RESUMEN
BACKGROUND: Inactivating mutations of SMAD4 are frequent in metastatic colorectal carcinomas. In previous analyses, we were able to show that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas in vitro cell growth was not affected. Using this cellular model system, we searched for new Smad4 targets comparing nuclear subproteomes derived from Smad4 re-expressing and Smad4 negative SW480 cells. METHODS: High resolution two-dimensional (2D) gel electrophoresis was applied to identify novel Smad4 targets in the nuclear subproteome of Smad4 re-expressing SW480 cells. The identified candidate protein Keratin 23 was further characterized by tandem affinity purification. Immunoprecipitation, subfractionation and immunolocalization studies in combination with RNAi were used to validate the Keratin 23-14-3-3ε interaction. RESULTS: We identified keratins 8 and 18, heat shock proteins 60 and 70, plectin 1, as well as 14-3-3ε and γ as novel proteins present in the KRT23-interacting complex. Co-immunoprecipitation and subfractionation analyses as well as immunolocalization studies in our Smad4-SW480 model cells provided further evidence that KRT23 associates with 14-3-3ε and that Smad4 dependent KRT23 up-regulation induces a shift of the 14-3-3ε protein from a nuclear to a cytoplasmic localization. CONCLUSION: Based on our findings we propose a new regulatory circuitry involving Smad4 dependent up-regulation of KRT23 (directly or indirectly) which in turn modulates the interaction between KRT23 and 14-3-3ε leading to a cytoplasmic sequestration of 14-3-3ε. This cytoplasmic KRT23-14-3-3 interaction may alter the functional status of the well described 14-3-3 scaffold protein, known to regulate key cellular processes, such as signal transduction, cell cycle control, and apoptosis and may thus be a previously unappreciated facet of the Smad4 tumor suppressive circuitry.
Asunto(s)
Proteínas 14-3-3/metabolismo , Queratinas Tipo I/metabolismo , Proteína Smad4/metabolismo , Proteínas 14-3-3/genética , Western Blotting , Línea Celular Tumoral , Núcleo Celular/metabolismo , Chaperonina 60/metabolismo , Citoplasma/metabolismo , Electroforesis en Gel Bidimensional , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Inmunoprecipitación , Queratina-18/metabolismo , Queratina-8/metabolismo , Queratinas Tipo I/genética , Microscopía Confocal , Plectina/metabolismo , Unión Proteica , Interferencia de ARN , Proteína Smad4/genética , Regulación hacia ArribaRESUMEN
Rac proteins (Rac1, 1b, 2, 3) belong to the GTP-binding proteins (or GTPases) of the Ras superfamily and thus act as molecular switches cycling between an active GTP-bound and an inactive GDP-bound form through nucleotide exchange and hydrolysis. Like most other GTPases, these proteins adopt different conformations depending on the bound nucleotide, the main differences lying in the conformation of two short and flexible loop structures designated as the switch I and switch II region. The three distinct mammalian Rac isoforms, Rac1, 2 and 3, share a very high sequence identity (up to 90%), with Rac1b being an alternative splice variant of Rac1 with a 19 amino acid insertion in vicinity to the switch II region. We have demonstrated that Rac1 and Rac3 are very closely related with respect to their biochemical properties, such as effector interaction, nucleotide binding, and hydrolysis. In contrast, Rac2 displays a slower nucleotide association and is more efficiently activated by the Rac-GEF Tiam1. Modeling and normal mode analysis corroborate the hypothesis that the altered molecular dynamics of Rac2, in particular at the switch I region, may be responsible for different biochemical properties. On the other hand, our structural and biochemical analysis of Rac1b has shown that, compared with Rac1, Rac1b has an accelerated GEF-independent GDP/GTP-exchange and an impaired GTP-hydrolysis, accounting for a self-activating GTPase. This chapter discusses the use of fluorescence spectroscopic methods, allowing real-time monitoring of the interaction of nucleotides, regulators, and effectors with the Rac proteins at submicromolar concentrations and quantification of the kinetic and equilibrium constants.
