RESUMEN

The PDZ-binding kinase (PBK) protein is localised exclusively in spermatogenic cells, such as spermatogonia, spermatocytes and round spermatids, of the adult testis. However, its role in male fertility remains unknown. Analysis of adult Pbk-knockout (KO) male mice showed no significant difference in the weight of the testes, epididymis and seminal vesicle compared with adult wild-type (WT) mice. There were no significant differences in testis morphology, tubule diameter and the number of offspring born to females mated with KO or WT male mice. Sperm number, motility and morphology did not differ significantly between KO and WT mice. The oocyte fertilisation rate and embryo development following IVF were comparable between groups fertilised using spermatozoa from KO versus WT mice (P>0.05). Further analysis revealed that the phosphorylation of the mitogen-activated protein kinases (MAPKs) p38 kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases was dysregulated in the testis of KO mice. In conclusion, Pbk-KO male mice are fertile and their spermatozoa and testis do not show any morphological and functional abnormalities despite the dysregulated phosphorylation of MAPKs. It is likely that functional redundancy of PBK and overlapping substrate specificities of the MAPK superfamily compensated for the loss of PBK from the testis.

Asunto(s)

Fertilidad/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Animales , Femenino , Fertilización , Masculino , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oocitos/fisiología , Tamaño de los Órganos , Fosforilación , Espermatozoides/enzimología , Espermatozoides/fisiología , Testículo/anatomía & histología , Testículo/enzimología

RESUMEN

PfPK7 is an "orphan" kinase displaying regions of homology to multiple protein kinase families. PfPK7 functions in regulating parasite proliferation/development as evident from the phenotype analysis of knockout parasites. Despite this regulatory role, the functions of PfPK7 in signaling pathways are not known. To better understand PfPK7-regulated phosphorylation events, we performed isobaric tag-based quantitative comparative phosphoproteomics of the schizont and segmenter stages from wild-type and pfpk7 - parasite lines. This analysis identified 3,875 phosphorylation sites on 1,047 proteins. Among these phosphorylation events, 146 proteins with 239 phosphorylation sites displayed reduction in phosphorylation in the absence of PfPK7. Further analysis of the phosphopeptides revealed three motifs whose phosphorylation was down regulated in the pfpk7 - cell line in both schizonts and segmenters. Decreased phosphorylation following loss of PfPK7 indicates that these proteins may function as direct substrates of PfPK7. We demonstrated that PfPK7 is active toward three of these potential novel substrates; however, PfPK7 did not phosphorylate many of the other proteins, suggesting that decreased phosphorylation in these proteins is an indirect effect. Our phosphoproteomics analysis is the first study to identify direct substrates of PfPK7 and reveals potential downstream or compensatory signaling pathways.

Asunto(s)

Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Fosfoproteínas/metabolismo , Fosforilación , Plasmodium falciparum/química , Plasmodium falciparum/enzimología , Proteínas Quinasas , Proteínas Protozoarias/fisiología , Esquizontes/química , Esquizontes/metabolismo , Transducción de Señal , Especificidad por Sustrato

RESUMEN

Arabidopsis genome contains 20 genes encoding mitogen-activated protein kinases (MAPKs, or MPKs), and ten genes encoding MAPK kinases (MAPKKs, or MKKs), the upstream kinases that activate MAPKs in the signaling cascades. They play critical roles in many different biological processes ranging from growth/development to response to environmental stimuli and pathogen invasion. T-DNA knockout lines are not currently available for all these genes. There is also functional redundancy at both MAPK and MAPKK levels. In addition, embryo lethality is associated with some double mutant combinations, which makes it difficult to investigate their specific functions in plants. In such situation, the use of RNA interference technology by which mRNA of interested gene is targeted by double-stranded RNA (dsRNA) for degradation and gene silencing provides a powerful tool for loss-of-function analyses. In this chapter, we describe the hairpin-RNA interference (hpRNAi) method we employed to silence MPK3/MPK6 and their upstream MKK4/MKK5 in the model plant Arabidopsis, with particular emphasis on the generation of hpRNAi constructs for single gene RNAi, tandem RNAi of two MAPKK genes, and tissue-specific RNAi.

