Phosphorylation of sites 3 and 4 in rabbit skeletal muscle glycogen synthase by cAMP-dependent protein kinase.
Rabbit skeletal muscle glycogen synthase (synthase a) can be phosphorylated by 0.2 to 2.5 microM catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.5 to 3 mol of 32PO4/subunit (90,000 X g). When a complete tryptic digest of this 32P-synthase was chromatographed on reverse phase high performance liquid chromatography, it was observed that, in addition to sites 1a, 1b, and 2, site 3 was phosphorylated. The peptide containing site 5 also contained 32PO4, but sequence analysis identified a new phosphorylation site, site 4 (Arg-His-Ser-Ser(PO4)-) which precedes site 5. Phosphorylation of sites 3 and 4 became significant when the total phosphorylation stoichiometry exceeded 1.5 mol/subunit. The heat-stable protein kinase inhibitor protein or the regulatory subunit of cAMP-dependent protein kinase decreased the phosphorylation of all sites, including sites 3 and 4. Phosphorylation of all sites by the holoenzyme form of cAMP-dependent kinase was highly dependent on the presence of cAMP. These results establish that phosphorylation of these sites is due to the cAMP-dependent protein kinase itself and not to a contaminating kinase(s). Synthase b from control rabbit muscle, containing 2 to 2.5 mol of phosphate/subunit, incorporated up to 2.0 mol of 32PO4 catalyzed in vitro by the cAMP-dependent protein kinase. Again, significant 32PO4 was detected in sites 3 and 4 as well as in 1a, 1b, and 2. These results suggest that the in vivo phosphorylation of sites 1a, 1b, 2, and 3 observed after injection of epinephrine in rabbits could be due solely to the known activation of the cAMP-dependent protein kinase.
Sheorain
V S
VS
Corbin
J D
JD
Soderling
T R
TR
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology*; D006006:Glycogen Synthase / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D010710:Phosphates; D047428:Protein Kinase Inhibitors; D000242:Cyclic AMP; D006006:Glycogen Synthase; D011494:Protein Kinases; D014357:Trypsin