RESUMEN

Bub1 is a conserved mitotic kinase involved in signaling of the spindle assembly checkpoint. Multiple phosphorylation sites on Bub1 have been characterized, yet it is challenging to understand the interplay between the multiple phosphorylation sites due to the limited availability of phosphospecific antibodies. In addition, phosphoregulation of Bub1 in Schizosaccharomyces pombe is poorly understood. Here we report the identification of a new Mph1/Mps1-mediated phosphorylation site, i.e., Ser532, of Bub1 in Schizosaccharomyces pombe. A phosphospecific antibody against phosphorylated Bub1-Ser532 was developed. Using the phosphospecific antibody, we demonstrated that phosphorylation of Bub1-Ser352 was mediated specifically by Mph1/Mps1 and took place during early mitosis. Moreover, live-cell microscopy showed that inhibition of the phosphorylation of Bub1 at Ser532 impaired the localization of Bub1, Mad1, and Mad2 to the kinetochore. In addition, inhibition of the phosphorylation of Bub1 at Ser532 caused anaphase B lagging chromosomes. Hence, our study constitutes a model in which Mph1/Mps1-mediated phosphorylation of fission yeast Bub1 promotes proper kinetochore localization of Bub1 and faithful chromosome segregation.

Asunto(s)

Segregación Cromosómica , Cinetocoros , Proteínas Serina-Treonina Quinasas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Transducción de Señal , Anafase , Anticuerpos Fosfo-Específicos/inmunología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Mitosis , Fosforilación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/inmunología , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/metabolismo

RESUMEN

The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of the two tyrosine residues (N-terminal Y47 and C-terminal Y58) in the ITAM sequence is crucial for the recruitment and activation of Syk, a cytoplasmic tyrosine kinase with central signaling roles in mast cells. Using a reconstitution system in which individual tyrosine-to-phenylalanine substituted γ chains were expressed in γ-chain-deficient mast cells, we previously reported differential dephosphorylation of these tyrosines. Herein, we developed monoclonal antibodies highly specific to the phosphorylated Y47 and Y58 residues, which enables monitoring their phosphorylation under more physiological conditions. Using these antibodies, preferential dephosphorylation of Y58 following FcεRI stimulation was confirmed. Furthermore, Y58 is potentially more susceptible to phosphorylation than is Y47. Consistent with this, an in vitro kinase assay using these phospho-specific antibodies demonstrated that the Src family kinase Lyn, which is primarily responsible for ITAM phosphorylation, phosphorylates Y58 more efficiently than Y47. These results indicate that Y58 is more susceptible to dephosphorylation and phosphorylation than is Y47. Because a phosphate group on Y58 is more important for Syk binding than is a phosphate group on Y47, the preferential phosphorylation and dephosphorylation of Y58 may contribute to the fine tuning of Syk activity by promoting rapid recruitment and reducing excessive activation.

Asunto(s)

Anticuerpos Monoclonales/metabolismo , Anticuerpos Fosfo-Específicos/metabolismo , Motivo de Activación del Inmunorreceptor Basado en Tirosina , Mastocitos/inmunología , Receptores de IgG/metabolismo , Quinasa Syk/metabolismo , Tirosina/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Fosfo-Específicos/inmunología , Células Cultivadas , Mastocitos/metabolismo , Ratones Endogámicos C57BL , Fosforilación , Receptores de IgG/química , Transducción de Señal , Tirosina/química

