RESUMEN
Phosphorylation of proteins on tyrosine residues is regulated by the activities of protein tyrosine kinases and protein tyrosine phosphatases. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) essentially and transiently expressed during development of the central and peripheral nervous systems. ALK has been identified as a major neuroblastoma predisposition gene and activating mutations have been identified in a subset of sporadic neuroblastoma tumors. We previously established that the mutated receptors were essentially retained in the endoplasmic reticulum/Golgi compartments due to their constitutive activity. Intriguingly we demonstrated a stronger phosphorylation for the minor pool of receptor addressed to the plasma membrane. We decided to investigate the potential involvement of tyrosine phosphatase in dephosphorylation of this intracellular pool. In this study we first showed that general inhibition of tyrosine phosphatases resulted in a dramatic increase of the tyrosine phosphorylation of the wild type but also of the mutated receptors. This increase not only required the intrinsic kinase activity of the ALK receptor but also involved the Src tyrosine kinase family. Second we provided strong evidences that the endoplasmic reticulum associated phosphatase PTP1B is key player in the control of ALK phosphorylation. Our data shed a new light on the biological significance of the basal phosphorylation levels of both wild type and mutated ALK receptors and could be essential to further understand their roles in malignancies.
Asunto(s)
Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Quinasa de Linfoma Anaplásico , Animales , Dimerización , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Células 3T3 NIH , Fosforilación/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Proteínas Tirosina Quinasas Receptoras/genética , Transfección , Tirosina/metabolismo , Vanadatos/farmacología , Familia-src Quinasas/metabolismoRESUMEN
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK), which is transiently expressed during development of the central and peripheral nervous system. ALK has been recently identified as a major neuroblastoma predisposition gene and activating mutations have also been identified in a subset of sporadic neuroblastoma tumors. Two hot spots of ALK mutations have been observed at positions F1174 and R1275. Here, we studied stably transfected cell lines expressing wild-type or F1174L- or R1275Q-mutated ALK in parallel with a neuroblastoma cell line (CLB-GE) in which the allele mutated at position F1174 is amplified. We observed that the mutated ALK variants were essentially intracellular and were largely retained in the reticulum/Golgi compartments. This localization was corroborated by a defect of N-linked glycosylation. Although the mutated receptors exhibited a constitutive activation, the minor pool of receptor addressed to the plasma membrane was much more tyrosine phosphorylated than the intracellular pool. The use of antagonist monoclonal antibodies suggested that the constitutive activity of the mutated receptors did not require the dimerization of the receptor, whereas adequate dimerization triggered by agonist monoclonal antibodies increased this activity. Finally, kinase inactivation of the mutated receptors restored maturation and cell-surface localization. Our results show that constitutive activation of ALK results in its impaired maturation and intracellular retention. Furthermore, they provide a rationale for the potential use of kinase inhibitors and antibodies in ALK-dependent tumors.
Asunto(s)
Arginina , Mutación , Fenilalanina , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Quinasa de Linfoma Anaplásico , Animales , Línea Celular Tumoral , Membrana Celular/enzimología , Retículo Endoplásmico/enzimología , Activación Enzimática , Glicosilación , Aparato de Golgi/enzimología , Humanos , Ratones , Peso Molecular , Células 3T3 NIH , Pliegue de Proteína , Transporte de Proteínas/genética , Proteínas Tirosina Quinasas Receptoras/genéticaRESUMEN
Recent studies demonstrated that the chemokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 and its receptor, CCR2, play important roles in various brain diseases. In this study, using quantitative autoradiography, we studied the pharmacological properties of [125l]MCP-1/CCL2 binding in rat brain and we clearly showed the distribution of CCR2 receptors in cerebral cortex, nucleus accumbens, striatum, amygdala, thalamus, hypothalamus, hippocampus, substantia nigra, mammillary bodies and raphe nuclei. Moreover, using double fluorescent immunohistochemistry, we showed that CCR2 receptors were constitutively expressed on neurons and astrocytes. Using RT-PCR methods, we demonstrated that CCR2 mRNA is present in various brain areas described above. Four hours after an acute intraperitoneal lipopolysaccharide injection, we showed that MCP-1/CCL2 binding was up-regulated in several brain structures; this effect took place on both CCR2B labelled neurons and astrocytes and to a lesser extent on activated microglia. To explore neurobiological function of CCR2, actimetric study was carried out. After intracerebroventricular injections of MCP-1/CCL2, we showed that motor activity was markedly decreased. Our results provide the first evidence for constitutive CCR2 receptor expression with precise neuroanatomical and cellular localizations in the brain, and its regulation during an inflammatory process, suggesting that MCP-1/CCL2 and CCR2 play important physiological and pathophysiological role(s) in the CNS.
