Expression of active Akt inhibits proteasome-mediated degradation of estrogen receptor alpha

Abstract

In an attempt to investigate the effect of Akt (protein kinase B) on the protein level of estrogen receptor alpha(ERα), transient transfection studies were performed in 293T cells using constitutively active Akt and ERα expression constructs. Co-expression of constitutively active Akt resulted in up-regulation of ERα protein without changes in mRNA level, suggesting that Akt-mediated up-regulation of ERα occurs at the translational level. Studies using phosphatidylinositol 3-kinase (PI3K) inhibitors and Akt inhibitor revealed that treatment of these inhibitors effectively decreased the protein level of ERα, implicating that kinase activity of Akt is essential for ERα up-regulation. Cycloheximide decay assay and studies with proteasome inhibitor showed that Akt-mediated up-regulation of ERα was maintained by inhibiting proteasome-mediated degradation of ERα. When Akt-consensus phosphorylation site mutant, ERα S167A was tested for Akt-mediated up-regulation, ERα S167A protein level was also increased by Akt comparable to wild type ERα. This result may suggest that up-regulation of ERα is not mediated by direct phosphorylation of ERα on Ser167 by Akt. In coimmunoprecipitation assays, a 90 kDa protein detected by western blot using Akt-substrate antibody was shown to be associated with ERα from the sample expressing both active Akt and ERα. This result might suggest that phosphorylation of unknown 90 kDa protein by Akt, may increase its association with ERα and promotes stabilization of ERα.