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O-GlcNAcylation of the human epidermal growth factor receptor

AutorStateva, Silvia R.; Villalobo, Antonio
Fecha de publicación2015
EditorRoyal Society of Chemistry (Great Britain)
CitaciónOrganic and Biomolecular Chemistry 13(30): 8196-8204 (2015)
ResumenThe 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.
Versión del editorhttps://doi.org/10.1039/C5OB00443H
URIhttp://hdl.handle.net/10261/125079
DOI10.1039/C5OB00443H
Identificadoresdoi: 10.1039/C5OB00443H
issn: 1477-0520
e-issn: 1477-0539
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