Phosphorylation of rabbit liver glycogen synthase by multiple protein kinases

M. Camici, Z. Ahmad, Anna De Paoli-Roach, Peter Roach

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Purified rabbit liver glycogen synthase was found to be a substrate for six different protein kinases: (i) cyclic AMP-dependent protein kinase, (ii) two Ca2+-stimulated protein kinases, phosphorylase kinase (from muscle) and a calmodulin-dependent glycogen synthase kinase, and (iii) three members of a Ca2+ and cyclic nucleotide independent class, PC(0.7), F(A)/GSK-3, and casein kinase-1. Greatest inactivation accompanied phosphorylation by cyclic AMP-dependent protein kinase (to 0.5-0.7 phosphate/subunit, x̄glucose-6-P activity ratio reduced from approximately 1 to 0.6) or F(A)/GSK-3 (to ~1 phosphate/subunit, activity ratio, 0.46). Phosphorylation by the combination F(A)/GSK-3 plus PC(0.7) was synergistic, and more extensive inactivation was achieved. The phosphorylation reactions just described caused significant reduction in the V(max) of the glycogen synthase with little effect on the S(0.5) (substrate concentration corresponding to V(max)/2). Phosphorylase kinase achieved a lesser inactivation, to an activity ratio of 0.75 at 0.6 phosphate/subunit. PC(0.7) acting alone, casein kinase-1, and the calmodulin-dependent protein kinase did not cause inactivation of liver glycogen synthase with the conditions used. Analysis of CNBr fragments of phosphorylated glycogen synthase indicated that the phosphate was distributed primarily between two polypeptides, with apparent M(r) = 12,300 (CB-I) and 16,0000-17,0000 (CB-II). PC(0.7) and casein kinase-1 displayed a decided specificity for CB-II, and the calmodulin-dependent protein kinase was specific for CB-II. The other protein kinases were able, to some extent, to introduce phosphate into both CB-I and CB-II. Studies using limited proteolysis indicated that CB-II was located at a terminal region of the subunit. CB-I contains a minimum of one phosphorylation site and CB-II at least three sites. Liver glycogen synthase is therefore potentially subject to the same type of multisite regulation as skeletal muscle glycogen synthase although the muscle and liver enzymes display significant differences in both structural and kinetic properties.

Original languageEnglish
Pages (from-to)2466-2473
Number of pages8
JournalJournal of Biological Chemistry
Volume259
Issue number4
StatePublished - 1984

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Glycogen Synthase
Liver Glycogen
Phosphorylation
Protein Kinases
Casein Kinase I
Rabbits
Glycogen Synthase Kinase 3
Phosphates
Phosphorylase Kinase
Muscle
Cyclic AMP-Dependent Protein Kinases
Phosphorylase a
Proteolysis
Glycogen Synthase Kinases
Calcium-Calmodulin-Dependent Protein Kinase Type 2
Muscles
Calcium-Calmodulin-Dependent Protein Kinases
Cyclic Nucleotides
Substrates
Calmodulin

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation of rabbit liver glycogen synthase by multiple protein kinases. / Camici, M.; Ahmad, Z.; De Paoli-Roach, Anna; Roach, Peter.

In: Journal of Biological Chemistry, Vol. 259, No. 4, 1984, p. 2466-2473.

Research output: Contribution to journalArticle

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N2 - Purified rabbit liver glycogen synthase was found to be a substrate for six different protein kinases: (i) cyclic AMP-dependent protein kinase, (ii) two Ca2+-stimulated protein kinases, phosphorylase kinase (from muscle) and a calmodulin-dependent glycogen synthase kinase, and (iii) three members of a Ca2+ and cyclic nucleotide independent class, PC(0.7), F(A)/GSK-3, and casein kinase-1. Greatest inactivation accompanied phosphorylation by cyclic AMP-dependent protein kinase (to 0.5-0.7 phosphate/subunit, x̄glucose-6-P activity ratio reduced from approximately 1 to 0.6) or F(A)/GSK-3 (to ~1 phosphate/subunit, activity ratio, 0.46). Phosphorylation by the combination F(A)/GSK-3 plus PC(0.7) was synergistic, and more extensive inactivation was achieved. The phosphorylation reactions just described caused significant reduction in the V(max) of the glycogen synthase with little effect on the S(0.5) (substrate concentration corresponding to V(max)/2). Phosphorylase kinase achieved a lesser inactivation, to an activity ratio of 0.75 at 0.6 phosphate/subunit. PC(0.7) acting alone, casein kinase-1, and the calmodulin-dependent protein kinase did not cause inactivation of liver glycogen synthase with the conditions used. Analysis of CNBr fragments of phosphorylated glycogen synthase indicated that the phosphate was distributed primarily between two polypeptides, with apparent M(r) = 12,300 (CB-I) and 16,0000-17,0000 (CB-II). PC(0.7) and casein kinase-1 displayed a decided specificity for CB-II, and the calmodulin-dependent protein kinase was specific for CB-II. The other protein kinases were able, to some extent, to introduce phosphate into both CB-I and CB-II. Studies using limited proteolysis indicated that CB-II was located at a terminal region of the subunit. CB-I contains a minimum of one phosphorylation site and CB-II at least three sites. Liver glycogen synthase is therefore potentially subject to the same type of multisite regulation as skeletal muscle glycogen synthase although the muscle and liver enzymes display significant differences in both structural and kinetic properties.

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