Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 98
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
Proc Natl Acad Sci U S A ; 121(11): e2308570121, 2024 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-38442170

RESUMEN

Cytokinesis is the last step of cell division and is regulated by the small GTPase RhoA. RhoA activity is required for all steps of cytokinesis, including prior to abscission when daughter cells are ultimately physically separated. Like germ cells in all animals, the Caenorhabditis elegans embryonic germline founder cell initiates cytokinesis but does not complete abscission, leaving a stable intercellular bridge between the two daughter cells. Here, we identify and characterize C. elegans OSGN-1 as a cytokinetic regulator that promotes RhoA activity during late cytokinesis. Sequence analyses and biochemical reconstitutions reveal that OSGN-1 is a flavin-containing monooxygenase (MO). Genetic analyses indicate that the MO activity of OSGN-1 is required to maintain active RhoA at the end of cytokinesis in the germline founder cell and to stabilize the intercellular bridge. Deletion of OSGIN1 in human cells results in an increase in binucleation as a result of cytokinetic furrow regression, and this phenotype can be rescued by expressing a catalytically active form of C. elegans OSGN-1, indicating that OSGN-1 and OSGIN1 are functional orthologs. We propose that OSGN-1 and OSGIN1 are conserved MO enzymes required to maintain RhoA activity at the intercellular bridge during late cytokinesis and thus favor its stability, enabling proper abscission in human cells and bridge stabilization in C. elegans germ cells.


Asunto(s)
Citocinesis , Dermatitis , Oxigenasas , Animales , Humanos , Citocinesis/genética , Caenorhabditis elegans/genética , División Celular
2.
Cell ; 145(7): 1075-87, 2011 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-21683433

RESUMEN

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.


Asunto(s)
Aminoácidos/farmacología , Compuestos de Bifenilo/farmacología , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidores , Sitio Alostérico , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Análisis Mutacional de ADN , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Enzimas Ubiquitina-Conjugadoras , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genética
3.
Pharmacol Rev ; 73(4): 263-296, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34732541

RESUMEN

Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling pathways that play essential roles in transducing extracellular environmental signals into diverse cellular responses to maintain homeostasis. These pathways are classically organized into an architecture of three sequentially acting protein kinases: a MAPK kinase kinase that phosphorylates and activates a MAPK kinase, which in turn phosphorylates and activates the effector MAPK. The activity of MAPKs is tightly regulated by phosphorylation of their activation loop, which can be modulated by positive and negative feedback mechanisms to control the amplitude and duration of the signal. The signaling outcomes of MAPK pathways are further regulated by interactions of MAPKs with scaffolding and regulatory proteins. Accumulating evidence indicates that, in addition to these mechanisms, MAPK signaling is commonly regulated by ubiquitin-proteasome system (UPS)-mediated control of the stability and abundance of MAPK pathway components. Notably, the biologic activity of some MAPKs appears to be regulated mainly at the level of protein turnover. Recent studies have started to explore the potential of targeted protein degradation as a powerful strategy to investigate the biologic functions of individual MAPK pathway components and as a new therapeutic approach to overcome resistance to current small-molecule kinase inhibitors. Here, we comprehensively review the mechanisms, physiologic importance, and pharmacological potential of UPS-mediated protein degradation in the control of MAPK signaling. SIGNIFICANCE STATEMENT: Accumulating evidence highlights the importance of targeted protein degradation by the ubiquitin-proteasome system in regulating and fine-tuning the signaling output of mitogen-activated protein kinase (MAPK) pathways. Manipulating protein levels of MAPK cascade components may provide a novel approach for the development of selective pharmacological tools and therapeutics.


Asunto(s)
Complejo de la Endopetidasa Proteasomal , Ubiquitina , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Transducción de Señal
4.
Int J Mol Sci ; 25(14)2024 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-39062801

