Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells.
In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity. The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA. Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT. Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants. Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells). Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue. The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity. A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant. Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively). These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism.
Eldar-Finkelman
H
H
Department of Pharmacology, University of Washington, Seattle 98195, USA.
Argast
G M
GM
Foord
O
O
Fischer
E H
EH
Krebs
E G
EG
D000409:Alanine; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D018698:Glutamic Acid; D006006:Glycogen Synthase / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012694:Serine; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012694:Serine; D006006:Glycogen Synthase; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D000409:Alanine