Alleviating the suppression of glycogen synthase kinase-3β by Akt leads to the phosphorylation of cAMP-response element-binding protein and its transactivation in intact cell nuclei

Glycogen synthase kinase-3beta (GSK-3beta) activity is suppressed when it becomes phosphorylated on serine 9 by protein kinase B (Akt). To determine how GSK-3beta activity opposes Akt function we used various methods to alleviate GSK-3beta suppression in prostate carcinoma cells. In some experiments, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase ( a kinase involved in activating Akt) and tumor necrosis factor-alpha (TNF-alpha) were used to activate GSK-3beta. In other experiments mutant forms of GSK-3beta, GSK-3beta(Delta9) (a constitutively active deletion mutant of GSK-3beta) and GSK-3beta(Y216F) ( an inactive point mutant of GSK-3beta) were used to alter GSK-3beta activity. LY294002, TNF-alpha, and overexpression of wild-type GSK-3beta or of GSK-3beta(Delta9), but not GSK-3beta(Y216F), alleviated the suppression of GSK-3beta activity in prostate carcinoma cells and enhanced the turnover of beta-catenin. Forced expression of wild-type GSK-3beta or of GSK-3beta(Delta9), but not GSK-3beta(Y216F), suppressed cell growth and showed that the phosphorylation status of GSK-3beta can affect its intracellular distribution. When transcription factors activator protein-1 and cyclic AMP-response element (CRE)-binding protein were analyzed as targets of GSK-3beta activity, overexpression of wild-type GSK-3beta suppressed AP1-mediated transcription and activated CRE-mediated transcription. Overexpression of GSK-3beta(Delta9) caused an (80-fold) increase in CRE-mediated transcription, which was further amplified ( up to 130-fold) by combining GSK-3betaDelta9 overexpression with the suppression of Jun activity. This study also demonstrated for the first time that expression of constitutively active GSK-3beta(Delta9) results in the phosphorylation of CRE-binding protein on serine 129 and enhancement of CRE-mediated transcription in intact cell nuclei.