Autophosphorylation-based Calcium (Ca 2+) Sensitivity Priming and Ca 2+/Calmodulin Inhibition of Arabidopsis thaliana Ca 2+-dependent Protein Kinase 28 (CPK28)

J Biol Chem. 2017 Mar 10;292(10):3988-4002. doi: 10.1074/jbc.M116.763243. Epub 2017 Jan 30.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / drug effects
  • Arabidopsis / growth & development
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / metabolism
  • Calcium / pharmacology*
  • Calmodulin / pharmacology*
  • Gene Expression Regulation, Plant / drug effects*
  • Kinetics
  • Phosphorylation / drug effects
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Kinases / chemistry*
  • Protein Kinases / metabolism
  • Sequence Homology, Amino Acid

Substances

  • Arabidopsis Proteins
  • Calmodulin
  • Protein Kinases
  • calcium-dependent protein kinase
  • Calcium