REQUIREMENTS FOR CALCIUM AND CALMODULIN IN THE CALMODULIN KINASE ACTIVATION CASCADE

We have previously purified and cloned rat brain Ca2+/calmodulin-depen dent 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 (Tokumit su, H., Enslen, H., and Soderling, T. R. (1995) J. Biol. Chem. 270, 19 320-19324), In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr(196) by CaM-KK is triggered b y elevated intracellular Ca2+ in intact cells and requires binding of Ca2+/CaM to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV1 78-246), which contains the activating phosphorylation site (Thr(196)) but not the autoinhibitory domain or the CaM-binding domain, still re quired Ca2+/CaM for phosphorylation by wild-type CaM-KK. A truncated m utant of CaM-KK (CaM-KK1-434) phosphorylated CaM-K IV178-246 in a Ca2/CaM-independent manner, but this constitutively active CaM-KK1-434 re quired Ca2+/CaM for phosphorylation and activation of wild-type CaM-K IV, These results demonstrate that binding of Ca2+/CaM to both CaM-K I V and CaM-KK is required for the CaM-kinase cascade, Both CaM-KK and C aM-K IV appear to have similar Ca2+/CaM requirements with EC(50) value s of approximately 100 nM. Studies using co-expression of CaM-K IV wit h CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated bot h total and Ca2+/CaM-independent activities of wild-type CaM-K IV, but not the Thr(196) --> Ala mutant, upon ionomycin stimulation.