Abstract
The enzyme glycogen synthase kinase-3 (GSK-3) has been implicated in the control of several metabolic enzymes and transcription factors in response to extracellular signals. In the past, the enzyme has been considered to be a protein Ser/Thr kinase although it was recently reported to contain Tyr(P) (Hughes, K., Nikolakaki, E., Plyte, S. E., Totty, N. F., and Woodgett, J. R. (1993) EMBO J. 12, 803-808). A cDNA encoding rabbit skeletal muscle GSK-3 beta was cloned and expressed in Escherichia coli as an active protein kinase, with apparent M(r) 46,000, capable of phosphorylating several known GSK-3 substrates. Recombinant GSK-3 beta autophosphorylated on Ser, Thr, and Tyr residues although the enzyme already contained Tyr(P) as judged by its recognition by anti-Tyr(P) antibodies. The net result of the autophosphorylation was a 3-5-fold reduction in enzyme activity. GSK-3 alpha, purified from rabbit muscle, also underwent autophosphorylation but only on Ser and Thr residues. In this case, the autophosphorylation stabilized the enzyme activity compared with the control lacking ATP/Mg2+. Of several phosphatases tested, the lambda-phage phosphatase was the most effective in dephosphorylating at Ser and Thr residues but did not dephosphorylate at Tyr residues. The action of the lambda-phosphatase caused a reactivation of GSK-3 beta to approximately 80% of the starting activity. The protein tyrosine phosphatase PTP1B was able to dephosphorylate at Tyr residues leading to a reduction in enzyme activity. A truncated form of GSK-3 beta, apparent M(r) 40,000, had a significantly higher specific activity, was defective in autophosphorylation, and was not inactivated in the autophosphorylation reaction. We conclude that GSK-3 beta is a dual specificity protein kinase in the same sense as the mitogen-activated protein kinase/ERK family of enzymes. Phosphorylation at different residues differentially controls enzyme activity, Ser/Thr phosphorylation causing inactivation and Tyr phosphorylation resulting in increased activity.
Highlights
The enzyme glycogen synthase kinase-3 (GSK-3) has been implicated in the control of several metabolic enzymes and transcription factors in response to extracellular signals
Recombinant GSK-30 autophosphorylated on Ser, Thr, and Tyr residues the enzyme already contained Tyr(P)as judged by its recognition by anti-Tyr(P) antibodies
PTPlB wasable to dephosphorylate at Tyr residues named for its involvement in the control of glycogen metaboleading to a reduction in enzyme activity.A truncated lism (Woodgett, 1991) and has been implicated in regulating form of GSK3P, apparent M,40,000, had a significantly metabolicenzymes such as glycogen synthase
Summary
Molecular Cloningof GSK-3f3 cDNA-Based on known rabbit muscle A. Kinase activity was eluted with a linear gradieonft 50-500 mM NaCl in buffer A (tota3l 60 ml). Fractions (0.75 ml) containing GSK-30 activity werpeooled, concentrated to ml (PY20) and peroxidase-conjugated polyclonal anti-mouse IgG antibodies were purchased from ICN and Sigrmesap,ectively. Themembraneswerethenincubatedwith as described above, diluted with 9 molf buffer A, and chromato- anti-GSK-3 or anti-Tyr(P) antibody PY20 and further probed with a graphed on an S-Sepharose Fast Flow The isolationof the OtherMaterials and Methods-For phosphoamino acid analysis, proteolytic fragmentof GSK-3j3 was performed in the samweay except phosphorylated proteins were resolved by SDS-PAGE using 12% polythat several protease inhibitors Protein Kinase Assays-GSK-3 kinase activity was measured rou- one-dimensional thin layer electrophoresis and detectebdy autoradiogtinely by assay of phospho-CREB peptide phosphorylation.
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