O-Linked N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification abundant on nuclear and cytoplasmic proteins. Recently, we demonstrated that the murine estrogen receptor-β (mER-β) is alternatively O-GlcNAcylated or O-phosphorylated at Ser16. Analyses of mER-βs containing mutations in the three adjacent hydroxyl amino acids at this locus confirmed that Ser16 is the major site of O-GlcNAc modification on mER-β and that mutants lacking hydoxyl amino acids at this locus are glycosylation-deficient. Pulse-chase studies in transfected Cos-1 cells demonstrate that the turnover rate of the mutant containing a glutamic acid moiety at Ser16, which mimics constitutive phosphorylation at this locus, is faster than that of the wild type receptor. Whereas, the mutant without hydroxyl amino acids at this locus is degraded at a slower rate, indicating that O-GlcNAc/O-phosphate at Ser16 modulates mER-β protein stability. Luciferase reporter assays also show that the Ser 16 locus mutants have abnormal transactivation activities, suggesting that the two alternative modifications at Ser16 on mER-β may also be involved in transcriptional regulation. DNA mobility shift assays show that the mutants do not have altered DNA binding. Green fluorescence protein constructs of both wild type and mutant forms of mER-β show that the receptor is nearly exclusively localized within the nucleus. It appears that reciprocal occupancy of Ser16 by either O-phosphate or O-GlcNAc modulates the degradation and activity of mER-β.
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