Asunto(s)
Proteínas de Unión al GTP rac/aislamiento & purificación , GTP Fosfohidrolasas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Hidrólisis , Cinética , Espectrometría de Fluorescencia/métodos , ortoaminobenzoatos/metabolismo , Proteínas de Unión al GTP rac/química , Proteína de Unión al GTP rac1/aislamiento & purificación , Proteína RCA2 de Unión a GTPRESUMEN
Large-scale gene expression analyses of microdissected primary tissue are still difficult because generally only a limited amount of mRNA can be obtained from microdissected cells. The introduction of the T7-based RNA amplification technique was an important step to reduce the amount of RNA needed for such analyses. This amplification technique produces amplified antisense RNA (aRNA), which so far has precluded its direct use for serial analysis of gene expression (SAGE) library production. We describe a method, termed 'aRNA-longSAGE', which is the first to allow the direct use of aRNA for standard longSAGE library production. The aRNA-longSAGE protocol was validated by comparing two aRNA-longSAGE libraries with two Micro-longSAGE libraries that were generated from the same RNA preparations of two different cell lines. Using a conservative validation approach, we were able to verify 68% of the differentially expressed genes identified by aRNA-longSAGE. Furthermore, the identification rate of differentially expressed genes was roughly twice as high in our aRNA-longSAGE libraries as in the standard Micro-longSAGE libraries. Using our validated aRNA-longSAGE protocol, we were able to successfully generate longSAGE libraries from as little as 40 ng of total RNA isolated from 2000-3000 microdissected pancreatic ductal epithelial cells or cells from pancreatic intraepithelial neoplasias.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Microdisección , Técnicas de Amplificación de Ácido Nucleico , ARN sin Sentido/biosíntesis , Células CACO-2 , ADN Complementario/biosíntesis , Células HeLa , Humanos , Páncreas/citología , Páncreas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa , ARN/químicaRESUMEN
The nonsteroidal anti-inflammatory drug Sulindac has chemopreventive and antitumorigenic properties. Its metabolites induce apoptosis and inhibit signaling pathways critical for malignant transformation, including the Ras pathway. Here we show that the new Sulindac derivative IND 12 reverses the phenotype of Ras-transformed MDCK-f3 cells and restores an untransformed epithelioid morphology characterized by growth in monolayers with regular cell-cell adhesions. Moreover, IND 12 treatment induces the expression at membranes of the cell adhesion protein E-cadherin and increases the level of the E-cadherin-bound beta-catenin. As a consequence, IND 12-treated MDCK-f3 cells lose their invasion capacity and regain the ability to aggregate. In the presence of IND 12, MDCK-f3 cells show regenerated expression and activity ratios of the small GTPases Rac and Rho normally found in untransformed MDCK cells. Strikingly, IND 12 treatment decreases the levels of phosphorylated mitogen-activated protein kinases, which are downstream substrates of the Ras-regulated Raf/mitogen-activated protein kinase pathway, and the level of Ras-induced activation of gene expression. Our findings identify a novel drug with high potential in cancer therapy by targeting Ras-induced cell transformation.
Asunto(s)
Antineoplásicos/farmacología , Transformación Celular Neoplásica , Genes ras/efectos de los fármacos , Sulindac/farmacología , Transactivadores , Animales , Antiinflamatorios no Esteroideos/farmacología , Cadherinas/metabolismo , Agregación Celular/efectos de los fármacos , Línea Celular Transformada , Proteínas del Citoesqueleto/metabolismo , Perros , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Genes ras/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Sulindac/análogos & derivados , beta CateninaRESUMEN
In confluent primary cultures of rat hepatocytes,hypertonic stress leads to cell shrinkage and activates non-selective cation channels as the main mechanism of regulatory cell volume increase. The process is found to employ the exocytotic insertion of channels into the plasma membrane and (in addition to PKC) PLC, tyrosine kinases and G proteins, but not PI 3-kinase are part of the signalling network. Furthermore, hypertonic stress leads to the formation of stress fibres and significantly alters the activity of RhoA, Rac and Cdc42. These latter effects, however, are likely to reflect the restoration of cell shape rather than the regulation of cell volume, both most probably converging at the level of focal adhesions and integrins.