Asunto(s)

Arabidopsis/enzimología , Arabidopsis/genética , Ingeniería Genética/métodos , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Interferencia de ARN , Agrobacterium/genética , Arabidopsis/citología , Intrones/genética , Secuencias Invertidas Repetidas/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/deficiencia , Especificidad de Órganos , Fenotipo , Plantas Modificadas Genéticamente , Transformación Genética

RESUMEN

Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth and pathogenicity, and in responses to the environment. In this study, we characterized a MAP kinase kinase FgMkk1 in Fusarium graminearum, the causal agent of wheat head blight. Phenotypic analyses of the FgMKK1 mutant (ΔFgMKK1) showed that FgMkk1 is involved in the regulation of hyphal growth, pigmentation, conidiation, deoxynivalenol biosynthesis and virulence of F. graminearum. ΔFgMKK1 also showed increased sensitivity to cell wall-damaging agents, and to osmotic and oxidative stresses, but exhibited decreased sensitivity to the fungicides iprodione and fludioxonil. In addition, the mutant revealed increased sensitivity to a biocontrol agent, Trichoderma atroviride. Western blot assays revealed that FgMkk1 positively regulates phosphorylation of the MAP kinases Mgv1 and FgOs-2, the key component in the cell wall integrity (CWI) and high-osmolarity glycerol (HOG) signalling pathway respectively. Yeast two-hybrid assay indicated that Mgv1 interacts with a transcription factor FgRlm1. The FgRLM1 mutant (ΔFgRLM1) showed increased sensitivity to cell wall-damaging agents and exhibited decreased virulence. Taken together, our data indicated that FgMkk1 is an upstream component of Mgv1, and regulates vegetative differentiation, multiple stress response and virulence via the CWI and HOG signalling pathways. FgRlm1 may be a downstream component of Mgv1 in the CWI pathway in F. graminearum.

Asunto(s)

Proteínas Fúngicas/genética , Fusarium/patogenicidad , Regulación Fúngica de la Expresión Génica , Hifa/patogenicidad , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Agentes de Control Biológico , Pared Celular/genética , Pared Celular/metabolismo , Proteínas Fúngicas/metabolismo , Fungicidas Industriales , Fusarium/efectos de los fármacos , Fusarium/genética , Fusarium/metabolismo , Eliminación de Gen , Glicerol/metabolismo , Hifa/efectos de los fármacos , Hifa/genética , Hifa/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Concentración Osmolar , Presión Osmótica , Fosforilación , Enfermedades de las Plantas/microbiología , Transducción de Señal , Trichoderma/patogenicidad , Triticum/microbiología , Virulencia

RESUMEN

The plant cell wall is composed of a matrix of cellulose fibers, flexible pectin polymers, and an array of assorted carbohydrates and proteins. The receptor-like Wall-Associated Kinases (WAKs) of Arabidopsis bind pectin in the wall, and are necessary both for cell expansion during development and for a response to pathogens and wounding. Mitogen Activated Protein Kinases (MPKs) form a major signaling link between cell surface receptors and both transcriptional and enzyme regulation in eukaryotes, and Arabidopsis MPK6 and MPK3 indeed have important roles in development and the response to stress and pathogens. A dominant allele of WAK2 requires kinase activity and activates a stress response that includes an increased ROS accumulation and the up-regulation of numerous genes involved in pathogen resistance, wounding, and cell wall biogenesis. This dominant allele requires a functional pectin binding and kinase domain, indicating that it is engaged in a WAK signaling pathway. A null mutant of the major plasma membrane ROS-producing enzyme complex, rbohd/f does not suppress the WAK2cTAP-induced phenotype. A mpk6, but not a mpk3, null allele is able to suppress the effects of this dominant WAK2 mutation, thus distinguishing MPK3 and MPK6, whose activity previously was thought to be redundant. Pectin activation of gene expression is abated in a wak2-null, but is tempered by the WAK-dominant allele that induces elevated basal stress-related transcript levels. The results suggest a mechanism in which changes to the cell wall can lead to a large change in cellular responses and help to explain how pathogens and wounding can have general effects on growth.

