Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle

Identification of the sites phosphorylated by casein kinase-I

Authors


  • Note. Dr. J. Kuret and Dr J. R. Woodgett have made equally important contributions to this work.

Correspondence to P. Cohen, Department of Biochemistry, Medical Science Institute, The University, Dundee, Tayside, Scotland DD1 4HN

Abstract

A casein kinase was highly purified from rabbit skeletal muscle whose substrate specificity and enzymatic properties were virtually identical to those of casein kinase-I from rabbit reticulocytes. Prolonged incubation of glycogen synthase with high concentrations of skeletal muscle casein kinase-I and Mg-ATP resulted in the incorporation of >6 mol phosphate/mol subunit and decreased the activity ratio (∓ glucose-6P) from 0.8 to <0.02. The sites phosphorylated by casein kinase-I were all located in the N and C-terminal cyanogen bromide peptides, termed CB-1 and CB-2. At an incorporatio of 6 mol phosphate/mol subunit, ∼2 mol/mol was present in CB-1 and ∼4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated the serines that were 3, 7 and 10 residues from the N-terminus of glycogen synthase, with minor phosphorylation at threonine-5. Within CB-2, ∼9% of the phosphate incorporated was located between residues 28 and 53, and at least five of the seven serine residues in this region were phosphorylated. The remaining 10% of phosphate incorporated into CB-2 was located between residues 98 and 123, mainly at a serine residue(s).

Two of the major sites labelled by casein kinase-I (serine-3 and serine-10 of CB-1) are not phosphorylated by any other protein kinase. This will enable the role of casein kinase-I as a glycogen synthase kinase in vivo to be evaluated.

Abbreviations
SDS

sodium dodecyl sulphate

HPLC

high-performance liquid chromatography

Enzymes
 

Glycogen phosphorylase (EC 2.4.1.1)

 

glycogen synthase (EC 2.4.1.11)

 

phosphorylase kinase (EC 2.7.1.38)

 

glycogen synthase kinase and casein kinases-I and II (EC 2.7.1.37)

 

acetyl-CoA carboxylase (EC 6.4.1.2)

 

ATP-citrate lyase (EC 4.1.3.8)

 

trypsin (EC 3.4.21.4)

 

chymotrypsin (EC 3.4.21.1)

 

lysine-specific proteinase (EC 3.4.99.3)

Ancillary