Inactivation of Ca²⁺/Calmodulin-Dependent Protein Kinase II by Ca²⁺/Calmodulin

Abstract

Incubation of calmodulin-dependent protein kinase II with Ca²⁺ and calmodulin resulted in a marked inactivation of the enzyme. Chelation of Ca²⁺ by EGTA or addition of calmodulin antagonists, W-7 or trifluoperazine, completely blocked this inactivation. The concentration required for the half-maximal inactivation, 127 μM, is three to four orders of magnitude higher than that for the half-maximal activation of the enzyme. The Ca²⁺/ calmodulin-independent activity of the proteolytic fragment of the enzyme, whose calmodulin-binding site involved in the enzyme activation was deleted, was also decreased by incubation with Ca²⁺ and calmodulin. These results suggest that calmodulin-dependent protein kinase II possesses a second, low-affinity calmodulin-binding site, which is distinct from the calmodulin-binding site involved in the activation of the enzyme, and that the binding of calmodulin to the second binding site causes the inactivation of the enzyme. The inactivation by Ca²⁺/calmodulin was temperature-dependent. The addition of both 500 μM ADP and 10 mM MgCl₂ markedly protected the enzyme against the inactivation, while such a marked protection was not observed after the addition of either of the two alone. The addition of 5 μM autocamtide-2, a synthetic substrate peptide containing the amino acid sequence of the autophosphorylation site (Thr²⁸⁶/Thr²⁸⁷ in α/β, γ, and δ isoforms) lying within the autoinhibitory domain, also protected the enzyme against the inactivation by Ca²⁺/calmodulin, while syntide-2, another synthetic substrate peptide corresponding to a phosphorylation site of glycogen synthase, did not protect it even at a concentration as high as 304 μM.