Eur. J. Biochem. 119, 443-451 (1981) zyxwvutsr '0 FEBS 1981 zyxwvutsrqpon Purification of Glycogen Synthase Kinase 3 from Rabbit Skeletal Muscle Copurification with the Activating Factor (FA) of the (Mg-ATP) Dependent Protein Phosphatase Brian A. HEMMINGS, David YELLOWLEES, John C. KERNOHAN, and Philip COHEN Department of Biochemistry, University of Dundee, Dundee (Received June 9, 1981) Glycogen synthase kinase 3 was purified 80- 100000-fold from extracts of rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate, chromatography on phosphocellulose and Affigel Blue, and affinity chromatography on (glycogen synthase)-Agarose. 0.08 mg of protein was isolated from 5000 g muscle corresponding to a yield of 8 zyxwvuts %. The preparations were estimated to be 50 % pure, and the results suggest that glycogen synthase kinase 3 is a monomeric enzyme, zyxwvu M, = 54000 .+ 3000. Glycogen synthase kinase 3 phosphorylated three serine residues in glycogen synthase, all contained within nine amino acids in the same tryptic peptide. The rate of formation of the mono-, di- and triphosphorylated derivatives of the peptide showed that the three sites were not phosphorylated either randomly or in a simple sequential manner. The results could be fitted to a model in which either of two serine residues were phosphorylated initially, the phosphorylation of one of these residues being linked to an extremely rapid phosphorylation of the third serine. Glycogen synthase kinase 3 phosphorylated glycogen synthase much more effectively than all other proteins tested. Casein was phosphorylated at a very low rate and with a much higher K,. There was no significant phosphorylation of glycogen phosphorylase, phosphorylase kinase, protein phosphatase inhibitors 1 and 2, 1,-pyruvate kinase, acetyl-CoA carboxylase, ATP-citrate lyase, and histones HI and H2B. The enzyme was also unable to catalyse the inactivation of hydroxymethylglutaryl-CoA reductase. Glycogen synthase kinase 3 underwent an 'autophosphorylation' reaction following incubation with Mg-ATP and up to 4 mol of phosphate could be incorporated per mol of enzyme. The activating factor (FA) of the (Mg-ATP)-dependent protein phosphatase was found to copurify with glycogen synthase kinase 3 throughout the isolation procedure. It also proved impossible to separate the two activities by chromatography on CM-Sephadex or hydroxyapatite, or by gel filtration on Sephadex G-100. The two activities had similar K, values for ATP and GTP. The results suggest that glycogen synthase kinase 3 and factor FA activities reside in the same protein. Neither the catalytic subunit of cyclic-AMP-dependent protein kinase nor phosphorylase kinase were able to activate the (Mg-ATP)-dependent protein phosphatase. The implications of these findings for the regulation of glycogen metabolism, and the mechanism of activation of the (Mg-ATP)-dependent protein phosphatase are discussed. Glycogen synthase can be phosphorylated and inactivated zyxwv in zyxwvutsrqponmlkji vitlv by at least three different protein kinases; cyclic-AMP- dependent protein kinase, phosphorylase kinase and an en- zyme termed glycogen synthase kinase 3 111. The activity of glycogen synthase kinase 3 is unaffected by cyclic AMP, cyclic GMP, calcium ions, calmodulin or the specific protein inhibitor of cyclic-AMP-dependent protein kinase [I]. It has been partially purified from rabbit skeletal muscle and shown to phosphorylate three serine residues on glycogen synthase, all located within nine amino acids in the primary structure [2]. These residues are distinct from the sites phosphorylated by either cyclic-AMP-dependent protein kinase or phos- phorylase kinase [2,3]. Glycogen synthase kinase 3 does not phosphorylate other enzymes involved in the regulation of glycogen metabolism (phosphorylase, phosphorylase kinase, protein phosphatase inhibitor 1) at a significant rate [I]. This paper is dedicated to Professor Hclinut IIolzer on the occasion Enzymc. Glycogen synthase kinase (EC 2.7.1.37). of his 60th birthday. Partially purified preparations of glycogen synthase ki- nase 3 were found to be contaminated with the activating factor (FA) of an enzyme termed the (Mg-ATP)-dependent protein phosphatase [4,5). This enzyme is only an active protein phosphatase after it has been preincubated with factor FA and Mg-ATP [5-81. It has a broad substrate specificity and dephosphorylates glycogen phosphorylase, phosphorylase kinase, glycogen synthase and other proteins [3,5]. Its properties are remarkably similar to those of protein phosphatase 1, the major protcin phosphatase involved in the regulation of glycogen metabolism in rabbit skeletal muscle [5]. Since glycogen synthase kinase 3 catalyses the phos- phorylation of glycogen synthase while factor FA promotes the dephosphorylation of a number of phosphoproteins including glycogen synthase, it was anticipated that these two activities would reside in distinct proteins. The results described in this paper demonstrate that this is not the casc, however, and glycogen synthase kinase 3 and factor FA appear to be one and the same protein.