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 alpha 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 Kd 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 Km 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.