Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

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

Date

09/13/2012

Pubmed ID

22967762

DOI

10.1016/j.bbagen.2012.08.024

Scopus ID

2-s2.0-84866531577   47 Citations

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.

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.

Author List

Drougat L, Olivier-Van Stichelen S, Mortuaire M, Foulquier F, Lacoste AS, Michalski JC, Lefebvre T, Vercoutter-Edouart AS

Author

Stephanie Olivier-Van Stichelen PhD Assistant Professor in the Biochemistry department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

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