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-3β (GSK-3β) activity is suppressed when it becomes phosphorylated on serine 9 by protein kinase B (Akt). To determine how GSK-3β activity opposes Akt function we used various methods to alleviate GSK-3β 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-α (TNF-α) were used to activate GSK-3β. In other experiments mutant forms of GSK-3β, GSK-3βΔ9 (a constitutively active deletion mutant of GSK-3β) and GSK-3βY216F (an inactive point mutant of GSK-3β) were used to alter GSK-3β activity. LY294002, TNF-α, and overexpression of wild-type GSK-3β or of GSK-3βΔ9, but not GSK-3βY216F, alleviated the suppression of GSK-3β activity in prostate carcinoma cells and enhanced the turnover of β-catenin. Forced expression of wild-type GSK-3β or of GSK-3βΔ9, but not GSK-3βY216F, suppressed cell growth and showed that the phosphorylation status of GSK-3β 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-3β activity, overexpression of wild-type GSK-3β suppressed AP1-mediated transcription and activated CRE-mediated transcription. Overexpression of GSK-3βΔ9 caused an (80-fold) increase in CRE-mediated transcription, which was further amplified (up to 130-fold) by combining GSK-3βΔ9 overexpression with the suppression of Jun activity. This study also demonstrated for the first time that expression of constitutively active GSK-3βΔ9 results in the phosphorylation of CRE-binding protein on serine 129 and enhancement of CRE-mediated transcription in intact cell nuclei.