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

Bender KW, et al., 292(10):3988-4002, J Biol Chem, 2017

Calcium (Ca2+)-dependent protein kinases (CPKs) are proteins that play a key role in Ca2+ signaling and protein phosphorylation in plants. Described herein is a screening of a panel of plant CPKs for calmodulin (CaM) binding in order to explore a possible role of CaM in regulation of CPKs. Furthermore, the contribution of autophosphorylation to CPK function has been investigated. Recombinant Arabidopsis thaliana CPK28 was identified as a high affinity Ca2+/CaM binding protein. Bio-Layer Interferometry (BLI) was used to perform the binding analysis of the CaM6-CPK28 binding interaction. A Pall ForteBio Octet instrument equipped with biotinylated CaM6 immobilized Streptavidin Biosensor probes were used. After immobilization of CaM6, the Biosensor tips were immersed in a dilution series of purified recombinant His6-CPK28. The KD value of CPK28-CaM interaction was determined using a steady-state binding model equation. The study has revealed new scenarios pertaining to the regulation of CPK28 by Ca2+, autophosphorylation, and CaM binding.

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