Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Identification of the sites phosphorylated by casein kinase-I.
A casein kinase was highly purified from rabbit skeletal muscle whose substrate specificity and enzymatic properties were virtually identical to those of casein kinase-I from rabbit reticulocytes. Prolonged incubation of glycogen synthase with high concentrations of skeletal muscle casein kinase-I and Mg-ATP resulted in the incorporation of greater than 6 mol phosphate/mol subunit and decreased the activity ratio (+/- glucose-6P) from 0.8 to less than 0.02. The sites phosphorylated by casein kinase-I were all located in the N and C-terminal cyanogen bromide peptides, termed CB-1 and CB-2. At an incorporation of 6 mol phosphate/mol subunit, approximately equal to 2 mol/mol was present in CB-1 and approximately equal to 4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated the serines that were 3, 7 and 10 residues from the N-terminus of glycogen synthase, with minor phosphorylation at threonine-5. Within CB-2, approximately equal to 90% of the phosphate incorporated was located between residues 28 and 53, and at least five of the seven serine residues in this region were phosphorylated. The remaining 10% of phosphate incorporated into CB-2 was located between residues 98 and 123, mainly at a serine residue(s). Two of the major sites labelled by casein kinase-I (serine-3 and serine-10 of CB-1) are not phosphorylated by any other protein kinase. This will enable the role of casein kinase-I as a glycogen synthase kinase in vivo to be evaluated.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D047388:Casein Kinases; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006006:Glycogen Synthase / Q000378:metabolism*; D066298:In Vitro Techniques; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology
D006006:Glycogen Synthase; D011494:Protein Kinases; D047388:Casein Kinases