Asunto(s)
Encéfalo/metabolismo , Receptores de Quimiocina/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Autorradiografía , Sitios de Unión , Unión Competitiva/efectos de los fármacos , Encéfalo/anatomía & histología , Encéfalo/efectos de los fármacos , Quimiocina CCL2/administración & dosificación , Quimiocina CCL2/metabolismo , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Interleucina-1/administración & dosificación , Lipopolisacáridos/farmacología , Masculino , Microglía/citología , Microglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores CCR2 , Receptores de Quimiocina/análisis , Receptores de Quimiocina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder among Caucasians and is caused by abnormalities in the cystic fibrosis transmembrane conductance regulator gene (CFTR). CFTR gene encodes a chloride channel that regulates secretion in many exocrine tissues especially pancreatic and pulmonary tissues. The clinical presentation of cystic fibrosis is highly variable with isolated CAVD (congenital absence of vas deferens) and/or typical pancreatic and pulmonary manifestations. Over 500 mutations in the CFTR gene have been described and vary among different geographic locations. The severity of clinical manifestations and specially the pulmonary disease is poorly correlated with genotype. It is interesting to collect clinical and genetical data by analysing a larger cohort of CF patients. These results are likely to improve our understanding of the physiopathology of CF and the genetic counselling; particular biochemical defect could lead to more specific treatments in the future. From our 110 patients selected in Champagne-Ardenne country, we analysed the entire coding sequence of CFTR gene and detected 95% of CF mutations and in fact, 89.5% if we include the CAVD patients; 59.4% of CF mutations were detected for these patients. Three new mutations have been here reported. We found numerous CF mutations with a large distribution throughout the gene. Nevertheless, three exons are mainly involved: 10, 11 and 21. Relationships between the genotype and phenotype are difficult to assess.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/epidemiología , Fibrosis Quística/genética , Adolescente , Adulto , Niño , Preescolar , Fibrosis Quística/diagnóstico , Francia/epidemiología , Genotipo , Humanos , Lactante , Mutación , FenotipoRESUMEN
The authors report here the use of the PhastSystem (Pharmacia) to perform the single strand conformation polymorphism analysis of polymerase chain reaction products of the exon 11 of the CFTR gene. It provides a rapid (2 hours) safe and reliable technique for the development of carrier testing for individuals or couples with a family history of cystic fibrosis.
Asunto(s)
Fibrosis Quística/genética , ADN de Cadena Simple/genética , Exones/genética , Genes/genética , Conformación de Ácido Nucleico , ADN de Cadena Simple/análisis , Electroforesis en Gel de Poliacrilamida , Humanos , Reacción en Cadena de la Polimerasa/métodosRESUMEN
A novel technique using a two-step polymerase chain reaction (PCR) with specific primers detecting human papillomavirus (HPV) DNA of types 6/11, 16, and 18 and a final nonisotopic colorimetric detection has been developed. Sixty formalin-fixed and paraffin-embedded sections were treated with this methodology and the results compared with those obtained with in situ hybridization (ISH). Twenty cases displaying HPV DNA with ISH were positive with PCR. Seven (35%) of 20 cases negative for ISH but evocative of HPV infection with classic histology displayed HPV DNA with the two-step PCR. Only one case (5%) of 20 normal tissues and/or inflammatory lesions not evocative of HPV infection and negative upon ISH showed HPV DNA. This original technique allows rapid, highly sensitive, and specific detection of HPV DNA and is suitable for most laboratories.
