Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition

Biochim Biophys Acta. 2012 Dec;1820(12):1839-48. doi: 10.1016/j.bbagen.2012.08.024. Epub 2012 Sep 3.


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.

Methods and 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.

General significance: Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / metabolism*
  • Blotting, Western
  • Electrophoresis, Gel, Two-Dimensional
  • Fluorescent Antibody Technique
  • G1 Phase / physiology*
  • Humans
  • Immunoprecipitation
  • MCF-7 Cells
  • N-Acetylglucosaminyltransferases / metabolism*
  • Phosphorylation
  • Protein Processing, Post-Translational*
  • Proteomics*
  • S Phase / physiology*
  • Signal Transduction
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • N-Acetylglucosaminyltransferases
  • O-GlcNAc transferase
  • Acetylglucosamine