RESUMEN
Endocytosis of activated growth factor receptors regulates spatio-temporal cellular signaling. In the case of the EGF receptor, sorting into multivesicular bodies (MVBs) controls signal termination and subsequently leads to receptor degradation in lysosomes. Annexin A1, a Ca(2+)-regulated membrane binding protein often deregulated in human cancers, interacts with the EGF receptor and is phosphorylated by internalized EGF receptor on endosomes. Most relevant for EGF receptor signal termination, annexin A1 is required for the formation of internal vesicles in MVBs that sequester ligand-bound EGF receptor away from the limiting membrane. To elucidate the mechanism underlying annexin A1-dependent EGF receptor trafficking we employed an N-terminally truncated annexin A1 mutant that lacks the EGF receptor phosphorylation site and the site for interaction with its protein ligand S100A11. Overexpression of this dominant-negative mutant induces a delay in EGF-induced EGF receptor transport to the LAMP1-positive late endosomal/lysosomal compartment and impairs ligand-induced EGF receptor degradation. Consistent with these findings, EGF-stimulated EGF receptor and MAP kinase pathway signaling is prolonged. Importantly, depletion of S100A11 also results in a delayed EGF receptor transport and prolonged MAP kinase signaling comparable to the trafficking defect observed in cells expressing the N-terminally truncated annexin A1 mutant. These results strongly suggest that the function of annexin A1 as a regulator of EGF receptor trafficking, degradation and signaling is critically mediated through an N-terminal interaction with S100A11 in the endosomal compartment. This interaction appears to be essential for lysosomal targeting of the EGF receptor, possibly by providing a physical scaffold supporting inward vesiculation in MVBs. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.
Asunto(s)
Anexina A1/metabolismo , Movimiento Celular , Proliferación Celular , Receptores ErbB/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Proteínas S100/metabolismo , Anexina A1/antagonistas & inhibidores , Anexina A1/genética , Compartimento Celular , Ensayo de Unidades Formadoras de Colonias , Endocitosis/fisiología , Endosomas/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Células HeLa , Humanos , Técnicas para Inmunoenzimas , Proteínas de Membrana de los Lisosomas/genética , Lisosomas/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Transporte de Proteínas , Proteínas S100/antagonistas & inhibidores , Proteínas S100/genética , Resonancia por Plasmón de SuperficieRESUMEN
Skp2B, an F-box protein of unknown function, is frequently overexpressed in breast cancer. In order to determine the function of Skp2B and whether it has a role in breast cancer, we performed a two-hybrid screen and established transgenic mice expressing Skp2B in the mammary glands. We found that Skp2B interacts with the repressor of estrogen receptor activity (REA) and that overexpression of Skp2B leads to a reduction in REA levels. In the mammary glands of MMTV-Skp2B mice, REA levels are also low. Our results show that in virgin transgenic females, Skp2B induces lobuloalveolar development and differentiation of the mammary glands normally observed during pregnancy. As this phenotype is identical to what was observed for REA heterozygote mice, our observations suggest that the Skp2B-REA interaction is physiologically relevant. However, in contrast to REA(+/-) mice, MMTV-Skp2B mice develop mammary tumors, suggesting that Skp2B affects additional proteins. These results indicate that the observed expression of Skp2B in breast cancer does contribute to tumorigenesis at least in part by modulating the activity of the estrogen receptor.
