Phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1). Stoichiometry and distribution of the phosphorylation sites on the glycogen synthase subunit.
The stoichiometry of the phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1) depended on the concentration of protein kinase in the assay and reached values of 7-8 mol/mol subunit at high concentrations. Phosphorylation by CK-1 above 4 mol/mol subunit promoted a further decrease of glycogen synthase activity when determined by the low glucose-6-phosphate/high glucose-6-phosphate activity ratio assay. Analysis by limited proteolysis with trypsin and chymotrypsin showed that all of the regions in glycogen synthase phosphorylated by casein/glycogen synthase kinase-2 (CK-2), the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase), FA/glycogen synthase kinase-3 (FA/GSK-3) and phosphorylase b kinase were also phosphorylated by CK-1. Digestion with CNBr of glycogen synthase phosphorylated by CK-1 revealed the presence of the two phosphopeptides also labeled by the other protein kinases, the largest phosphopeptide (CB2) containing more phosphorylation sites for CK-1 than the smallest one (CB1). Three phosphopeptides (CB2-c, CB2-d and CB2-e) were obtained by trypsinization of CB2 phosphorylated by CK-1. None of them coincided with those labeled by A-kinase, a fact that was confirmed by the additivity of the effect of both protein kinases. In contrast, CB2-d comigrated with the peptide phosphorylated by FA/GSK-3, and CB2-e with that labeled by CK-2, whereas CB2-c would correspond to a new phosphopeptide.
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002364:Caseins / Q000378:metabolism*; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D006006:Glycogen Synthase / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D014357:Trypsin
D002364:Caseins; D010446:Peptide Fragments; D006006:Glycogen Synthase; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002918:Chymotrypsin; D014357:Trypsin; D003488:Cyanogen Bromide