Asunto(s)

Alelos , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Proteínas Quinasas/genética , Estrés Fisiológico/genética , Arabidopsis/enzimología , Arabidopsis/metabolismo , Eliminación de Gen , Glucósidos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/deficiencia , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

The cell wall integrity (CWI) pathway, best characterized in S. cerevisiae, is strikingly conserved in Aspergillus species. We analyzed the importance of AfMkk2, a CWI signaling kinase, for virulence and antifungal therapy in the human pathogen A. fumigatus. A mutant lacking AfMkk2 is less adherent to glass and plastic surfaces and shows increased sensitivity to alkaline pH stress and antifungals. Rather than AfMpkA, the target kinase of AfMkk2, AfMpkB is activated in the mutant under cell wall stress. Interestingly, the mutant lacking AfMkk2 shows an enhanced sensitivity to posaconazole and voriconazole. And in agreement with its sensitivity to moderate temperatures, it is less virulent in a murine infection model. Our data underline the importance of mkk2 for the fitness, but also for the pathogenicity of A. fumigatus.

Asunto(s)

Aspergillus fumigatus/enzimología , Aspergillus fumigatus/patogenicidad , Pared Celular/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Transducción de Señal , Factores de Virulencia/metabolismo , Animales , Antifúngicos/farmacología , Aspergilosis/microbiología , Aspergilosis/mortalidad , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/crecimiento & desarrollo , Adhesión Celular , Modelos Animales de Enfermedad , Vidrio , Humanos , Concentración de Iones de Hidrógeno , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Plásticos , Pirimidinas/farmacología , Análisis de Supervivencia , Temperatura , Triazoles/farmacología , Virulencia , Factores de Virulencia/deficiencia , Voriconazol

RESUMEN

The mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate (cAMP) signal transduction pathways have critical roles in the consolidation of hippocampus-dependent memory. We found that extracellular regulated kinase 1/2 MAPK phosphorylation and cAMP underwent a circadian oscillation in the hippocampus that was paralleled by changes in Ras activity and the phosphorylation of MAPK kinase and cAMP response element-binding protein (CREB). The nadir of this activation cycle corresponded with severe deficits in hippocampus-dependent fear conditioning under both light-dark and free-running conditions. Circadian oscillations in cAMP and MAPK activity were absent in memory-deficient transgenic mice that lacked Ca2+ -stimulated adenylyl cyclases. Furthermore, physiological and pharmacological interference with oscillations in MAPK phosphorylation after the cellular memory consolidation period impaired the persistence of hippocampus-dependent memory. These data suggest that the persistence of long-term memories may depend on reactivation of the cAMP/MAPK/CREB transcriptional pathway in the hippocampus during the circadian cycle.

Asunto(s)

Ritmo Circadiano/fisiología , AMP Cíclico/metabolismo , Regulación de la Expresión Génica/fisiología , Hipocampo/metabolismo , Memoria/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Adenilil Ciclasas/metabolismo , Análisis de Varianza , Animales , Aprendizaje por Asociación/efectos de los fármacos , Aprendizaje por Asociación/fisiología , Butadienos/farmacología , Proteína de Unión a CREB/metabolismo , Calcio/administración & dosificación , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Clásico/fisiología , AMP Cíclico/deficiencia , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Miedo , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/anatomía & histología , Hipocampo/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Actividad Motora/fisiología , Nitrilos/farmacología , Transducción de Señal/fisiología , Estadísticas no Paramétricas , Factores de Tiempo