RESUMEN

Hepatocellular carcinoma (HCC) is the most prevalent primary liver malignancy and is a major cause of cancer-related mortality in the world. This study aimed to characterize glutamine amino acid transporter expression profiles in HCC compared to those of normal liver cells. In vitro and in vivo models of HCC were studied using qPCR, whereas the prognostic significance of glutamine transporter expression levels within patient tumors was analyzed through RNAseq. Solute carrier (SLC) 1A5 and SLC38A2 were targeted through siRNA or gamma-p-nitroanilide (GPNA). HCC cells depended on exogenous glutamine for optimal survival and growth. Murine HCC cells showed superior glutamine uptake rate than normal hepatocytes (p < 0.0001). HCC manifested a global reprogramming of glutamine transporters compared to normal liver: SLC38A3 levels decreased, whereas SLC38A1, SLC7A6, and SLC1A5 levels increased. Also, decreased SLC6A14 and SLC38A3 levels or increased SLC38A1, SLC7A6, and SLC1A5 levels predicted worse survival outcomes (all p < 0.05). Knockdown of SLC1A5 and/or SLC38A2 expression in human Huh7 and Hep3B HCC cells, as well as GPNA-mediated inhibition, significantly decreased the uptake of glutamine; combined SLC1A5 and SLC38A2 targeting had the most considerable impact (all p < 0.05). This study revealed glutamine transporter reprogramming as a novel hallmark of HCC and that such expression profiles are clinically significant.


Asunto(s)
Carcinoma Hepatocelular , Regulación Neoplásica de la Expresión Génica , Glutamina , Neoplasias Hepáticas , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Humanos , Animales , Pronóstico , Ratones , Línea Celular Tumoral , Glutamina/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Masculino , Femenino , Proteínas Portadoras , Sistema de Transporte de Aminoácidos ASC
5.
J Cell Physiol ; 2022 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-36576983

RESUMEN

Extracellular signal-regulated kinase 3 (ERK3) is a poorly characterized member of the mitogen-activated protein (MAP) kinase family. Functional analysis of the ERK3 signaling pathway has been hampered by a lack of knowledge about the substrates and downstream effectors of the kinase. Here, we used large-scale quantitative phosphoproteomics and targeted gene silencing to identify direct ERK3 substrates and gain insight into its cellular functions. Detailed validation of one candidate substrate identified the gelsolin/villin family member supervillin (SVIL) as a bona fide ERK3 substrate. We show that ERK3 phosphorylates SVIL on Ser245 to regulate myosin II activation and cytokinesis completion in dividing cells. Depletion of SVIL or ERK3 leads to increased cytokinesis failure and multinucleation, a phenotype rescued by wild type SVIL but not by the non-phosphorylatable S245A mutant. Our results unveil a new function of the atypical MAP kinase ERK3 in cell division and the regulation of cell ploidy.

6.
J Cell Physiol ; 237(4): 2271-2287, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35141958

RESUMEN

The physiological functions and downstream effectors of the atypical mitogen-activated protein kinase extracellular signal-regulated kinase 3 (ERK3) remain to be characterized. We recently reported that mice expressing catalytically-inactive ERK3 (Mapk6KD/KD ) exhibit a reduced postnatal growth rate as compared to control mice. Here, we show that genetic inactivation of ERK3 impairs postnatal skeletal muscle growth and adult muscle regeneration after injury. Loss of MAPK-activated protein kinase 5 (MK5) phenocopies the muscle phenotypes of Mapk6KD/KD mice. At the cellular level, genetic or pharmacological inactivation of ERK3 or MK5 induces precocious differentiation of C2C12 or primary myoblasts, concomitant with MyoD activation. Reciprocally, ectopic expression of activated MK5 inhibits myogenic differentiation. Mechanistically, we show that MK5 directly phosphorylates FoxO3, promoting its degradation and reducing its association with MyoD. Depletion of FoxO3 rescues in part the premature differentiation of C2C12 myoblasts observed upon inactivation of ERK3 or MK5. Our findings reveal that ERK3 and its substrate MK5 act in a linear signaling pathway to control postnatal myogenic differentiation.


Asunto(s)
Proteína Forkhead Box O3/metabolismo , Transducción de Señal , Animales , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteína Quinasa 6 Activada por Mitógenos/metabolismo , Músculos , Proteínas Serina-Treonina Quinasas/metabolismo
7.
Genes Dev ; 27(8): 900-15, 2013 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-23599344

RESUMEN

Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence.


