RESUMEN
Cu ion (Cu) entry into human cells is mediated by CTR1 (also known as SLC31A1), the high-affinity Cu transporter. When extracellular Cu is raised, the cell is protected against excess accumulation by rapid internalization of the transporter. When Cu is lowered, the transporter returns to the membrane. We show in HEK293 cells overexpressing CTR1 that expression of either the C-terminal domain of AP180 (also known as SNAP91), a clathrin-coat assembly protein that sequesters clathrin, or a dominant-negative mutant of dynamin, decreases Cu-induced endocytosis of CTR1, as does a dynamin inhibitor and clathrin knockdown using siRNA. Utilizing imaging, siRNA techniques and a new high-throughput assay for endocytosis employing CLIP-tag methodology, we show that internalized CTR1 accumulates in early sorting endosomes and recycling compartments (containing Rab5 and EEA1), but not in late endosomes or lysosomal pathways. Using live cell fluorescence, we find that upon extracellular Cu removal CTR1 recycles to the cell surface through the slower-recycling Rab11-mediated pathway. These processes enable cells to dynamically alter transporter levels at the plasma membrane and acutely modulate entry as a safeguard against excess cellular Cu.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Clatrina/metabolismo , Cobre/metabolismo , Dinaminas/metabolismo , Endosomas/metabolismo , Clatrina/genética , Transportador de Cobre 1 , Dinaminas/genética , Endocitosis , Células HEK293 , Homeostasis , Humanos , Transporte Iónico , Proteínas de Ensamble de Clatrina Monoméricas/genética , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Mutación/genética , Transporte de Proteínas , ARN Interferente Pequeño , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Cardiotonic steroids (CTS), specific inhibitors of Na,K-ATPase activity, have been widely used for treating cardiac insufficiency. Recent studies suggest that low levels of endogenous CTS do not inhibit Na,K-ATPase activity but play a role in regulating blood pressure, inducing cellular kinase activity, and promoting cell viability. Higher CTS concentrations inhibit Na,K-ATPase activity and can induce reactive oxygen species, growth arrest, and cell death. CTS are being considered as potential novel therapies in cancer treatment, as they have been shown to limit tumor cell growth. However, there is a lack of information on the relative toxicity of tumor cells and comparable non-tumor cells. We have investigated the effects of CTS compounds, ouabain, digitoxin, and bufalin, on cell growth and survival in cell lines exhibiting the full spectrum of non-cancerous to malignant phenotypes. We show that CTS inhibit membrane Na,K-ATPase activity equally well in all cell lines tested regardless of metastatic potential. In contrast, the cellular responses to the drugs are different in non-tumor and tumor cells. Ouabain causes greater inhibition of proliferation and more extensive apoptosis in non-tumor breast cells compared to malignant or oncogene-transfected cells. In tumor cells, the effects of ouabain are accompanied by activation of anti-apoptotic ERK1/2. However, ERK1/2 or Src inhibition does not sensitize tumor cells to CTS cytotoxicity, suggesting that other mechanisms provide protection to the tumor cells. Reduced CTS-sensitivity in breast tumor cells compared to non-tumor cells indicates that CTS are not good candidates as cancer therapies.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Glicósidos Cardíacos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Glándulas Mamarias Humanas/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Glándulas Mamarias Humanas/patología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas de Neoplasias/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Restoration of the functional potency of pancreatic islets either through enhanced proliferation (hyperplasia) or increase in size (hypertrophy) of beta cells is a major objective for intervention in diabetes. We have obtained experimental evidence that global knock-out of a small, single-span regulatory subunit of Na,K-ATPase, FXYD2, alters glucose control. Adult Fxyd2(-/-) mice showed significantly lower blood glucose levels, no signs of peripheral insulin resistance, and improved glucose tolerance compared with their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the Fxyd2(-/-) mice compared with the wild type littermates, compatible with an observed increase in the level of circulating insulin. No changes were seen in the exocrine compartment of the pancreas, and the mice had only a mild, well-adapted renal phenotype. Morphometric analysis revealed an increase in beta cell mass in KO compared with WT mice. This appears to explain a phenotype of hyperinsulinemia. By RT-PCR, Western blot, and immunocytochemistry we showed the FXYD2b splice variant in pancreatic beta cells from wild type mice. Phosphorylation of Akt kinase was significantly higher under basal conditions in freshly isolated islets from Fxyd2(-/-) mice compared with their WT littermates. Inducible expression of FXYD2 in INS 832/13 cells produced a reduction in the phosphorylation level of Akt, and phosphorylation was restored in parallel with degradation of FXYD2. Thus we suggest that in pancreatic beta cells FXYD2 plays a role in Akt signaling pathways associated with cell growth and proliferation.
