Regulation of Ca²⁺/calmodulin-dependent cyclic nucleotide phosphodiesterase by the autophosphorylated form of Ca²⁺/calmodulin-dependent protein kinase II

The 63-kDa subunit, but not the 60-kDa subunit, of brain calmodulin-dependent cyclic nucleotide phosphodiesterase was phosphorylated in vitro by the autophosphorylated form of Ca²⁺/calmodulin-dependent protein kinase II. When calmodulin was bound to the phosphodiesterase, 1.33 ± 0.20 mol of phosphate was incorporated per mol of the 63-kDa subunit within 5 min with no significant effect on enzyme activity. Phosphorylation in the presence of low concentrations of calmodulin resulted in a phosphorylation stoichiometry of 2.11 ± 0.21 and increased about 6-fold the concentration of calmodulin necessary for half-maximal activation of the phosphodiesterase. Peptide mapping analyses of complete tryptic digests of the 63-kDa subunit revealed two major (P₁, P₄) and two minor (P₂, P₃) ³²P-peptides. Calmodulin-binding to the phosphodiesterase almost completely inhibited phosphorylation of P₁ and P₂ with reduced phosphorylation rates of P₃ and P₄, suggesting the affinity change of the enzyme for calmodulin may be caused by phosphorylation of P₁ and/or P₂. When Ca²⁺/calmodulin-dependent protein kinase II was added without prior autophosphorylation, there was no phosphorylation of the 63-kDa phosphodiesterase subunit or of the kinase itself in the presence of a low concentration of calmodulin, and with excess calmodulin the phosphodiesterase subunit was phosphorylated only at P₃ and P₄. Thus the 63-kDa subunit of phosphodiesterase has a regulatory phosphorylation site(s) that is phosphorylated by the autophosphorylated form of Ca²⁺/calmodulin-dependent protein kinase II and blocked by Ca²⁺/calmodulin binding to the subunit.