In vivo phosphorylation of liver glycogen synthase. Effect of glucose and glucagon treatment of liver cells on the distribution of the [32P]phosphate.
Glycogen synthase was phosphorylated in vivo by perfusing rat liver or incubating liver cells with [32P]phosphate. It was then isolated by immunoprecipitation and subjected to exhaustive tryptic proteolysis. The trypsin-derived [32P]phosphopeptides were separated by high pressure liquid chromatography (HPLC). Incubation of in vivo phosphorylated synthase with endogenous synthase phosphatase to convert synthase D to synthase R resulted in removal of phosphate from all of the labeled phosphopeptides. In prelabeled liver cells treated with glucagon or glucose, the activities of synthase and phosphorylase changed in the direction expected. The total labeling in the immunoprecipitated synthase was found to be increased to 126% and decreased to 67% of the control with glucagon and glucose treatment, respectively. When the HPLC [32P]phosphopeptide profile of synthase from glucagon-treated animals was compared with that of controls, there were only minor differences in the two profiles. All the peaks were present and the proportion of labeling in each remained similar. There also was only a modest change in the [32P]phosphopeptide profile with glucose treatment when compared with that of controls. These results indicate that regulation of synthase activity in the hepatocyte involves changes in phosphorylation at multiple sites. Indeed, in 32P-labeled liver cells, all of the labeled sites appeared to be involved.
Metabolic-Endocrine Section, Veterans Affairs Medical Center, Minneapolis, Minn. 55417.
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D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D005934:Glucagon / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006006:Glycogen Synthase / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D010446:Peptide Fragments / Q000302:isolation & purification; D010748:Phosphopeptides / Q000302:isolation & purification; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D010446:Peptide Fragments; D010748:Phosphopeptides; D005934:Glucagon; D006006:Glycogen Synthase; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D005947:Glucose