RESUMEN
Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.
Asunto(s)
Mitocondrias/enzimología , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Tirosina/metabolismo , Animales , Femenino , Ratones , Ratones Desnudos , Mitocondrias/metabolismo , Trasplante de Neoplasias , Neoplasias/patología , Fosforilación , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Trasplante HeterólogoRESUMEN
To better understand the signaling properties of oncogenic FGFR3, we performed phospho-proteomics studies to identify potential downstream signaling effectors that are tyrosine phosphorylated in hematopoietic cells expressing constitutively activated leukemogenic FGFR3 mutants. We found that FGFR3 directly tyrosine phosphorylates the serine/threonine kinase p90RSK2 at Y529, which consequently regulates RSK2 activation by facilitating inactive ERK binding to RSK2 that is required for ERK-dependent phosphorylation and activation of RSK2. Moreover, inhibition of RSK2 by siRNA or a specific RSK inhibitor fmk effectively induced apoptosis in FGFR3-expressing human t(4;14)-positive myeloma cells. Our findings suggest that FGFR3 mediates hematopoietic transformation by activating RSK2 in a two-step fashion, promoting both the ERK-RSK2 interaction and subsequent phosphorylation of RSK2 by ERK.
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Transformación Celular Neoplásica/metabolismo , Sistema de Señalización de MAP Quinasas , Mieloma Múltiple/enzimología , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/fisiología , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Apoptosis , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Mieloma Múltiple/metabolismo , Fosforilación , Interferencia de ARN , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Tirosina/metabolismoRESUMEN
Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3(A572V), JAK3(V722I), and JAK3(P132T) each transform Ba/F3 cells to factor-independent growth, and JAK3(A572V) confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations.
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Leucemia Experimental/genética , Leucemia Megacarioblástica Aguda/genética , Proteínas Tirosina Quinasas/genética , Alelos , Animales , Apoptosis/efectos de los fármacos , Benzamidas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Mesilato de Imatinib , Janus Quinasa 2 , Janus Quinasa 3 , Células K562 , Leucemia Experimental/metabolismo , Leucemia Experimental/patología , Leucemia Megacarioblástica Aguda/metabolismo , Leucemia Megacarioblástica Aguda/patología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Pirimidinas/farmacología , ARN Interferente Pequeño/genética , TYK2 QuinasaRESUMEN
Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.
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Péptidos y Proteínas de Señalización Intracelular/química , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional , Animales , Encéfalo/metabolismo , Línea Celular , Cromatografía de Afinidad , Cromatografía Liquida , Embrión de Mamíferos/metabolismo , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico/métodos , Fosfoproteínas/química , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosforilación , Mapas de Interacción de Proteínas , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/aislamiento & purificación , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
14-3-3 proteins play critical roles in controlling multiple aspects of the cellular response to stress and DNA damage including regulation of metabolism, cell cycle progression, cell migration, and apoptotic cell death by binding to protein substrates of basophilic protein kinases following their phosphorylation on specific serine/threonine residues. Although over 200 mammalian proteins that bind to 14-3-3 have been identified, largely through proteomic studies, in many cases the relevant protein kinase responsible for conferring 14-3-3-binding to these proteins is not known. To facilitate the identification of kinase-specific 14-3-3 clients, we developed a biochemical approach using high-density protein filter arrays and identified the translational regulatory molecule PABPC1 as a substrate for Chk1 and MAPKAP Kinase-2 (MK2) in vitro, and for MK2 in vivo, whose phosphorylation results in 14-3-3-binding. We identify Ser-470 on PABPC1 within the linker region connecting the RRM domains to the PABC domain as the critical 14-3-3-binding site, and demonstrate that loss of PABPC1 binding to 14-3-3 results in increased cell proliferation and decreased cell death in response to UV-induced DNA damage.
RESUMEN
Constitutively active JAK2V617F and thrombopoietin receptor (TpoR) W515L/K mutants are major determinants of human myeloproliferative neoplasms (MPNs). We show that a TpoRW515 mutation (W515A), which we detected in 2 myelofibrosis patients, and the Delta5TpoR active mutant, where the juxtamembrane R/KW(515)QFP motif is deleted, induce a myeloproliferative phenotype in mouse bone marrow reconstitution experiments. This phenotype required cytosolic Y112 of the TpoR. Phosphotyrosine immunoprofiling detected phosphorylated cytosolic TpoR Y78 and Y112 in cells expressing TpoRW515A. Mutation of cytosolic Y112 to phenylalanine prevented establishment of the in vivo phenotype and decreased constitutive active signaling by Delta5TpoR and TpoRW515A, especially via the mitogen-activated protein (MAP)-kinase pathway, without decreasing Janus kinase 2 (JAK2) activation. In contrast, mutation of cytosolic Y78 to phenylalanine enhanced the myeloproliferative syndrome induced by the TpoRW515 mutants, by enhancing receptor-induced JAK2 activation. We propose that TpoR cytosolic phosphorylated Y112 and flanking sequences could become targets for pharmacologic inhibition in MPNs.
