RESUMEN
The Hippo pathway fulfills a crucial function in controlling the balance between proliferation, differentiation and apoptosis in cells. Recent studies showed that G protein-coupled receptors (GPCRs) serve as upstream regulators of Hippo signaling, that either activate or inactivate the Hippo pathway via the large tumor suppressor kinase (LATS) and its substrate, the co-transcription factor Yes-associated protein (YAP). In this study, we focused on the Angiotensin II type 1 receptor (AT1R), which belongs to the GPCR family and has an essential role in the control of blood pressure and water homeostasis. We found that Angiotensin II (Ang II) inactivates the pathway by decreasing the activity of LATS kinase; therefore, leading to an enhanced nuclear shuttling of unphosphorylated YAP in HEK293T cells. This shuttling of YAP is actin-dependent as disruption of the actin cytoskeleton inhibited dephosphorylation of LATS and YAP. Interestingly, in contrast to HEK293T cells, podocytes, which are a crucial component of the glomerular filtration barrier, display a predominant nuclear YAP localization in vivo and in vitro. Moreover, stimulation with Ang II did not alter Hippo pathway activity in podocytes, which show a deactivated pathway. Reactivation of the LATS kinase activity in podocytes resulted in an increased cytoplasmic YAP localization accompanied by a strong induction of apoptosis. Thus, our work indicates that the control of LATS activation and subsequent YAP localization is important for podocyte homeostasis and survival.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Fosfoproteínas/genética , Podocitos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Angiotensina II/farmacología , Animales , Apoptosis/genética , Línea Celular Transformada , Supervivencia Celular , Regulación de la Expresión Génica , Células HEK293 , Vía de Señalización Hippo , Homeostasis , Humanos , Ratones , Ratones Transgénicos , Especificidad de Órganos , Fosfoproteínas/metabolismo , Fosforilación , Podocitos/citología , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Transducción de Señal , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
The human KIBRA gene has been linked to human cognition through a lead intronic single-nucleotide polymorphism (SNP; rs17070145) that is associated with episodic memory performance and the risk to develop Alzheimer's disease. However, it remains unknown how this relates to the function of the KIBRA protein. Here, we identified two common missense SNPs (rs3822660G/T [M734I], rs3822659T/G [S735A]) in exon 15 of the human KIBRA gene to affect cognitive performance, and to be in almost complete linkage disequilibrium with rs17070145. The identified SNPs encode variants of the KIBRA C2 domain with distinct Ca(2+) dependent binding preferences for monophosphorylated phosphatidylinositols likely due to differences in the dynamics and folding of the lipid-binding pocket. Our results further implicate the KIBRA protein in higher brain function and provide direction to the cellular pathways involved.
Asunto(s)
Cognición/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación Missense/genética , Fosfatidilinositoles/metabolismo , Fosfoproteínas/genética , Exones/genética , Exones/fisiología , Femenino , Genotipo , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Desequilibrio de Ligamiento/genética , Desequilibrio de Ligamiento/fisiología , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Masculino , Persona de Mediana Edad , Mutación Missense/fisiología , Pruebas Neuropsicológicas , Fosfoproteínas/fisiología , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
LASP-1 is a multidomain protein predominantly localized at focal contacts, where it regulates cytoskeleton dynamics and cell migration. However, in different tumor entities, a nuclear LASP-1 accumulation is observed, thought to have an important role in cancer progression. Until now, the molecular mechanisms that control LASP-1 nuclear import were not elucidated. Here, we identified a novel LASP-1-binding partner, zona occludens protein 2 (ZO-2), and established its role in the signal transduction pathway of LASP-1 nucleo-cytoplasmatic shuttling. Phosphorylation of LASP-1 by PKA at serine 146 induces translocation of the LASP-1/ZO-2 complex from the cytoplasm to the nucleus. Interaction occurs within the carboxyterminal proline-rich motif of ZO-2 and the SH3 domain in LASP-1. In situ proximity ligation assay confirmed the direct binding between LASP-1 and ZO-2 and visualized the shuttling. Nuclear export is mediated by Crm-1 and a newly identified nuclear export signal in LASP-1. Finally, dephosphorylation of LASP-1 by phosphatase PP2B is suggested to relocalize the protein back to focal contacts. In summary, we define a new pathway for LASP-1 in tumor progression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteína de la Zonula Occludens-2/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas del Citoesqueleto/biosíntesis , Humanos , Proteínas con Dominio LIM/biosíntesis , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
The rs17070145 polymorphism (C â T substitution, intron 9) of the KIBRA gene has recently been associated with episodic memory and cognitive flexibility. These findings were inconsistent across reports though, and largely lacked gene-gene or gene-environment interactions. The aim of the present study was to determine the impact of the rs17070145 polymorphism on clinically relevant cognitive domains and its interaction with the modifiers 'lifestyle' and 'cardiovascular risk factors'. Five-hundred forty-five elderly volunteers (mean age 64 years, ±7 years, 56% women) accomplished a comprehensive cognitive testing. Principal component analysis was used to reveal the internal structure of the data, rendering four composite scores: verbal memory, word fluency, executive function/psychomotor speed, and working memory. Lifestyle was assessed with a detailed questionnaire, age-associated risk factors by clinical interview and examination. There was no main effect of the rs17070145 genotype on any cognitive composite scores. However, we found worse performance in executive functions for T-allele carriers in the presence of arterial hypertension (ß=-0.365, p=0.0077 and 0.031 after Bonferroni correction). This association was further modified by gender, showing the strongest association in hypertensive females (ß=-0.500, p=0.0072 and 0.029 after Bonferroni correction). The effect of KIBRA on cognitive function seems to be complex and modified by gender and arterial hypertension.