Asunto(s)
Senescencia Celular/genética , Sistema de Señalización de MAP Quinasas/fisiología , Proteolisis , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/enzimología , Técnicas de Silenciamiento del Gen , Humanos , Sistema de Señalización de MAP Quinasas/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas ras/metabolismo
8.
Blood ; 127(24): 3054-61, 2016 06 16.
Artículo en Inglés | MEDLINE | ID: mdl-27034432

RESUMEN

In this study, we analyzed RNA-sequencing data of 14 samples characterized by biallelic CEBPA (CEBPA(bi)) mutations included in the Leucegene collection of 415 primary acute myeloid leukemia (AML) specimens, and describe for the first time high frequency recurrent mutations in the granulocyte colony-stimulating factor receptor gene CSF3R, which signals through JAK-STAT proteins. Chemical interrogation of these primary human specimens revealed a uniform and specific sensitivity to all JAK inhibitors tested irrespective of their CSF3R mutation status, indicating a general sensitization of JAK-STAT signaling in this leukemia subset. Altogether, these results identified the co-occurrence of mutations in CSF3R and CEBPA in a well-defined AML subset, which uniformly responds to JAK inhibitors and paves the way to personalized clinical trials for this disease.


Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Resistencia a Antineoplásicos/genética , Quinasas Janus/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores del Factor Estimulante de Colonias/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Análisis Mutacional de ADN/métodos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Perfilación de la Expresión Génica , Frecuencia de los Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucemia Mieloide Aguda/clasificación , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Mutación , Medicina de Precisión , Transcriptoma , Células Tumorales Cultivadas , Adulto Joven
9.
Bioconjug Chem ; 28(6): 1677-1683, 2017 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-28449575

RESUMEN

The RAS-RAF-MEK-ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels-Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and "on-target" staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus.


Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/análisis , Sondas Moleculares/química , Línea Celular , Reacción de Cicloadición , Fosfatasas de Especificidad Dual/análisis , Colorantes Fluorescentes , Humanos , Inhibidores de Proteínas Quinasas
10.
Blood ; 124(15): 2362-9, 2014 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-25185265

RESUMEN

Multipotent long-term repopulating hematopoietic stem cells (LT-HSCs) can self-renew or differentiate into the less primitive short-term repopulating stem cells (ST-HSCs), which themselves produce progenitors that ensure the daily supply of all essential blood components. The Polycomb group (PcG) protein BMI1 is essential for the activity of both HSCs and progenitor cells. Although BMI1 operates by suppressing the Ink4a/Arf locus in progenitors and ST-HSCs, the mechanisms through which this gene regulates the activity of LT-HSCs remain poorly understood. Toward this goal, we isolated BMI1-containing protein complexes and identified UBAP2L as a novel BMI1-interacting protein. We also showed that UBAP2L is preferentially expressed in mouse and human HSC-enriched populations when compared with more mature cell types, and that this gene is essential for the activity of LT-HSCs. In contrast to what is observed for Bmi1 knockdown, we found that UBAP2L depletion does not affect the Ink4a/Arf locus. Given that we demonstrated that BMI1 overexpression is able to rescue the deleterious effects of Ubap2l downregulation on LT-HSC activity and that UBAP2L is part of a PcG subcomplex comprising BMI1, we propose a model in which at least 2 different BMI1-containing PcG complexes regulate HSC activity, which are distinguishable by the presence of UBAP2L.


Asunto(s)
Proteínas Portadoras/metabolismo , Células Madre Hematopoyéticas/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Regulación hacia Abajo , Eliminación de Gen , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Leucemia Mieloide Aguda/metabolismo , Ratones Endogámicos C57BL , Proteínas del Grupo Polycomb/metabolismo , Unión Proteica , ARN Interferente Pequeño/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo
11.
Am J Obstet Gynecol ; 215(3): 384.e1-384.e89, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27143398

