RESUMEN
In epithelial cells, the tight junction divides the plasma membrane into distinct apical and basolateral domains. Polarization is essential for epithelial cell function, and apico-basal cell polarity is lost during the epithelial to mesenchymal transition (EMT), a program of events characterized not only by loss of cell polarity, but also by enhanced cell motility and increased cell invasion. Among several apically localized protein complexes, the Crumbs and Par protein complexes have pivotal roles in control of epithelial polarity and apical membrane formation. Here, we demonstrate that the Snail transcriptional repressor antagonizes expression of the Crumbs polarity complex. We show that Snail abolishes localization of the Crumbs and Par complexes to the tight junction, decreases Crumbs complex protein levels and suppresses Crumbs3 transcription. Evidence that Snail acts directly to antagonize Crumbs3 promoter activity is presented. Strikingly, we note that reexpression of exogenous Crumbs3 in Snail-expressing Madin-Darby Canine Kidney cells partially restores cell-cell junctions. Moreover, we find that the EMT inducer transforming growth factor-beta elicits transcriptional repression of Crumbs3 and results in a measurable loss of Crumbs3 protein. Our findings provide new insights into the links between the transcriptional repression function of Snail and its role in antagonizing key apico-basal polarity factors during EMT.
Asunto(s)
Glicoproteínas de Membrana/genética , Factores de Transcripción/metabolismo , Animales , Línea Celular , Membrana Celular/fisiología , Movimiento Celular/fisiología , Polaridad Celular/genética , Perros , Células Epiteliales/citología , Células Epiteliales/fisiología , Regulación Neoplásica de la Expresión Génica , Riñón , Mesodermo/citología , Mesodermo/fisiología , Factores de Transcripción de la Familia Snail , Uniones Estrechas/genética , Uniones Estrechas/fisiologíaRESUMEN
Mammalian adipose tissue serves a number of functions, including storage of nutrients for periods of fasting and control of organismal metabolism. Critical to these functions is the capacity of the fat cell to respond to insulin with a significant increase in glucose uptake. It is now generally recognized that the major site of action of insulin in this tissue is the mobilization of a pool of latent, intracellular transport proteins. Nonetheless, the precise signaling pathways which mediate the insulin-stimulated increase in glucose transport remain uncertain. In recent years, the serine/threonine protein kinase Akt/PKB has emerged as an important candidate signaling molecule. Considerable current effort is being directed at trying to definitively establish whether Akt/PKB is an important intermediate in insulin signaling to glucose transport in muscle and fat.
Asunto(s)
Adipocitos/citología , Tejido Adiposo/metabolismo , Insulina/fisiología , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Humanos , Resistencia a la Insulina , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-aktRESUMEN
A critical component of vertebrate cellular differentiation is the acquisition of sensitivity to a restricted subset of peptide hormones and growth factors. This accounts for the unique capability of insulin (and possibly insulin-like growth factor-1), but not other growth factors, to stimulate glucose uptake and anabolic metabolism in heart, skeletal muscle, and adipose tissue. This selectivity is faithfully recapitulated in the cultured adipocyte line, 3T3-L1, which responds to insulin, but not platelet-derived growth factor (PDGF), with increased hexose uptake. The serine/threonine protein kinases Akt1 and Akt2, which have been implicated as mediators of insulin-stimulated glucose uptake, as well as glycogen, lipid, and protein synthesis, were shown to mirror this selectivity in this tissue culture system. This was particularly apparent in 3T3-L1 adipocytes overexpressing an epitope-tagged form of Akt2 in which insulin activated Akt2 10-fold better than PDGF. Similarly, in 3T3-L1 adipocytes, only insulin stimulated phosphorylation of Akt's endogenous substrate, GSK-3beta. Other signaling molecules, including phosphatidylinositol 3-kinase, pp70 S6-kinase, mitogen-activated protein kinase, and PHAS-1/4EBP-1, did not demonstrate this selective responsiveness to insulin but were instead activated comparably by both insulin and PDGF. Moreover, concurrent treatment with PDGF and insulin did not diminish activation of phosphatidylinositol 3-kinase, Akt, or glucose transport, indicating that PDGF did not simultaneously activate an inhibitory mechanism. Interestingly, PDGF and insulin comparably stimulated both Akt isoforms, as well as numerous other signaling molecules, in undifferentiated 3T3-L1 preadipocytes. Collectively, these data suggest that differential activation of Akt in adipocytes may contribute to insulin's exclusive mediation of the metabolic events involved in glucose metabolism. Moreover, they suggest a novel mechanism by which differentiation-dependent hormone selectivity is conferred through the suppression of specific signaling pathways operational in undifferentiated cell types.
