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

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.

Protein kinases represent one of the largest eukaryotic protein superfamilies. While roughly 500 protein kinases have been identified in humans (1), the genomes of Arabidopsis thaliana (hereafter, Arabidopsis) (2) and Oryza sativa (3) encode more than 1,000 and 1,500 protein kinases, respectively, including several families unique to plants. Among these protein kinases are the receptor-like kinases (RLKs), receptor-like cytoplasmic kinases (RLCKs), and calcium-dependent protein kinases (CDPKs or CPKs) that have emerged as key regulators of plant immunity (4–6). Despite encompassing only 2% of most eukaryotic genomes, protein kinases phosphorylate more than 40% of cellular proteins (7, 8), reflecting their diverse roles in coordinating intracellular signaling events. Reversible phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues can serve an array of functions including changes in protein conformation and activation state (9, 10), protein stability and degradation (11, 12), subcellular localization (13–15), and interaction with protein substrates (16–18).Calcium (Ca²⁺) is a ubiquitous secondary messenger that acts cooperatively with protein phosphorylation to propagate intracellular signals. Spatial and temporal changes in intracellular Ca²⁺ levels occur in response to environmental and developmental cues (19–23). In plants, Ca²⁺ transients are decoded by four major groups of calcium sensor proteins, which possess one or more Ca²⁺-binding EF-hand motifs (24, 25): calmodulins (CaM), CaM-like proteins, calcineurin B–like proteins, CDPKs, and Ca²⁺/CaM-dependent protein kinases.At the intersection of phosphorylation cascades and Ca²⁺ signaling are CDPKs, a unique family of Ca²⁺ sensor/kinase-effector proteins. CDPKs have been identified in all land plants and green algae, as well as certain protozoan ciliates and apicomplexan parasites (26, 27). CDPKs have a conserved domain architecture, consisting of a canonical Ser/Thr protein kinase domain and an EF-hand containing Ca²⁺-binding CaM-like domain (CLD), linked together by an autoinhibitory junction (AIJ) and flanked by variable regions on both the amino (N) and carboxyl (C) termini (28, 29). As their name implies, most CDPKs require Ca²⁺ for their activation (30). Upon Ca²⁺ binding to all EF-hands in the CaM-like domain, a dramatic conformational change occurs, freeing the AIJ from the catalytic site of the kinase, rendering the enzyme active (31–33). CDPKs vary in their sensitivity to Ca²⁺ (30), presumably allowing proteins to perceive distinct stimuli through differences in Ca²⁺-binding affinity. For example, Arabidopsis CPK4 displays half maximal kinase activity in the presence of ∼3 μM free Ca²⁺ (30) while CPK5 only requires ∼100 nM (34). Importantly, CDPKs are signaling hubs with documented roles in multiple distinct pathways (4, 24, 35–38) and are therefore likely regulated beyond Ca²⁺ activation.Subfunctionalization is at least partially mediated by protein localization and interaction with pathway-specific binding partners, as is well documented for Arabidopsis CPK3 which functions in response to biotic and abiotic stimuli in distinct cellular compartments (39). Recent attention has been drawn to site-specific phosphorylation as a mechanism to regulate the activity of multifunctional kinases. For example, phosphorylation sites on the RLK BRASSINOSTEROID INSENSITIVE 1–ASSOCIATED KINASE 1 (BAK1) are differentially required for its function as a coreceptor with a subset of leucine-rich repeat –RLKs (40). Phosphoproteomic analyses indicate that CDPKs are differentially phosphorylated following exposure to distinct stimuli (41–48); however, the biochemical mechanisms by which site-specific phosphorylation regulates multifunctional CDPKs is still poorly understood.Arabidopsis CPK28 is a plasma membrane–localized protein kinase with dual roles in plant immune homeostasis (49–51) and phytohormone-mediated reproductive growth (52, 53). In vegetative plants, CPK28 serves as a negative regulator of immune signal amplitude by phosphorylating and activating two PLANT U-BOX–type E3 ubiquitin ligases, PUB25 and PUB26, which target the key immune RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) for proteasomal degradation (50). Owing to elevated levels of BIK1, CPK28 null plants (cpk28-1) have heightened immune responses and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) (51). Upon transition to the reproductive stage, cpk28-1 plants additionally present shorter leaf petioles, enhanced anthocyanin production, and a reduction in stem elongation (52, 53). The molecular basis for developmental phenotypes in the cpk28-1 knockout mutant, beyond hormonal imbalance (52, 53), are comparatively unknown.Our recent work demonstrated that autophosphorylation status dictates Ca²⁺-sensitivity of CPK28 peptide kinase activity in vitro (54). While dephosphorylated CPK28 is stimulated by the addition of 100 μM CaCl₂ compared to untreated protein, hyperphosphorylated CPK28 displayed similar levels of activity at basal Ca²⁺ concentrations (54). These results highlight the interesting possibility that phosphorylation status may control the activation of multifunctional kinases in distinct pathways by allowing CDPKs to respond to stimulus-specific Ca²⁺ signatures.In the present study, we identify a single autophosphorylation site, Ser318, that decouples the activity of CPK28 in immune signaling from its role in reproductive growth. We show that expression of a nonphosphorylatable Ser-to-Ala variant (CPK28^S318A) is unable to complement the immune phenotypes of cpk28-1 mutants but is able to complement defects in stem growth. Further, we uncover a functional role for phosphorylation of Ser318 in priming CPK28 for activation at low free Ca²⁺]. Together, we demonstrate that site-specific phosphorylation can direct the activity of a multifunctional kinase in distinct pathways and provide evidence for a conserved mechanism in orthologous group IV CDPKs.