RESUMEN

BACKGROUND: Neonatal respiratory distress syndrome in preterm infants is a leading cause of neonatal death. Pulmonary insufficiency-related infant mortality rates have improved with antenatal glucocorticoid treatment and neonatal surfactant replacement. However, the mechanism of glucocorticoid-promoted fetal lung maturation is not understood fully, despite decades of clinical use. We previously have shown that genetic deletion of Erk3 in mice results in growth restriction, cyanosis, and early neonatal lethality because of pulmonary immaturity and respiratory distress. Recently, we demonstrated that the addition of postnatal surfactant administration to antenatal dexamethasone treatment resulted in enhanced survival of neonatal Erk3-null mice. OBJECTIVE: To better understand the molecular underpinnings of corticosteroid-mediated lung maturation, we used high-throughput transcriptomic and high-resolution morphologic analysis of the murine fetal lung. We sought to examine the alterations in fetal lung structure and function that are associated with neonatal respiratory distress and antenatal glucocorticoid treatment. STUDY DESIGN: Dexamethasone (0.4 mg/kg) or saline solution was administered to pregnant dams on embryonic days 16.5 and 17.5. Fetal lungs were collected and analyzed by microCT and RNA-seq for differential gene expression and pathway interactions with genotype and treatment. Results from transcriptomic analysis guided further investigation of candidate genes with the use of immunostaining in murine and human fetal lung tissue. RESULTS: Erk3(-/-) mice exhibited atelectasis with decreased overall porosity and saccular space relative to wild type, which was ameliorated by glucocorticoid treatment. Of 596 differentially expressed genes (q < 0.05) that were detected by RNA-seq, pathway analysis revealed 36 genes (q < 0.05) interacting with dexamethasone, several with roles in lung development, which included corticotropin-releasing hormone and surfactant protein B. Corticotropin-releasing hormone protein was detected in wild-type and Erk3(-/-) lungs at E14.5, with significantly temporally altered expression through embryonic day 18.5. Antenatal dexamethasone attenuated corticotropin-releasing hormone at embryonic day 18.5 in both wild-type and Erk3(-/-) lungs (0.56-fold and 0.67-fold; P < .001). Wild type mice responded to glucocorticoid administration with increased pulmonary surfactant protein B (P = .003). In contrast, dexamethasone treatment in Erk3(-/-) mice resulted in decreased surfactant protein B (P = .012). In human validation studies, we confirmed that corticotropin-releasing hormone protein is present in the fetal lung at 18 weeks of gestation and increases in expression with progression towards viability (22 weeks of gestation; P < .01). CONCLUSION: Characterization of whole transcriptome gene expression revealed glucocorticoid-mediated regulation of corticotropin-releasing hormone and surfactant protein B via Erk3-independent and -dependent mechanisms, respectively. We demonstrated for the first time the expression and temporal regulation of corticotropin-releasing hormone protein in midtrimester human fetal lung. This unique model allows the effects of corticosteroids on fetal pulmonary morphologic condition to be distinguished from functional gene pathway regulation. These findings implicate Erk3 as a potentially important molecular mediator of antenatal glucocorticoid action in promoting surfactant protein production in the preterm neonatal lung and expanding our understanding of key mechanisms of clinical therapy to improve neonatal survival.


Asunto(s)
Dexametasona/administración & dosificación , Glucocorticoides/administración & dosificación , Pulmón/patología , Proteína Quinasa 6 Activada por Mitógenos/deficiencia , Animales , Animales Recién Nacidos , Hormona Liberadora de Corticotropina/metabolismo , Modelos Animales de Enfermedad , Femenino , Factor II del Crecimiento Similar a la Insulina/metabolismo , Pulmón/diagnóstico por imagen , Pulmón/metabolismo , Pulmón/fisiopatología , Ratones Noqueados , Embarazo , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Síndrome de Dificultad Respiratoria del Recién Nacido/patología , Microtomografía por Rayos X
12.
Immunology ; 145(1): 161-9, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25521218

RESUMEN

Extracellular signal-regulated kinase 3 (ERK3 )is an atypical member of the mitogen-activated protein kinase (MAPK) family. We have previously shown that ERK3 is expressed during thymocyte differentiation and that its expression is induced in mature peripheral T cells following activation of ERK1/2 by T-cell receptor (TCR) signalling. Herein, we have investigated whether ERK3 expression is required for proper T-cell selection. Using a knock-in mouse model in which the coding sequence of ERK3 is replaced by the gene encoding for the ß-galactosidase reporter, we show that ERK3 is expressed by double-positive (DP) thymocytes undergoing positive selection. In ERK3-deficient mice with a polyclonal TCR repertoire, we observe a decrease in positive selection. This reduction in positive selection was also observed when ERK3-deficient mice were backcrossed to class I- and class II-restricted TCR transgenic mice. Furthermore, the response of DP thymocytes to in vitro TCR stimulation was strongly reduced in ERK3-deficient mice. Together, these results show that ERK3 expression following TCR signalling is critical for proper thymic positive selection.