Asunto(s)
Mutación , Trastornos Mieloproliferativos/genética , Mielofibrosis Primaria/genética , Receptores de Trombopoyetina/genética , Tirosina/genética , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Línea Celular , Proliferación Celular , Humanos , Immunoblotting , Janus Quinasa 2/metabolismo , Ratones , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Fosfoproteínas/metabolismo , Fosforilación , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Mielofibrosis Primaria/metabolismo , Mielofibrosis Primaria/patología , Receptores de Trombopoyetina/metabolismo , Transfección , Tirosina/metabolismoRESUMEN
Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA.
Asunto(s)
Apoptosis/efectos de la radiación , Cromatografía Liquida/métodos , Daño del ADN , Procesamiento Proteico-Postraduccional/efectos de la radiación , Transducción de Señal/efectos de la radiación , Espectrometría de Masas en Tándem/métodos , Rayos Ultravioleta , Secuencia de Aminoácidos , Autofagia/efectos de la radiación , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Humanos , Indicadores y Reactivos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Mapeo de Interacción de ProteínasRESUMEN
Antibodies to SARS-CoV-2 are central to recovery and immunity from COVID-19. However, the relationship between disease severity and the repertoire of antibodies against specific SARS-CoV-2 epitopes an individual develops following exposure remains incompletely understood. Here, we studied seroprevalence of antibodies to specific SARS-CoV-2 and other betacoronavirus antigens in a well-annotated, community sample of convalescent and never-infected individuals obtained in August 2020. One hundred and twenty-four participants were classified into five groups: previously exposed but without evidence of infection, having no known exposure or evidence of infection, seroconverted without symptoms, previously diagnosed with symptomatic COVID-19, and recovered after hospitalization with COVID-19. Prevalence of IgGs specific to the following antigens was compared between the five groups: recombinant SARS-CoV-2 and betacoronavirus spike and nucleocapsid protein domains, peptides from a tiled array of 22-mers corresponding to the entire spike and nucleocapsid proteins, and peptides corresponding to predicted immunogenic regions from other proteins of SARS-CoV-2. Antibody abundance generally correlated positively with severity of prior illness. A number of specific immunogenic peptides and some that may be associated with milder illness or protection from symptomatic infection were identified. No convincing association was observed between antibodies to Receptor Binding Domain(s) (RBDs) of less pathogenic betacoronaviruses HKU1 or OC43 and COVID-19 severity. However, apparent cross-reaction with SARS-CoV RBD was evident and some predominantly asymptomatic individuals had antibodies to both MERS-CoV and SARS-CoV RBDs. Findings from this pilot study may inform development of diagnostics, vaccines, and therapeutic antibodies, and provide insight into viral pathogenic mechanisms.
Asunto(s)
COVID-19 , SARS-CoV-2 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Epítopos , Humanos , Proyectos Piloto , Estudios Seroepidemiológicos , Glicoproteína de la Espiga del CoronavirusRESUMEN
Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)-ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.
