Inactivation of glycogen-synthase kinase-3-beta by phosphorylation: new kinase connections in insulin and growth-factor signaling

The beta-isoform of glycogen synthase kinase-3 (GSK3beta) isolated from rabbit skeletal muscle was inactivated 90-95 % following incubation with MgATP and either MAP kinase-activated protein kinase-1 (MAPKAP kinase-1, also termed RSK-2) or p70 S6 kinase (p70S6K), and re-activated with protein phosphatase 2A. MAPKAP kinase-1 and p70S6K phosphorylated the same tryptic peptide on GSK3beta, and the site of phosphorylation was identified as the serine located nine residues from the N-terminus of the protein. The inhibitory effect of Ser-9 phosphorylation on GSK3beta activity was observed with three substrates, (inhibitor-2, c-jun and a synthetic peptide), and also with glycogen synthase provided that 0.15 M KCl was added to the assays. The results suggest that Ser-9 phosphorylation underlies the reported inhibition of GSK3beta by insulin and that GSK3 may represent a point of convergence of two major growth-factor-stimulated protein kinase cascades.