Phosphorylation of the Type‐II Regulatory Subunit of Cyclic‐AMP‐Dependent Protein Kinase by Glycogen Synthase Kinase 3 and Glycogen Synthase Kinase 5

The regulatory (R II ) subunit of type‐II cyclic‐AMP‐dependent protein kinase from bovine heart is phosphorylated at a significant rate in vitro by glycogen synthase kinase 3 and glycogen synthase 5, but not by glycogen synthase kinase 4 or phosphorylase kinase. The regulatory (R I ) subunit of type‐I cyclic‐AMP‐dependent protein kinase from rabbit skeletal muscle is not phosphorylated by any of these four protein kinases. Glycogen synthase kinase 3 phosphorylates two serine residues on the R II subunit located 44 and 47 amino acids from the N terminus of the polypeptide chain. Glycogen synthase kinase 5 phosphorylates serine‐74 and serine‐76. These sites are distinct from the residue phosphorylated by the catalytic subunit of cyclic‐AMP‐dependent protein kinase (serine‐95). The R II subunit, as normally isolated, contains 1.5–1.8 mol alkali‐labile phosphate per subunit. At least 80% of this phosphate (∼ 1.3 mol/subunit) is located in the thermolytic peptide containing serine‐74 and serine‐76, demonstrating that phosphorylation of the R II subunit by glycogen synthase kinase 5 occurs in vivo. Only small amounts of phosphate (∼ 0.1 mol/subunit) are associated with the thermolytic peptides containing serine‐44/serine‐47 and serine‐95. The phosphorylation sites on the R II subunit are organised in a strikingly similar manner to those of glycogen synthase, the amino acid sequences in the immediate vicinity of the phosphorylation sites showing a particular resemblance. These include the presence of a number of proline residues near the sites phosphorylated by glycogen synthase kinase 3, five consecutive acidic residues C‐terminal to the sites phosphorylated by glycogen synthase kinase 5, and two adjacent arginine residues just N‐terminal to the sites phosphorylated by the catalytic subunit of cyclic‐AMP‐dependent protein kinase. Glycogen synthase kinase 5 is very similar or identical to the enzyme that has been variously termed casein kinase TS, casein kinase 2, casein kinase G, casein kinase N‐II or troponin‐T kinase. The biological role of this enzyme is reviewed.