Further evidence that the inhibition of glycogen synthase kinase-3β by IGF-1 is mediated by PDK1/PKB-induced phosphorylation of Ser-9 and not by dephosphorylation of Tyr-216

293 cells were transfected with wild-type GSK3β (WT-GSK3β) or a mutant in which the PKB phosphorylation site (Ser-9) was altered to Ala (A9-GSK3β). Upon stimulation with IGF-1 or insulin, WT-GSK3β was inhibited 75% or 60%, respectively, whereas the activity of the A9-GSK3β mutant was unaffected. Incubation of WT-GSK3β with PP2A1 (a Ser/Thr-specific phosphatase) completely reversed the IGF-1- or insulin-induced inhibition. IGF-1 stimulation did not induce any tyrosine dephosphorylation of WT-GSK3β or A9-GSK3β. Coexpression of WT-GSK3β in 293 cells with either PKBα (also known as AKT) or PDK1 (the 'upstream' activator of PKB) mimicked the IGF-1- or insulin-induced phosphorylation of Ser-9 and inactivation of GSK3β.