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-depen dent protein kinase IV (CaM-kinase IV) is activated through the bindin g of Ca2+/CaM and by phosphorylation. Studies with the synthetic autoi nhibitory 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 (HMDT(308) to DEDD and FN317 to DD) generated fully active Ca2+/CaM-independent kinases. Tot al activities of purified, baculovirus-expressed wild type and mutant kinases were increased 2-fold by intramolecular autophosphorylation, b ut this reaction was extremely slow (1-2 h) and probably not physiolog ical. However, CaM-kinase IV can be activated by brain CaM-kinase IV k inase resulting in large increases in both total (5-7-fold) and Ca2+/C aM-independent (>20-fold) CaM-kinase IV activities. This activation re action 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 eff ect 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 resul ts of this study demonstrate that CaM-kinase IV does have an autoinhib itory domain within residues His(305)-Lys(321) that suppresses kinase activity in the absence of Ca2+/CaM. CaM-kinase IV is not significantl y 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 t ranscriptional regulation through phosphorylation of cAMP responsive e lement binding protein (Enslen, H., Sun, P., Brickey, D., Soderling, S . H., Klamo, E., and Soderling, T. R. (1994) J. Biol. Chem. 269, 15520 -15527).