An ultrasensitive Ca2+/calmodulin-dependent protein kinase II-protein phosphatase 1 switch facilitates specificity in postsynaptic calcium signaling -- Data Supplement - HTML Page - 2759Fig8Legend.htslp -- Proceedings of the National Academy of Sciences

Supporting Figure 8 Fig. 8.
Nonlinear peptide phosphorylation by Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) at low Ca²⁺. (A and B) Plotted is the amount of phosphorylated CaMKII substrate peptide AC-2 (nmol/μg CaMKII) versus time at 1 μM (A) or 10 μM (B) Ca²⁺. Circles and diamonds represent experiments with wild-type and T286A mutant CaMKII, respectively. (C) Plotted is the CaM-dependent CaMKII(T286A) activity versus [Ca²⁺] (± SD, n = 2). Here, [CaM] = 5 μM. We observed that a plot of phosphorylated CaMKII substrate vs. time was not linear but rather an increasing function, indicating that CaMKII activity at low Ca²⁺ increases as the reaction proceeds (see A). This behavior was not observed with a CaMKII mutant, CaMKII(T286A), which cannot autophosphorylate (Fig. 8A). It was also not observed for either wild-type CaMKII or CaMKII(T286A) at a [Ca²⁺] where CaMKII is maximally active (see B). Hence, we attribute the increased activity as the reaction progresses to the slow autophosphorylation of CaMKII during the assay, which converts CaMKII to its more active autonomous form. The nonlinear time course of product formation did not alter the shape of the percent Ca²⁺/CaM activity versus [Ca²⁺] curve significantly (Fig. 2B) given that the same curve obtained with CaMKII(T286A) gave a nearly identical n_H value (see C).