Asunto(s)
Selección Clonal Mediada por Antígenos , Sistema de Señalización de MAP Quinasas/inmunología , Proteína Quinasa 6 Activada por Mitógenos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Timocitos/inmunología , Timo/inmunología , Animales , Regulación Enzimológica de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/inmunología , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Noqueados , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Proteína Quinasa 6 Activada por Mitógenos/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/citología , Timocitos/citología , Timo/citología
13.
Blood ; 121(26): 5203-7, 2013 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-23687087

RESUMEN

Oncogenic NRAS mutations are frequently identified in human myeloid leukemias. In mice, expression of endogenous oncogenic Nras (Nras(G12D/+)) in hematopoietic cells leads to expansion of myeloid progenitors, increased long-term reconstitution of bone marrow cells, and a chronic myeloproliferative neoplasm (MPN). However, acute expression of Nras(G12D/+) in a pure C57BL/6 background does not induce hyperactivated granulocyte macrophage colony-stimulating factor signaling or increased proliferation in myeloid progenitors. It is thus unclear how Nras(G12D/+) signaling promotes leukemogenesis. Here, we show that hematopoietic stem cells (HSCs) expressing Nras(G12D/+) serve as MPN-initiating cells. They undergo moderate hyperproliferation with increased self-renewal. The aberrant Nras(G12D/+) HSC function is associated with hyperactivation of ERK1/2 in HSCs. Conversely, downregulation of MEK/ERK by pharmacologic and genetic approaches attenuates the cycling of Nras(G12D/+) HSCs and prevents the expansion of Nras(G12D/+) HSCs and myeloid progenitors. Our data delineate critical mechanisms of oncogenic Nras signaling in HSC function and leukemogenesis.


Asunto(s)
GTP Fosfohidrolasas/fisiología , Células Madre Hematopoyéticas/patología , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/patología , MAP Quinasa Quinasa 1/metabolismo , Proteínas de la Membrana/fisiología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación/genética , Animales , Citometría de Flujo , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mielomonocítica Crónica/metabolismo , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Ratones , Ratones Transgénicos , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Fosforilación , Transducción de Señal
14.
PLoS Pathog ; 8(7): e1002747, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22792062

RESUMEN

Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNß, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Inmunidad Innata , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/metabolismo , Línea Celular , ADN/metabolismo , Perfilación de la Expresión Génica , Proteínas de la Matriz de Golgi , Células HEK293 , Células HeLa , Humanos , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/biosíntesis , Interferón beta/genética , Mitocondrias/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteoma , Interferencia de ARN , ARN Bicatenario/metabolismo , ARN Interferente Pequeño , Proteínas de Unión al ARN , Transducción de Señal , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
15.
Mol Syst Biol ; 9: 669, 2013 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-23712012

RESUMEN

The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases.


Asunto(s)
Células Epiteliales/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfoproteínas/metabolismo , Proteoma , Animales , Línea Celular , Ensamble y Desensamble de Cromatina , Citoesqueleto/genética , Citoesqueleto/metabolismo , Células Epiteliales/citología , Regulación de la Expresión Génica , Humanos , Uniones Intercelulares/genética , Uniones Intercelulares/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Empalme del ARN , Ratas , Transducción de Señal , Especificidad de la Especie , Especificidad por Sustrato , Transcripción Genética
16.
Blood ; 120(8): e17-27, 2012 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-22802335

RESUMEN

We recently generated 2 phenotypically similar Hoxa9+Meis1 overexpressing acute myeloid leukemias that differ by their in vivo biologic behavior. The first leukemia, named FLA2, shows a high frequency of leukemia stem cells (LSCs; 1 in 1.4 cells), whereas the second, FLB1, is more typical with a frequency of LSCs in the range of 1 per several hundred cells. To gain insights into possible mechanisms that determine LSC self-renewal, we profiled and compared the abundance of nuclear and cytoplasmic proteins and phosphoproteins from these leukemias using quantitative proteomics. These analyses revealed differences in proteins associated with stem cell fate, including a hyperactive p38 MAP kinase in FLB1 and a differentially localized Polycomb group protein Ezh2, which is mostly nuclear in FLA2 and predominantly cytoplasmic in FLB1. Together, these newly documented proteomes and phosphoproteomes represent a unique resource with more than 440 differentially expressed proteins and 11 543 unique phosphopeptides, of which 80% are novel and 7% preferentially phosphorylated in the stem cell-enriched leukemia.


Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Activación Enzimática , N-Metiltransferasa de Histona-Lisina/análisis , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Mapas de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Represoras/análisis , Proteínas Represoras/metabolismo , Factores de Transcripción/análisis , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por Mitógenos/análisis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
17.
Pediatr Res ; 76(1): 24-32, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24732107

RESUMEN

BACKGROUND: Respiratory distress syndrome (RDS) persists as a prevalent cause of infant morbidity and mortality. We have previously demonstrated that deletion of Erk3 results in pulmonary immaturity and neonatal lethality. Using RNA sequencing, we identified corticotrophin releasing hormone (CRH) and surfactant protein B (SFTPB) as potential molecular mediators of Erk3-dependent lung maturation. In this study, we characterized the impact of antenatal glucocorticoids and postnatal surfactant on neonatal survival of Erk3 null mice. METHODS: In a double crossover design, we administered dexamethasone (dex) or saline to pregnant dams during the saccular stage of lung development, followed by postnatal surfactant or saline via inhalation intubation. Survival was recorded, and detailed lung histological analysis and staining for CRH and SFTPB protein expression were performed. RESULTS: Without treatment, Erk3 null pups die within 6 h of birth with reduced aerated space, impaired thinning of the alveolar septa, and abundant glycogen stores, as described in human RDS. The administration of dex and surfactant improved RDS-associated lethality of Erk3(-/-) pups and partially restored functional fetal lung maturation by accelerating the downregulation of pulmonary CRH and partially rescuing the production of SFTPB. CONCLUSION: These findings emphasize that Erk3 is integral to terminal differentiation of type II cells, SFTPB production, and fetal pulmonary maturity.


Asunto(s)
Glucocorticoides/administración & dosificación , Pulmón/embriología , Pulmón/crecimiento & desarrollo , Surfactantes Pulmonares/administración & dosificación , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológico , Animales , Diferenciación Celular , Hormona Liberadora de Corticotropina/metabolismo , Estudios Cruzados , Dexametasona/administración & dosificación , Dexametasona/química , Modelos Animales de Enfermedad , Femenino , Glucocorticoides/química , Pulmón/patología , Masculino , Exposición Materna , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 6 Activada por Mitógenos/genética , Embarazo , Preñez , Proteína B Asociada a Surfactante Pulmonar/metabolismo , Síndrome de Dificultad Respiratoria del Recién Nacido/genética , Factores de Tiempo
18.
Dis Model Mech ; 17(7)2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-39051113

RESUMEN

Hepatocellular carcinoma (HCC) is a disease of high unmet medical need that has become a global health problem. The development of targeted therapies for HCC has been hindered by the incomplete understanding of HCC pathogenesis and the limited number of relevant preclinical animal models. We recently unveiled a previously uncharacterized YES kinase (encoded by YES1)-dependent oncogenic signaling pathway in HCC. To model this subset of HCC, we established a series of syngeneic cell lines from liver tumors of transgenic mice expressing activated human YES. The resulting cell lines (referred to as HepYF) were enriched for expression of stem cell and progenitor markers, proliferated rapidly, and were characterized by high SRC family kinase (SFK) activity and activated mitogenic signaling pathways. Transcriptomic analysis indicated that HepYF cells are representative of the most aggressive proliferation class G3 subgroup of HCC. HepYF cells formed rapidly growing metastatic tumors upon orthotopic implantation into syngeneic hosts. Treatment with sorafenib or the SFK inhibitor dasatinib markedly inhibited the growth of HepYF tumors. The new HepYF HCC cell lines provide relevant preclinical models to study the pathogenesis of HCC and test novel small-molecule inhibitor and immunotherapy approaches.


