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Title: Regulatory O-GlcNAcylation sites on FoxO1 are yet to be identified

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. - Highlights: • We mutate four previously identified O-GlcNAcylationmore » sites on Foxo1. • Unexpectedly, these mutations do not reduce Foxo1 O-GlcNAcylation. • These mutation do not reduce Foxo1 transcriptional activity. • We identify a new O-GlcNAcylation site on Foxo1 by mass spectrometry. • Mutation of this site increases Foxo1 transcriptional activity.« less
Authors:
 [1] ;  [2] ;  [2] ;  [3] ;  [4] ;  [5] ;  [2] ;  [2] ;  [2] ;  [1] ;  [2] ;  [2] ;  [3] ;  [1] ;  [2] ;  [2]
  1. INSERM, U1016, Institut Cochin, Paris (France)
  2. (France)
  3. Structural and Functional Glycobiology Unit, Lille 1 University, CNRS (UMR 8576), IFR 117, Villeneuve d'Ascq (France)
  4. (Mexico)
  5. INSERM, U1068, CRCM, Marseille Protéomique IBiSA, Marseille, F-13009 (France)
Publication Date:
OSTI Identifier:
22462094
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 462; Journal Issue: 2; Other Information: Copyright (c) 2015 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; DISSOCIATION; FLUORESCENCE; GLUCOSAMINE; HUMAN POPULATIONS; LIQUIDS; MASS SPECTROSCOPY; MICE; MODIFICATIONS; MUTAGENESIS; MUTATIONS; PEPTIDES; RESIDUES; TRANSCRIPTION FACTORS