Separation and Characterisation of Glycogen Synthase Kinase 3,Glycogen Synthase Kinase 4 and Glycogen Synthase Kinase 5 from Rabbit Skeletal Muscle
Authors: COHEN Philip , YELLOWLEES David , AITKEN Alastair , DONELLA‐DEANA Aridnna , HEMMINGS Brian A. , PARKER Peter J. 
Published: March 2005

Three glycogen synthase kinases termed glycogen synthase kinase 3, glycogen synthase kinase 4 and glycogen synthase kinase 5, which are distinct from cyclic‐AMP‐dependent protein kinase and phosphorylase kinase,have been purified and characterised. Their activities are unaffected by Ca2+, calmodulin, cyclic nucleotides and the inhibitor protein of cyclic‐AMP‐dependent protein kinase.

Glycogen synthase kinase 3 was eluted from phosphocellulose at 0.2 M NaCl and the apparent M, of the partially purified enzyme determined by gel filtration in the presence of 0.5 M NaCl was 72000. Glycogen synthase kinase 3 used GTP as a substrate (VGTp/VATp= 0.65) but the apparent affinity for GTP (Km= 0.4 mM) was lower than that for ATP (Km= 0.02 mM). Glycogen synthase kinase 3 phosphorylated three serine residues (sites 3a, 3b and 3c) on glycogen synthase, but seven other proteins that are phosphorylated rapidly by cyclic‐AMP‐dependent protein kinase were not substrates for glycogen synthase kinase 3. Glycogen synthase kinase 3 phosphorylated casein at a very low rate and did not phosphorylate phosvitin, although a protein of M1= 35000 which contaminated phosvitin to avariable extent was a good substrate. Glycogen synthase kinase 3 was the only enzyme capable of activating the (Mg‐ATP)‐dependent protein phosphatase.

Glycogen synthase kinase 4 was eluted from phosphocellulose at 0.4 M NaCl and its apparent M, was 115000.This enzyme was absolutely specific for ATP as phosphoryl donor (Km=0.01 mM), and it phosphorylated glycogen synthase at the same serine residue as phosphorylase kinase (site 2). Glycogen synthase kinase 4 was distinct from phosphorylase kinase since its activity was unaffected by Ca2+ or calmodulin, it had a quite different M, and nucleoside triphosphate specificity, and it could not phosphorylate glycogen phosphorylase.Glycogen synthase kinase 4 did not phosphorylate any other proteins tested at a significant rate.

Glycogen synthase kinase 5 was eluted from phosphocellulose at 0.7 M NaCl and its apparent M, was 170000–180000. This enzyme could use GTP as a substrate (YGTP VATP= 0.85) and the apparent affinities for GTP(K, = 0.04 mM) and ATP (Km = 0.01 mM) were similar. Glycogen synthase kinase 5 phosphorylated a single serine residue on glycogen synthase, termed site 5 and the sequence surrounding this site was found to be His‐Ser‐Ser‐Pro‐His‐Gln‐Ser (PGlu‐Asp‐Glu‐Glu‐Glu‐Pro.

The phosphorylation of site 5 did not decrease the activity of glycogen synthase in contrast to the phosphorylation by other glycogen synthase kinases. Glycogen synthase kinase 5 phosphorylated casein, phosvitin and acetyl‐CoA carboxylase more rapidly than glycogen synthase, but did not phosphorylate other proteins tested. Glycogen synthase kinase 5 was activated sixfold by spermine at 5.7 mM Mg2+ and 40–50‐fold at 1.2 mM Mg2+, and was powerfully inhibited by heparin (Ki= 0.04 pg/ml).

40–50% of the glycogen synthase kinase 5 was precipitated when muscle extracts were acidified to pH 6.1, whereas glycogen synthase kinase 3 remained in the supernatant. When the supernatant was chromatographed on phosphocellulose, glycogen synthase kinase 3 accounted for 84–92%, glycogen synthase kinase 4 for 5 – 7 and glycogen synthase kinase 5 for 0.2 ‐10% of the glycogen synthase kinase activity that was eluted from the column, depending on the concentration of NaCl, Mg2+ and spermine used in the assays.

Purified phosphorylase kinase contained detectable amounts of glycogen synthase kinase 5, and contamination by this enzyme accounts for reports that phosphorylase kinase can phosphorylate glycogen synthase at a site distinct from site 2.

Most, if not all, of the glycogen synthase kinases reported in the literature (other than cyclic‐AMP‐dependent protein kinase and phosphorylase kinase), can now be identified as glycogen synthase kinases 3, 4 or 5, or combinations of two of these enzymes. The properties of glycogen synthase kinase 5 demonstrate that it is identical to the enzyme that has been termed casein kinase TS, casein kinase 2 or troponin‐T kinase.