Phosphorylation-dependent subfunctionalization 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 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.

Flg22‐induced Ca2+ increases undergo desensitization and resensitization

“…Especially for BIKI, which differs from the polyubiquitination regulated by PUB25 and PUB26, the monoubiquitination of BIKI induced by flg22 facilitates the dissociation of BIKI from the FLS2, which precedes FLS2 endocytosis (Ma et al, 2020). The phosphorylation state and/or stability of BIK1 are also negatively regulated by Ca 2+ ‐dependent protein kinase CPK28 (Bredow et al, 2021; Bredow & Monaghan, 2021; Monaghan et al, 2014; Monaghan, Matschi, Romeis, & Zipfel, 2015), which may be further activated through the PTI‐induced Ca 2+ influx. Therefore, this negative feed‐back regulation module may play an important role in the sensitive adaption of FICA.…”

Ubiquitination of Receptorsomes, Frontline of Plant Immunity

“…ATL31/6 positively regulates immune responses through the ubiquitination of CPK28 and relieving the CPK28-mediated negative regulation of BIK1 ( Figure 1 ) [ 94 ]. Additionally, CPK28 undergoes intermolecular autophosphorylation on Ser318, which is required for the activation of CPK28 under low intracellular [Ca 2+ ] to prevent the initiation of an immune response in the absence of infection [ 95 ]. Whether this residual phosphorylation status affects the polyubiquitination of CPK28 remains to be determined.…”

A DEK domain-containing protein GhDEK2D mediated Gossypium hirsutum enhanced resistance to Verticillium dahliae

“… 12 Reversible phosphorylation of serine, threonine, and tyrosine residues performs many important functions, such as altering protein conformation and activation states, protein stability and degradation, subcellular localization, and interaction with protein substrates. 13 Mining resistance genes has become the major way to sustain cotton industry. Protein post-translational modifications is major defense mechanism for regulating plant immune.…”