Asunto(s)
Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Infecciones Tumorales por Virus/diagnóstico , Secuencia de Bases , Carcinoma in Situ/microbiología , Condiloma Acuminado/microbiología , Cartilla de ADN/genética , ADN Viral/genética , Estudios de Evaluación como Asunto , Femenino , Humanos , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Infecciones por Papillomavirus/microbiología , Enfermedades del Pene/microbiología , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Sensibilidad y Especificidad , Infecciones Tumorales por Virus/microbiología , Enfermedades del Cuello del Útero/microbiología , Neoplasias del Cuello Uterino/microbiologíaRESUMEN
Deletion of the amino acid residue Phe 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein represents the most common mutation identified in cystic fibrosis (CF) patients. A monoclonal and a polyclonal antibody directed against different regions of CFTR were used to localize the CFTR protein in normal and CF airway epithelium derived from polyps of non-CF and CF subjects homozygous for the delta Phe 508 CFTR mutation. To identify the cellular and subcellular localization of CFTR, immunofluorescent light microscopy, confocal scanning microscopy, and immunogold transmission electron microscopy were performed on cryofixed tissue. A markedly different subcellular distribution was identified between normal and CF airway epithelial cells. In normal epithelium, labeling was restricted to the surface apical compartment of the ciliated cells. In contrast, in the epithelium from homozygous delta Phe 508 CF patients, CFTR markedly accumulated in the cytosol of all the epithelial cells. These findings are consistent with the concept that the CFTR delta Phe 508 mutation modifies the intracellular maturation and trafficking of the protein, leading to an altered subcellular distribution of the delta Phe 508 mutant CFTR.
Asunto(s)
Fibrosis Quística/metabolismo , Proteínas de la Membrana/metabolismo , Tráquea/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Epitelio/metabolismo , Epitelio/ultraestructura , Homocigoto , Humanos , Microscopía Electrónica , Microscopía Fluorescente , Proteínas Recombinantes/metabolismo , Tráquea/ultraestructuraRESUMEN
The present study reports results obtained with DNA ploidy measurement by Feulgen cytophotometry and in situ hybridization (ISH) on the same sections of 36 cervical lesions containing human papillomavirus (HPV) DNA. Fifteen of 16 low grade lesions [11 flat condylomas and five condylomatous with intraepithelial neoplasias (CIN)-1, 1 with condylomatous features] were diploid. "Oncogenic" HPV (types 16 and 33) were detected in three of these cases, while the aneuploid case expressed HPV type 11. By contrast, in the 12 CIN-2 and eight CIN-3 with condylomas, there was a good correlation between presence of an aneuploid DNA pattern and detection of "oncogenic" HPVs. Demonstration of an aneuploid profile and "oncogenic" HPV as found in all our CIN-3, may indicative of a poor prognosis in low grade lesions. Hence, the combination of the two techniques (ISH and Feulgen cytophotometry) may provide significant prognostic information for condylomas and CINs.
Asunto(s)
Condiloma Acuminado/genética , ADN/análisis , Enfermedades del Cuello del Útero/genética , Aneuploidia , Carcinoma in Situ/química , Carcinoma in Situ/genética , Carcinoma in Situ/microbiología , Condiloma Acuminado/microbiología , ADN/genética , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Diploidia , Femenino , Humanos , Hibridación in Situ , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Pronóstico , Enfermedades del Cuello del Útero/microbiología , Neoplasias del Cuello Uterino/química , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/microbiologíaRESUMEN
Human papillomavirus DNA was detected in 40 condylomatous lesions of various sites (vulva, cervix, larynx, penis and anus) by in situ hybridization using 35S-labelled probes and four non-radioactive probes to compare the various sensitivities of these techniques on the same material (formalin-fixed and paraffin-embedded sections). Radioactive probes yielded 28 positive results out of 40 (70%). Sulphonated probes (HybriCyte kit) also gave 28 positive results with a fine pattern of hybridization grains and equal sensitivity to 35S-labelled probes. Biotinylated and digoxigenin-labelled probes gave analogous results (25 positive reactions with the PathoGene kit, 26 with the Viratype kit, and 25 with digoxigenin-labelled probes) but are slightly less sensitive than radiolabelled and sulphonated probes especially when the signal is weak.
