GLYCOGEN-SYNTHASE KINASE-3-BETA IS A DUAL-SPECIFICITY KINASE DIFFERENTIALLY REGULATED BY TYROSINE AND SERINE THREONINE PHOSPHORYLATION/

The enzyme glycogen synthase kinase-3 (GSK-3) has been implicated in t he control of several metabolic enzymes and transcription factors in r esponse to extracellular signals. In the past, the enzyme has been con sidered to be a protein Ser/Thr kinase although it was recently report ed 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 encodi ng rabbit skeletal muscle GSK-3 beta was cloned and expressed in Esche richia coli as an active protein kinase, with apparent M(r) 46,000, ca pable of phosphorylating several known GSK-3 substrates. Recombinant G SK-3 beta autophosphorylated on Ser, Thr, and Tyr residues although th e 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 re sidues. In this case, the autophosphorylation stabilized the enzyme ac tivity compared with the control lacking ATP/Mg2+. Of several phosphat ases tested, the lambda-phage phosphatase was the most effective in de phosphorylating at Ser and Thr residues but did not dephosphorylate at Tyr residues. The action of the lambda-phosphatase caused a reactivat ion of GSK-3 beta to similar to 80% of the starting activity. The prot ein tyrosine phosphatase PTP1B was able to dephosphorylate at Tyr resi dues leading to a reduction in enzyme activity. A truncated form of GS K-3 beta, apparent M(r) 40,000, had a significantly higher specific ac tivity, 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-activ ated protein kinase/ERK family of enzymes. Phosphorylation at differen t residues differentially controls enzyme activity, Ser/Thr phosphoryl ation causing inactivation and Tyr phosphorylation resulting in increa sed activity.