Proteínas de Ciclo Celular/biosíntesis , Línea Celular Transformada , ADN Complementario/análisis , Cemento Dental/citología , Cemento Dental/metabolismo , Dinoprost/farmacología , Fosfatasa 1 de Especificidad Dual , Inducción Enzimática , Perfilación de la Expresión Génica , Proteínas Inmediatas-Precoces/biosíntesis , Ratones , Hibridación de Ácido Nucleico , Fosfoproteínas Fosfatasas/biosíntesis , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/biosíntesis , ARN Mensajero/análisis , Activación TranscripcionalRESUMEN
The transmembrane protein-tyrosine phosphatase (PTP) DEP-1 (density-enhanced phosphatase) is a candidate tumor suppressor in the colon epithelium. We have explored the function of DEP-1 in colon epithelial cells by inducible re-expression in a DEP-1-deficient human colon cancer cell line. Density-enhanced phosphatase-1 re-expression led to profound inhibition of cell proliferation and cell migration, and was associated with cytoskeletal rearrangements. These effects were dependent on the PTP activity of DEP-1 as they were not observed with cells expressing the catalytically inactive DEP-1 C1239S variant. shRNA-mediated suppression of DEP-1 in a colon epithelial cell line with high endogenous DEP-1 levels enhanced proliferation, further supporting the antiproliferative function of DEP-1. Nutrients, which are considered to be chemoprotective with respect to colon cancer development, including butyrate, green tea and apple polyphenols, had the capacity to elevate transcription of endogenous DEP-1 mRNA and expression of DEP-1 protein. Upregulation of DEP-1 expression, and in turn inhibition of cell growth and migration may present a previously unrecognized mechanism of chemoprevention by nutrients.
Asunto(s)
Adenocarcinoma/patología , Adenoma/patología , Anticarcinógenos/farmacología , Colon/citología , Neoplasias del Colon/patología , Células Epiteliales/efectos de los fármacos , Proteínas de Neoplasias/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Adenocarcinoma/enzimología , Adenoma/enzimología , Butiratos/farmacología , División Celular/efectos de los fármacos , Línea Celular Tumoral/citología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Movimiento Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Colon/enzimología , Neoplasias del Colon/enzimología , Regulación hacia Abajo , Inducción Enzimática/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/enzimología , Flavonoides/farmacología , Humanos , Lisofosfolípidos/farmacología , Malus/química , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fenoles/farmacología , Extractos Vegetales/farmacología , Polifenoles , Proteína Fosfatasa 1 , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , ARN Interferente Pequeño/farmacología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Té/química , Transcripción Genética/efectos de los fármacos , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The genomic sequence of the bracovirus associated with the wasp Toxoneuron nigriceps (Hymenoptera, Braconidae) (TnBV), an endophagous parasitoid of the tobacco budworm larvae, Heliothis virescens (Lepidoptera, Noctuidae), contains a large gene family coding for protein tyrosine phosphatases (PTPs). Here we report the characterization of cDNAs for two of the viral PTPs isolated by screening a cDNA library from haemocytes of parasitized host larvae. The two encoded proteins show 70% amino acid identity and are expressed in the fat body of parasitized hosts. In addition, one was expressed in inactivated prothoracic glands (PTGs), 24 h after parasitoid oviposition. The rapid block of ecdysteroidogenesis does not appear to be due to inhibition of general protein synthesis, as indirectly indicated by the unaltered S6 kinase activity in the cytosolic extracts of basal PTGs from parasitized host larvae. Rather, TnBV PTP over-expression in inactivated host PTGs suggests that gland function may be affected by the disruption of the phosphorylation balance of key proteins regulating points upstream from the ribosomal S6 phosphorylation in the PTTH signaling cascade.
