Preparation and characterization of calmodulin-dependent protein kinase IV (CaM-kinase IV) free of CaM-kinase IV kinase from rat cerebral cortex.

Our earlier finding [Kameshita, I. and Fujisawa, H. (1993) J. Biochem. 113, 583-590] that calmodulin-dependent protein kinase IV (CaM-kinase IV) from rat cerebral cortex is markedly activated through autophosphorylation was reexamined in the light of a more recent study [Okuno, S. and Fujisawa, H. (1993) J. Biochem. 114, 167-170] suggesting the involvement of Ca2+/calmodulin-dependent CaM-kinase IV kinase in the activation of CaM-kinase IV in rat brain. Further purification of the previous CaM-kinase IV preparation by HPLC on a DEAE-NPR column abolished its marked activation on incubation under Ca2+/calmodulin-dependent phosphorylation conditions, and the marked activation was restored by the addition of the fraction separated on a DEAE-NPR column, suggesting that CaM-kinase IV was separated from CaM-kinase IV kinase involved in its activation by HPLC on DEAE-NPR. The HPLC-purified CaM-kinase IV still underwent a slow autophosphorylation, which was an intramolecular reaction. Some kinetic properties of CaM-kinase IV before and after autophosphorylation were examined for comparison. In addition, the purification by HPLC on DEAE-NPR revealed that a novel Ca(2+)-dependent calmodulin-binding protein, designated as CaMBP64, is present as a major contaminant in the previous purified CaM-kinase IV preparation.