Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition |
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Authors: | Ludivine Drougat Stéphanie Olivier-Van Stichelen Marlène Mortuaire François Foulquier Anne-Sophie Lacoste Jean-Claude Michalski Tony Lefebvre Anne-Sophie Vercoutter-Edouart |
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Institution: | 1. Unité de Glycobiologie Structurale et Fonctionnelle, UMR CNRS/USTL no 8576, IFR 147, Avenue Mendeleïev, Université des Sciences et Technologies de Lille, 59655 Villeneuve d''Ascq, France;2. Plateforme de Protéomique, IFR 147, Université des Sciences et Technologies de Lille, 59655 Villeneuve d''Ascq, France |
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Abstract: | BackgroundDNA 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 resultsQuiescent 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.ConclusionsThe 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 significanceOur results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation. |
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Keywords: | CDK Cyclin dependent kinase CHX Cycloheximide CK Cytokeratin hnRNP Heterogeneous nuclear ribonucleoprotein HU Hydroxyurea MCM Minichromosome maintenance O-GlcNAc O-linked beta-N-acetylglucosaminylation OGA -GlcNAcase or beta-N-acetylglucosaminidase" target="_blank">O-GlcNAcase or beta-N-acetylglucosaminidase OGT O-GlcNAc transferase PTM Post-translational modification pRb Retinoblastoma protein UDP-GlcNAc Uridine diphosphate N-acetylglucosamine |
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