Asunto(s)
Forma de la Célula , Tamaño de la Célula , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Soluciones Hipertónicas/farmacología , Actinas/metabolismo , Potenciales de Acción , Animales , Cationes/farmacología , Células Cultivadas , Citoesqueleto/metabolismo , Adhesiones Focales , Hepatocitos/metabolismo , Canales Iónicos/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Ratas , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismoRESUMEN
OBJECTIVES: The aim of the study was to analyze the molecular mechanisms activated during postinfarction remodeling in human hearts. BACKGROUND: The molecular mechanisms of initial response to ischemic insult in the heart and the pathways involved in compensation and remodeling are still largely unknown. METHODS: Up-regulation or down-regulation of gene expression in the human viable peri-infarct (vs. remote) myocardial region was investigated by complementary deoxyribonucleic acid array technology and confirmed at a single-gene/protein level with reverse transcriptase polymerase chain reaction and immunohistochemistry. An in vitro model of cardiomyocyte hypoxia in HL1 cells was used to validate anti-apoptotic effects of the candidate gene/protein and to assess the associated downstream cascade. Finally, a mouse model of myocardial infarction was used to test the in vivo effects of exogenous transfection with the candidate gene/protein. RESULTS: Protein disulfide isomerase (PDI), a member of the unfolded protein response, is 3-fold up-regulated in the viable peri-infarct myocardial region, and in a postmortem model, its expression is significantly inversely correlated with apoptotic rate and with presence of heart failure (HF) and biventricular dilatation. Induced PDI expression in HL1 cells conferred protection from hypoxia-induced apoptosis. Adenoviral-mediated PDI gene transfer to the mouse heart resulted in 2.5-fold smaller infarct size, significantly reduced cardiomyocyte apoptosis in the peri-infarct region, and smaller left ventricular end-diastolic diameter versus mice treated with a transgene-null adenoviral vector. CONCLUSIONS: These results suggest that PDI promotes survival after ischemic damage and that zinc-superoxide dismutase is one of the PDI molecular targets. Pharmacological modulation of this pathway might prove useful for future prevention and treatment of HF.
Asunto(s)
Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Miocitos Cardíacos/fisiología , Proteína Disulfuro Isomerasas/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/fisiología , Técnicas de Cultivo de Célula , Hipoxia de la Célula/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteína Disulfuro Isomerasas/genética , ARN Mensajero/metabolismo , Remodelación Ventricular/fisiologíaRESUMEN
The Rho family of small GTPases plays a central role in intracellular signal transduction, particularly in reorganization of the actin cytoskeleton. Rho activity induces cell contractility, whereas Rac promotes cellular protrusion, which counteracts Rho signaling. In this regard, the reciprocal balance between these GTPases determines cell morphology and migratory behavior. Here we demonstrate that Tiam1/Rac1 signaling is able to antagonize Rho activity directly at the GTPase level in COS-7 cells. p190-RhoGAP plays a central regulatory role in this signaling pathway. Interfering with its activation by Src-kinase-dependent tyrosine phosphorylation or its recruitment to the membrane through interaction with the SH2 domains of p120-RasGAP blocks the Tiam1-mediated rapid downregulation of Rho. This process is mediated by Rac1, but not by Rac2 or Rac3 isoforms. Our data provide evidence for a biochemical pathway of the reciprocal regulation of two related small GTPases, which are key elements in cell migration.
Asunto(s)
Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Actinas/metabolismo , Animales , Células COS , Movimiento Celular , Chlorocebus aethiops , Citoesqueleto/metabolismo , Regulación hacia Abajo , GTP Fosfohidrolasas/metabolismo , Fosforilación , Isoformas de Proteínas/metabolismo , Transducción de Señal/fisiología , Tirosina/química , Tirosina/metabolismo , Proteína Activadora de GTPasa p120/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down-regulated by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTP-hydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 10(5)-fold by Rap1GAP and has a k(cat) of 6 s(-1) at 25 degrees C. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V(max) of the GTP-hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25- and 100-fold, most likely by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61. The Rap1(G12V) mutant which in Ras reduces catalysis 10(6)-fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP-catalyzed GTP-hydrolysis reaction on Rap1.
