Calmodulin binding to the cytoskeletal neuronal calmodulin-dependent protein kinase is regulated by autophosphorylation

A brain cytoskeletal preparation that is highly enriched in calmodulin-dependent protein kinase facilitated the study of the binding of ¹²⁵I-labeled calmodulin to the native enzyme. The binding was specific, saturable, Ca²⁺-dependent, and inhibited by trifluoperazine. Stoichiometric analysis revealed that the ratio of bound calmodulin to the α subunit of the protein kinase was about 1:10 (±30%), indicating that in the native state not all of the enzyme subunits were accessible to bind calmodulin. The K(d) for the binding reaction was 7 x 10^-9 M and was subject to regulation by divalent cations other than Ca²⁺, decreasing to 1.7 x 10^-9 M in the presence of 7 mM MgCl₂. Activation of the protein kinase in the presence of Ca²⁺ and calmodulin resulted in marked autophosphorylation of the enzyme subunits. The autophosphorylation was accompanied by a 2-fold decrease in the affinity and number of ¹²⁵I-labeled calmodulin binding sites. This effect was also reflected by an increase in the apparent K(m) for Ca²⁺ from 90 to 200 x 10^-9 M. Thus, enzyme autophosphorylation appears to represent a negative feedback signal, rendering the enzyme less sensitive to subsequent stimulation by physiologic increases in the intracellular Ca²⁺ concentration. These results help to clarify the mode of neuronal intracellular Ca²⁺ signaling.