FIGURE 8.

Autophosphorylation enhances basal levels, Ca²⁺ responsiveness, and Ca²⁺/CaM sensitivity of CPK28 peptide kinase activity. A, peptide kinase activity of the three different CPK28 phosphoforms toward the ACSM+1 peptide substrate (10 μm). Activity was measured in the presence of 500 μm EGTA, 100 μm CaCl₂, or no treatment (Untreated). Activities are reported relative to Ca²⁺-stimulated P⁻-CPK28. B, analysis of P⁻- and P⁺-CPK28 peptide kinase activity across a range of free Ca²⁺ concentrations. Open circles, P⁺-CPK28; closed circles, P⁻-CPK28. Gray region represents the physiological range of free Ca²⁺ in plants (∼100 to 1000 nm); inset, fold activation of P⁻-CPK28 and P⁺-CPK28 by Ca²⁺ across the physiological range. Fold activation was calculated relative to the activity at 100 nm free Ca²⁺. Point estimates are mean with 95% confidence intervals. C, peptide kinase activity of the three different CPK28 phosphoforms in the presence of 100 μm CaCl₂ with or without (untreated) the addition of 1 μm CaM6. For each phosphoform, activity is reported relative to the untreated control. Point estimates in A and C represent means with standard deviation of three technical replicates. Data shown are representative of at least two independent preparations of recombinant CPK28.