RESUMEN

Antibodies raised against defined phosphorylation sites of the microtubule-associated protein tau are widely used in scientific research and being applied in clinical assays. However, recent studies have revealed an alarming degree of non-specific binding found in these antibodies. In order to quantify and compare the specificity phospho-tau antibodies and other post-translational modification site-specific antibodies in general, a measure of specificity is urgently needed. Here, we report a robust flow cytometry assay using human embryonic kidney cells that enables the determination of a specificity parameter termed Φ, which measures the fraction of non-specific signal in antibody binding. We validate our assay using anti-tau antibodies with known specificity profiles, and apply it to measure the specificity of seven widely used phospho-tau antibodies (AT270, AT8, AT100, AT180, PHF-6, TG-3, and PHF-1) among others. We successfully determined the Φ values for all antibodies except AT100, which did not show detectable binding in our assay. Our results show that antibodies AT8, AT180, PHF-6, TG-3, and PHF-1 have Φ values near 1, which indicates no detectable non-specific binding. AT270 showed Φ value around 0.8, meaning that approximately 20% of the binding signal originates from non-specific binding. Further analyses using immunocytochemistry and western blotting confirmed the presence of non-specific binding of AT270 to non-tau proteins found in human embryonic kidney cells and the mouse hippocampus. We anticipate that the quantitative approach and parameter introduced here will be widely adopted as a standard for reporting the specificity for phospho-tau antibodies, and potentially for post-translational modification targeting antibodies in general. Cover Image for this issue: doi: 10.1111/jnc.14727.

Asunto(s)

Anticuerpos Fosfo-Específicos/inmunología , Especificidad de Anticuerpos/inmunología , Proteínas tau/inmunología , Animales , Citometría de Flujo/métodos , Colorantes Fluorescentes , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteínas Fluorescentes Verdes , Células HEK293 , Hipocampo/química , Humanos , Inmunohistoquímica , Riñón/química , Ratones , Fosforilación , Transfección

RESUMEN

Hypothalamic AMPK plays a key role in the control of energy homeostasis by regulating energy intake and energy expenditure, particularly modulating brown adipose tissue (BAT) thermogenesis. The function of AMPK can be assayed by analyzing its phosphorylated protein levels in tissues, since AMPK is activated when it is phosphorylated at Thr-172. Here, we describe a method to obtain hypothalamic (nuclei-specific) protein extracts and the suitable conditions to assay AMPK phosphorylation by Western blotting.

Asunto(s)

Proteínas Quinasas Activadas por AMP/análisis , Activación Enzimática/efectos de los fármacos , Pruebas de Enzimas/métodos , Hipotálamo/metabolismo , Isoenzimas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/inmunología , Proteínas Quinasas Activadas por AMP/metabolismo , Adenoviridae/genética , Animales , Anticuerpos Fosfo-Específicos/inmunología , Activación Enzimática/genética , Activadores de Enzimas/farmacología , Pruebas de Enzimas/instrumentación , Inhibidores Enzimáticos/farmacología , Vectores Genéticos/genética , Isoenzimas/genética , Isoenzimas/inmunología , Ratones , Fosforilación/efectos de los fármacos , Fosforilación/genética , Fosforilación/inmunología , Ratas , Técnicas Estereotáxicas/instrumentación , Treonina/inmunología , Treonina/metabolismo

RESUMEN

A complex composed of goat anti-rabbit secondary antibody conjugated to a polymer coated with horseradish peroxidase (HRP) molecules was used to develop rapid and highly sensitive immunostaining protocol for the detection of phosphorylated p27/Kip1 (T157) in human tissues. This polymer-HRP complex produced much better sensitivity detection compared to conventional biotin-streptavidin-HRP chemistry. Using polymer-HRP made it possible to reduce primary antibody concentration, eliminate some incubation steps such as avidin-biotin blocking and incubation with separate biotinylated secondary antibodies, and shorten the incubation time with primary antibody. Specificity of the detection was confirmed by eliminating labeling after treating tissues with lambda phosphatase to remove phosphate groups from p27/Kip1. Secondary antibodies conjugated to polymer-HRP is a reagent of choice in both research and diagnostic pathology allowing detecting low abundant and weakly expressed tissue targets.

Asunto(s)

Anticuerpos Fosfo-Específicos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Peroxidasa de Rábano Silvestre , Inmunoconjugados , Inmunohistoquímica/métodos , Fosfoproteínas/metabolismo , Anticuerpos Fosfo-Específicos/inmunología , Especificidad de Anticuerpos/inmunología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/inmunología , Peroxidasa de Rábano Silvestre/química , Humanos , Fosfoproteínas/inmunología , Fosforilación , Sensibilidad y Especificidad