Asunto(s)
ADN Viral/análisis , Papillomaviridae/aislamiento & purificación , Adolescente , Anciano , Anciano de 80 o más Años , Biotina , Niño , Preescolar , Condiloma Acuminado/microbiología , Sondas de ADN , Digoxigenina , Femenino , Humanos , Lactante , Masculino , Métodos , Persona de Mediana Edad , Papillomaviridae/genética , Radioisótopos de AzufreRESUMEN
We previously identified a set of soluble proteins whose phosphorylation could be originally related to the multihormonal regulations of anterior pituitary cells. Among these proteins, stathmin (proteins 7 and 8) was found to be ubiquitous and mostly abundant in neurons. Interestingly, stathmin and some other phosphoproteins of the same set could be identified also in PC12 cells in culture. Their phosphorylation was stimulated in these cells by nerve growth factor (NGF) in a way associated with its short term actions, probably corresponding to the early steps of its neuronal differentiating activity. In addition, the same proteins had their phosphorylation stimulated in the presence of fibroblast growth factor, known to stimulate PC12 cell differentiation in a way similar to NGF. A pharmacological analysis allowed us to distinguish three characteristic subsets of phosphoproteins, respectively, affected by cAMP-dependent agents, by cAMP-independent ones, or by both types of agents. Moreover, phosphorylation of stathmin and some other proteins was additive in the presence of NGF and of the cAMP-promoting agent forskolin. Altogether, the present results unravel some intracellular mechanisms related to the regulation of PC12 cells by extracellular effectors. They extend to the regulation of cell differentiation in our recent model for stathmin (Sobel, A., Boutterin, M-C., Beretta, L., Chneiweiss, H., Doye, V., and Peyro-Saint-Paul, H. (1989) J. Biol. Chem. 264, 3765-3772) as an ubiquitous intracellular relay possibly integrating the actions of diverse second messenger pathways involved in cell regulations.
Asunto(s)
Proteínas de Microtúbulos , Proteínas de Neoplasias/metabolismo , Factores de Crecimiento Nervioso/farmacología , Fosfoproteínas/metabolismo , Células Tumorales Cultivadas/metabolismo , Adenosina/farmacología , Neoplasias de las Glándulas Suprarrenales , Animales , Línea Celular , Colforsina/farmacología , Electroforesis en Gel Bidimensional , Feocromocitoma , Fosfoproteínas/aislamiento & purificación , Fosforilación , Ratas , Estatmina , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Stathmin is a 19-kDa phosphoprotein presumably involved in regulations of cell proliferation, differentiation, and functions as an intracellular relay for extracellular signals activating diverse second messenger pathways. Antisera prepared against the whole protein or against two peptides (residues 15-27 and 134-149) recognized the two isoforms (alpha and beta) of stathmin in their different phosphorylated states on immunoblots. Also, the possible existence of a family of stathmin-related proteins is suggested by the detection with some sera of proteins of 17, 21, and 60 kDa in brain. Stathmin and its diverse molecular forms were detected in all mouse tissues tested, in varying concentrations. Depending on the tissue, it is 2-100 times more abundant in the neonate than in the adult. It is most abundant in brain at both developmental stages, the protein levels being paralleled by the expression of the corresponding mRNA as detected with a specific cDNA probe. Antibodies directed against the rat protein also reacted with stathmin-like proteins in the brain of other mammals, birds, reptiles, amphibians, and some fish species, and the various isoforms could be recognized on immunoblots. In conclusion, our results suggest that stathmin is most likely involved in two distinct types of regulations: 1) "developmental" regulations, related to cell proliferation, differentiation, and maturation, and 2) "functional" regulations mostly at the adult stage, and typically in the nervous system. In addition, stathmin is also phylogenetically well conserved at least in vertebrates. Together, these observations support the proposed ubiquitous nature and general importance of stathmin in biological regulations.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Microtúbulos , Fosfoproteínas/genética , Filogenia , Envejecimiento , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Encéfalo/crecimiento & desarrollo , Sondas de ADN , Electroforesis en Gel Bidimensional , Expresión Génica , Humanos , Sueros Inmunes , Lagartos , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos , Fosfoproteínas/biosíntesis , Fosfoproteínas/aislamiento & purificación , ARN Mensajero/genética , Conejos , Ranidae , Ratas , Ratas Endogámicas , Estatmina , TorpedoRESUMEN
A technique of detection of human papillomaviruses by in situ hybridization in paraffin-embedded formalin-fixed sections of genito-anal lesions is described. The probes are labeled by incorporation of digoxigenin-labeled nucleotides. The hybrids are revealed by immunohistochemistry with an anti-digoxigenin antibody coupled with alkaline phosphatase. This technique is sensitive, cheap and may be used in routine in laboratories of pathology.
