Requirements for calcium and calmodulin in the calmodulin kinase activation cascade

We have previously purified and cloned rat brain Ca²⁺/calmodulin- dependent protein kinase kinase (CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19320-19324). In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr¹⁹⁶ by CaM-KK is triggered by elevated intracellular Ca²⁺ in intact cells and requires binding of Ca²⁺/CaM to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV_178-246), which contains the activating phosphorylation site (Thr¹⁹⁶) but not the autoinhibitory domain or the CaM-binding domain, still required Ca²⁺/CaM for phosphorylation by wild-type CaM-KK. A truncated mutant of CaM-KK (CaM-KK_1-434) phosphorylated CaM-K IV_178-246 in a Ca²⁺/CaM-independent manner, but this constitutively active CaM-KK_1-434 required Ca²⁺/CaM for phosphorylation and activation of wild-type CaM-K IV. These results demonstrate that binding of Ca²⁺/CaM to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade. Both CaM-KK and CaM-K IV appear to have similar Ca²⁺/CaM requirements with EC₅₀ values of approximately 100 nM. Studies using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated both total and Ca²⁺/CaM- independent activities of wild-type CaM-K IV, but not the Thr¹⁹⁶ → Ala mutant, upon ionomycin stimulation.