RESUMEN

Phospho-specific primary antibodies are used in immunohistochemistry (IHC) to detect phosphorylated sequences in proteins, in some cases they may also cross-react with non- or de-phosphorylated sequences. To rule out nonspecific staining, and to determine that the staining pattern is specific it is necessary to employ a so-called absorption control: phospho-specific primary antibodies are first incubated with phospho-peptide immunogen to block antibody binding sites, and this mixture is applied to tissue sections. If the antibody pre-blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific. However, if the staining does occur, it indicates that the antibody is nonspecific. The drawback of doing absorption by mixing the peptide with the antibody is that in solution such peptide-antibody complexes can dissociate unblocking the antibody which becomes capable of binding to cell and tissue targets, producing unwanted staining. To overcome this problem, we have developed a simple absorption control technique allowing for efficient blocking of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of peptide-antibody complex from the incubation mixture eliminating the risk of un-blocking primary antibodies via their dissociation from the blocking peptide.

Asunto(s)

Técnica del Anticuerpo Fluorescente , Inmunohistoquímica/métodos , Técnicas de Inmunoadsorción , Fosfopéptidos/metabolismo , Animales , Anticuerpos Fosfo-Específicos/inmunología , Ratones , Microscopía Confocal , Células 3T3 NIH , Fosfopéptidos/inmunología

RESUMEN

Numerous bacterial pathogens employ specialized protein secretion machineries to directly inject anti-host proteins, termed effector proteins, into eukaryotic cells. Effector proteins carrying small phosphorylatable tags can be used to detect and quantify effector protein injection. Here, we describe the use of the ELK- and GSK-tags to detect the translocation of the Y. pestis YopE effector protein into RAW 264.7 macrophage-like cells using immunoblot analysis with phospho-specific antibodies.

Asunto(s)

Anticuerpos Fosfo-Específicos , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Epítopos , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Fosfo-Específicos/inmunología , Proteínas Bacterianas/química , Western Blotting , Línea Celular , Epítopos/química , Epítopos/inmunología , Orden Génico , Ratones , Fosforilación , Plásmidos/genética , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Yersinia pestis/inmunología , Yersinia pestis/metabolismo

RESUMEN

Increasing evidence has shown that inflammation is involved in pressure overload-induced cardiac remodeling. Monocyte chemoattractant protein-1 (MCP-1) plays a pivotal role in the inflammatory process. However, the mechanisms underlying the upregulation of MCP-1 expression remain poorly understood. In the present study, we examined the hypothesis that an increased production of reactive oxygen species (ROS) mediates the upregulation of MCP-1. In a pressure-overloaded rat heart model with abdominal aortic coarctation (AC), superoxide dismutase-inhibitable cytochrome C reduction assay showed that ROS generation in the myocardium increased significantly at 1 week by 61% (n=8, P<0.01), peaked at 2 weeks and maintained these high levels for 4 weeks. The elevation of ROS was paralleled by the increased expression of MCP-1 and left ventricular remodeling (cardiac hypertrophy, perivascular and interstitial fibrosis). The oral administration of the antioxidant, N-acetylcysteine (NAC, 0.2 g/kg/day), for 2 or 4 weeks, significantly attenuated ROS production by 69 and 68%, respectively (n=8, P<0.01), as well as left ventricular remodeling. NAC treatment for 2 weeks also significantly reduced the MCP-1 mRNA and protein levels by 52 and 60%, respectively (n=4-8, both P<0.01), but had no effect on blood pressure. In the rats with AC at 2 weeks, when MCP-1 expression and inflammation changes were overt, immunoblotting with phospho-specific antibodies revealed that extracellular regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK), but not p38 mitogen-activated protein kinase, were activated. NAC administration attenuated JNK activation, but had no effect on ERK. Our results suggest that increased ROS production may play an important role in the increased expression of MCP-1 in pressure overload-induced cardiac remodeling. JNK is likely involved in the signaling pathway.

