Phosphorylation-dependent sub-functionalization of the calcium-dependent protein kinase CPK28

Abstract

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 intra- and inter-molecular auto-phosphorylation on Ser318 and can additionally be trans-phosphorylated 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.