Asunto(s)
Glucemia/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/sangre , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Empalme Alternativo , Animales , Western Blotting , Línea Celular Tumoral , Femenino , Regulación Enzimológica de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Hiperplasia , Inmunohistoquímica , Células Secretoras de Insulina/patología , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
To catalyze ion transport, the Na,K-ATPase must contain one α and one ß subunit. When expressed by transfection in various expression systems, each of the four α subunit isoforms can assemble with each of the three ß subunit isoforms and form an active enzyme, suggesting the absence of selective α-ß isoform assembly. However, it is unknown whether in vivo conditions the α-ß assembly is random or isoform-specific. The α(2)-ß(2) complex was selectively immunoprecipitated by both anti-α(2) and anti-ß(2) antibodies from extracts of mouse brain, which contains cells co-expressing multiple Na,K-ATPase isoforms. Neither α(1)-ß(2) nor α(2)-ß(1) complexes were detected in the immunoprecipitates. Furthermore, in MDCK cells co-expressing α(1), ß(1), and ß(2) isoforms, a greater fraction of the ß(2) subunits was unassembled with α(1) as compared with that of the ß(1) subunits, indicating preferential association of the α(1) isoform with the ß(1) isoform. In addition, the α(1)-ß(2) complex was less resistant to various detergents than the α(1)-ß(1) complex isolated from MDCK cells or the α(2)-ß(2) complex isolated from mouse brain. Therefore, the diversity of the α-ß Na,K-ATPase heterodimers in vivo is determined not only by cell-specific co-expression of particular isoforms, but also by selective association of the α and ß subunit isoforms.
Asunto(s)
Multimerización de Proteína , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Unión Competitiva , Encéfalo/enzimología , Membrana Celular/enzimología , Células Cultivadas , Perros , Estabilidad de Enzimas , Humanos , Inmunoprecipitación , Isoenzimas/metabolismo , Riñón , Proteínas Luminiscentes/metabolismo , Ratones , Modelos Moleculares , Especificidad de Órganos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Nervio Ciático/enzimología , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificaciónRESUMEN
The Na,K-ATPase is an alphabeta heterodimer responsible for maintaining fluid and electrolyte homeostasis in mammalian cells. We engineered Madin-Darby canine kidney cell lines expressing alpha(1)FLAG, beta(1)FLAG, or beta(2)MYC subunits via a tetracycline-regulated promoter and a line expressing both stable beta(1)MYC and tetracycline-regulated beta(1)FLAG to examine regulatory mechanisms of sodium pump subunit expression. When overexpression of exogenous beta(1)FLAG increased total beta subunit levels by >200% without changes in alpha subunit abundance, endogenous beta(1) subunit (beta(1)E) abundance decreased. beta(1)E down-regulation did not occur during beta(2)MYC overexpression, indicating isoform specificity of the repression mechanism. Measurements of RNA stability and content indicated that decreased beta subunit expression was not accompanied by any change in mRNA levels. In addition, the degradation rate of beta subunits was not altered by beta(1)FLAG overexpression. Cells stably expressing beta(1)MYC, when induced to express beta(1)FLAG subunits, showed reduced beta(1)MYC and beta(1)E subunit abundance, indicating that these effects occur via the coding sequences of the down-regulated polypeptides. In a similar way, Madin-Darby canine kidney cells overexpressing exogenous alpha(1)FLAG subunits exhibited a reduction of endogenous alpha(1) subunits (alpha(1)E) with no change in alpha mRNA levels or beta subunits. The reduction in alpha(1)E compensated for alpha(1)FLAG subunit expression, resulting in unchanged total alpha subunit abundance. Thus, regulation of alpha subunit expression maintained its native level, whereas beta subunit was not as tightly regulated and its abundance could increase substantially over native levels. These effects also occurred in human embryonic kidney cells. These data are the first indication that cellular sodium pump subunit abundance is modulated by translational repression. This mechanism represents a novel, potentially important mechanism for regulation of Na,K-ATPase expression.