RESUMEN

MEK/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase signaling is imperative for proper chemotaxis. Dictyostelium mek1(-) (MEK1 null) and erk1(-) cells exhibit severe defects in cell polarization and directional movement, but the molecules responsible for the mek1(-) and erk1(-) chemotaxis defects are unknown. Here, we describe a novel, evolutionarily conserved gene and protein (smkA and SMEK, respectively), whose loss partially suppresses the mek1(-) chemotaxis phenotypes. SMEK also has MEK1-independent functions: SMEK, but not MEK1, is required for proper cytokinesis during vegetative growth, timely exit from the mound stage during development, and myosin II assembly. SMEK localizes to the cell cortex through an EVH1 domain at its N terminus during vegetative growth. At the onset of development, SMEK translocates to the nucleus via a nuclear localization signal (NLS) at its C terminus. The importance of SMEK's nuclear localization is demonstrated by our findings that a mutant lacking the EVH1 domain complements SMEK deficiency, whereas a mutant lacking the NLS does not. Microarray analysis reveals that some genes are precociously expressed in mek1(-) and erk1(-) cells. The misexpression of some of these genes is suppressed in the smkA deletion. These data suggest that loss of MEK1/ERK1 signaling compromises gene expression and chemotaxis in a SMEK-dependent manner.

Asunto(s)

Polaridad Celular , Quimiotaxis , Regulación de la Expresión Génica/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Núcleo Celular/metabolismo , Secuencia Conservada , Dictyostelium/citología , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/química , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Datos de Secuencia Molecular , Mutación/genética , Miosinas/metabolismo , Fenotipo , Transporte de Proteínas , Alineación de Secuencia

RESUMEN

The multisite phosphorylation of the transcription factor ATF-2 was investigated using transformed embryonic fibroblasts from wild-type mice and mice deficient in c-Jun N-terminal kinases (JNK)1 and 2, and in the presence and absence of inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) and the classical MAP kinase cascade. In wild-type cells, p38 MAPK and extracellular signal-regulated protein kinase (ERK)1/2 were not rate limiting for the phosphorylation of Thr69, Thr71 or Ser90. In JNK-deficient cells, p38 MAPK substituted for JNK partially in the phosphorylation of Thr69 and p38 MAPK or ERK1/2 in the phosphorylation of Thr71. JNK was the only MAP kinase that phosphorylated Ser90 under the conditions examined.

Asunto(s)

Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 2 , Animales , Anisomicina/farmacología , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , MAP Quinasa Quinasa 7 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Noqueados , Proteína Quinasa 8 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/deficiencia , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.

Asunto(s)

Mesangio Glomerular/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Tirosina Quinasas/fisiología , Factor de Crecimiento Transformador beta/farmacología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Células Cultivadas , Colágeno/genética , Activación Enzimática/efectos de los fármacos , Fibronectinas/genética , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Mesangio Glomerular/enzimología , Humanos , Cinética , MAP Quinasa Quinasa 3 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 13 Activada por Mitógenos , Proteína Quinasa 14 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Mutación , Isoformas de Proteínas/análisis , Proteínas Tirosina Quinasas/deficiencia , ARN Mensajero/análisis , Proteínas Recombinantes , Transfección , Factor de Crecimiento Transformador beta1 , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/genética

RESUMEN

During the development of multicellular organisms, concerted actions of molecular signalling networks determine whether cells undergo proliferation, differentiation, death or ageing. Here we show that genetic inactivation of the stress signalling kinase, MKK7, a direct activator of JNKs in mice, results in embryonic lethality and impaired proliferation of hepatocytes. Beginning at passage 4-5, mkk7(-/-) mouse embryonic fibroblasts (MEFs) display impaired proliferation, premature senescence and G2/M cell cycle arrest. Similarly, loss of c-Jun or expression of a c-JunAA mutant in which the JNK phosphorylation sites were replaced with alanine results in a G2/M cell-cycle block. The G2/M cell-cycle kinase CDC2 was identified as a target for the MKK7-JNK-c-Jun pathway. These data show that the MKK7-JNK-c-Jun signalling pathway couples developmental and environmental cues to CDC2 expression, G2/M cell cycle progression and cellular senescence in fibroblasts.

