Purified glycogen synthase from rabbit muscle was phosphorylated by cyclic AMP-independent glycogen synthase kinase-1 (GSK-1), which incorporates up to four Pi/ subunit; cyclic AMP-dependent protein kinase (cAMPdPK), which incorporates up to two Pi/subunit; and phosphorylase kinase (PhosbK), which incorporates 0.6 Pi/subunit. In order to define the sites phosphorylated by these kinases, phosphorylated glycogen synthase was treated with CNBr and the peptides obtained were subjected to electrophoresis in 8% polyacrylamide gels in the presence of 0.1% sodium dodecyl sulfate and 8 m urea. Two phosphopeptides (I and II) were obtained from synthase which had been phosphorylated by GSK-1 and the same pattern was obtained from synthase phosphorylated using cAMPdPK. However, glycogen synthase phosphorylated by PhosbK presented only phosphopeptide II. Analysis of CNBr phosphopeptides was carried further by producing tryptic peptides, which were electrophoretically separated on 12.5% polyacrylamide-sodium dodecyl sulfate-urea gels. When phosphopeptide I resulting from the action of GSK-1 was treated with trypsin, three phosphopeptides were observed, whereas when phosphopeptide I proceeding from the action of cAMPdPK was subjected to tryptic hydrolysis, two new phosphopeptides appeared which were different from those originated by glycogen synthase phosphorylated by GSK-1. The tryptic analysis of phosphopeptide II resulting from the action of GSK-1, cAMPdPK, and PhosbK yielded in all cases the same phosphopeptide. We report that GSK-1 is able to phosphorylate at least four sites and that three of those sites are different from two of the three sites phosphorylated by cAMPdPK. In addition GSK-1 and cAMPdPK phosphorylated the same site as PhosbK. It may thus be concluded that these kinases phosphorylated at least six different sites in glycogen synthase. © 1982.
|Journal||Archives of Biochemistry and Biophysics|
|Publication status||Published - 1 Oct 1982|