Phosphorylation of bovine heart glycogen synthase by two protein kinases. Kinetic properties of phosphorylated forms of the enzyme.

A glycogen synthase kinase, partially purified from bovine heart, was used to phosphorylate bovine heart glycogen synthase I.The kinase was not affected by CAMP, the heat-stable CAMP-dependent protein kinase inhibitor, or calcium ion.This enzyme had no activity on mixed histones or skeletal muscle phosphorylase b and only slight activity with a-casein and phosvitin.The K,,, for ATP"&+, when phosphorylating glycogen synthase, was 0.36 m, and GTP, ITP, CTP, and UTP did not serve as phosphoryl donors.One phosphate was incorporated per glycogen synthase subunit.Phosphorylation decreased the activity of glycogen synthase to less than 5% of the maximum activity in the presence of glucose 6-phosphate after a single phosphate was incorporated per subunit.This phosphorylated form of glycogen synthase was designated GS-3.Simultaneous phosphorylation of heart glycogen synthase by both the glycogen synthase kinase and CAMPdependent protein kinase incorporated 3 phosphates/ glycogen synthase subunit with a subsequent decrease in the activity of glycogen synthase to less than 5%.This unusual stoichiometry is discussed extensively.This phosphorylated form of glycogen synthase was designated GS-1,2,3.32P-labeled peptides produced by CNBr cleavage of GS-3 and GS-1,2,3 showed that the glycogen synthase kinase phosphorylated a single, relatively small peptide.This peptide was easily distinguishable on polyacrylamide gels from the two peptides phosphorylated by the catalytic subunit of CAMP-dependent protein kinase.The kinetic properties of each form of heart glycogen synthase, produced by site-specific phosphorylation with two protein kinases, were examined.Site-specific effects on K,,, (UDP-glucose), AI12 (Na2S04), and All2 (glucose-6-P) were demonstrated.Phosphorylation by CAMP-dependent protein kinase produced enzyme forms that could be nearly inactive in the absence of glucose-6-P, but even the most highly phosphorylated form was completely reactivated at relatively low con- centrations of glucose-6-P.