Asunto(s)
Proteínas de Unión al GTP rap1/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Catálisis , Hidrólisis , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rap1/química , Proteínas de Unión al GTP rap1/aislamiento & purificaciónRESUMEN
Rac1b was recently identified in malignant colorectal tumors as an alternative splice variant of Rac1 containing a 19-amino acid insertion next to the switch II region. The structures of Rac1b in the GDP- and the GppNHp-bound forms, determined at a resolution of 1.75 A, reveal that the insertion induces an open switch I conformation and a highly mobile switch II. As a consequence, Rac1b has an accelerated GEF-independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored partially by GTPase-activating proteins. Interestingly, Rac1b is able to bind the GTPase-binding domain of PAK but not full-length PAK in a GTP-dependent manner, suggesting that the insertion does not completely abolish effector interaction. The presented study provides insights into the structural and biochemical mechanism of a self-activating GTPase.
Asunto(s)
Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Empalme Alternativo , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Unión al ADN , Activación Enzimática , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Técnicas In Vitro , Cinética , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/metabolismo , Proteína de Unión al GTP rac1/químicaRESUMEN
Recent human genetic approaches showed that mutations in three genes encoding OPHN1, PAK3, and alphaPIX cause nonspecific X-linked mental retardation. These three proteins act to modulate Rho GTPase signaling pathways and may participate in neuronal morphogenesis by regulating the actin cytoskeleton. Here we showed that the Oligophrenin-1 gene is expressed in the developing spinal cord and later in brain areas that are characterized by high synaptic plasticity. At the cellular level OPHN1 is expressed in both glial and neuronal cells where it colocalizes with actin, notably at the tip of growing neurites. This interaction seems to be direct through a novel uncharacterized domain in the carboxyl-terminal end of OPHN1. Overexpression experiments in fibroblasts showed that the OPHN1 RhoGAP domain regulates in vivo the actin cytoskeleton by inhibition of Rho pathways. Interestingly the amino-terminal domain of OPHN1 inhibits the RhoGAP activity through an as yet unknown mechanism, suggesting that OPHN1 may be tightly regulated in vivo.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Sistema Nervioso Central/anomalías , Sistema Nervioso Central/metabolismo , Proteínas del Citoesqueleto , Proteínas Activadoras de GTPasa/metabolismo , Neuritas/metabolismo , Neuroglía/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Células 3T3 , Animales , Células COS , Sistema Nervioso Central/citología , Retroalimentación Fisiológica/genética , Feto , Proteínas Activadoras de GTPasa/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Ratones , Neuritas/ultraestructura , Neuroglía/ultraestructura , Proteínas Nucleares/genética , Fosfoproteínas/genética , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Ratas , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Rho-like GTPases such as RhoA, Rac1 and Cdc42 are key regulators of actin-dependent cell functions including cell morphology, adhesion and migration. Tiam1 (T lymphoma invasion and metastasis 1), a guanine nucleotide exchange factor that activates Rac, is an important regulator of cell shape and invasiveness in epithelial cells and fibroblasts. Overexpression of Tiam1 in metastatic melanoma cells converted the constitutive mesenchymal phenotype into an epithelial-like phenotype. This included the induction of stringent cell-cell contacts mediated by the Ig-like receptor ALCAM (activated leukocyte cell adhesion molecule) and actin redistribution to cell-cell junctions. This phenotypic switch was dependent on increased Rac but not Rho activity, and on the redistribution and adhesive function of ALCAM, whereas cadherins were not involved. Although cell proliferation was significantly enhanced, the gain of cell-cell junctions strongly counteracted cell motility and invasion as shown for two- and three-dimensional collagen assays as well as invasion into human skin reconstructs. The reverse transition from mesenchymal invasive to a resident epithelial-like phenotype implicates a role for Tiam1/Rac signaling in the control of cell-cell contacts through a novel ALCAM-mediated mechanism.