Alternative O-Glycosylation/O-Phosphorylation of the Murine Estrogen Receptor β

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Abstract
Estrogen receptor β, a homologue to estrogen receptor α, is a new member of the steroid hormone receptor family. Recently, we documented that estrogen receptor α, like other transcription factors, is modified by O-linked N-acetylglucosamine (O-GlcNAc), a ubiquitous transitory posttranslational modification on nuclear and cytoplasmic proteins. Here, we report that estrogen receptor β is alternatively modified by either O-GlcNAc or O-phosphate. Lectin chromatography of in vitro translated protein first suggested that murine estrogen receptor β (mER-β) is O-GlcNAcylated. Structural characterization of the carbohydrate moieties on mER-β, overexpressed in insect Sf9 cells, confirmed the presence of O-GlcNAc. mER-β, overexpressed in mammalian cells, is also O-GlcNAcylated. The major site of O-GlcNAc on mER-β from Sf9 cells is Ser16 near the N-terminus. Concomitant analyses also documented the O-phosphorylation of mER-β at Ser16. MALDI-TOF mass spectrometry showed alternative occupancy of this locus by these two abundant and dynamic posttranslational modifications. The localization of a major O-GlcNAc/O-phosphate site in proximity of the transactivation domain and as part of a PEST region (target sequences for rapid protein degradation) on mER-β suggests that these modifications may play a role in regulating estrogen receptor β transactivation and turnover.