Effects of epinephrine, diabetes, and insulin on rabbit skeletal muscle glycogen synthase. Phosphorylation site occupancies.
Phosphorylation site stoichiometries were determined for skeletal muscle glycogen synthase purified from control, alloxan-diabetic, and epinephrine-treated rabbits. One method of analysis was direct determination of the total in vivo phosphate content of each site after reverse phase high performance liquid chromatography separation of a complete tryptic digest of the purified synthase. The second method of analysis, in vitro phosphorylation, was based on the premise that in vitro 32P incorporation into each site would be inversely related to the in vivo phosphate content of that site. Glycogen synthase from control rabbits had the following distribution of in vivo phosphate (mole of phosphate/mol of site): site 1a, 0.29 +/- 0.08; site 5, 0.62 +/- 0.07; site 3, 0.46 +/- 0.06; site 1b, 0.23 +/- 0.03; and site 2, 0.43 +/- 0.07. Synthase from diabetic rabbits had 2-fold elevations of in vivo phosphate contents of sites 2 and 3. Epinephrine resulted in increased phosphorylation in vivo of site 1b (2.0-fold), site 2 (2.0-fold), and site 3 (1.5-fold). The in vitro phosphorylation analysis showed decreased 32P incorporation in vitro (indicative of increased in vivo phosphorylation) as follows: epinephrine, site 1a, site 3, site 1b, site 2; diabetic, site 3, site 2. The effect of diabetes on the in vitro phosphorylation of sites 2 and 3 was reversed by insulin treatment. We conclude that the major effect of epinephrine, phosphorylation of sites 1a, 1b, and 2, is mediated by the activation of the cAMP-dependent kinase. The mechanisms accounting for the phosphorylation of site 3 in response to epinephrine and phosphorylation of sites 2 and 3 in the diabetic state are under investigation.
D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002851:Chromatography, High Pressure Liquid; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D004837:Epinephrine / Q000494:pharmacology*; D006006:Glycogen Synthase / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits
D007328:Insulin; D006006:Glycogen Synthase; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004837:Epinephrine