Asunto(s)
Lepidópteros/fisiología , Lepidópteros/parasitología , Polydnaviridae/enzimología , Proteínas Tirosina Fosfatasas/fisiología , Avispas/virología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Cuerpo Adiposo/fisiología , Datos de Secuencia Molecular , Polydnaviridae/genética , Biosíntesis de Proteínas/fisiología , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas S6 Ribosómicas/fisiología , Alineación de Secuencia , Análisis de Secuencia de ADN , Avispas/genéticaRESUMEN
Despite dramatic advances in adjuvant therapies, patients with malignant glioma face a bleak prognosis. Because many adjuvant therapies seek to induce glioma apoptosis, strategies that lower thresholds for the induction of apoptosis may improve patient outcomes. Therefore, elucidation of the biological mechanisms that underlie resistance to current therapies is needed to develop new therapeutic strategies. Here we proposed a novel mechanism of proapoptotic effect induced by a pharmacological peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist, troglitazone, that facilitates caspase signaling in human glioma cells. Troglitazone activates protein-tyrosine phosphatase (PTP)-1B, which subsequently reduces phosphotyrosine 705 STAT3 (pY705-STAT3) via a PPARgamma-independent pathway. Reduction of pY705-STAT3 in glioma cells caused down-regulation of FLIP (FADD-like IL-1beta-converting enzyme-inhibitory protein) and Bcl-2. Furthermore, troglitazone induced Ser-392 phosphorylation of p53 via a PPARgamma-dependent pathway and up-regulation of Bax in a p53 wild-type glioma. When given with tumor necrosis factor-related apoptosis-inducing ligand or caspase-dependent chemotherapeutic agents, such as etoposide and paclitaxel, troglitazone exhibited a synergistic effect by facilitating caspase-8/9 activities. A PPARgamma antagonist, GW9662, did not block this effect, although a PTP inhibitor abrogated it. Knockdown of STAT3 by STAT3-small interfering RNA negated the inhibitory effect of PTP inhibitor on troglitazone, indicating that troglitazone uses a STAT3 inactivation mechanism that makes caspase-8/9 activities susceptible to cytotoxic agents in glioma cells and that PTP1B plays a critical role in the down-regulation of activated STAT3, as well as FLIP and Bcl-2. When taken with caspase-dependent anti-neoplastic agents, troglitazone may be a promising drug for use against malignant gliomas because it facilitates the caspase cascade, thereby lowering thresholds for the apoptosis induction of glioma cells.
Asunto(s)
Antineoplásicos/farmacología , Caspasas/metabolismo , Cromanos/farmacología , Glioma/enzimología , PPAR gamma/agonistas , Proteínas Tirosina Fosfatasas/metabolismo , Tiazolidinedionas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/fisiología , Caspasa 8 , Caspasa 9 , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Glioma/tratamiento farmacológico , Glioma/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , Factor de Transcripción STAT3/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF , Troglitazona , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.
Asunto(s)
Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/química , Calmodulina/metabolismo , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Compuestos de Anilina/química , Arabidopsis , Sitios de Unión , Unión Competitiva , Western Blotting , Calcio/química , Calcio/metabolismo , Calmodulina/química , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Fosfatasas de Especificidad Dual , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Glutatión Transferasa/metabolismo , Cinética , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Compuestos Organofosforados/química , Péptidos/química , Hidrolasas Diéster Fosfóricas/metabolismo , Fosforilación , Fosfotirosina/química , Unión Proteica , Proteína Fosfatasa 1 , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , Alelos , Animales , Arterias/metabolismo , Northern Blotting , Southern Blotting , Encéfalo/metabolismo , Capilares/metabolismo , Adhesión Celular , Comunicación Celular , ADN Complementario/metabolismo , Endotelio Vascular/metabolismo , Femenino , Genes Reporteros , Heterocigoto , Operón Lac , Hígado/metabolismo , Ratones , Ratones Transgénicos , Modelos Genéticos , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Bazo/metabolismo , Factores de Tiempo , Distribución Tisular , beta-Galactosidasa/metabolismoRESUMEN
