O-GlcNAcylation of the human epidermal growth factor receptor

The reversible O-linked attachment of single β-D-N-acetylglucosamine (GlcNAc) moieties to serine/threonine residues in target proteins is a frequently occurring post-translational modification affecting the functionality of many cellular systems. In this report we present experimental evidence sugge...

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Autores: Stateva, Silvia R., Villalobo, Antonio
Tipo de recurso: artículo
Estado:Versión aceptada para publicación
Fecha de publicación:2015
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/125079
Acceso en línea:http://hdl.handle.net/10261/125079
Access Level:acceso abierto
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spelling O-GlcNAcylation of the human epidermal growth factor receptorStateva, Silvia R.Villalobo, AntonioThe reversible O-linked attachment of single β-D-N-acetylglucosamine (GlcNAc) moieties to serine/threonine residues in target proteins is a frequently occurring post-translational modification affecting the functionality of many cellular systems. In this report we present experimental evidence suggesting that the epidermal growth factor receptor (EGFR) is subjected to O-GlcNAcylation in human carcinoma epidermoid A431 cells and human lung carcinoma A549 cells. However, no signal was detected in human cervix adenocarcinoma HeLa cells or in mouse EGFR-T17 fibroblasts ectopically expressing the human EGFR. We detected a positive O-GlcNAcylation signal in the immunoprecipitated EGFR by Western blotting using two distinct specific anti-O-GlcNAc antibodies even after N-deglycosylation of the receptor using peptide-N-glycosidase F (PNGase F). Conversely, the presence of EGFR was detected by Western blotting using an anti-EGFR antibody in the immunocomplex of O-GlcNAcylated proteins immunoprecipitated with an anti-O-GlcNAc antibody. These signals were enhanced when the O-linked β-N-acetylglucosaminidase (OGA) inhibitor Thiamet G was added to prevent the deglycosylation of the GlcNAc moiety(ies). Moreover, we also detected a positive signal in the immunoprecipitated and N-deglycosylated EGFR using PNGase F, and tunicamycin when the cells were metabolically labeled with azido-GlcNAc (GlcNAz), biotinylated and probed with a streptavidin-labeled peroxidase. Finally, EGFR and O-linked β-N-acetylglucosamine transferase (OGT) co-immunoprecipitate, and incubation of the immunoprecipitated EGFR with the immunoprecipitated OGT in the presence of uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) resulted in a significant enhancement of the EGFR O-GlcNAcylation signal as detected by Western blotting using an anti-O-GlcNAc antibody. We conclude that the human EGFR is subjected to O-GlcNAcylation in the A431 and A549 tumor cell lines.This work was funded by grants to AV from the European Commission (contract no. PITN-GA-2011-289033), the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494 and SAF2014-52048-R), and the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349). SRS received funding from the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7/2007-2013 under REA grant agreement PITN-GA-2011-289033.Peer ReviewedRoyal Society of Chemistry (UK)Ministerio de Economía y Competitividad (España)European CommissionComunidad de MadridConsejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]2015201520152015info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Postprintinfo:eu-repo/semantics/acceptedVersionhttp://hdl.handle.net/10261/125079reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/EC/FP7/289033S2011/BMD-2349/I2M2info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/SAF2014-52048-Rhttps://doi.org/10.1039/C5OB00443HSíinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1250792026-05-22T06:33:51Z
dc.title.none.fl_str_mv O-GlcNAcylation of the human epidermal growth factor receptor
title O-GlcNAcylation of the human epidermal growth factor receptor
spellingShingle O-GlcNAcylation of the human epidermal growth factor receptor
Stateva, Silvia R.
title_short O-GlcNAcylation of the human epidermal growth factor receptor
title_full O-GlcNAcylation of the human epidermal growth factor receptor
title_fullStr O-GlcNAcylation of the human epidermal growth factor receptor
title_full_unstemmed O-GlcNAcylation of the human epidermal growth factor receptor
title_sort O-GlcNAcylation of the human epidermal growth factor receptor
dc.creator.none.fl_str_mv Stateva, Silvia R.
Villalobo, Antonio
author Stateva, Silvia R.
author_facet Stateva, Silvia R.
Villalobo, Antonio
author_role author
author2 Villalobo, Antonio
author2_role author
dc.contributor.none.fl_str_mv Ministerio de Economía y Competitividad (España)
European Commission
Comunidad de Madrid
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
description The reversible O-linked attachment of single β-D-N-acetylglucosamine (GlcNAc) moieties to serine/threonine residues in target proteins is a frequently occurring post-translational modification affecting the functionality of many cellular systems. In this report we present experimental evidence suggesting that the epidermal growth factor receptor (EGFR) is subjected to O-GlcNAcylation in human carcinoma epidermoid A431 cells and human lung carcinoma A549 cells. However, no signal was detected in human cervix adenocarcinoma HeLa cells or in mouse EGFR-T17 fibroblasts ectopically expressing the human EGFR. We detected a positive O-GlcNAcylation signal in the immunoprecipitated EGFR by Western blotting using two distinct specific anti-O-GlcNAc antibodies even after N-deglycosylation of the receptor using peptide-N-glycosidase F (PNGase F). Conversely, the presence of EGFR was detected by Western blotting using an anti-EGFR antibody in the immunocomplex of O-GlcNAcylated proteins immunoprecipitated with an anti-O-GlcNAc antibody. These signals were enhanced when the O-linked β-N-acetylglucosaminidase (OGA) inhibitor Thiamet G was added to prevent the deglycosylation of the GlcNAc moiety(ies). Moreover, we also detected a positive signal in the immunoprecipitated and N-deglycosylated EGFR using PNGase F, and tunicamycin when the cells were metabolically labeled with azido-GlcNAc (GlcNAz), biotinylated and probed with a streptavidin-labeled peroxidase. Finally, EGFR and O-linked β-N-acetylglucosamine transferase (OGT) co-immunoprecipitate, and incubation of the immunoprecipitated EGFR with the immunoprecipitated OGT in the presence of uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) resulted in a significant enhancement of the EGFR O-GlcNAcylation signal as detected by Western blotting using an anti-O-GlcNAc antibody. We conclude that the human EGFR is subjected to O-GlcNAcylation in the A431 and A549 tumor cell lines.
publishDate 2015
dc.date.none.fl_str_mv 2015
2015
2015
2015
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Postprint
info:eu-repo/semantics/acceptedVersion
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/125079
url http://hdl.handle.net/10261/125079
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
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#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/EC/FP7/289033
S2011/BMD-2349/I2M2
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/SAF2014-52048-R
https://doi.org/10.1039/C5OB00443H
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Royal Society of Chemistry (UK)
publisher.none.fl_str_mv Royal Society of Chemistry (UK)
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
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