Asunto(s)
ADN Viral/análisis , Digoxigenina , Papillomaviridae/aislamiento & purificación , Sondas de ADN , Humanos , Inmunohistoquímica , Hibridación de Ácido Nucleico , Papillomaviridae/genéticaRESUMEN
Stathmin is a ubiquitous soluble protein (Mr approximately 19,000; pI approximately 6.2-5.5) whose phosphorylation is associated with the intracellular mechanisms involved in the regulations of cell differentiation and functions by extracellular effectors. It is present in various tissues and cell types and has several nonphosphorylated and increasingly phosphorylated forms, and it is particularly abundant in brain. Very high concentrations of stathmin were also detected in mouse embryo striatal neurons grown in primary culture, whereas stathmin was barely detectable in astrocytes from the same source. Stathmin appeared in neurons as a major substrate for protein phosphorylation and, in particular, for the cyclic AMP (cAMP)-dependent protein kinase, because its phosphorylation was stimulated by cAMP in cell-free preparations and in intact cells by forskolin, a potent activator of adenylate cyclase. During brain ontogenesis, stathmin was first detected at embryonic day 12; its concentration increased until birth and then decreased from postnatal day 10 to adulthood. In parallel, its molecular forms shifted from the least phosphorylated to the more phosphorylated ones. This result may reflect the evolution of the activity of stathmin during development and the subsequent maturation of the brain. In conclusion, our results substantiate the likely role of stathmin as an intracellular relay of extracellular regulations, as they point out its specific importance related to neuronal functions and brain differentiation.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/crecimiento & desarrollo , AMP Cíclico/farmacología , Proteínas de Microtúbulos , Neuronas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Células Cultivadas , Colforsina/farmacología , Cuerpo Estriado/citología , Cuerpo Estriado/embriología , Electroforesis en Gel Bidimensional , Ratones , Fosforilación , EstatminaRESUMEN
We previously identified a group of cytoplasmic phosphoproteins whose phosphorylation could be related to the multihormonal regulation of PRL in the homogeneous tumor-derived GH cell lines (set of proteins 1-11) and in heterogeneous normal anterior pituitary cells in culture (set of proteins 1-15). In normal cells, a mixture of hypothalamic hormones induced, like the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, stronger phosphorylation changes than TRH alone. Proteins of the set 1-15 are therefore likely to be present also in the nonmammotrophic anterior pituitary cell types, where their phosphorylation can be regulated by the hormones specific of the cell type considered. This interpretation is confirmed by the presence of the same proteins in cells of the corticotroph-like AtT-20/D16 cell line and the regulation of their phosphorylation by CRF. The same phosphoproteins were also detected in non dissociated anterior pituitary tissue from ovariectomized rats. Their phosphorylation was regulated by various hormones and other extracellular agents in a way similar to their regulation in anterior pituitary cells in culture. A 5-day estradiol implant pretreatment of the ovariectomized rats, which stimulates the anterior pituitary gland both directly and indirectly, resulted in a very high level of basal phosphorylation of proteins 1-15. Only very little further stimulation was achieved by the addition of exogenous hypothalamic hormones, indicating that the actual physiological regulatory pathways are the same as those unraveled in the various cell culture model systems. In conclusion, phosphorylation of proteins 1-15 1) can be related to the multihormonal regulation of the various anterior pituitary cell types in culture and 2) corresponds to intracellular molecular mechanisms actually involved in the physiological regulations of pituitary functions in the intact anterior pituitary gland.
