Dennis B. Rylatt

Publisher Summary This chapter discusses glycogen synthase kinase-3 from rabbit skeletal muscle. Glycogen synthase kinase-3 is one of the five glycogen synthase kinases that are identified in skeletal muscle, and is of major importance in determining the kinetic properties of glycogen synthase in vivo. It catalyzes the phosphorylation of three serine residues on glycogen synthase, converting the enzyme from a form that is almost fully active in the absence of glucose-6P, to one that is largely dependent on this allosteric activator. Glycogen synthase kinase-3 also has a second activity that is not shared by any other protein kinase—namely, the ability to activate an enzyme termed the MgATP-dependent protein phosphatase. Glycogen synthase may also contain traces of a modified form of phosphorylase kinase that has lost its sensitivity to regulation by calcium ions, and is therefore, no longer inhibited by ethylene glycol tetraacetic acid (EGTA). This is largely removed by passing glycogen synthase through phosphocellulose.

Glycogen synthase kinase-3 phosphorylates three serine residues on glycogen synthase (sites 3a, 3b and 3c) which are all located in the same nine-amino-acid segment of the polypeptide chain. The sequence in this region is: Arg-Tyr-Pro-Arg-Pro-Ala-Ser(P)-Val-Pro-Pro-Ser(P)-Pro-Ser-Leu-Ser(P)-Arg-. These serine residues are distinct from the sites phosphorylated preferentially by cyclic-AMP-dependent protein kinase (sites 1a and 1b) and phosphorylase kinase (site 2). The N-terminal sequence of glycogen synthase containing the serine residue phosphorylated by phosphorylase kinase has been extended. The sequence in this region is: Pro-Leu-Ser-Arg-Thr-Leu-Ser(P)-Val-Ser-Ser-Leu-Pro-Gly-Leu-Glu-Asp-Trp-Glu-Asp- Glu-Phe-Asp-Leu-Glu-Asn-Ser-Val-Leu-Phe-(Asx2,Glx2,Ala2,Val2,Lys)-. The similarity to the N-terminal sequence of phosphorylase is confined to the immediate vicinity of the phosphorylation site (residues 4--15). The relationship of glycogen synthase kinase-3 to glycogen synthase kinases that have been described by other laboratories is discussed.

Glycogen synthase kinase‐3 phosphorylates three serine residues on glycogen synthase (sites 3a, 3b and 3c) which are all located in the same nine‐amino‐acid segment of the polypeptide chain. The sequence in this region is: Arg‐Tyr‐Pro‐Arg‐Pro‐Ala‐Ser(P)‐Val‐Pro‐Pro‐Ser(P)‐Pro‐Ser‐Leu‐Ser(P)‐Arg‐. These serine residues are distinct from the sites phosphorylated preferentially by cyclic‐AMP‐dependent protein kinase (sites 1a and 1b) and phosphorylase kinase (site 2). The N‐terminal sequence of glycogen synthase containing the serine residue phosphorylated by phosphorylase kinase has been extended. The sequence in this region is: Pro‐Leu‐Ser‐Arg‐Thr‐Leu‐Ser(P)‐Val‐Ser‐Ser‐Leu‐Pro‐Gly‐Leu‐Glu‐Asp‐Trp‐Glu‐Asp‐Glu‐Phe‐Asp‐Leu‐Glu‐Asn‐Ser‐Val‐Leu‐Phe‐(Asx 2 ,Glx 2 ,Ala 2 ,Val 2 ,Lys)‐. The similarity to the N‐terminal sequence of phosphorylase is confined to the immediate vicinity of the phosphorylation site (residues 4–15). The relationship of glycogen synthase kinase‐3 to glycogen synthase kinases that have been described by other laboratories is discussed.

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Homogeneous preparations of phosphorylase kinase from rabbit skeletal muscle catalyse a calcium-dependent phosphorylation of glycogen synthase a isolated from the same tissue. The calcium-dependent glycogen synthase kinase activity copurifies with phosphorylase kinase throughout the standard procedure for the isolation of the latter enzyme. At the final step of the purification, gel filtration on Sepharose 4B, the elution profiles for glycogen synthase kinase and phosphorylase kinase activities are identical. ICR/IAn mice, which lack muscle phosphorylase kinase activity, do not contain detectable calcium-dependent glycogen synthase kinase activity. These results indicate that the calcium-dependent phosphorylation of glycogen synthase is catalysed by phosphorylase kinase and not by another calcium-dependent protein kinase that might be contaminating the preparation. The phosphorylation of glycogen synthase a by phosphorylase kinase reaches a plateau in the range 0.73 ± 0.1 molecules of phosphate incorporated per subunit and is accompanied by a 2-fold decrease in the activity in the absence of glucose 6-phosphate. The phosphorylation takes place on a unique serine residue located seven amino acids from the N-terminus of the polypeptide chain. The amino acid sequence surrounding serine-7 is similar to the amino acid sequence surrounding the phosphoserine in phosphorylase a. Glycogen synthase kinase-2 is the name given to a protein kinase present as a trace contaminant in highly purified preparations of glycogen synthase [Nimmo, H. G. and Cohen, P. (1974) FEBS Lett. 47, 162–167]. Glycogen synthase kinase-2 also phosphorylates serine-7 exclusively, and evidence is presented which demonstrates that glycogen synthase kinase-2 is merely a modified form of phosphorylase kinase which has lost its ability to be regulated by calcium ions at pH 6.8. The rate of phosphorylation of glycogen synthase a by phosphorylase kinase is 2–3-fold slower than the rate of phosphorylation of phosphorylase b when identical concentrations of the two protein substrates are used (6 μM). At physiological concentrations of glycogen synthase (0.3 mg/ml) and phosphorylase (8.0 mg/ml), the time required for half-maximal phosphorylation of each enzyme by phosphorylase kinase is similar. These results suggest that the phosphorylation of glycogen synthase by phosphorylase kinase may be physiologically significant, and the implications of these findings are considered.