Asunto(s)

Senescencia Celular/genética , Fase G2/genética , Hepatocitos/enzimología , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Mitosis/genética , Estrés Fisiológico/enzimología , Animales , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Feto , Fibroblastos/citología , Fibroblastos/enzimología , Genes Letales/genética , Hepatocitos/citología , Proteínas Quinasas JNK Activadas por Mitógenos , Hígado/anomalías , Hígado/patología , MAP Quinasa Quinasa 7 , Sistema de Señalización de MAP Quinasas/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación/genética , Fosforilación , Proteínas Proto-Oncogénicas c-jun/deficiencia , Proteínas Proto-Oncogénicas c-jun/genética , Estrés Fisiológico/genética

RESUMEN

The p38 mitogen-activated protein kinase (MAPK) is activated in vitro by three different protein kinases: MKK3, MKK4, and MKK6. To examine the relative roles of these protein kinases in the mechanism of p38 MAP kinase activation in vivo, we examined the effect of disruption of the murine Mkk3, Mkk4, and Mkk6 genes on the p38 MAPK signaling pathway. We show that MKK3 and MKK6are essential for tumor necrosis factor-stimulated p38 MAPK activation. In contrast, ultraviolet radiation-stimulated p38 MAPK activation was mediated by MKK3, MKK4, and MKK6. Loss of p38 MAPK activation in the mutant cells was associated with defects in growth arrest and increased tumorigenesis. These data indicate that p38 MAPK is regulated by the coordinated and selective actions of three different protein kinases in response to cytokines and exposure to environmental stress.

Asunto(s)

Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Activación Enzimática/efectos de la radiación , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Masculino , Ratones , Ratones Noqueados , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/efectos de la radiación , Mutación , Isoformas de Proteínas/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/efectos de la radiación , Transactivadores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Rayos Ultravioleta/efectos adversos , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

During its life cycle, the parasitic protozoon Leishmania mexicana differentiates from a flagellated form, the promastigote, to an amastigote form carrying a rudimentary flagellum. Besides biochemical changes, this process involves a change in overall cell morphology including flagellar shortening. A mitogen-activated protein kinase kinase homologue designated LmxMKK was identified in a homology screening and found to be critically involved in the regulation of flagellar assembly and cell size. LmxMKK is exclusively expressed in the promastigote stage and is likely to be regulated by posttranslational mechanisms such as phosphorylation. A deletion mutant for the single-copy gene revealed motile flagella dramatically reduced in length and lacking the paraflagellar rod, a structure adjacent to the axoneme in kinetoplastid flagella. Moreover, a fraction of the cells showed perturbance of the axonemal structure. Complementation of the deletion mutant with the wild-type gene restored typical promastigote morphology. We propose that LmxMKK influences anterograde intraflagellar transport to maintain flagellar length in Leishmania promastigotes; as such, it is the first protein kinase known to be involved in organellar assembly.

Asunto(s)

Flagelos/enzimología , Leishmania mexicana/enzimología , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Proteínas Protozoarias/fisiología , Animales , Movimiento Celular/genética , Tamaño de la Célula/genética , Células Cultivadas , ADN Complementario/análisis , ADN Complementario/genética , Flagelos/ultraestructura , Regulación de la Expresión Génica/genética , Leishmania mexicana/ultraestructura , Ratones , Microscopía Electrónica , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/aislamiento & purificación , Datos de Secuencia Molecular , Mutación/genética , Fosforilación , Procesamiento Proteico-Postraduccional/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico

RESUMEN

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Tirosina Quinasas/genética , Animales , Fertilidad/genética , Marcación de Gen , Ingeniería Genética/métodos , Activación de Linfocitos/genética , MAP Quinasa Quinasa 2 , Ratones , Ratones Mutantes , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/metabolismo , Recombinación Genética , Linfocitos T/fisiología , Timo/citología , Timo/fisiología