Asunto(s)

Quimiocina CCL2/metabolismo , Corazón/fisiopatología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Miocardio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Administración Oral , Animales , Anticuerpos Fosfo-Específicos/inmunología , Coartación Aórtica/fisiopatología , Quimiocina CCL2/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Depuradores de Radicales Libres/farmacología , Regulación de la Expresión Génica , Masculino , Presión , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos

RESUMEN

Recommendations for the use of heparin for preventing miscarriage are recently rapidly changing based on evidenced based prospective studies. At present either heparin or low molecular weight heparin (LMWH) is recommended for the antiphospholipid syndrome (APS). However criteria for diagnosing APS have become much stricter. The exact timing of the heparin is still being evaluated since it is not clear if the main therapeutic effect is in inhibition of thrombosis when the heparin could be started at the time in the first trimester when the platelets become thrombophilic or does its main role in improving implantation in which it would be started shortly before or shortly after ovulation. Possibly heparin is superior to LMWH in improving the implantation process though more studies are needed to corroborate or refute this suggestion. At present inherited thrombophilias are not considered a cause of first trimester miscarriage and thus measuring these factors are not recommended. There is no evidence that heparin has any benefit in preventing miscarriage from unexplained causes. Heparin is effective alone and there does not appear to be any extra benefit from adding aspirin (or even aspirin may negate some of its benefits).

Asunto(s)

Aborto Espontáneo/tratamiento farmacológico , Aborto Espontáneo/prevención & control , Heparina/uso terapéutico , Aborto Habitual/tratamiento farmacológico , Aborto Habitual/prevención & control , Anticuerpos Fosfo-Específicos/inmunología , Anticoagulantes/uso terapéutico , Síndrome Antifosfolípido/diagnóstico , Síndrome Antifosfolípido/tratamiento farmacológico , Femenino , Humanos , Recién Nacido , Embarazo , Complicaciones Hematológicas del Embarazo/tratamiento farmacológico , Trombofilia/genética , Trombofilia/inmunología , Trombofilia/patología

RESUMEN

The reversible phosphorylation of tyrosine residues is one of the most frequent post-translational modifications regulating enzymatic activities and protein-protein interactions in eukaryotic cells. Cells responding to internal or external regulatory inputs modify their phosphorylation status and diseased cells can often be diagnosed by observing alterations in their qualitative or quantitative phosphorylation profile. As a consequence the ability to describe the phosphorylation profile of a cell is central to many approaches aiming at the characterisation of signalling pathways. Anti-phosphotyrosine (pY) antibodies are widely used as experimental tools to monitor the phosphorylation status of a cell. By using peptide microarray technology we have characterised the substrate specificity of three widely used pY antibodies. We report that they are more sensitive to sequence context than is generally assumed and that their sequence preferences differ.

Asunto(s)

Anticuerpos Fosfo-Específicos/inmunología , Especificidad de Anticuerpos/inmunología , Análisis por Matrices de Proteínas/métodos , Proteómica/métodos , Secuencia de Aminoácidos , Células HEK293 , Humanos , Datos de Secuencia Molecular , Fosfopéptidos/química , Fosfopéptidos/inmunología , Fosforilación , Reproducibilidad de los Resultados , Procesamiento de Señales Asistido por Computador

RESUMEN

In response to stimulation with proinflammatory cytokines, the deubiquitinase A20 inducibly interacts with the regulatory molecules TAX1BP1, Itch and RNF11 to form the A20 ubiquitin-editing complex. However, the molecular signal that coordinates the assembly of this complex has remained elusive. Here we demonstrate that TAX1BP1 was inducibly phosphorylated on Ser593 and Ser624 in response to proinflammatory stimuli. The kinase IKKα, but not IKKß, was required for phosphorylation of TAX1BP1 and directly phosphorylated TAX1BP1 in response to stimulation with tumor necrosis factor (TNF) or interleukin 1 (IL-1). TAX1BP1 phosphorylation was pivotal for cytokine-dependent interactions among TAX1BP1, A20, Itch and RNF11 and downregulation of signaling by the transcription factor NF-κB. IKKα therefore serves a key role in the negative feedback of NF-κB canonical signaling by orchestrating assembly of the A20 ubiquitin-editing complex to limit inflammatory gene activation.

