Purification and characterization of a novel protein activator of Ca2+/calmodulin-dependent protein kinase I

Abstract

A protein activator of Ca2+/calmodulin (CaM)-dependent protein kinase I was purified from rat brain. The activator was retained on a CaM-Sepharose column in the presence of Ca2+ 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 Ca2+/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 Km values of CaM kinase I for both ATP and syntide-2 without a change in Vmax 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.