RESUMEN

The ischemic activation of p38alpha mitogen-activated protein kinase (p38alpha-MAPK) is thought to contribute to myocardial injury. Under other circumstances, activation is through dual phosphorylation by MAPK kinase 3 (MKK3). Therefore, the mkk3-/- murine heart should be protected during ischemia. In retrogradely perfused mkk3-/- and mkk3+/+ mouse hearts subjected to 30 minutes of global ischemia and 120 minutes of reperfusion, infarction/risk volume was similar (50+/-5 versus 51+/-4, P=0.93, respectively), as was intraischemic p38-MAPK phosphorylation (10 minutes ischemia as percent basal, 608+/-224 versus 384+/-104, P=0.43, respectively). This occurred despite undetectable activation of MKK3/6 in mkk3-/- hearts. However, tumor necrosis factor (TNF)-induced p38-MAPK phosphorylation was markedly diminished in mkk3-/- vs mkk3+/+ hearts (percent basal, 127+/-23 versus 540+/-267, respectively, P=0.04), suggesting an MKK-independent activation mechanism by ischemia. Hence, we examined p38-MAPK activation by TAB1-associated autophosphorylation. In wild-type mice and mkk3-/- mice, the p38-MAPK catalytic site inhibitor SB203580 (1 micromol/L) diminished phosphorylation during ischemia versus control (10 minutes ischemia as percent basal, 143+/-2 versus 436+/-96, P=0.003, and 122+/-25 versus 623+/-176, P=0.05, respectively) and reduced infarction volume (infarction/risk volume, 57+/-5 versus 36+/-3, P<0.001, and 50+/-5 versus 29+/-3, P=0.003, respectively) but did not alter TNF-induced activation, although in homogenates of ischemic hearts but not TNF-exposed hearts, p38-MAPK was associated with TAB1. Furthermore, adenovirally expressed wild-type and drug-resistant p38alpha-MAPK, lacking the SB203580 binding site, was phosphorylated when H9c2 myoblasts were subjected to simulated ischemia. However, SB203580 (1 micromol/L) did not prevent the phosphorylation of resistant p38alpha-MAPK. These findings suggest the ischemic activation of p38-MAPK contributing to myocardial injury is by TAB1-associated autophosphorylation.

Asunto(s)

Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Isquemia Miocárdica/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Células Cultivadas , Progresión de la Enfermedad , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Técnicas In Vitro , MAP Quinasa Quinasa 3 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Mioblastos/citología , Mioblastos/metabolismo , Infarto del Miocardio/etiología , Infarto del Miocardio/patología , Isquemia Miocárdica/complicaciones , Isquemia Miocárdica/patología , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

OBJECTIVE: We investigated the comparative roles of mitogen-activated protein (MAP) kinases, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38, in vascular smooth muscle cell (VSMC) proliferation, migration, and gene expression. METHODS AND RESULTS: VSMCs were infected with recombinant adenovirus containing dominant-negative mutants of ERK, p38, and JNK (Ad-DN-ERK, Ad-DN-p38, and Ad-DN-JNK, respectively) to specifically inhibit the respective MAP kinases and then stimulated with platelet-derived growth factor (PDGF)-BB. Ad-DN-ERK attenuated PDGF-BB-induced VSMC proliferation more potently than Ad-DN-p38 or Ad-DN-JNK, indicating the dominant role of ERK in VSMC proliferation. Ad-DN-ERK, Ad-DN-p38, and Ad-DN-JNK similarly inhibited PDGF-induced VSMC migration. Ad-DN-ERK and Ad-DN-JNK suppressed PDGF-BB-induced downregulation of cyclin-dependent kinase inhibitor p27Kip1, whereas Ad-DN-p38 decreased PDGF-BB-induced upregulation of p21Cip1. Ad-DN-ERK inhibited PDGF-BB-induced plasminogen activator inhibitor type-1 (PAI-1), monocyte chemoattractant protein-1, and transforming growth factor-beta1 expressions, Ad-DN-p38 blocked monocyte chemoattractant protein-1 and transforming growth factor-beta1 expression but not PAI-1, whereas Ad-DN-JNK suppressed only PAI-1 expression. Moreover, in vivo gene transfer of Ad-DN-p38 to rat carotid artery caused the inhibition of intimal hyperplasia by balloon injury, indicating the involvement of p38 in vascular remodeling in vivo. CONCLUSIONS: We propose that these 3 MAP kinases participate in vascular diseases via differential molecular mechanisms and are new therapeutic targets for treatment of vascular diseases.

