Phosphorylation and activation of Ca²⁺-calmodulin-dependent protein kinase IV by Ca²⁺-calmodulin-dependent protein kinase Ia kinase: Phosphorylation of threonine 196 is essential for activation

Purified pig brain Ca²⁺-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca²⁺-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca²⁺-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca²⁺-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of ∼3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase TV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase FV, demonstrating that Thr-196 phosphorylation is essential for activation.