Activation mechanisms for Ca²⁺/calmodulin-dependent protein kinase IV. Identification of a brain CaM-kinase IV kinase

This manuscript examines the mechanisms by which Ca²⁺/calmodulin- dependent protein kinase IV (CaM-kinase IV) is activated through the binding of Ca²⁺/CaM and by phosphorylation. Studies with the synthetic autoinhibitory domain peptides of CaM-kinase II indicate that CaM-kinase IV has a similarly located autoinhibitory domain, and this was confirmed since site-directed mutagenesis of this region (HMDT³⁰⁸ to DEDD and FN³¹⁷ to DD) generated fully active Ca²⁺/CaM-independent kinases. Total activities of purified, baculovirus-expressed wild type and mutant kinases were increased 2-fold by intramolecular autophosphorylation, but this reaction was extremely slow (1-2 h) and probably not physiological. However, CaM-kinase IV can be activated by brain CaM-kinase IV kinase resulting in large increases in both total (5-7-fold) and Ca²⁺/CaM-independent (>20-fold) CaM-kinase IV activities. This activation reaction required Mg²⁺/ATP and Ca²⁺/CaM, was intermolecularly catalyzed, and was reversed by protein phosphatase 2A. Activation of CaM-kinase IV resulted in a 10-fold decrease in K(m) for syntide-2 with little effect on K(m) for ATP or V(max). CaM-kinase IV kinase was highly purified from rat brain extract and was shown to be a 68-kDa monomer. The results of this study demonstrate that CaM-kinase IV does have an autoinhibitory domain within residues His³⁰⁵-Lys³²¹ that suppresses kinase activity in the absence of Ca²⁺/CaM. CaM-kinase IV is not significantly activated by autophosphorylation, but it can be activated 10- fold by a CaM-kinase IV kinase. This kinase cascade activation mechanism may be important for the physiological function of CaM-kinase IV such as transcriptional regulation through phosphorylation of cAMP responsive element binding protein (Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T. R. (1994) J. Biol. Chem. 269, 15520-15527).