Asunto(s)

Proteínas Portadoras/inmunología , Cisteína Endopeptidasas/inmunología , Quinasa I-kappa B/inmunología , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/inmunología , FN-kappa B/inmunología , Proteínas de Neoplasias/inmunología , Fosforilación/efectos de los fármacos , Proteínas Recombinantes/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Anticuerpos Fosfo-Específicos/inmunología , Anticuerpos Fosfo-Específicos/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Clonación Molecular , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al ADN , Escherichia coli , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/farmacología , Interleucina-1/inmunología , Interleucina-1/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacos

RESUMEN

By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.

Asunto(s)

Anticuerpos Fosfo-Específicos/metabolismo , Cromatina/efectos de los fármacos , Fijadores/química , Histocitoquímica/métodos , Sales (Química)/química , Coloración y Etiquetado/métodos , Zinc/química , Anticuerpos Fosfo-Específicos/inmunología , Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada , Camptotecina/farmacología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina/química , Cromatina/ultraestructura , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/metabolismo , Fluorescencia , Formaldehído/química , Histonas/metabolismo , Humanos , Indoles/análisis , Citometría de Barrido por Láser , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Inhibidores de Topoisomerasa I/farmacología , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

Protein phosphorylation is a universal key posttranslational modification that affects the activity and other properties of intracellular proteins. Phosphosite-specific antibodies can be produced as polyclonals or monoclonals in different animal species, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is key for their use in proteomics and profiling of disease.

Asunto(s)

Anticuerpos Fosfo-Específicos/análisis , Inmunohistoquímica/métodos , Proteínas/análisis , Animales , Anticuerpos Fosfo-Específicos/inmunología , Western Blotting/métodos , Descubrimiento de Drogas/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo/métodos , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas/inmunología , Proteínas/metabolismo , Proteómica/métodos , Estudios de Validación como Asunto

RESUMEN

The in situ expression levels and subcellular localization of molecules involved in signal transduction using specific antibodies can be useful for prognosis and diagnosis of human diseases such as cancer. In addition, it has the potential to be helpful in monitoring biologic response to targeted therapies. The increasing availability of such antibodies makes these studies feasible. However, compared to typical immunohistochemical stains in which stabile molecules such as cytokeratins are targeted, additional -validation may be required for signal transduction immunohistochemistry.

Asunto(s)

Anticuerpos Fosfo-Específicos/análisis , Inmunohistoquímica/métodos , Neoplasias/diagnóstico , Fosfoproteínas/análisis , Animales , Anticuerpos Fosfo-Específicos/inmunología , Línea Celular Tumoral , Humanos , Neoplasias/patología , Fosfoproteínas/inmunología , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-akt/inmunología , Transducción de Señal

RESUMEN

Phosphoproteins are considered to be among the most important proteins in the body. They are the proteins that regulate almost all cell processes from cell division in cancer to neuronal signal transduction in learning and memory. This review will describe the development of a revolutionary immunochemical technique that produces antibodies that bind to target proteins only when the protein is in the phosphorylated state. These phospho-specific antibodies can thus be used to track the activity of a protein, not simply its level of expression. In this review, we will discuss both the design of the phosphopeptide immunogen and immunization. The affinity purification of the phospho-specific antibody as well as the methods most suitable for characterizing the phosphospecificity of the antibody will be described here. Taken together, these methods will cover the key procedures and protocols required to produce a phospho-specific antibody that works.

Asunto(s)

Anticuerpos Fosfo-Específicos/inmunología , Anticuerpos Fosfo-Específicos/aislamiento & purificación , Western Blotting/métodos , Cromatografía de Afinidad/métodos , Inmunohistoquímica/métodos , Fosfoproteínas/inmunología , Animales , Humanos