Asunto(s)
Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/genética , Trastornos del Conocimiento/epidemiología , Trastornos del Conocimiento/genética , Hipertensión/epidemiología , Hipertensión/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoproteínas/genética , Anciano , Cognición/fisiología , Estudios de Cohortes , Comorbilidad/tendencias , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The ability of the mammalian brain to store and recall information is based on synaptic plasticity due to constant remodeling of synaptic contacts. Although various classes of proteins such as neurotransmitter receptors, cytoskeletal components and protein kinases were already identified as modulators of memory formation, their specific interactions and crosstalks are still poorly understood. Genetic variants of the scaffolding protein KIBRA (kidney brain) a substrate of the memory-related protein kinase C zeta and component of the neuronal cytoskeleton, were recently shown to be associated with human memory performance. However, the function of KIBRA on the cellular and physiological level is still unclear. To gain more insights into the temporal and spatial expression of KIBRA, we performed in situ hybridization assays and immunohistological staining of human and rodent (rat) brain. Our data demonstrate that KIBRA is mainly expressed in memory-related regions of the brain (hippocampus, cortex) but is also found in the cerebellum and the hypothalamus. In primary hippocampal neurons, KIBRA displays a somatodendritic distribution and an enrichment at the postsynaptic density. Binding studies further show that KIBRA is able to form head-to-tail homodimers and that dimerization is mediated by the internal C2-like domain. Our data indicate that KIBRA is involved in brain development and memory formation as a postsynaptic scaffold protein connecting cytoskeletal and signaling molecules.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Expresión Génica/fisiología , Proteínas/metabolismo , Animales , Animales Recién Nacidos , Línea Celular Transformada , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Intercambio de Guanina Nucleótido , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Masculino , Persona de Mediana Edad , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas , Proteínas/genética , ARN Mensajero/metabolismo , Ratas , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The gene encoding brain-specific dendritic BC200 small non-messenger RNA is limited to the primate order and arose from a monomeric Alu element. It is present and neuronally expressed in all Anthropoidea examined. By comparing the human sequence of about 13.2 kb with each of the prosimian (lemur 14.6 kb, galago 12 kb, and tarsier 13.8 kb) orthologous loci, we could establish that the BC200 RNA gene is absent from the prosimian lineages. In Strepsirhini (lemurs and lorises), a dimeric AluJ-like element integrated very close to the BC200 insertion point, while the corresponding tarsier region is devoid of any repetitive element. Consequently, insertion of the Alu monomer that gave rise to the BC200 RNA gene must have occurred after the anthropoid lineage diverged from the prosimian lineage(s). Shared insertions of other repetitive elements favor proximity of simians and tarsiers in support of their grouping into Haplorhini and the omomyid hypothesis. On the other hand, the nucleotide sequences in the segment that is available for comparison in all four species reveal less exchanges between Strepsirhini (lemur and galago) and human than between tarsier and human. Our data imply that the early activity of dimeric Alu sequences must have been concurrent with the activity of monomeric Alu elements that persisted longer than is usually thought. As BC200 RNA gave rise to more than 200 pseudogenes, we used their consensus sequence variations as a molecular archive recording the BC200 RNA sequence changes in the anthropoid lineage leading to Homo sapiens and timed these alterations over the past 35-55 million years.