Asunto(s)

Proteínas Quinasas JNK Activadas por Mitógenos , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Músculo Liso Vascular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Animales , Becaplermina , Traumatismos de las Arterias Carótidas/patología , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Ciclinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hiperplasia , MAP Quinasa Quinasa 4 , Masculino , Proteína Quinasa 1 Activada por Mitógenos/deficiencia , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/deficiencia , Proteínas Quinasas Activadas por Mitógenos/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/enzimología , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Inhibidor 1 de Activador Plasminogénico/genética , Proteínas Proto-Oncogénicas c-sis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/fisiología , Transducción Genética , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1 , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , Túnica Íntima/patología , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

Elevated levels of mitogen-activated protein kinase/extracellular regulatory kinase (MAPK/ERK) activity are frequently found in some cancer cells. In efforts to reduce tumor growth, attempts have been made to develop cancer therapeutic agents targeting the MAPK. Here, by use of biologic, biochemical, and gene manipulation methods in human polymorphonuclear neutrophils (PMNs), we have identified a key pathway important in normal cell function involving MAPK/ERK in PMNs for growth inhibition of Candida albicans. Contact with C albicans triggered MAPK/ERK activation in PMNs within 5 minutes, and blocking of MAPK/ERK activation, either by the pharmacologic reagent PD098059 or by dominant-negative MAPK kinase (MEK) expression via vaccinia viral delivery, suppressed antimicrobial activity. Rac and Cdc42, but not Ras or Rho, were responsible for this MAPK/ERK activation. Expression of dominant-negative Rac (N17Rac) or Cdc42 (N17Cdc42) eliminated not only C albicans- mediated ERK phosphorylation but also phagocytosis and granule migration toward the ingested microbes, whereas dominant-negative Ras (N17Ras) and Rho (N19Rho) did not. PAK1 (p21-activated kinase 1) activation is induced by C albicans, suggesting that PAK1 may also be involved in the Rac1 activation of MAPK/ERK. We conclude from these data that Rac/Cdc42-dependent activation of MAPK/ERK is a critical event in the immediate phagocytic response of PMNs to microbial challenge. Therefore, use of MAPK pharmacologic inhibitors for the treatment of cancer may result in the interruption of normal neutrophil function. A balance between therapeutic outcome and undesirable side effects must be attained to achieve successful and safe anticancer therapy.

Asunto(s)

Gránulos Citoplasmáticos/fisiología , Sistema de Señalización de MAP Quinasas , Neutrófilos/fisiología , Fagocitosis/fisiología , Proteína de Unión al GTP cdc42/fisiología , Proteína de Unión al GTP rac1/fisiología , Candida albicans , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Genes Dominantes , Humanos , MAP Quinasa Quinasa 1 , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/fisiología , Transducción Genética , Proteína de Unión al GTP cdc42/genética , Quinasas p21 Activadas , Proteína de Unión al GTP rac1/genética , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/fisiología

RESUMEN

Mice lacking the stress-signaling kinase SEK1 die from embryonic day 10.5 (E10.5) to E12.5. Although a defect in liver formation is accompanied with the embryonic lethality of sek1(-/-) mice, the mechanism of the liver defect has remained unknown. In the present study, we first produced a monoclonal antibody specifically recognizing murine hepatoblasts for the analysis of liver development and further investigated genetic interaction ofsek1 with tumor necrosis factor-alpha receptor 1 gene (tnfr1) and protooncogene c-jun, which are also responsible for liver formation and cell apoptosis. The defective liver formation in sek1(-/-) embryos was not protected by additionaltnfr1 mutation, which rescues the embryonic lethality of mice lacking NF-kappaB signaling components. There was a progressive increase in the hepatoblast cell numbers of wild-type embryos from E10.5 to E12.5. Instead, impaired hepatoblast proliferation was observed in sek1(-/-) livers from E10.5, though fetal liver-specific gene expression was normal. The impaired phenotype in sek1(-/-) livers was more severe than in c-jun(-/-) embryos, and sek1(-/-) c-jun(-/-) embryos died more rapidly before E8.5. The hepatoblast proliferation required no hematopoiesis, since liver development was not impaired in AML1(-/-) mice that lack hematopoietic functions. Stimulation of stress-activated protein kinase/c-Jun N-terminal kinase by hepatocyte growth factor was attenuated in sek1(-/-) livers. Thus, SEK1 appears to play a crucial role in hepatoblast proliferation and survival in a manner apparently different from NF-kappaB or c-Jun.