RESUMEN

Psychiatric disorders including anxiety, depression, and addiction are both precipitated and exacerbated by severe or chronic stress exposure. While acutely, stress responses are adaptive, repeated exposure to stress can dysregulate the brain in such a way as to predispose the organism to both physiological and mental illness. Understanding the neuronal chemicals, cell types, and circuits involved in both normal and pathological stress responses are essential in developing new therapeutics for psychiatric diseases. Varying degrees of stressor exposure cause the release of a constellation of chemicals, including neuropeptides such as dynorphin. Neuropeptidergic release can be very difficult to directly measure with adequate spatial and temporal resolution. Moreover, the downstream consequences following release and receptor binding are numerous and also difficult to measure with cellular resolution. Following repeated stressor exposure, dynorphin is released, binds to the kappa opioid receptor (KOR), and causes activation of KOR. Agonist-activated KOR becomes a substrate for G protein receptor kinase (GRK), which phosphorylates the Ser369 residue at the C-terminal tail of the receptor in the first step in the ß-Arrestin-dependent desensitization cascade. Through the use of phospho--selective antibodies developed and validated in the laboratory, we have the tools, to assess with fine cellular resolution, the strength of behavioral stimulus required for release, time course of the release, and regional location of release. We have gone on to show that following KOR activation, both ERK 1/2 and p38 MAP kinase phosphorylation are increased through use of commercially available phospho-selective antibodies. Finally, we have identified that one effector of KOR/p38MAP kinase is K(IR) 3.1 and have developed a phospho-selective antibody against the Y12 motif of this channel. Much like KOR and p38 MAP kinase, phosphorylation of this potassium channel increases following repeated stress. The following chapter discusses immunohistochemical and quantification methods used for phospho-selective antibodies used in various brain regions following behavioral manipulations.

Asunto(s)

Anticuerpos Fosfo-Específicos/análisis , Inmunohistoquímica/métodos , Canales de Potasio de Rectificación Interna/metabolismo , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Anticuerpos Fosfo-Específicos/inmunología , Anticuerpos Fosfo-Específicos/aislamiento & purificación , Encéfalo/metabolismo , Encéfalo/ultraestructura , Línea Celular , Cromatografía de Afinidad/métodos , Humanos , Ratones , Microscopía/métodos , Proteína Quinasa 1 Activada por Mitógenos/análisis , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/análisis , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Canales de Potasio de Rectificación Interna/análisis , Canales de Potasio de Rectificación Interna/inmunología , Ratas , Receptores Opioides kappa/análisis , Receptores Opioides kappa/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología

RESUMEN

Although phospho-specific primary antibodies used in immunohistochemistry (IHC) are expected to detect phosphorylated proteins, in some cases these antibodies may also cross-react with nonphosphorylated proteins. Therefore, it is of ultimate importance to employ a control to determine that the staining pattern is specific. One of the frequently used controls in IHC is a so-called absorption control: phospho-specific primary antibodies are first incubated with a phospho-peptide immunogen to block antibody-binding sites, and this mixture is subsequently applied to tissue sections. If the antibody blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific, but if staining is obtained, the antibody is considered nonspecific. Unfortunately, bound peptide can dissociate from the antibody allowing unblocked antibody to bind to tissue targets, producing unwanted staining. We have developed a simple absorption-control protocol allowing for the efficient neutralization of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of antibody-peptide complex from the incubation solution, minimizing the risk of formation of unblocked antibodies capable of producing tissue staining.

Asunto(s)

Anticuerpos Fosfo-Específicos/inmunología , Inmunohistoquímica/métodos , Separación Inmunomagnética/métodos , Péptidos/inmunología , Células 3T3 , Animales , Ratones , Péptidos/química , Fosforilación

RESUMEN

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine (Ser/Thr) kinase comprising two isoforms, GSK-3α and GSK-3ß. Both enzymes are similarly inactivated by serine phosphorylation (GSK-3α at Ser21 and GSK-3ß at Ser9) and activated by tyrosine phosphorylation (GSK-3α at Tyr279 and GSK-3ß at Tyr216). Antibodies raised to phosphopeptides containing the sequences around these phosphorylation sites are frequently used to provide an indication of the activation state of GSK-3 in cell and tissue extracts. These antibodies have further been used to determine the subcellular localisation of active and inactive forms of GSK-3, and the results of those studies support roles for GSK-3 phosphorylation in diverse cellular processes. However, the specificity of these antibodies in immunocytochemistry has not been addressed in any detail. RESULTS: Taking advantage of gene silencing technology, we examined the specificity of several commercially available anti-phosphorylated GSK-3 antibodies. We show that antibodies raised to peptides containing the phosphorylated Ser21/9 epitope crossreact with unidentified antigens that are highly expressed by mitotic cells and that mainly localise to spindle poles. In addition, two antibodies raised to peptides containing the phosphorylated Tyr279/216 epitope recognise an unidentified protein at focal contacts, and a third antibody recognises a protein found in Ki-67-positive cell nuclei. While the phosphorylated Ser9/21 GSK-3 antibodies also recognise other proteins whose levels increase in mitotic cells in western blots, the phosphorylated Tyr279/216 antibodies appear to be specific in western blotting. However, we cannot rule out the posssibility that they recognise very large or very small proteins that might not be detected using a standard western blotting approach. CONCLUSIONS: Our findings indicate that care should be taken when examining the subcellular localisation of active or inactive GSK-3 and, furthermore, suggest that the role of GSK-3 phosphorylation in some cellular processes be reassessed.

