Phosphorylation-dependent subfunctionalization of the calcium-dependent protein kinase CPK28

Abstract

Significance Calcium-dependent protein kinases (CDPKs) are a family of Ca2+ sensor proteins that contribute to various aspects of plant growth and development. One of these, CPK28, regulates immune homeostasis and reproductive-stage transition in several plant species, including Arabidopsis thaliana. Here, we show that phosphorylation on a single residue, Ser318, results in conformational changes in CPK28 that allow the kinase to be active at low Ca2+ concentrations, enabling it to respond quickly to an immune trigger. Intriguingly, phosphorylation on this residue is not required for its role in development, demonstrating pathway-specific regulation. We further provide evidence that this is a conserved biochemical mechanism in related CDPKs, highlighting the potential for biotechnological applications. Calcium (Ca2+)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca2+ sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca2+ activation at physiological concentrations of Ca2+, possibly through stabilization of the Ca2+-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca2+] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific requirements for phosphorylation-based Ca2+-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.