Amino acid sequence at the site on rabbit skeletal muscle glycogen synthase phosphorylated by the endogenous glycogen synthase kinase-2 activity

Abstract

Purified preparations of glycogen synthase (EC 2.4.1.11) have been shown to be contaminated with two types of protein kinase that phosphorylate the enzyme. One of these kinases was cyclic AMPdependent protein kinase (EC 2.7.1.37), while the other was an activity termed glycogen synthase kinase-2. The latter enzyme could be distinguished from cyclic AMP-dependent protein kinase in a number of ways. Its activity was unaffected by cyclic AMP or by the specific protein inhibitor of cyclic AMP-dependent protein kinase, it had a higher Km for ATP, and a different nucleoside triphosphate specificity [ 11. Following digestion of the native enzyme with trypsin, the sites phosphorylated by cyclic AMPdependent protein kinase were found to be released much more rapidly than those phosphorylated by glycogen synthase kinase-2, and this suggested that the two kinases phosphorylated different sites on the enzyme [ 11. The amino acid sequences at the two sites phosphorylated by cyclic AMP-dependent protein kinase have been determined [2-41. In this paper, we show that the endogenous glycogen synthase kinase-2 activity, preferentially phosphorylates a serine residue distinct from either of the sites labelled by cyclic AMP-dependent protein kinase. In conjunction with the information presented in [4], it is concluded that this serine is located seven residues from the N-terminus of the polypeptide chain. 2. Materials and methods