Asunto(s)

Anticuerpos Fosfo-Específicos/inmunología , Células/enzimología , Glucógeno Sintasa Quinasa 3/inmunología , Antígenos/inmunología , Núcleo Celular/inmunología , Activación Enzimática , Adhesiones Focales/inmunología , Silenciador del Gen , Glucógeno Sintasa Quinasa 3 beta , Proteínas Fluorescentes Verdes/metabolismo , Isoenzimas/inmunología , Mitosis , Fosfoserina/inmunología , Fosfotirosina/inmunología , Plásmidos/genética , ARN Interferente Pequeño/metabolismo

RESUMEN

Recent efforts have identified the p38α Ser/Thr kinase as a potential target for the treatment of inflammatory diseases as well as non-small cell lung carcinoma. Despite the significance of p38α, no direct activity probe compatible with cell lysate analysis exists. Instead, proxies for kinase activation, such as phosphospecific antibodies, which do not distinguish between p38 isoforms, are often used. Our laboratory has recently developed a sulfonamido-oxine (Sox) fluorophore that undergoes a significant increase in fluorescence in response to phosphorylation at a proximal residue, allowing for real-time activity measurements. Herein we report the rational design of a p38α-selective chemosensor using this approach. We have validated the selectivity of this sensor using specific inhibitors and immunodepletions and show that p38α activity can be monitored in crude lysates from a variety of cell lines, allowing for the potential use of this sensor in both clinical and basic science research applications.

Asunto(s)

Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Péptidos Cíclicos/química , Animales , Anticuerpos Fosfo-Específicos/química , Anticuerpos Fosfo-Específicos/inmunología , Extractos Celulares/análisis , Extractos Celulares/química , Extractos Celulares/inmunología , Línea Celular Tumoral , Inhibidores Enzimáticos/inmunología , Células HeLa , Humanos , Isoenzimas/inmunología , Ratones , Proteína Quinasa 14 Activada por Mitógenos/inmunología , Células 3T3 NIH , Péptidos Cíclicos/metabolismo , Fosforilación , Especificidad por Sustrato

RESUMEN

Analysis of signalling events by classical biochemical approaches is limited as the outcome is an averaged readout for protein activation of a single protein within a cell population. This is a clear restriction when addressing signalling events in mixed populations or subpopulations of cells. By combining flow cytometry with a panel of phosphospecific antibodies against several signal molecules simultaneously in a multi-parameter phospho flow cytometry analysis it is possible to obtain a higher level of understanding of the signal transduction dynamics at a single cell level. In addition, analysis of mixed cell populations makes it possible to study cells ex vivo in a state more closely resembling the in vivo situation. The multimeric analysis yields information on combinations of signals turned on and off in specific settings such as disease (signal nodes) that can be used for biomarker analysis and for development of drug screening strategies. Prostaglandin E(2) (PGE(2)) is known to signal through four G-protein coupled transmembrane receptors, EP1-4, activating a multitude of potential signalling pathways. The analysis of the PGE(2) signalling network elicited by activation of the four EP receptors in lymphoid cells revealing several signalling nodes is reviewed as an example.

Asunto(s)

Transducción de Señal , Anticuerpos Fosfo-Específicos/inmunología , Dinoprostona/metabolismo , Citometría de Flujo , Fosforilación , Receptores de Prostaglandina E/metabolismo