Asunto(s)
Carcinoma Hepatocelular , Proliferación Celular , Modelos Animales de Enfermedad , Neoplasias Hepáticas , Metástasis de la Neoplasia , Animales , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Proliferación Celular/efectos de los fármacos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Humanos , Línea Celular Tumoral , Sorafenib/farmacología , Sorafenib/uso terapéutico , Dasatinib/farmacología , Dasatinib/uso terapéutico , Ratones Transgénicos , Ratones , Familia-src Quinasas/metabolismo , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Transducción de Señal/efectos de los fármacos , Niacinamida/análogos & derivados , Niacinamida/farmacología
19.
Physiol Rep ; 12(11): e16108, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38872461

RESUMEN

ERK3/MAPK6 activates MAP kinase-activated protein kinase (MK)-5 in selected cell types. Male MK5 haplodeficient mice show reduced hypertrophy and attenuated increase in Col1a1 mRNA in response to increased cardiac afterload. In addition, MK5 deficiency impairs cardiac fibroblast function. This study determined the effect of reduced ERK3 on cardiac hypertrophy following transverse aortic constriction (TAC) and fibroblast biology in male mice. Three weeks post-surgery, ERK3, but not ERK4 or p38α, co-immunoprecipitated with MK5 from both sham and TAC heart lysates. The increase in left ventricular mass and myocyte diameter was lower in TAC-ERK3+/- than TAC-ERK3+/+ hearts, whereas ERK3 haploinsufficiency did not alter systolic or diastolic function. Furthermore, the TAC-induced increase in Col1a1 mRNA abundance was diminished in ERK3+/- hearts. ERK3 immunoreactivity was detected in atrial and ventricular fibroblasts but not myocytes. In both quiescent fibroblasts and "activated" myofibroblasts isolated from adult mouse heart, siRNA-mediated knockdown of ERK3 reduced the TGF-ß-induced increase in Col1a1 mRNA. In addition, intracellular type 1 collagen immunoreactivity was reduced following ERK3 depletion in quiescent fibroblasts but not myofibroblasts. Finally, knocking down ERK3 impaired motility in both atrial and ventricular myofibroblasts. These results suggest that ERK3 plays an important role in multiple aspects of cardiac fibroblast biology.


Asunto(s)
Fibroblastos , Animales , Masculino , Ratones , Fibroblastos/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Miocardio/metabolismo , Miocardio/citología , Proteína Quinasa 6 Activada por Mitógenos/metabolismo , Proteína Quinasa 6 Activada por Mitógenos/genética , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Células Cultivadas , Cardiomegalia/metabolismo , Cardiomegalia/patología , Cardiomegalia/genética , Miocitos Cardíacos/metabolismo
20.
Gastroenterology ; 142(1): 130-139.e4, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21945831

RESUMEN

BACKGROUND & AIMS: Development of fibrosis is part of the pathophysiologic process of chronic liver disease. Although it is considered deleterious, it also represents a form of tissue repair. Deposition of extracellular matrix changes the cellular environment of the liver; we investigated whether it increases resistance to noxious stimuli and the role of changes in intracellular signaling to hepatocytes in mediating this effect. METHODS: Primary cultures of mouse hepatocytes were exposed to type I collagen (COL1); cell injury was assessed by morphologic and biochemical criteria. The expression of Bcl-2 family members was evaluated by immunoblot analyses. Activation of extracellular signal-regulated kinase (ERK) was assessed using phospho-specific antibodies. Liver fibrosis was induced by repeated administration of thioacetamide or carbon tetrachloride to mice; mice were then exposed to Fas antibodies. RESULTS: Hepatocytes exposed to COL1 were more resistant to a variety of hepatotoxins, in a dose-dependent manner, and had lower levels of Bad, Bid, and Bax proapoptotic proteins compared with control hepatocytes. Activation of ERK1/2 was stronger and quicker in hepatocytes exposed to COL1. The MEK1/2 inhibitors U0126 and PD98059 reversed the protective effects of COL1 and the decrease in proapoptotic proteins. Hepatocytes isolated from ERK1(-/-) mice were insensitive to the protective effect of COL1. Fibrotic livers from wild-type mice had high levels of phospho-ERK1 and were resistant to Fas-induced cell death. ERK1(-/-) mice lost this effect. CONCLUSIONS: Production of COL1 during liver fibrosis induces a hepatoprotective response that is mediated by activation of ERK1 signaling.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Cirrosis Hepática Experimental/patología , Hígado/patología , Enfermedad Aguda , Animales , Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Western Blotting , Tetracloruro de Carbono , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Colágeno Tipo I/metabolismo , Citoprotección , Activación Enzimática , Hígado/efectos de los fármacos , Hígado/metabolismo , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Tioacetamida , Factores de Tiempo , Proteína X Asociada a bcl-2/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Receptor fas/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA