Purification and Characterization of a Novel Protein Activator of Ca 2+/Calmodulin-Dependent Protein Kinase I

Abstract

A protein activator of Ca 2+/calmodulin (CaM)-dependent protein kinase I was purified from rat brain. The activator was retained on a CaM-Sepharose column in the presence of Ca 2+ and kinase assay of renatured gel revealed the 64 kDa molecule in the purified activator fraction to be autophosphorylated and to phosphorylate recombinant CaM kinase I in the presence of Ca 2+/calmodulin. These results suggest that this activator of CaM kinase I is also a CaM-dependent protein kinase. Phosphorylation of CaM kinase I by the activator resulted in drastic potentiation of its CaM-dependent activity. Furthermore, kinetic analyses demonstrated that the activation decreases the K m values of CaM kinase I for both ATP and syntide-2 without a change in V max values. Considering the quite low enzymatic activity of recombinant CaM kinase I without activation, the 64 kDa species might be essential for CaM kinase I function in vivo.