Asunto(s)
Adipocitos/fisiología , Proteínas Musculares , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/fisiología , Células 3T3 , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Diferenciación Celular , Transportador de Glucosa de Tipo 4 , Glucógeno Sintasa Quinasa 3 , Humanos , Insulina/farmacología , Ratones , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/fisiología , Fosforilación , Proteínas Proto-Oncogénicas c-akt , Receptores del Factor de Crecimiento Derivado de Plaquetas/fisiologíaRESUMEN
The current studies investigated the contribution of phosphatidylinositol 3-kinase (PI3-kinase) isoforms to insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation. Experiments involving the microinjection of antibodies specific for the p110 catalytic subunit of class I PI3-kinases demonstrated an absolute requirement for this form of the enzyme in GLUT4 translocation. This finding was confirmed by the demonstration that the PI3-kinase antagonist wortmannin inhibits GLUT4 and insulin-responsive aminopeptidase translocation with a dose response identical to that required to inhibit another class I PI3-kinase-dependent event, activation of pp70 S6-kinase. Interestingly, wortmannin inhibited insulin-stimulated glucose uptake at much lower doses, suggesting the existence of a second, higher affinity target of the drug. Subsequent removal of wortmannin from the media shifted this dose-response curve to one resembling that for GLUT4 translocation and pp70 S6-kinase. This is consistent with the lower affinity target being p110, which is irreversibly inhibited by wortmannin. Wortmannin did not reduce glucose uptake in cells stably expressing Myr-Akt, which constitutively induced GLUT4 translocation to the plasma membrane; this demonstrates that wortmannin does not inhibit the transporters directly. In addition to elucidating a second wortmannin-sensitive pathway in 3T3-L1 adipocytes, these studies suggest that the presence of GLUT4 on the plasma membrane is not sufficient for activation of glucose uptake.
Asunto(s)
Androstadienos/farmacología , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Células 3T3 , Adipocitos , Aminopeptidasas/metabolismo , Animales , Cromonas/farmacología , Cistinil Aminopeptidasa , Desoxiglucosa/metabolismo , Técnica del Anticuerpo Fluorescente , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Insulina/farmacología , Isoenzimas/metabolismo , Ratones , Microinyecciones , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , WortmaninaRESUMEN
The protein kinase Akt/PKB is a crucial regulator of cell survival in response to mitogenic signals. The increased kinase activity of v-akt, an oncogenic form of Akt/PKB, causes mouse T cell lymphoma, and overexpression of Akt/PKB is associated with progression of several tumor types in human. In this study, we demonstrate that ligation of B cell antigen receptor (BCR) leads to activation of Akt/PKB in B lymphocytes. BCR-induced activation of Akt/PKB required the tyrosine kinase Syk, which was not previously known to regulate Akt/PKB. In contrast, BCR crosslinking of Lyn-deficient B cells resulted in markedly enhanced hyperphosphorylation and activation of Akt/PKB compared with wild-type B cells, indicating that this Src-family kinase acts as an endogenous antagonist of BCR-induced Akt/PKB activation. Lyn inhibited Akt/PKB additively with an okadaic acid-sensitive endogenous phosphatase(s). Expression of exogenous Lyn in mutant cells restored normal BCR-induced phosphorylation of Akt/PKB. Negative regulation of Akt/PKB by Lyn was not dependent on the protein phosphatases SHP-1, SHP-2, or SHIP. Our results show that Lyn provides a mechanism for negative regulation and opposes the effect of Syk on BCR-mediated activation of Akt/PKB. Deregulation of Akt/PKB correlates with the hyperresponsiveness of B cells from Lyn-deficient mice stimulated by BCR crosslinking and may contribute to the autoimmune syndrome that develops in Lyn-deficient animals.
