Figure 5

Regulation of β-catenin by t-DARPP is AKT-dependent. (A) t-DARPP expression was induced in tetracycline-inducible AGS-t-DARPP cells following treatment with doxycycline for a period of 48 h. Consistent with findings in cells stably overexpressing t-DARPP, induction of t-DARPP expression led to marked accumulation of β-catenin, c-MYC, Cyclin D1, pGSK-3β (Ser9), and pAKT (Ser473). (B) Proteins obtained from MKN45 cells that express endogenous t-DARPP transfected with either control scrambled siRNA or t-DARPP specific siRNA oligonucleotides were subjected to Western blot analysis. As shown, knockdown of endogenous t-DARPP led to a marked decrease in protein levels of β-catenin, c-MYC, Cyclin D1, pAKT (Ser473), and pGSK-3β (Ser9). (C) AGS cells stably overexpressing t-DARPP were treated with dimethyl sulfoxide (DMSO) as control and LY294002 (40 uM), a potent PI3 kinase inhibitor, for 30 min and 2 h. As shown by Western blot analysis, treatment with LY294002 led to complete abrogation of downstream AKT and GSK-3β phosphorylation in t-DARPP expressing AGS cells. Inhibition of PI3 kinase in AGS-t-DARPP cells resulted in significant downregulation of β-catenin, c-MYC and Cyclin D1.