Purification of glycogen synthase kinase 3 from rabbit skeletal muscle. Copurification with the activating factor (FA) of the (Mg-ATP) dependent protein phosphatase.

TLDR: Neither the catalytic subunit of cyclic-AMP-dependent protein kinase nor pphosphorylase kinase were able to activate the (Mg-ATP)-dependent protein phosphatase, and the results suggest that glycogen synthase Kinase 3 and factor FA activities reside in the same protein.

Abstract: 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%. The preparations were estimated to be 50%pure, and the results suggest that glycogen synthase kinase 3 is a monomeric enzyme, Mr=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 rersidues were phosphorylated initially, thephosphorylation of one of these residues being liked 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 Km. There was no significant phosphorylation of glycogen phosphorylase, phosphorylase kinase, protein phosphatase inhibitors 1 and 2, l-pyruvate kinase, acetyl-CoA vstnocylsdr, ATP-citrate lyase, and histones H1 and H2B. The enzyme was also unable to catalyse the inactivation of hydroxymethylglutaryl-CoA reductase. Glycogen synthase kinase 3 underwent an ‘autophosphorylaaaation’ 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 CCCM-Sephadex or hydroxyapatite, or by gel filtration on Sephadex g-100. The two activities had similar Km 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 pphosphorylase 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.