title: Regulatory O-GlcNAcylation sites on FoxO1 are yet to be identified.
abstract: O-GlcNAcylation is a reversible post-translational modification that regulates cytosolic and nuclear proteins. We and others previously demonstrated that FoxO1 is O-GlcNAcylated in different cell types, resulting in an increase in its transcriptional activity. Four O-GlcNAcylation sites were identified in human FOXO1 but directed mutagenesis of each site individually had modest (T317) or no effect (S550, T648, S654) on its O-GlcNAcylation status and transcriptional activity. Moreover, the consequences of mutating all four sites had not been investigated. In the present work, we mutated these sites in the mouse Foxo1 and found that mutation of all four sites did not decrease Foxo1 O-GlcNAcylation status and transcriptional activity, and would even tend to increase them. In an attempt to identify other O-GlcNAcylation sites, we immunoprecipitated wild-type O-GlcNAcylated Foxo1 and analysed the tryptic digest peptides by mass spectrometry using High-energy Collisional Dissociation. We identified T646 as a new O-GlcNAcylation site on Foxo1. However, site directed mutagenesis of this site individually or together with all four previously identified residues did not impair Foxo1 O-GlcNAcylation and transcriptional activity. These results suggest that residues important for the control of Foxo1 activity by O-GlcNAcylation still remain to be identified.
authors.affiliation: INSERM, U1016, Institut Cochin, Paris, France; CNRS, UMR8104, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Fardini
authors.affiliation: Structural and Functional Glycobiology Unit, Lille 1 University, CNRS (UMR 8576), IFR 117, Villeneuve d'Ascq, France; Facultad de Odontología, Universidad Autónoma Benito Juárez de Oaxaca, Oaxaca, Mexico.
authors.forename: Yobana
authors.initials: Y
authors.lastname: Perez-Cervera
authors.affiliation: INSERM, U1068, CRCM, Marseille Protéomique IBiSA, Marseille, F-13009, France; Institut Paoli-Calmettes Team, Cell Polarity, Cell Signaling and Cancer, Marseille, F-13009, France; Aix-Marseille Université, F-13284, Marseille, France; CNRS, UMR7258, CRCM, Marseille, F-13009, France.
authors.forename: Luc
authors.initials: L
authors.lastname: Camoin
authors.affiliation: INSERM, U1016, Institut Cochin, Paris, France; CNRS, UMR8104, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Pagesy
authors.affiliation: Structural and Functional Glycobiology Unit, Lille 1 University, CNRS (UMR 8576), IFR 117, Villeneuve d'Ascq, France.
authors.forename: Tony
authors.initials: T
authors.lastname: Lefebvre
authors.affiliation: INSERM, U1016, Institut Cochin, Paris, France; CNRS, UMR8104, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France. Electronic address: tarik.issad@inserm.fr.
authors.forename: Tarik
authors.initials: T
authors.lastname: Issad
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
chemical_list: C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D011993:Recombinant Fusion Proteins; D000117:Acetylglucosamine
keywords: Forkhead transcription factor box O; O-GlcNAc glycosylation; O-GlcNAc transferase; Post-translational modification; Site-directed mutagenesis; Transcriptional activity