Glycogen synthase kinase-2 and phosphorylase kinase are the same enzyme.

Abstract

Homogeneous preparations of phosphorylase kinase from rabbit skeletal muscle catalyse a calcium-dependent phosphorylation of glycogen synthase a isolated from the same tissue. The calcium-dependent glycogen synthase kinase activity copurifies with phosphorylase kinase throughout the standard procedure for the isolation of the latter enzyme. At the final step of the purification, gel filtration on Sepharose 4B, the elution profiles for glycogen synthase kinase and phosphorylase kinase activities are identical. ICR/IAn mice, which lack muscle phosphorylase kinase activity, do not contain detectable calcium-dependent glycogen synthase kinase activity. These results indicate that the calcium-dependent phosphorylation of glycogen synthase is catalysed by phosphorylase kinase and not by another calcium-dependent protein kinase that might be contaminating the preparation. The phosphorylation of glycogen synthase a by phosphorylase kinase reaches a plateau in the range 0.73 ± 0.1 molecules of phosphate incorporated per subunit and is accompanied by a 2-fold decrease in the activity in the absence of glucose 6-phosphate. The phosphorylation takes place on a unique serine residue located seven amino acids from the N-terminus of the polypeptide chain. The amino acid sequence surrounding serine-7 is similar to the amino acid sequence surrounding the phosphoserine in phosphorylase a. Glycogen synthase kinase-2 is the name given to a protein kinase present as a trace contaminant in highly purified preparations of glycogen synthase [Nimmo, H. G. and Cohen, P. (1974) FEBS Lett. 47, 162–167]. Glycogen synthase kinase-2 also phosphorylates serine-7 exclusively, and evidence is presented which demonstrates that glycogen synthase kinase-2 is merely a modified form of phosphorylase kinase which has lost its ability to be regulated by calcium ions at pH 6.8. The rate of phosphorylation of glycogen synthase a by phosphorylase kinase is 2–3-fold slower than the rate of phosphorylation of phosphorylase b when identical concentrations of the two protein substrates are used (6 μM). At physiological concentrations of glycogen synthase (0.3 mg/ml) and phosphorylase (8.0 mg/ml), the time required for half-maximal phosphorylation of each enzyme by phosphorylase kinase is similar. These results suggest that the phosphorylation of glycogen synthase by phosphorylase kinase may be physiologically significant, and the implications of these findings are considered.