Asunto(s)
Glándulas Mamarias Animales/crecimiento & desarrollo , Receptores de Estrógenos/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Diferenciación Celular , Quistes/patología , Femenino , Humanos , Hiperplasia , Glándulas Mamarias Animales/patología , Virus del Tumor Mamario del Ratón , Ratones , Ratones Transgénicos , Neoplasias/patología , Prohibitinas , Unión Proteica , Receptores de Estrógenos/genética , Conducta Sexual , Maduración Sexual , Transcripción Genética , Ubiquitina/metabolismoRESUMEN
Skp2 is an F-box protein involved in the ubiquitination and subsequent degradation of the cyclin-dependent kinase (Cdk) inhibitor p27. Skp2 has been reported to be overexpressed in a variety of cancer types and to correlate with poor prognosis. We have identified a novel isoform of Skp2 we named Skp2B, which differs from Skp2 only in the C-terminal domain and unlike Skp2 localizes to the cytoplasm. Here, we describe the relative expression of both Skp2 and Skp2B in breast cancer cell lines and in primary breast cancers using quantitative real time RT-PCR. We show that Skp2B mRNA is expressed 10-fold less than Skp2 mRNA in the immortalized but non-transformed breast cell line, 184B5. However, Skp2B is overexpressed as frequently as Skp2, and to higher levels than Skp2 in breast cancer cell lines and primary cancers. Further, we show that cytoplasmic staining is frequent in primary breast cancers. In addition, we found that xenografts expressing Skp2B grow faster than xenografts expressing low levels of Skp2B, and that this effect is independent of p27 degradation. These findings therefore suggest that Skp2B overexpression is also observed in breast cancers and identify Skp2B as a putative oncogene.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/fisiopatología , Proteínas F-Box/metabolismo , Perfilación de la Expresión Génica , Proteínas Quinasas Asociadas a Fase-S/genética , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Oncogenes , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Proteínas Quinasas Asociadas a Fase-S/farmacología , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/metabolismo , UbiquitinasRESUMEN
Phosphorylation of the Ca2+ and membrane-binding protein annexin 1 by epidermal growth factor (EGF) receptor tyrosine kinase has been thought to be involved in regulation of the EGF receptor trafficking. To elucidate the interaction of annexin 1 during EGF receptor internalization, we followed the distribution of annexin 1-GFP fusion proteins at sites of internalizing EGF receptors. The observed association of annexin 1 with EGF receptors was confirmed by immunoprecipitation. We found that this interaction was independent of a functional phosphorylation site in the annexin 1 N-terminal domain but mediated through the Ca2+ binding core domain.
Asunto(s)
Anexina A1/metabolismo , Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Anexina A1/química , Anexina A1/genética , Calcio/metabolismo , Endocitosis/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
We report here that blocking the activity of the 26 S proteasome results in drastic changes in the morphology of the mitochondria and accumulation of intermembrane space (IMS) proteins. Using endonuclease G (endoG) as a model IMS protein, we found that accumulation of wild-type but to a greater extent mutant endoG leads to changes in the morphology of the mitochondria similar to those observed following proteasomal inhibition. Further, we show that wild-type but to a greater extent mutant endoG is a substrate for ubiquitination, suggesting the presence of a protein quality control. Conversely, we also report that wild-type but not mutant endoG is a substrate for the mitochondrial protease Omi but only upon inhibition of the proteasome. These findings suggest that although elimination of mutant IMS proteins is strictly dependent on ubiquitination, elimination of excess or spontaneously misfolded wild-type IMS proteins is monitored by ubiquitination and as a second checkpoint by Omi cleavage when the proteasome function is deficient. One implication of our finding is that in the context of attenuated proteasomal function, accumulation of IMS proteins would contribute to the collapse of the mitochondrial network such as that observed in neurodegenerative diseases. Another implication is that such collapse could be accelerated either by mutations in IMS proteins or by mutations in Omi itself.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Línea Celular , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Humanos , Microscopía Electrónica , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/ultraestructura , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/ultraestructura , Transporte de Proteínas , UbiquitinaciónRESUMEN
The human Golgi-associated PR-1-related protein (GAPR-1) is closely related to plant pathogenesis-related (PR-1) proteins, which are upregulated in response to pathogen attack. Family members have been identified in a variety of organisms, together constituting the superfamily of PR-1 proteins. GAPR-1 is found within lipid-enriched microdomains on the cytosolic side of the endomembrane system. GAPR-1 is tightly anchored to membranes and absent from the cytosol, although it does not possess a membrane-spanning domain. Crystals of recombinantly expressed GAPR-1 have been grown that diffract to high (1.5 A) resolution. Complete data sets have been collected on a trigonal crystal form (P3(1)21/P3(2)21), with unit-cell parameters a = b = 73.5, c = 63.2 A. Molecular replacement using the NMR coordinates of tomato pathogenesis-related protein (28% identity) was unsuccessful and a search for heavy-metal derivatives or alternative phasing methods has been initiated.