Autophosphorylation-based Calcium (Ca2+) Sensitivity Priming and Ca2+/Calmodulin Inhibition of Arabidopsis thaliana Ca2+-dependent Protein Kinase 28 (CPK28)

Abstract

Plant calcium (Ca²⁺)-dependent protein kinases (CPKs) represent the primary Ca²⁺-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca²⁺ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca²⁺/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca²⁺/CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca²⁺-responsive and was inhibited by Ca²⁺/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca²⁺ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca²⁺ activation and might also allow CPK28 to remain active when Ca²⁺ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca²⁺/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca²⁺ signaling specificity through Ca²⁺ sensor priming.

Keywords: Arabidopsis thaliana; Ca2+-dependent protein kinase; autophosphorylation; calcium; calmodulin (CaM); protein-protein interaction.