title: Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition.
abstract: BACKGROUND
  DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood.
  
  
  RESULTS
  Quiescent MCF7 cells were stimulated by serum mitogens and cell cycle progression was determined by flow cytometry. The levels of O-GlcNAc modified proteins, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) were examined by Western blotting and OGA activity was measured during the progression of cells towards S phase. A global decrease in O-GlcNAcylation was observed at S phase entry, concomitantly to an increase in the activity of OGA. A combination of two-dimensional electrophoresis, Western blotting and mass spectrometry was then used to detect and identify cell cycle-dependent putative O-GlcNAcylated proteins. 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated through G1/S transition were identified and the O-GlcNAc variations of Cytokeratin 8, hnRNP K, Caprin-1, Minichromosome Maintenance proteins MCM3, MCM6 and MCM7 were validated by immunoprecipitation.
  
  
  CONCLUSIONS
  The dynamics of O-GlcNAc is regulated during G1/S transition and observed on key proteins involved in the cytoskeleton networks, mRNA processing, translation, protein folding and DNA replication.
  
  
  CONCLUSIONS
  Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.
authors.affiliation: Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
authors.forename: Ludivine
authors.initials: L
authors.lastname: Drougat
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Olivier-Van Stichelen
authors.forename: Marlène
authors.initials: M
authors.lastname: Mortuaire
authors.forename: François
authors.initials: F
authors.lastname: Foulquier
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Lacoste
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Michalski
authors.forename: Tony
authors.initials: T
authors.lastname: Lefebvre
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Vercoutter-Edouart
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D005455:Fluorescent Antibody Technique; D016193:G1 Phase / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D061986:MCF-7 Cells; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D016196:S Phase / Q000502:physiology*; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D000117:Acetylglucosamine