Asunto(s)

Hígado/embriología , MAP Quinasa Quinasa 4 , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Animales , Anticuerpos Monoclonales , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptosis , Biomarcadores , Recuento de Células , División Celular , Activación Enzimática/efectos de los fármacos , Femenino , Expresión Génica , Hematopoyesis , Factor de Crecimiento de Hepatocito/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos , Hígado/citología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/deficiencia , Proteínas Quinasas Activadas por Mitógenos/genética , Modelos Biológicos , Embarazo , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo

RESUMEN

Borrelia burgdorferi, the Lyme disease agent, causes joint inflammation in an experimental murine model. Inflammation occurs, in part, due to the ability of B. burgdorferi to induce the production of proinflammatory cytokines and a strong CD4(+) T helper type 1 response. The mechanisms by which spirochetes induce these responses are not completely known, although transcription factors, such as NF-kappa B in phagocytic cells, initiate the proinflammatory cytokine burst. We show here that the mitogen-activated protein (MAP) kinase of 38 kDa (p38 MAP kinase) is involved in the proinflammatory cytokine production elicited by B. burgdorferi Ags in phagocytic cells and the development of murine Lyme arthritis. B. burgdorferi Ags activated p38 MAP kinase in vitro, and the use of a specific inhibitor repressed the spirochete-induced production of TNF-alpha. The infection of mice that are deficient for a specific upstream activator of the kinase, MAP kinase kinase 3, resulted in diminished proinflammatory cytokine production and the development of arthritis, without compromising the ability of CD4(+) T cells to respond to borrelial Ags or the production of specific Abs. Overall, these data indicated that the p38 MAP kinase pathway plays an important role in B. burgdorferi-elicited inflammation and point to potential new therapeutic approaches to the treatment of inflammation induced by the spirochete.

Asunto(s)

Artritis Infecciosa/enzimología , Artritis Infecciosa/etiología , Borrelia burgdorferi/inmunología , Enfermedad de Lyme/enzimología , Enfermedad de Lyme/etiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Animales , Antígenos Bacterianos/inmunología , Artritis Infecciosa/genética , Artritis Infecciosa/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/microbiología , Línea Celular , Activación Enzimática/genética , Activación Enzimática/inmunología , Inflamación/enzimología , Inflamación/etiología , Inflamación/genética , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interferón gamma/metabolismo , Enfermedad de Lyme/genética , Enfermedad de Lyme/inmunología , MAP Quinasa Quinasa 3 , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fagocitos/inmunología , Fagocitos/metabolismo , Fagocitos/microbiología , Fosforilación , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , Receptores de Interferón/antagonistas & inhibidores , Receptores de Interferón/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos , Receptor de Interferón gamma

RESUMEN

We investigated the potential of mouse embryonic stem (ES) cells to differentiate into hepatocytes in vitro. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, transthyretin, alpha 1-anti-trypsin and albumin, when cultured without additional growth factors and late differential markers of hepatic development, such as tyrosine aminotransferase (TAT) and glucose-6-phosphatase (G6P), when cultured in the presence of growth factors critical for late embryonic liver development. Further, induction of TAT and G6P expression was induced regardless of expression of the functional SEK1 gene, which is thought to provide a survival signal for hepatocytes during an early stage of liver morphogenesis. The data indicate that the in vitro ES differentiation system has a potential to generate mature hepatocytes. The system has also been found useful in analyzing the role of growth factors and intracellular signaling molecules in hepatic development.

Asunto(s)

Hepatocitos/citología , Hígado/citología , Hígado/embriología , MAP Quinasa Quinasa 4 , Células Madre/citología , Células Madre/metabolismo , Animales , Antígenos de Diferenciación/biosíntesis , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Sustancias de Crecimiento/farmacología , Hepatocitos/efectos de los fármacos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/deficiencia , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , ARN Mensajero/metabolismo , Células Madre/efectos de los fármacos