Asunto(s)
Linfocitos B/enzimología , Precursores Enzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Linfocitos B/inmunología , Línea Celular , Pollos , Activación Enzimática/inmunología , Precursores Enzimáticos/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Tirosina Quinasas/inmunología , Transducción de Señal/inmunología , Quinasa Syk , Familia-src Quinasas/inmunologíaRESUMEN
A major action of insulin is to accelerate the rate of uptake of sugar into muscle and adipose cells following a meal. The biochemical mechanism by which this is accomplished has been a subject of intense experimentation, although elucidation of the pathways has remained elusive. In recent years, numerous signaling molecules and cascades modulated by insulin have been identified, although few have been definitively established as important to the metabolic actions of the hormone. An exception to this is the lipid kinase phosphatidylinositide 3'-kinase, which, under many conditions, appears absolutely required for insulin to stimulate hexose uptake into adipocytes. Akt/PKB, a serine/threonine protein kinase activated by insulin in a phosphatidylinositide 3'-kinase-dependent manner, has been implicated as a critical mediator of insulin's actions on metabolism and cell survival. Nonetheless, Akt/PKB's role in many insulin effects, particularly accelerated glucose transport, remains controversial. Interestingly, soluble analogues of ceramide antagonize both insulin's activation of Akt/PKB as well as its stimulation of glucose transport, consistent with a causal relationship between the two.
Asunto(s)
Ceramidas/efectos adversos , Glucosa/metabolismo , Insulina/metabolismo , Proteínas Musculares , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Animales , Transporte Biológico Activo , Diabetes Mellitus Tipo 2/metabolismo , Transportador de Glucosa de Tipo 4 , Humanos , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Proto-Oncogénicas c-akt , Especificidad por SustratoRESUMEN
In order to characterize the structural and dynamic factors that determine the assembly in b hemoproteins, the solution structure of the 98-residue protein apocytochrome b5 was determined by NMR methods. Over 800 experimental restraints derived from a series of two- and three-dimensional experiments were used. Holocytochrome b5, the protein with iron protoporphyrin-IX liganded to His-39 and His-63, contains in sequence the following elements of secondary structure: beta 1-alpha 1-beta 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5-beta 2-alpha 6 [Mathews, F.S., Czerwinski, E. W., & Argos, P. (1979) The Porphyrins, Vol. 7, pp. 107-147, Academic Press, New York]. The folded holoprotein possesses two hydrophobic cores: an extensive, functional core around the heme (core 1), and a smaller, structural core remote from the heme (core 2). The apoprotein was found to contain a stable four-stranded beta-sheet encompassing beta 1, beta 2, beta 3, and beta 4 and three alpha-helices, corresponding to alpha 1, alpha 2, and alpha 6. Two short alpha-helices (alpha 3 and alpha 5) appear to form partially, and alpha 4 is not detected. These three helices and beta 5 border the heme binding pocket and are disordered in the apoprotein NMR structure. According to backbone 1H-15N NOE results, the most flexible region of the apoprotein, except for the termini, extends from Ala-50 (in beta 5) to Glu-69 (in alpha 5). The polypeptide segment bearing His-63 (located immediately prior to alpha 5) exhibits faster internal motions than that bearing His-39 (at the C-terminal end of alpha 2). The latter imidazole samples a restricted region of space, whereas the former can adopt many orientations with respect to the stable core. It was concluded that heme removal affects the structure and dynamics of most of core 1 whereas it leaves core 2 largely intact. The results provide guidelines for the rational design of b hemoproteins: a modular structure including a packed, stable core and a partially folded binding site is anticipated to present strong kinetic and thermodynamic advantages compared to approaches relying on the complete formation of secondary structure prior to heme binding.