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.
D000818:Animals; D001921:Brain / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D003599:Cytoskeleton / Q000201:enzymology; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D009474:Neurons / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D046911:Macromolecular Substances; D014268:Trifluoperazine; D011494:Protein Kinases; D002118:Calcium