Alternative O-glycosylation/O-phosphorylation of serine-16 in murine estrogen receptor beta: post-translational regulation of turnover and transactivation activity.

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-beta (mER-beta) is alternatively O-GlcNAcylated or O-phosphorylated at Ser(16). Analyses of mER-betas containing mutations in the three adjacent hydroxyl amino acids at this locus confirmed that Ser(16) is the major site of O-GlcNAc modification on mER-beta and that mutants lacking hydroxyl 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 Ser(16), 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 Ser(16) modulates mER-beta protein stability. Luciferase reporter assays also show that the Ser(16) locus mutants have abnormal transactivation activities, suggesting that the two alternative modifications at Ser(16) on mER-beta 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-beta show that the receptor is nearly exclusively localized within the nucleus. It appears that reciprocal occupancy of Ser(16) by either O-phosphate or O-GlcNAc modulates the degradation and activity of mER-beta.