Akt stabilizes estrogen receptor alpha with the concomitant reduction in its transcriptional activity

Abstract

We have investigated the effect of Akt on estrogen receptor (ER) a protein level and its transcriptional activity. Transient transfection studies revealed that constitutively active AM up-regulated ER alpha at the post-transcriptional level. Studies using Akt inhibitor and dominant-negative Akt1 showed that Akt1 kinase activity is required for the up-regulation of ERa. Cycloheximicle decay assays and studies with proteasome inhibitor indicated that Akt1-mediated up-regulation of ER alpha was maintained by inhibiting proteasome-mediated degradation of ERa. When Akt consensus phosphorylation site mutant, ER alpha S167A was tested for Akt1-mediated up-regulation, increase of ER alpha S167A by Akt1 was significantly impaired as compared to wild type ERa. In addition, dominant-negative glycogen synthase kinase (GSK) 3 beta and LiCl could also partially up-regulate ERa protein level, suggesting that concerted action of Akt1-mediated phosphorylation on S167 and kinase activity of Akt-downstream GSK3 beta could affect ERa protein level. Paradoxically, co-expression of Akt1 could down-regulate transcriptional activity of ER alpha. The inhibitory effect of Akt1 on ERa transcriptional activity was not attributable to changes in subcellular distribution of ERa. Transfection studies using increasing amount of Akt1 and ERa indicated that the transcriptional activity of ER alpha was negatively regulated by ERa protein quantities at higher ER alpha concentrations. Chromatin immunoprecipitation assays revealed that at Akt1 concentration high enough to induce up-regulation of ERa, association of ERa to promoter region of ERa target pS2 gene was impaired. Taken together, these data suggest that Akt1 could increase ER alpha protein level with simultaneous reduction in its transcriptional activity, possibly by modulating association of ERa to the target gene promoters.