α-Ca²⁺/calmodulin-dependent protein kinase II (αCaMKII) is a highly expressed neuronal kinase that processes patterns of repeated electrophysiological signals transduced by the intracellular messengers Ca²⁺ and calmodulin. Autophosphorylation of αCaMKII at residue Thr₂₈₆ results in a persistent kinase partial activity, which is thought to be Ca²⁺ independent (Braun & Schulman, 1995). In this study, the mechanisms of activation of unphosphorylated αCaMKII and phospho-Thr₂₈₆-αCaMKII by Ca²⁺ were investigated. Steady-state rates of phosphorylation of smooth muscle myosin light chain (MLC) were measured over the range of [Ca²⁺] 2.5 nM to 500 µM using a spectrofluorimetric coupled assay (Török et al. 1998). Smooth muscle myosin regulatory light chain (MLC) was overexpressed in E. coli BL21 cells. At 5 µM calmodulin and 100 µM MLC, Thr₂₈₆-autophosphorylation was inhibited. Phospho-Thr₂₈₆-αCaMKII was generated by pre-incubation of αCaMKII with Ca²⁺/calmodulin and Mg²⁺ATP. αCaMKII required 425 nM (S.E.M. ± 175, number of experiments = 4) [Ca²⁺] for half-maximal activation with high co-operativity (Hill coefficient, n_H = 5.4 (S.E.M. ± 0.4)). The activity of αCaMKII was < 4 % below 300 nM [Ca²⁺] and reached maximum (k_cat 1.2 s^-1) at 800 nM [Ca²⁺]. The [Ca²⁺] required for half-maximal activation of phospho-Thr₂₈₆-αCaMKII was reduced 3-fold to 137 nM (S.E.M. ± 104, number of experiments = 2) with lower co-operativity (n_H = 1.8 (S.E.M. ± 0.3)). Ca²⁺-independent activity of phospho-Thr₂₈₆-αCaMKII ([Ca²⁺] < 25 nM) was 5.0 % (S.E.M. ± 3.7) of the maximal activity. Phospho-Thr₂₈₆-αCaMKII reached full activity (k_cat 0.8 s^-1) at 1 µM [Ca²⁺]. Our data suggest that activation of unphosphorylated αCaMKII occurs upon stimulus-induced increases in intracellular [Ca²⁺]. Phospho-Thr₂₈₆-αCaMKII, once formed, can however be activated at resting [Ca²⁺], albeit in a Ca²⁺/calmodulin-dependent manner.

This work is supported by The Wellcome Trust and the Medical Research Council.