Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast-atom-bombardment mass spectrometry.
The in vivo phosphorylation state of glycogen synthase was re-examined by fast-atom-bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S-ethylcysteine. In animals injected with the beta-adrenergic antagonist propranolol, the phosphorylation sites in the N-terminal (N) and C-terminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28-C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28-C39 from 0.7 to 1.2 mol/mol and C100 from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin. Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic-AMP-dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28-C39, C46, C87 and C100 in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed. Residue N3, a major site phosphorylated by casein kinase-I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase-I is not a glycogen synthase kinase in vivo.
Poulter
L
L
Department of Chemistry, University of Cambridge.
Ang
S G
SG
Gibson
B W
BW
Williams
D H
DH
Holmes
C F
CF
Caudwell
F B
FB
Pitcher
J
J
Cohen
P
P
D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004837:Epinephrine / Q000494:pharmacology; D005260:Female; D006006:Glycogen Synthase / Q000032:analysis*; D038341:Glycogen Synthase Kinases; D013058:Mass Spectrometry; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011433:Propranolol / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D014357:Trypsin
D010446:Peptide Fragments; D011433:Propranolol; D006006:Glycogen Synthase; D011494:Protein Kinases; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin; D004837:Epinephrine