Asunto(s)
Neuronas/metabolismo , Filogenia , Primates/genética , Seudogenes/genética , ARN/genética , Elementos Alu/genética , Animales , Secuencia de Bases , Encéfalo/metabolismo , Secuencia de Consenso/genética , Dendritas/metabolismo , Genoma , Hominidae/genética , Humanos , Datos de Secuencia Molecular , Mutación/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Idiopathic generalized epilepsy (IGE) comprises a heterogeneous group of disorders, in which a high genetic predisposition and a complex mode of inheritance have been suggested. However, genes, which confer liability to common IGE subtypes including juvenile myoclonic epilepsy (JME) and childhood absence epilepsy (CAE) have not been identified so far. Here, we tested the hypothesis that genetic variation in the human homolog of the <<<
Asunto(s)
Proteínas del Citoesqueleto/genética , Epilepsia Generalizada/genética , Mutación , Proteínas del Tejido Nervioso/genética , Adulto , Secuencia de Bases , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Cromosomas Humanos Par 8 , Femenino , Genes Inmediatos-Precoces , Predisposición Genética a la Enfermedad , Variación Genética , Heterocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Polimorfismo Conformacional Retorcido-Simple , Análisis de SecuenciaRESUMEN
The gene encoding BC200 RNA arose from a monomeric Alu element. Subsequently, the RNA had been recruited or exapted into a function of the nervous system. Here we confirm the presence of the BC200 gene in several primate species among the Anthropoidea. The period following the divergence of New World monkeys and Old World monkeys from their common ancestor is characterized by a significantly higher substitution rate in the examined 5' flanking region than in the BC200 RNA coding region itself. Furthermore, the conservation of CpG dimers in the RNA coding region (200 bp) is drastically increased compared to the 5' flanking region (approximately 400 bp) over all 12 species examined. Finally, the brain-specific expression pattern of BC200 RNA and its presence as a ribonucleoprotein particle (RNP) are conserved in Old World and New World monkeys. Our studies indicate that the gene encoding BC200 RNA was created at least 35-55 million years ago and its presence, mode of expression, and association with protein(s) as an RNP are under selective pressure.
Asunto(s)
Secuencia Conservada , Evolución Molecular , Haplorrinos/genética , ARN/genética , Animales , Secuencia de Bases , Encéfalo/metabolismo , Islas de CpG , Expresión Génica , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/química , Ribonucleoproteínas/genética , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Rodent BC1 RNA and primate BC200 RNA are small cytoplasmic non-messenger RNAs that are phylogenetically unrelated. Nevertheless, the two RNAs exhibit a large degree of parallelism. In addition to some sequence similarities in their 3' domains, they are prevalently expressed in a similar subset of neurons and belong to a small group of transcripts with a somatodendritic location. Both RNAs are complexed with proteins as ribonucleoprotein particles (RNPs). Their similarities may even extend to analogous functional roles, for example, in the regulation of decentralized dendritic translation. To shed further light on the physiological role(s) of the BC1/BC200 RNPs, we began to analyze protein components that specifically bind to these RNAs. Ultraviolet-crosslinking experiments and affinity purification techniques revealed that the human autoantigen La/SS-B is associated with BC1/BC200 RNA in vitro and in vivo. As with other RNA polymerase III transcripts, La protein binds with high affinity to the 3' end of BC200 RNA. Our results suggest that an additional function of La may be control of dendritic translation by providing a link between the 5' Alu domain of BC200 RNP and the ribosome via the La protein dimer. The fact that La binds both BC1 and BC200 RNAs further supports the notion that the RNAs are functional analogs despite the fact that they arose from two separate retroposition events in two different mammalian lineages.
Asunto(s)
Autoantígenos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Citoplasmáticas Pequeñas , Ribonucleoproteínas/metabolismo , Animales , Encéfalo , Extractos Celulares , Reactivos de Enlaces Cruzados , Femenino , Humanos , Unión Proteica , ARN/metabolismo , ARN Largo no Codificante , ARN no Traducido , Ratas , Ratas Sprague-Dawley , Rayos Ultravioleta , Antígeno SS-BRESUMEN
The 10Sa RNA gene of Thermus thermophilus was isolated and sequenced. The tRNA-like structure at the 5' and 3' ends and other secondary structure features of the T. thermophilus 10Sa RNA are similar to E. coli 10Sa RNA. A variant of the sequence motif coding for the tag peptide is located in the centre of T. thermophilus 10Sa RNA.
Asunto(s)
Genes Bacterianos , ARN Bacteriano/genética , Thermus thermophilus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligopéptidos/química , Operón , Regiones Promotoras Genéticas , ARN Bacteriano/química , ARN de Transferencia/química , ARN de Transferencia/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
We have established three new cell lines deriving from malignant human gliomas. The cell lines were described in terms of both morphology and growth characteristics. Most cells in all three cell lines expressed the neuroepithelial marker protein GFAP. In terms of growth characteristics, the cells showed only slight differences. The cell lines showed no expression of the neural form of the c-src gene, pp60c-srcN, but did express the ubiquitous form, pp60c-src. The established glioma cell lines were also examined for expression of members of the neuropoietic cytokine family, CNTF and LIF, and their respective receptor components CNTFRalpha, LIFRbeta and gp130. With the exception of CNTFRalpha both the ligands and their receptor components were expressed in similar amounts in all three cell lines. The presence of ligand and receptor prompted us to study the effects of exogenously supplied factors on the growth of the glioma cell lines. Whereas LIF induced a high c-fos expression, only low c-fos induction was observed upon CNTF treatment. Accordingly, CNTF did not have any noticeable effects on glioma cell growth in culture, while LIF mediated an inhibiting effect on the growth of the three glioma cell lines in culture.