The inhibitory forms of killer cell Ig-like receptors (KIR) are MHC class I-binding receptors that are expressed by human NK cells and prevent their attack of normal cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatase, Src homology region 2-containing protein tyrosine phosphatase (SHP)-1, to phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the functional significance of parallel recruitment of a SHP-1-related phosphatase, SHP-2, to KIR ITIMs has not been addressed. In the present study, our results with mutant forms of a classical KIR, KIR3DL1, show a direct correlation between SHP-2 recruitment and functional inhibition of target cell conjugation and cytotoxicity. In addition, KIR3DL1 inhibition of target cell cytotoxicity is blocked by overexpression of a dominant-negative form of SHP-2. Finally, KIR3DL1 fused directly with the catalytic domain of SHP-2 inhibits both target cell conjugation and cytotoxicity responses. These results strongly indicate that SHP-2 catalytic activity plays a direct role in inhibitory KIR functions, and SHP-2 inhibits NK cell activation in concert with SHP-1.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Células Asesinas Naturales/enzimología , Células Asesinas Naturales/inmunología , Proteínas Tirosina Fosfatasas/fisiología , Receptores Inmunológicos/fisiología , Dominios Homologos src/inmunología , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/genética , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Secuencia de Aminoácidos , Animales , Dominio Catalítico/genética , Dominio Catalítico/inmunología , Línea Celular , Pruebas Inmunológicas de Citotoxicidad , Citotoxicidad Inmunológica/genética , Citotoxicidad Inmunológica/inmunología , Vectores Genéticos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células Asesinas Naturales/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteína Fosfatasa 2 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Receptores KIR , Receptores KIR3DL1 , Proteínas Tirosina Fosfatasas con Dominio SH2 , Eliminación de Secuencia , Células Tumorales Cultivadas , Tirosina/genética , Dominios Homologos src/genéticaRESUMEN
We have isolated a cDNA homologous to known dual-specificity phosphatases from a mouse macrophage cDNA library and termed it MKP-M (for mitogen-activated protein kinase phosphatase isolated from macrophages). Three other presumed splice variant isoforms have also been identified for MKP-M. The longest and most abundant mRNA contains an open reading frame corresponding to 677 amino acids and produces an 80-kDa protein. The deduced amino acid sequence of MKP-M is most similar to those of hVH-5 (or mouse M3/6) and VHP1, a Caenorhabditis elegans tyrosine phosphatase. It includes an N-terminal rhodanase homology domain, the extended active-site sequence motif (V/L)X(V/I)HCXAG(I/V)SRSXT(I/V)XXAY(L/I)M (where X is any amino acid), and a C-terminal PEST sequence. Northern blot analysis revealed a dominant MKP-M mRNA species of approximately 5.5 kb detected ubiquitously among all tissues examined. MKP-M was constitutively expressed in mouse macrophage cell lines, and its expression levels were rapidly increased by lipopolysaccharide (LPS) stimulation but not by tumor necrosis factor alpha (TNF-alpha), gamma interferon, interleukin-2 (IL-2), or IL-15 stimulation. Immunocytochemical analysis showed MKP-M to be present within cytosol. When expressed in COS7 cells, MKP-M blocks activation of mitogen-activated protein kinases with the selectivity c-Jun N-terminal kinase (JNK) >> p38 = extracellular signal-regulated kinase. Furthermore, expression of a catalytically inactive form of MKP-M in a mouse macrophage cell line increased the intensity and duration of JNK activation and TNF-alpha secretion after LPS stimulation, suggesting that MKP-M is at least partially responsible for the desensitization of LPS-mediated JNK activation and cytokine secretion in macrophages.
