A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation.

The inhibition of GSK3 is required for the stimulation of glycogen and protein synthesis by insulin and the specification of cell fate during development. Here, we demonstrate that the insulin-induced inhibition of GSK3 and its unique substrate specificity are explained by the existence of a phosphate binding site in which Arg-96 is critical. Thus, mutation of Arg-96 abolishes the phosphorylation of "primed" glycogen synthase as well as inhibition by PKB-mediated phosphorylation of Ser-9. Hence, the phosphorylated N terminus acts as a pseudosubstrate, occupying the same phosphate binding site used by primed substrates. Significantly, this mutation does not affect phosphorylation of "nonprimed" substrates in the Wnt-signaling pathway (Axin and beta-catenin), suggesting new approaches to design more selective GSK3 inhibitors for the treatment of diabetes.