Asunto(s)
Neoplasias Encefálicas/patología , Glioma/patología , Inhibidores de Crecimiento/farmacología , Interleucina-6 , Linfocinas/farmacología , Aneuploidia , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Neoplasias Encefálicas/química , División Celular , Aberraciones Cromosómicas , Factor Neurotrófico Ciliar , Receptor gp130 de Citocinas , Regulación Neoplásica de la Expresión Génica , Genes fos , Proteína Ácida Fibrilar de la Glía/biosíntesis , Proteína Ácida Fibrilar de la Glía/genética , Glioma/genética , Glioma/metabolismo , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/genética , Humanos , Cariotipificación , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Linfocinas/biosíntesis , Linfocinas/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Desnudos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas pp60(c-src)/biosíntesis , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Receptor de Factor Neurotrófico Ciliar , Receptores de Citocinas/biosíntesis , Receptores de Citocinas/genética , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Receptores de Factor de Crecimiento Nervioso/genética , Receptores OSM-LIF , Células Tumorales CultivadasRESUMEN
BC200 RNA is a brain-specific, small non-messenger RNA with a somatodendritic localization in primate neurons and a constituent of a ribonucleoprotein (RNP) complex. The primary and secondary structure of the 5' domain of BC200 RNA resembles that of the Alu domain of 7SL RNA, which is an integral part of the signal recognition particle (SRP). This would predict that similar proteins bind to this defined domain of both RNA species in vitro and in vivo. The data presented in this paper reveal that a protein that binds BC200 RNA in vivo is immunoreactive with antibodies against SRP9. This further supports the notion that the 5' domain of the BC200 RNA can fold into structures similar to the SRP Alu domain and, as a result, bind identical or similar proteins in vivo. The SRP9 protein binds only as dimer with SRP14 protein to the Alu domain of 7SL RNA to form a subdomain that, in SRP, is functional in translation arrest. Therefore, our data also indicate that the neuronal BC200 RNP is a candidate for regulating decentralized protein biosynthesis in dendrites, possibly with a mechanism that resembles translation arrest of the SRP.
Asunto(s)
Dendritas/química , Biosíntesis de Proteínas/fisiología , ARN Nuclear Pequeño/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/genética , Partícula de Reconocimiento de Señal/genética , Animales , Extractos Celulares/química , Dendritas/fisiología , Perros , Humanos , Conformación de Ácido Nucleico , Primates , Unión Proteica/fisiología , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/farmacología , Ribonucleoproteínas/metabolismo , Partícula de Reconocimiento de Señal/inmunología , Partícula de Reconocimiento de Señal/farmacologíaRESUMEN
We developed a rapid method for quantification of tyrosine phosphorylation in immunophenotypically defined cell populations in specimens of whole blood and unprocessed bone marrow. Samples were formaldehyde-fixed and cells were permeabilized. Phosphotyrosine residues and surface antigens were simultaneously stained by monoclonal antibodies and visualized by flow cytometry. The accuracy of the method was confirmed by demonstration of an increase of phosphotyrosine levels in pp60v-src transformed fibroblasts. In blood of healthy donors, monocytes and granulocytes showed higher levels of phosphotyrosine than lymphocytes. CD34+ peripheral blood stem cells showed slightly increased tyrosine phosphorylation compared to autologous lymphocytes. Significantly elevated levels of phosphotyrosine were demonstrated in leukaemic blasts compared to lymphocytes (P = 0.01).
Asunto(s)
Médula Ósea/metabolismo , Pruebas Hematológicas/métodos , Fosfotirosina/metabolismo , Enfermedad Aguda , Citometría de Flujo , Humanos , Leucemia Mieloide/diagnóstico , Fosforilación , Fosfotirosina/sangreRESUMEN
Through the development of a new colorimetric and non-radioactive method it is possible to measure PTPase activity in cell extracts. Applying this method we were able to demonstrate that intracellular PTPase activity in chicken embryo fibroblasts increases significantly after RSV induced transformation of these cells. PTPase activity in normal and transformed cells is inhibited by microM concentrations of vanadate, molybdate and zinc ion, but is not affected by mM concentrations of calcium, magnesium and sodium fluoride. The transformation specific activation of the PTPase seems to represent an early parameter during the events of cellular transformation.