O-GlcNAcylation/phosphorylation cycling at Ser10 controls both transcriptional activity and stability of delta-lactoferrin.
Delta-lactoferrin (DeltaLf) 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 DeltaLf function are not yet understood. Here, using a series of glycosylation mutants, we showed that Ser(10) is O-GlcNAcylated and that this modification is associated with increased DeltaLf stability, achieved by blocking ubiquitin-dependent proteolysis, demonstrating that O-GlcNAcylation protects against polyubiquitination. We highlighted the (391)KSQQSSDPDPNCVD(404) sequence as a functional PEST motif responsible for DeltaLf degradation and defined Lys(379) as the main polyubiquitin acceptor site. We next investigated the control of DeltaLf transcriptional activity by the O-GlcNAc/phosphorylation interplay. Reporter gene analyses using the Skp1 promoter fragment containing a DeltaLf response element showed that O-GlcNAcylation at Ser(10) negatively regulates DeltaLf 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 Ser(10) by aspartate. Moreover, using double chromatin immunoprecipitation assays, we showed that the DeltaLf transcriptional complex binds to the DeltaLf response element and is phosphorylated and/or ubiquitinated, suggesting that DeltaLf transcriptional activity and degradation are concomitant events. Collectively, our results indicate that reciprocal occupancy of Ser(10) by either O-phosphate or O-GlcNAc coordinately regulates DeltaLf stability and transcriptional activity.
Hardivillé
Stéphan
S
Unité de Glycobiologie Structurale et Fonctionnelle, Unité Mixte de Recherche 8576 CNRS, Université des Sciences et Technologies de Lille, IFR 147, 59655 Villeneuve d'Ascq, France.
Hoedt
Esthelle
E
Mariller
Christophe
C
Benaïssa
Monique
M
Pierce
Annick
A
D000117:Acetylglucosamine / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry; D002241:Carbohydrates / Q000737:chemistry; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D017930:Genes, Reporter; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000379:methods; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012694:Serine / Q000737:chemistry*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D002241:Carbohydrates; D001224:Aspartic Acid; D012694:Serine; D004247:DNA; D007781:Lactoferrin; D000117:Acetylglucosamine
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