Asunto(s)
Lipopolisacáridos/metabolismo , Sistema de Señalización de MAP Quinasas , Macrófagos/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monoéster Fosfórico Hidrolasas/química , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Células COS , Catálisis , Línea Celular , ADN Complementario/metabolismo , Regulación hacia Abajo , Fosfatasas de Especificidad Dual , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Regulación Enzimológica de la Expresión Génica , Biblioteca de Genes , Genes Dominantes , Humanos , Immunoblotting , Inmunohistoquímica , Interferón gamma/farmacología , Interleucina-15/farmacología , Interleucina-2/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , Modelos Genéticos , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/metabolismo , Plásmidos/metabolismo , Pruebas de Precipitina , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Distribución Tisular , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Tirosina/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.
Asunto(s)
Proteína Quinasa C/metabolismo , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/metabolismo , Actinas/metabolismo , Secuencias de Aminoácidos , Animales , Basófilos/química , Secuencia Conservada , Cisteína/química , Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Biblioteca de Genes , Células HeLa , Humanos , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteína Tirosina Fosfatasa no Receptora Tipo 13 , Proteínas Tirosina Fosfatasas/biosíntesis , Ratas , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Activation of mitogen-activated protein kinases (MAPKs), their upstream activators MAPK kinases (MAPKKs or MEKs) and induction of MKP-1 (CL100/3CH134) and MKP-3 (Pyst1/rVH6) dual-specificity MAPK phosphatases (MKPs) were studied in the mouse embryonic stem cell line P19 during the 7 day induction of neuronal differentiation triggered by aggregation and retinoic acid. ERK (extracellular signal-regulated kinase), but not JNK (c-Jun N-terminal kinase), was found activated with biphasic kinetics: a first transient phase on days 1 and 2, followed by a second activation that was sustained until the appearance of a neuronal phenotype. MEK activation appeared coincident with ERK activation. Cytosolic MKP-3 was induced in parallel to ERK activation, the induction being dependent on ERK activation, as was shown using the MEK-1 inhibitor PD98059. In contrast, nuclear MKP-1 was transiently elevated at 48 h, coincident with ERK inactivation and independently of ERK activity. As shown by cell fractionation, activated ERK is translocated to the nucleus. The complementary induction of ERK-specific phosphatases MKP-1 and MKP-3 permits precise and independent control of cytoplasmic and nuclear ERK activity, most probably required to properly induce a complex cellular programme of differentiation.
Asunto(s)
Proteínas de Ciclo Celular , Diferenciación Celular , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfoproteínas Fosfatasas , Proteínas Tirosina Fosfatasas/biosíntesis , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Proteínas Sanguíneas/farmacología , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Citoplasma/efectos de los fármacos , Citoplasma/enzimología , Fosfatasa 1 de Especificidad Dual , Fosfatasa 6 de Especificidad Dual , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 1 , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Proteína Fosfatasa 1 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Especificidad por Sustrato , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
The biological activity of p53 in IW32 erythroleukemia cells was investigated. IW32 cells had no detectable levels of p53 mRNA and protein expression. By transfecting a temperature-sensitive mutant p53 cDNA, tsp53val135, into the cells, we have established several clones stably expressing the mutant p53 allele. At permissive temperature, these p53 transfectants were arrested in G1 phase and underwent apoptosis. Moreover, differentiation along the erythroid pathway was observed as evidenced by increased benzidine staining and mRNA expression of beta-globin and the erythroid-specific delta-aminolevulinic acid synthase (ALAS-E). Treatment of cells with protein tyrosine phosphatase inhibitor vanadate blocked the p53-induced differentiation, but not that of cell death or growth arrest. Increased protein tyrosine phosphatase activity as well as mRNA levels of PTPbeta2 and PTPepsilon could be observed by wildtype p53 overexpression. These results indicate that p53 induced multiple phenotypic consequences through separate signal pathways in IW32 erythroleukemia cells, and protein tyrosine phosphatase is required for the induced differentiation.
