Glycogen synthase kinase-3 protects estrogen receptor α from proteasomal degradation and is required for full transcriptional activity of the receptor

Glycogen synthase kinase- 3 ( GSK- 3) plays a key role in the regulation of transcription factors including steroid receptors. Having identified estrogen receptor-alpha ( ER alpha) as substrate for GSK- 3, the impact of GSK- 3 on ER alpha function and activity upon 17 beta- 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- 3 alpha or GSK- 3 beta results in the reduction of ER alpha levels and transcriptional activity in ER alpha- positive breast cancer cells. Using MCF- 7 cells we demonstrate that reduction of ER alpha levels upon GSK- 3 silencing was due to increased proteasomal degradation of ER alpha rather than inhibition of ER alpha protein synthesis. Indeed, under this condition, ER alpha protein was rescued using the proteasome inhibitor MG132 in presence of the protein synthesis inhibitor cycloheximide. In addition, strong accumulation of ubiquitinated ER alpha was obtained after GSK- 3 silencing in the presence of MG132. We conclude that GSK- 3 protects ER alpha from proteasomal degradation and plays a crucial role in ER alpha protein stabilization and turnover. Furthermore, in vitro kinase assay depicted that GSK- 3 beta phosphorylates ER alpha at Ser- 118. GSK- 3 silencing resulted in decrease of E2- induced nuclear ER alpha 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 estrogenresponsive genes ( pS2 and progesterone receptor) was decreased upon GSK- 3 silencing. These findings demonstrated that GSK- 3 is required for E2- induced ER alpha phosphorylation at Ser118 and full transcriptional activity of the receptor upon E2 stimulation.