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Subunidades de Proteína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Línea Celular , Perros , Humanos , Inmunoprecipitación , Isoenzimas/genética , Isoenzimas/metabolismo , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Subunidades de Proteína/genética , Estabilidad del ARN , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase alpha- and beta-subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of overexpressing Na-K-ATPase beta1-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged beta1-subunits (beta1flag) or Myc-tagged beta1-subunits (beta1myc) under the control of a tetracycline-dependent promoter. The induction of beta1flag subunit synthesis in MDCK cells, which increases beta1-subunit expression at the plasma membrane by more than twofold, while maintaining stable alpha1 expression levels, revealed that all mature beta1-subunits associate with alpha1-subunits, and no evidence of "free" beta1-subunits was obtained. Consequently, the ratio of assembled beta1- to alpha1-subunits is significantly increased when "extra" beta-subunits are expressed. An increased beta1/alpha1 stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein-protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of cocultured beta1myc-expressing and beta1flag-expressing MDCK cells show colocalization of beta1myc and beta1flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the beta-subunits. Immunoprecipitation using MDCK cells constitutively expressing beta1myc and tetracycline-regulated beta1flag subunits confirmed beta-beta-subunit interactions. These results demonstrate that the equimolar ratio of assembled beta1/alpha1-subunits of the Na-K-ATPase in kidney cells is not fixed by the inherent properties of the interacting subunits. It is likely that cellular mechanisms are present that regulate the individual Na-K-ATPase subunit abundance.
Asunto(s)
Células Epiteliales/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Western Blotting , Línea Celular , Membrana Celular/metabolismo , Perros , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/metabolismo , Células Epiteliales/citología , Expresión Génica , Glicosilación/efectos de los fármacos , Aparato de Golgi/metabolismo , Humanos , Inmunoprecipitación , Membranas Intracelulares/metabolismo , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Ovinos , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genética , Especificidad de la Especie , Transfección , Tunicamicina/farmacologíaRESUMEN
The exclusive basolateral localization of the Na-K-ATPase in kidney epithelium is a critical aspect of nephron function. It has been suggested that mislocalized delivery of the Na-K-ATPase to the apical surface in autosomal dominant polycystic kidney disease (ADPKD) is due to the inappropriate expression of an alternative isoform of the beta-subunit, the beta(2)-isoform. It has been reported that heterologous expression of this beta(2)-isoform in Madin-Darby canine kidney (MDCK) cells results in apical delivery of the Na-K-ATPase. We created a MDCK cell line containing a tetracycline-inducible promoter and expressed either myc-tagged beta(2)- or flag-tagged beta(1)-subunits to study the surface localization of these beta-subunit isoforms in polarized monolayers. We find that the beta(2)-isoform is targeted to the basolateral surface of the plasma membrane in a polarization pattern indistinguishable from the beta(1)-isoform. However, inclusion of butyrate in the growth medium leads to upregulation of overexpressed beta(1)- or beta(2)-subunits and to their appearance at the apical surface. The beta(2)-isoform expressed in MDCK cells does not assemble into alpha(1)beta(2) heterodimers with the endogenous alpha(1). Our findings demonstrate that expression of the beta(2)-isoform does not lead to apical localization of the Na-K-ATPase in MDCK cells and provides evidence for an unexpected effect of butyrate on the trafficking of Na pump subunits.