Figure 2. Submaximal stimulated activity of CaMKII wild type and its T286A mutant was induced by 0.1 µM Ca²⁺/CaM and measured by the phosphorylation rate of the substrate syntide 2 (in 1 min reactions at 30°C).

Error bars indicate mean ± s.e.m; **: p<0.01, n.s.: p>0.05 in two-tailed t-test. A, T286 autophosphorylation of CaMKII wild type was assessed by Western analysis (left), and quantified by arbitrary relative immuno-detection values (IDV; right). T286 autophosphorylation stimulated by 0.1 µM Ca²⁺/CaM was slower compared to stimulation by 1 µM Ca²⁺/CaM, but the same level of maximal autophosphorylation was still achieved within 1 min reaction time at 30°C. B, Submaximal activation by 0.1 µM Ca²⁺/CaM was verified by comparing one individual preparation of each CaMKII wild type and T286A mutant to the activity induced by 1 µM Ca²⁺/CaM (n = 4 individual assays). C, The average activity of CaMKII from multiple preparations (N = 5) stimulated by 0.1 µM Ca²⁺/CaM did not differ significantly between CaMKII wild type and the T286A mutant. While the ratio of the mean activities of T286A over wild type was similar as observed at maximal stimulation (compare Fig. 1B,C), the variability at submaximal stimulation was greater (with standard deviations of 35–36% of the mean at submaximal stimulation compared to 13–17% at maximal stimulation for syntide 2 substrate).