Alternative O-glycosylation/O-phosphorylation of serine-16 in murine estrogen receptor beta - Post-translational regulation of turnover and transactivation activity
Xg. Cheng et Gw. Hart, Alternative O-glycosylation/O-phosphorylation of serine-16 in murine estrogen receptor beta - Post-translational regulation of turnover and transactivation activity, J BIOL CHEM, 276(13), 2001, pp. 10570-10575
O-Linked N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational mod
ification abundant on nuclear and cytoplasmic proteins. Recently, we demons
trated that the murine estrogen receptor-beta (mER-beta) is alternatively O
-GlcNAcylated or O-phosphorylated at Ser(16). Analyses of mER-betas contain
ing mutations in the three adjacent hydroxyl amino acids at this locus conf
irmed that Ser(16) is the major site of O-GlcNAc modification on mER-beta a
nd 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 Ser(1
6), 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 re
porter assays also show that the Ser(16) locus mutants have abnormal transa
ctivation 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 bindin
g. 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-ph
osphate or O-GlcNAc modulates the degradation and activity of mER-beta.