Asunto(s)
Fosfoproteínas/biosíntesis , Adenohipófisis/metabolismo , Animales , Línea Celular , Células Cultivadas , Hormona Liberadora de Corticotropina/farmacología , Electroforesis en Gel Bidimensional , Estradiol/farmacología , Femenino , Ovariectomía , Fosfatos/metabolismo , Fosfoproteínas/aislamiento & purificación , Fosforilación , Adenohipófisis/citología , Adenohipófisis/efectos de los fármacos , Neoplasias Hipofisarias , Ratas , Ratas EndogámicasRESUMEN
Stathmin, a 19-kDa neuron-enriched soluble phosphoprotein, has been recently proposed as an ubiquitous intracellular relay for the diverse extracellular signals regulating cell proliferation, differentiation, and functions through various second messenger pathways (Sobel, A., Boutterin, M.C., Beretta, L., Chneiweiss, H., Doye, V., and peyro-Saint-Paul, H. (1989) J. Biol. Chem. 264, 3765-3772). Internal sequences of the protein from rat brain were determined after purification by two-dimensional polyacrylamide gel electrophoresis, electrotransfer onto Immobilon, and in situ proteolysis. Oligonucleotide mixtures based on these sequences were used to clone a cDNA for stathmin from a rat PC12 cell lambda gt 10 library. The deduced amino acid sequence reveals partial homologies with the coiled coil structural regions of several intracellular matrix phosphoproteins. Using this cDNA as a probe, we show that the expression of stathmin mRNA parallels that of the protein during brain ontogenesis, reaching a maximum at the neonatal stage. In vitro translation of the derived cRNA yielded all the known molecular forms of stathmin, namely its alpha and beta isoforms in their unphosphorylated and phosphorylated states. Thus, a single cDNA codes for both biologically relevant isoforms of the protein, indicating that they differ by co- or post-translational modifications.
Asunto(s)
ADN/genética , Proteínas de Microtúbulos , Proteínas del Tejido Nervioso/genética , Fosfoproteínas/genética , Neoplasias de las Glándulas Suprarrenales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Electroforesis en Gel Bidimensional , Datos de Secuencia Molecular , Peso Molecular , Feocromocitoma , Fosfoproteínas/aislamiento & purificación , Homología de Secuencia de Ácido Nucleico , EstatminaRESUMEN
We previously identified (Sobel, A., and Tashjian, A. H., Jr. (1983) J. Biol. Chem. 258, 10312-10324) a group of cytoplasmic proteins whose phosphorylation could be related to the regulation by extracellular effectors of cells as different as pituitary and muscle cells. Among these phosphoproteins, proteins "7" and "8" (Mr approximately 19,000, pI approximately 5.8-6.0), that we now designate P1 and P2, are very abundant in rat brain. Partial purification of these proteins was therefore achieved after 100 degrees C precipitation of a rat brain-soluble fraction and further fractionation of the supernatant by ion exchange chromatography. Several related non-phosphorylated (N1, N2) and phosphorylated (P3) proteins were also identified in the heat-resistant supernatant. Antisera raised against P2 extracted from nitrocellulose blots of semipreparative two-dimensional gels recognized all the proteins N1, N2, P1, P2, and P3, confirming that they belong to the same protein family, and suggesting that they are likely various forms of a single protein core. The same protein could be detected biochemically and immunologically at various concentrations in all the tissues or cell types from diverse mammalian and nonmammalian species tested. Together with our previous data relating its phosphorylation to the regulation of the proliferation, differentiation, and/or the functions of the cells considered, this observation leads us to suggest that it might be an ubiquitous regulatory phosphoprotein playing the role of an intracellular "relay" for extracellular signals, after their binding to specific membrane receptors and the generation of second messengers. We propose to name this protein stathmin, from the greek "stathmos" (relay).