Asunto(s)
Genes p53 , Leucemia Eritroblástica Aguda/patología , Proteínas de Neoplasias/fisiología , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Fosfatasas/fisiología , Proteína p53 Supresora de Tumor/fisiología , 5-Aminolevulinato Sintetasa/biosíntesis , 5-Aminolevulinato Sintetasa/genética , Animales , Apoptosis , Diferenciación Celular , ADN Complementario/genética , Inducción Enzimática , Fase G1 , Regulación Leucémica de la Expresión Génica , Globinas/biosíntesis , Globinas/genética , Leucemia Eritroblástica Aguda/enzimología , Leucemia Eritroblástica Aguda/genética , Ratones , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fenotipo , Fosforilación , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Proteínas Recombinantes de Fusión/fisiología , Transfección , Células Tumorales CultivadasRESUMEN
Expression of mRNA protein tyrosine phosphatases (PTPs) was surveyed in an esophageal cancer cell line by RT-PCR using degenerate primers. The mRNAs for eight kinds of PTPs were expressed in the cell line. We examined mRNA expression of these PTPs in 12 cases of esophageal cancer by Northern analysis. Significant signals were obtained for three kinds of PTPs, PTP1B, PTPH1, and PTPD1. The magnitude of expression of each PTP was measured as the ratio of the signal intensity of each PTP to that of a control gene (NADPH), and the ratio was then compared to normal mucosa around the cancer lesion. Among the three kinds of PTPs, the expression of PTP1B mRNA was significantly depressed in cancer lesions compared with that in the surrounding normal mucosa. In contrast, the expression of PTPH1 mRNA was significantly increased in cancer lesions compared with that in normal mucosa. PTPD1 did not show any significant trend in comparisons of cancer and surrounding normal mucosa. The results suggest that PTP1B and PTPH1 are engaged in opposing signaling pathways, the tumor-suppressive and tumor-promoting pathways, respectively, in esophageal carcinogenesis.
Asunto(s)
Carcinoma de Células Escamosas/enzimología , Neoplasias Esofágicas/enzimología , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Proteínas Tirosina Fosfatasas/biosíntesis , Adulto , Anciano , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Diferenciación Celular , ADN Complementario/genética , Inducción Enzimática , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Femenino , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Membrana Mucosa/enzimología , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 3 , Proteínas Tirosina Fosfatasas/deficiencia , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Tirosina Fosfatasas no Receptoras , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Células Tumorales CultivadasRESUMEN
The cDNA sequence and expression of a novel rat protein tyrosine phosphatase (PTP) gene is reported. The predicted amino acid sequence is similar to rat PRL-1, but is more closely related to human PTP4A, another member of the recently identified fourth group of PTPs. Therefore, multiple PTPs of this group are expressed in mammalian species. The novel rat PTP gene is expressed in the anterior pituitary gland in a sexually dimorphic pattern which is indicative of a specialized role in endocrine function.
Asunto(s)
Proteínas Tirosina Fosfatasas/genética , Ratas/genética , Glándulas Suprarrenales/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Corteza Cerebral/enzimología , Clonación Molecular , ADN Complementario , Humanos , Riñón/enzimología , Hígado/enzimología , Masculino , Datos de Secuencia Molecular , Miocardio/enzimología , Especificidad de Órganos , Adenohipófisis/enzimología , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The spectrum of protein tyrosine phosphatases (PTPs) expressed in bone marrow-derived murine macrophages (BMMs) was examined using reverse transcriptase-polymerase chain reaction. Ten different PTP cDNAs were isolated and in this study we focus on mDEP-1, a type III receptor PTP. Three mDEP-1 transcripts were expressed in primary macrophages and macrophage cell lines and were induced during macrophage differentiation of M1 myeloid leukemia cells. A variant mRNA was identified that encodes an alternate carboxyl-terminus and 3' UTR. The expression of mDEP-1 was down-regulated by CSF-1 (macrophage colony-stimulating factor) and up-regulated by bacterial lipopolysaccharide, an important physiological regulator of macrophage function that opposes CSF-1 action. Whole mount in situ hybridization, and immunolocalization of the protein, confirmed that mDEP-1 is expressed by a subset of embryonic macrophages in the liver and mesenchyme. mDEP-1 was also detected in the eye and peripheral nervous system of the developing embryo. Attempts to express mDEP-1 constitutively in the macrophage cell line RAW264 were unsuccessful, with results suggesting that the gene product inhibits cell proliferation.