Asunto(s)
Transferasas de Hidroximetilo y Formilo/metabolismo , Linfoma Anaplásico de Células Grandes/enzimología , Complejos Multienzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleótido Desaminasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Antimetabolitos Antineoplásicos/farmacología , Carbazoles/farmacología , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Humanos , Transferasas de Hidroximetilo y Formilo/antagonistas & inhibidores , Indazoles/farmacología , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Linfoma Anaplásico de Células Grandes/patología , Metotrexato/farmacología , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Nucleótido Desaminasas/antagonistas & inhibidores , Compuestos de Fenilurea/farmacología , Fosfoproteínas/metabolismo , Fosforilación , Fosfotirosina/análisis , Mapeo de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transcripción GenéticaRESUMEN
A major question regarding the sensitivity of solid tumors to targeted kinase inhibitors is why some tumors respond and others do not. The observation that many tumors express EGF receptor (EGFR), yet only a small subset with EGFR-activating mutations respond clinically to EGFR inhibitors (EGFRIs), suggests that responsive tumors uniquely depend on EGFR signaling for their survival. The nature of this dependence is not understood. Here, we investigate dependence on EGFR signaling by comparing non-small-cell lung cancer cell lines driven by EGFR-activating mutations and genomic amplifications using a global proteomic analysis of phospho-tyrosine signaling. We identify an extensive receptor tyrosine kinase signaling network established in cells expressing mutated and activated EGFR or expressing amplified c-Met. We show that in drug sensitive cells the targeted tyrosine kinase drives other RTKs and an extensive network of downstream signaling that collapse with drug treatment. Comparison of the signaling networks in EGFR and c-Met-dependent cells identify a "core network" of approximately 50 proteins that participate in pathways mediating drug response.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Proteómica/métodos , Proteínas Proto-Oncogénicas c-met/metabolismo , Línea Celular Tumoral , Gefitinib , Humanos , Modelos Biológicos , Metástasis de la Neoplasia , Fosfotirosina/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Quinazolinas/farmacología , Transducción de SeñalRESUMEN
Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/análisis , Proteínas de Ciclo Celular , Línea Celular , Núcleo Celular/química , Células Cultivadas , Embrión de Mamíferos/citología , Factor 4E Eucariótico de Iniciación/análisis , Factores Eucarióticos de Iniciación , Fibroblastos/química , Fibroblastos/citología , Ratones , Fosfoproteínas/análisis , Fosforilación , ARN Mensajero/metabolismoRESUMEN
PURPOSE: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non-small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry. EXPERIMENTAL DESIGN: We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing. RESULTS: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry-based DNA sequencing. CONCLUSIONS: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.
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Anticuerpos Monoclonales , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutación/inmunología , Animales , Bioensayo , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/secundario , Análisis Mutacional de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Inmunoglobulina G/inmunología , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Conejos , Sensibilidad y Especificidad , Eliminación de Secuencia , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
To ensure survival in the face of genomic insult, cells have evolved complex mechanisms to respond to DNA damage, termed the DNA damage checkpoint. The serine/threonine kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) activate checkpoint signaling by phosphorylating substrate proteins at SQ/TQ motifs. Although some ATM/ATR substrates (Chk1, p53) have been identified, the lack of a more complete list of substrates limits current understanding of checkpoint pathways. Here, we use immunoaffinity phosphopeptide isolation coupled with mass spectrometry to identify 570 sites phosphorylated in UV-damaged cells, 498 of which are previously undescribed. Semiquantitative analysis yielded 24 known and 192 previously uncharacterized sites differentially phosphorylated upon UV damage, some of which were confirmed by SILAC, Western blotting, and immunoprecipitation/Western blotting. ATR-specific phosphorylation was investigated by using a Seckel syndrome (ATR mutant) cell line. Together, these results provide a rich resource for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response.
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Proteínas de Ciclo Celular/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal/efectos de la radiación , Proteínas Supresoras de Tumor/fisiología , Rayos Ultravioleta , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/fisiología , Secuencias de Aminoácidos/efectos de la radiación , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/inmunología , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Daño del ADN/fisiología , Daño del ADN/efectos de la radiación , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Humanos , Inmunoprecipitación , Fosfopéptidos/inmunología , Fosfopéptidos/aislamiento & purificación , Fosfopéptidos/fisiología , Fosforilación/efectos de la radiación , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Especificidad por Sustrato/genética , Especificidad por Sustrato/efectos de la radiación , Proteínas Supresoras de Tumor/inmunología , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The ability to reproduce scientific findings is foundational in research; yet, it is compromised in part by poorly characterized reagents, including antibodies. In this report, we describe the application of complementary validation strategies tailored for use in immunohistochemical assays in the characterization of rabbit monoclonal antibodies against YAP and TAZ, homologous and sequentially similar transcriptional effectors of the Hippo signaling pathway. A lack of antibody reagents rigorously validated for immunohistochemistry has limited the Hippo signaling research community's ability to interrogate YAP and TAZ independently in tissue. In a series of normal and diseased human tissues, we were able to demonstrate differential expression patterns of YAP and TAZ, suggesting the potential for functional differences of these proteins. These differences can now be studied in greater detail with these highly validated tools.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anticuerpos Monoclonales/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Humanos , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.
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Transformación Celular Neoplásica/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Mutación/genética , Fosfotransferasas/metabolismo , Piperazinas/farmacología , Pirimidinas/farmacología , Secuencia de Aminoácidos , Animales , Benzamidas , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Modelos Animales de Enfermedad , Femenino , Proteínas de Fusión bcr-abl/química , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Células Progenitoras Mieloides/citología , Fosfotirosina/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal , Especificidad por SustratoRESUMEN
Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.