Asunto(s)
Encéfalo/fisiología , Proteínas de Microtúbulos , Proteínas del Tejido Nervioso/fisiología , Fosfoproteínas/fisiología , Animales , Western Blotting , Citoplasma/análisis , Humanos , Punto Isoeléctrico , Peso Molecular , Proteínas del Tejido Nervioso/aislamiento & purificación , Fosfoproteínas/aislamiento & purificación , Hipófisis/fisiología , Ratas , Especificidad de la Especie , Estatmina , Distribución TisularRESUMEN
We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and [32P] phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins. The phosphoproteins described here are therefore likely to be part, in normal anterior pituitary cells and possibly in other cell types, of the intracellular machinery involved in the cascade of transducing events leading from the binding of extracellular signals to the regulation of their target biological functions.
Asunto(s)
Dopamina/farmacología , Fosfatos/metabolismo , Adenohipófisis/metabolismo , Neoplasias Hipofisarias/metabolismo , Hormona Liberadora de Tirotropina/farmacología , Péptido Intestinal Vasoactivo/farmacología , Animales , Línea Celular , Células Cultivadas , Femenino , Radioisótopos de Fósforo , Fosforilación , Adenohipófisis/efectos de los fármacos , Prolactina/metabolismo , Ratas , Ratas Endogámicas , Valores de Referencia , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The specialized functions of mammotropic cells from the anterior pituitary and of the pituitary tumor derived GH cell lines can be regulated by a large number of neurohormones and other substances. Although the biological responses to many of these regulatory signals are well documented, much less is known about the intracellular mechanisms involved in their action. Various "second messengers" are generated, which in turn regulate other intracellular activities like protein phosphorylation reactions. Thyrotropin-releasing hormone stimulates both prolactin synthesis and release by mammotrophs of the anterior pituitary, or by GH(4)C(1) cells in culture. Thyrotropin-releasing hormone simultaneously enhances the phosphorylation of a small number of cytoplasmic proteins in GH(4)C(1) cells, as revealed by two-dimensional polyacrylamide gel electrophoresis after (32)PO(4)(3?) labelling. On a pharmacological basis, two distinct subsets were identified among the seven phosphoproteins affected by thyrotropin-releasing hormone: set I, proteins 1-6, whose phosphorylation could be related to the regulation of prolactin synthesis; set II, proteins 7 and 8, whose phosphorylation was related to the modulation of prolactin release (Sobel A. and Tashjian A. H. Jr. (1983) J. Biol. Chem.258, 10312-10324). Phosphorylation of all these proteins but protein 1 was alkali-resistant, indicating that it might take place on tyrosine residues. Protein 1, which could be visualized by silver-staining and whose phosphorylation resulted in a shift of its position on two-dimensional gels, was present also in normal rat pituitary cells in primary culture. Moreover, thyrotropin-releasing hormone induced its phosphorylation as in GH(4)C(1) cells, thus indicating that this reaction is a normal regulatory process, not related to the tumoral origin of GH(4)C(1) cells. This observation allowed us to study some physiologically important regulatory mechanisms which were lost in GH cells as, for example, the inhibition of prolactin synthesis and release by dopamine: in normal anterior pituitary cells in culture, dopamine also prevented the thyrotropin-releasing hormone induced phosphorylation of protein 1. This result is in good agreement with those obtained with GH(4)C(1) cells, and extends them. The same technique allowed us to detect a protein with two-dimensional gel migration properties identical to those of protein 1 in certain tissues, like the adrenal medulla, but not in others, like the adrenal cortex. This tissue specificity might be related to the specific regulatory function of protein 1 in the cell.