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O-GlcNAcylation/Phosphorylation Cycling at Ser10 Controls Both Transcriptional Activity and Stability of Δ-Lactoferrin*

By Stéphan Hardivillé, Esthelle Hoedt, Christophe Mariller, Monique Benaïssa and Annick Pierce

Abstract

Δ-Lactoferrin (ΔLf) is a transcription factor that up-regulates DcpS, Skp1, and Bax genes, provoking cell cycle arrest and apoptosis. It is post-translationally modified either by O-GlcNAc or phosphate, but the effects of the O-GlcNAc/phosphorylation interplay on ΔLf function are not yet understood. Here, using a series of glycosylation mutants, we showed that Ser10 is O-GlcNAcylated and that this modification is associated with increased ΔLf stability, achieved by blocking ubiquitin-dependent proteolysis, demonstrating that O-GlcNAcylation protects against polyubiquitination. We highlighted the 391KSQQSSDPDPNCVD404 sequence as a functional PEST motif responsible for ΔLf degradation and defined Lys379 as the main polyubiquitin acceptor site. We next investigated the control of ΔLf transcriptional activity by the O-GlcNAc/phosphorylation interplay. Reporter gene analyses using the Skp1 promoter fragment containing a ΔLf response element showed that O-GlcNAcylation at Ser10 negatively regulates ΔLf transcriptional activity, whereas phosphorylation activates it. Using a chromatin immunoprecipitation assay, we showed that O-GlcNAcylation inhibits DNA binding. Deglycosylation leads to DNA binding and transactivation of the Skp1 promoter at a basal level. Basal transactivation was markedly enhanced by 2–3-fold when phosphorylation was mimicked at Ser10 by aspartate. Moreover, using double chromatin immunoprecipitation assays, we showed that the ΔLf transcriptional complex binds to the ΔLf response element and is phosphorylated and/or ubiquitinated, suggesting that ΔLf transcriptional activity and degradation are concomitant events. Collectively, our results indicate that reciprocal occupancy of Ser10 by either O-phosphate or O-GlcNAc coordinately regulates ΔLf stability and transcriptional activity

Topics: Glycobiology and Extracellular Matrices
Publisher: American Society for Biochemistry and Molecular Biology
OAI identifier: oai:pubmedcentral.nih.gov:2885199
Provided by: PubMed Central
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