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

Abstract

⌬-Lactoferrin (⌬Lf) 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 ⌬Lf 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 ⌬Lf 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 ⌬Lf degradation and defined Lys 379 as the main polyubiquitin acceptor site. We next investigated the control of ⌬Lf transcriptional activity by the O-GlcNAc/phosphorylation interplay. Reporter gene analyses using the Skp1 promoter fragment containing a ⌬Lf response element showed that O-GlcNAcylation at Ser 10 negatively regulates ⌬Lf 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 ⌬Lf transcriptional complex binds to the ⌬Lf response element and is phosphorylated and/or ubiquitinated, suggesting that ⌬Lf 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 ⌬Lf stability and transcriptional activity.

O-GlcNAc occurs cotranslationally to stabilize nascent polypeptide chains

“…Answering such questions will depend on time-resolved mapping of O-GlcNAcylation sites on both coand post-translationally O-GlcNAcylated proteins. On a more functional level, O-GlcNAc is a cellular stress response 6,35 that is also nutrient responsive [39][40][41] , and so it seems likely that cotranslational O-GlcNAcylation is an adaptive response enabling protection of nascent chains during times of stress or high metabolic flux, a view that is consistent with the protective effects of increased O-GlcNAc in various models 42 and the effects of altered O-GlcNAc levels on the levels of some proteins [7][8][9]37 .…”

Regulation of Protein Degradation by O-GlcNAcylation: Crosstalk with Ubiquitination

“…O-GlcNAcylation stabilizes ⌬-lactoferrin and retains a basal level of transcriptional activity. Upon activation, ⌬-lactoferrin is phosphorylated at S10, which promotes transcription and subsequent degradation through K379 polyubiquitination (27). These studies point to the idea that protein functions can be precisely controlled by dynamic and coordinated changes in O-GlcNAcylation, phosphorylation, and ubiquitination.…”

“…⌬-lactoferrin expression is down-regulated in cancer cells, whereas its highlevel expression is correlated with a good prognosis in human breast cancer (25). It has been shown that ⌬-lactoferrin is reciprocally O-GlcNAcylated and phosphorylated at S10 (26,27). O-GlcNAcylation stabilizes ⌬-lactoferrin and retains a basal level of transcriptional activity.…”

O-GlcNAc cycling mutants modulate proteotoxicity in Caenorhabditis elegans models of human neurodegenerative diseases

“…Excessive O-GlcNAcylation has been shown to block proteasome activity and proteasomedependent degradation of Sp1, p53, and delta-lactoferrin (49)(50)(51)(52). Moreover, knocking down OGT activity leads to proteasome activation (51).…”