Activation State-Dependent Substrate Gating in Ca²⁺/Calmodulin-Dependent Protein Kinase II

Figure 5

CaMKII substrate utilization and selectivity are differentially altered by T²⁸⁷ autophosphorylation. (a) Schematic representation of various conditions used to preautophosphorylate CaMKII_m prior to Ca²⁺/CaM-stimulated substrate phosphorylation reactions on SPOT arrays (see Materials and Methods). Essentially, time and/or temperature was altered to generate different extents of T²⁸⁷ autophosphorylation (i.e., varying ratios of CaMKII_m^−P/CaMKII_m^+P) as graphically illustrated in (b). (c) As a readout of T²⁸⁷ autophosphorylation, the % autonomy (i.e., [Ca²⁺/CaM-independent]/[Ca²⁺/CaM-stimulated] activity) was measured in separate soluble kinase assays using the high-affinity CaMKII substrate AC-2. Autonomous activity was measured immediately after the prereaction (pre-SPOTs) and following the SPOT reaction (post-SPOTs); mean ± s.e.m. (). (d) Ca²⁺/CaM-stimulated SPOT substrate phosphorylation for various T²⁸⁷ autophosphorylation conditions; mean ± s.d. (). Results represent one-way ANOVA of log-normalized data with Holm-Šídák posttest compared to 0% autonomy. ; ; .