title: Glycogen synthase kinase-3 protects estrogen receptor alpha from proteasomal degradation and is required for full transcriptional activity of the receptor.
abstract: Glycogen synthase kinase-3 (GSK-3) plays a key role in the regulation of transcription factors including steroid receptors. Having identified estrogen receptor-alpha (ERalpha) as substrate for GSK-3, the impact of GSK-3 on ERalpha function and activity upon 17beta-estradiol (E2)-dependent activation remains to be clarified. Here we show by using small interfering technology in combination with immunoblot, gene expression analysis, and luciferase reporter assays that silencing of GSK-3alpha or GSK-3beta results in the reduction of ERalpha levels and transcriptional activity in ERalpha-positive breast cancer cells. Using MCF-7 cells we demonstrate that reduction of ERalpha levels upon GSK-3 silencing was due to increased proteasomal degradation of ERalpha rather than inhibition of ERalpha protein synthesis. Indeed, under this condition, ERalpha protein was rescued using the proteasome inhibitor MG132 in presence of the protein synthesis inhibitor cycloheximide. In addition, strong accumulation of ubiquitinated ERalpha was obtained after GSK-3 silencing in the presence of MG132. We conclude that GSK-3 protects ERalpha from proteasomal degradation and plays a crucial role in ERalpha protein stabilization and turnover. Furthermore, in vitro kinase assay depicted that GSK-3beta phosphorylates ERalpha at Ser-118. GSK-3 silencing resulted in decrease of E2-induced nuclear ERalpha phosphorylation at Ser-118 and E2-induced estrogen response element-dependent luciferase reporter gene expression. Neither Ser-118 phosphorylation nor luciferase activity was restored by use of MG132. Moreover, the expression of estrogen-responsive genes (pS2 and progesterone receptor) was decreased upon GSK-3 silencing. These findings demonstrated that GSK-3 is required for E2-induced ERalpha phosphorylation at Ser-118 and full transcriptional activity of the receptor upon E2 stimulation.
authors.affiliation: Hormones and Signal Transduction Group, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Jean
authors.initials: J
authors.lastname: Grisouard
authors.forename: Senad
authors.initials: S
authors.lastname: Medunjanin
authors.forename: Alexander
authors.initials: A
authors.lastname: Hermani
authors.forename: Ashish
authors.initials: A
authors.lastname: Shukla
authors.forename: Doris
authors.initials: D
authors.lastname: Mayer
mesh_terms: D045744:Cell Line, Tumor; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D025801:Ubiquitin / Q000037:antagonists & inhibitors
chemical_list: D047628:Estrogen Receptor alpha; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025801:Ubiquitin; D012694:Serine; D004958:Estradiol; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; C448021:glycogen synthase kinase 3 alpha; D046988:Proteasome Endopeptidase Complex