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Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Proteómica/métodos , Tirosina/química , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Cromatografía Liquida , Humanos , Células Jurkat , Espectrometría de Masas , Ratones , Células 3T3 NIH , Péptidos/química , Fosforilación , Fosfotirosina/química , Transducción de SeñalRESUMEN
Deregulated expression of both Myc and Bcl-X(L) are consistent features of human plasma cell neoplasms (PCNs). To investigate whether targeted expression of Myc and Bcl-X(L) in mouse plasma cells might lead to an improved model of human PCN, we generated Myc transgenics by inserting a single-copy histidine-tagged mouse Myc gene, Myc(His), into the mouse Ig heavy-chain Calpha locus. We also generated Bcl-X(L) transgenic mice that contain a multicopy Flag-tagged mouse Bcl-x(Flag) transgene driven by the mouse Ig kappa light-chain 3' enhancer. Single-transgenic Bcl-X(L) mice remained tumor free by 380 days of age, whereas single-transgenic Myc mice developed B cell tumors infrequently (4 of 43, 9.3%). In contrast, double-transgenic Myc/Bcl-X(L) mice developed plasma cell tumors with short onset (135 days on average) and full penetrance (100% tumor incidence). These tumors produced monoclonal Ig, infiltrated the bone marrow, and contained elevated amounts of Myc(His) and Bcl-X(L)(Flag) proteins compared with the plasma cells that accumulated in large numbers in young tumor-free Myc/Bcl-X(L) mice. Our findings demonstrate that the enforced expression of Myc and Bcl-X(L) by Ig enhancers with peak activity in plasma cells generates a mouse model of human PCN that recapitulates some features of human multiple myeloma.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Plasmacitoma/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Médula Ósea/patología , Humanos , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Plasmacitoma/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-myc/genética , Bazo/citología , Bazo/metabolismo , Bazo/patología , Proteína bcl-XRESUMEN
Receptor-interacting protein 2 (RIP2) is a serine-threonine kinase that mediates signaling for many receptors of the innate and adaptive immune systems. Toll like receptors (TLR) are an important component of the innate immune response. Stimulation of RIP2-deficient cells with ligands for TLR 2, 3 and 4 results in impaired cytokine production and decreased activation of NF-kB and MAP kinases compared to wild-type cells. Stimulation of TLR 4 with its ligand lipopolysaccaride (LPS) leads to the activation of RIP2 kinase activity and its autophosphorylation. Here we identify serine residue 176 as a site of autophosphorylation using a combination of mass spectrometry and mutational analysis. Mutation of S176 to alanine not only abolishes autophosphorylation of RIP2 but also significantly decreases its catalytic activity. A phospho-specific anti-S176 antibody detects wild-type RIP2 but not kinase-dead RIP2 or the RIP2 S176A mutant. Endogenous RIP2 in THP-1 cells and mouse bone marrow derived macrophages can be detected by the phospho-RIP2 (S176) antibody only after stimulation with LPS suggesting that the antibody recognizes activated RIP2. In summary, our results indicate that S176 is a regulatory autophosphorylation site for RIP2 and that S176 phosphorylation can be used to monitor the activation state of RIP2.
Asunto(s)
Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/química , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/inmunología , Sitios de Unión , Western Blotting , Línea Celular , Cromatografía Liquida , Humanos , Espectrometría de Masas/métodos , Ratones , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína/métodos , SpodopteraRESUMEN
Morphine analgesia is mediated principally by the micro -opioid receptor (MOR). Since morphine and other opiates have been shown to influence glucose homeostasis, we investigated the hypothesis of direct cross talk between the MOR and the insulin receptor (IR) signaling cascades. We show that prolonged morphine exposure of cell lines expressing endogenous or transfected MOR, IR, and the insulin substrate 1 (IRS-1) protein specifically desensitizes IR signaling to Akt and ERK cascades. Morphine caused serine phosphorylation of the IR and impaired the formation of the signaling complex among the IR, Shc, and Grb2. Morphine also resulted in IRS-1 phosphorylation at serine 612 and reduced tyrosine phosphorylation at the YMXM p85-binding motifs, weakening the association of the IRS-1/p85 phosphatidylinositol 3-kinase complex. However, the IRS-1/Grb2 complex was unaffected by chronic morphine treatment. These results suggest that morphine attenuates IR signaling to Akt by disrupting the IRS-1-p85 interaction but inhibits signaling to ERK by disruption of the complex among the IR, Shc, and Grb2. Finally, we show that systemic morphine induced IRS-1 phosphorylation at Ser612 in the hypothalamus and hippocampus of wild type, but not MOR knockout, mice. Our results demonstrate that opiates can inhibit insulin signaling through direct cross talk between the downstream signaling pathways of the MOR and the IR.