Asunto(s)
Células de la Médula Ósea/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Factor Estimulante de Colonias de Macrófagos/fisiología , Macrófagos/metabolismo , Proteínas Tirosina Fosfatasas/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Diferenciación Celular , División Celular , Linaje de la Célula , Clonación Molecular , ADN Complementario/genética , Ojo/embriología , Ojo/metabolismo , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Técnica del Anticuerpo Fluorescente Indirecta , Hibridación in Situ , Macrófagos/clasificación , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Nervios Periféricos/embriología , Nervios Periféricos/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a ReceptoresRESUMEN
Interleukin-1 (IL-1) and Tumor Necrosis Factor-a (TNFalpha) are potent mediators of inflammatory reactions in the brain. Although much is known about the effects of IL-1 on expression of secretory proteins, few studies have addressed the question of a selective, IL-1-dependent expression of genes involved in neuromodulatory effects of inflammation. Protein-tyrosine-phosphatases (PTP's) have been shown to regulate signal transduction and adhesion processes in the developing nervous system. They are candidates for inflammation-induced neuromodulation. Therefore, we investigated if IL-1 regulates expression of PTP's. We applied a DNA-fingerprinting method based on the PCR-amplification of conserved domains of gene families and observed IL-1-dependent induction of two PTP's, cytoplasmic PTPvarepsilon and receptor-PTPgamma, RPTPgamma, in human U373-MG astrocytoma cells. Using Northern blot analysis, we confirmed this result and also show that in addition to IL-1, TNFalpha but not IL-6 induces the transcription of cytoplasmic PTPvarepsilon and RPTPgamma in human astrocytoma cells. Given the important role for PTP's in neuromodulatory aspects such as axonal guidance and neurite outgrowth, cytokine-induced induction of PTP's may play an important pathenogenic role in the development of chronic inflammatory diseases in the brain.
Asunto(s)
Astrocitoma/enzimología , Citocinas/farmacología , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Astrocitoma/genética , Secuencia de Bases , Northern Blotting , ADN Complementario/análisis , Electroforesis en Gel de Poliacrilamida , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1/farmacología , Interleucina-6/farmacología , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/análisis , ARN Mensajero/efectos de los fármacos , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores , Receptores de Superficie Celular , Análisis de Secuencia de ADN , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The mesangial cell provides structural support to the kidney glomerulus. A polymerase chain reaction-based cDNA display approach identified a novel protein-tyrosine phosphatase, rPTP-GMC1, whose transcript expression is transiently and dramatically up-regulated during the period of mesangial cell migration and proliferation that follows mesangial cell injury in the anti-Thy 1 model of mesangial proliferative glomerulonephritis in the rat. In situ hybridization analysis confirmed that rPTP-GMC1 mRNA is up-regulated specifically by mesangial cells responding to the injury and is not detectable in other cells in the kidney or in many normal tissues. In cell culture, rPTP-GMC1 is expressed by mesangial cells but not by glomerular endothelial or epithelial cells (podocytes). The longest transcript (7.5 kilobases) encodes a receptor-like protein-tyrosine phosphatase consisting of a single catalytic domain, a transmembrane segment, and 18 fibronectin type III-like repeats in the extracellular segment. A splice variant predicts a truncated molecule missing the catalytic domain. rPTP-GMC1 maps to human chromosome 12q15 and to the distal end of mouse chromosome 10. The predicted structure of rPTP-GMC1 and its pattern of expression in vivo and in culture suggest that it plays a role in regulating the adhesion and migration of mesangial cells in response to injury.