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Recent studies suggest that immune-induced alternative splice variants of the Arabidopsis thaliana Ca 2+ -dependent protein kinase (CDPK) AtCPK28 may result in signal attenuation. We put forward the hypothesis that expression of alternative truncated variants may be a broadly conserved regulatory mechanism of CDPKs throughout the green lineage., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Calcium (Ca 2+ )-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca 2+ 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 Ca 2+ activation at physiological concentrations of Ca 2+ , possibly through stabilization of the Ca 2+ -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 [Ca 2+ ] 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 Ca 2+ -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., Competing Interests: The authors declare no competing interest.

Activation of Ca 2+ signaling is a universal response to stress that allows cells to quickly respond to environmental cues. Fluctuations in cytosolic Ca 2+ are decoded in plants by Ca 2+ -sensing proteins such as Ca 2+ -dependent protein kinases (CDPKs). The perception of microbes results in an influx of Ca 2+ that activates numerous CDPKs responsible for propagating immune signals required for resistance against disease-causing pathogens. This review describes our current understanding of CDPK activation and regulation, and provides a comprehensive overview of CDPK-mediated immune signaling through interaction with various substrates.

Plant pattern recognition receptors (PRRs) perceive microbial and endogenous molecular patterns to activate immune signaling. The cytoplasmic kinase BIK1 acts downstream of multiple PRRs as a rate-limiting component, whose phosphorylation and accumulation are central to immune signal propagation. Previous work identified the calcium-dependent protein kinase CPK28 and heterotrimeric G proteins as negative and positive regulators of BIK1 accumulation, respectively. However, mechanisms underlying this regulation remain unknown. Here we show that the plant U-box proteins PUB25 and PUB26 are homologous E3 ligases that mark BIK1 for degradation to negatively regulate immunity. We demonstrate that the heterotrimeric G proteins inhibit PUB25/26 activity to stabilize BIK1, whereas CPK28 specifically phosphorylates conserved residues in PUB25/26 to enhance their activity and promote BIK1 degradation. Interestingly, PUB25/26 specifically target non-activated BIK1, suggesting that activated BIK1 is maintained for immune signaling. Our findings reveal a multi-protein regulatory module that enables robust yet tightly regulated immune responses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Plant calcium (Ca 2+ )-dependent protein kinases (CPKs) represent the primary Ca 2+ -dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca 2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca 2+ /CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca 2+ /CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca 2+ -responsive and was inhibited by Ca 2+ /CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca 2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca 2+ activation and might also allow CPK28 to remain active when Ca 2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca 2+ /CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca 2+ signaling specificity through Ca 2+ sensor priming., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Plants are protected from microbial infection by a robust immune system. Two of the earliest responses mediated by surface-localized immune receptors include an increase in cytosolic calcium (Ca(2+)) and a burst of apoplastic reactive oxygen species (ROS). The Arabidopsis plasma membrane-associated cytoplasmic kinase BIK1 is an immediate convergent substrate of multiple surface-localized immune receptors that is genetically required for the PAMP-induced Ca(2+) burst and directly regulates ROS production catalyzed by the NADPH oxidase RBOHD. We recently demonstrated that Arabidopsis plants maintain an optimal level of BIK1 through a process of continuous degradation regulated by the Ca(2+)-dependent protein kinase CPK28. cpk28 mutants accumulate more BIK1 protein and display enhanced immune signaling, while plants over-expressing CPK28 accumulate less BIK1 protein and display impaired immune signaling. Here, we show that CPK28 additionally contributes to the PAMP-induced Ca(2+) burst, supporting its role as a negative regulator of BIK1.

Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen to identify loci involved in PTI and characterize the Arabidopsis calcium-dependent protein kinase CPK28 as a negative regulator of immune signaling. Genetic analyses demonstrate that CPK28 attenuates PAMP-triggered immune responses and antibacterial immunity. CPK28 interacts with and phosphorylates the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28 contributes to BIK1 turnover. Our results suggest a negative regulatory mechanism that continually buffers immune signaling by controlling the turnover of this key signaling kinase., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Summary: The calcium‐dependent protein kinase CPK28 regulates several stress pathways in multiple plant species. Here, we aimed to discover CPK28‐associated proteins in Arabidopsis thaliana.We used affinity‐based proteomics and identified several potential CPK28 binding partners, including the C7 Raf‐like kinases MRK1, RAF26, and RAF39. We used biochemistry, genetics, and physiological assays to gain insight into their function.We define redundant roles for these kinases in stomatal opening, immune‐triggered reactive oxygen species (ROS) production, and resistance to a bacterial pathogen. We report that CPK28 associates with and trans‐phosphorylates RAF26 and RAF39, and that MRK1, RAF26, and RAF39 are active kinases that localize to endomembranes. Although Raf‐like kinases share some features with mitogen‐activated protein kinase kinase kinases (MKKKs), we found that MRK1, RAF26, and RAF39 are unable to trans‐phosphorylate any of the 10 Arabidopsis mitogen‐activated protein kinase kinases (MKKs).Overall, our study suggests that C7 Raf‐like kinases associate with and are phosphorylated by CPK28, function redundantly in stomatal opening and immunity, and possess substrate specificities distinct from canonical MKKKs. [ABSTRACT FROM AUTHOR]

Mechanisms to sense and respond to calcium have evolved in all organisms. Calmodulin is a universal calcium sensor across eukaryotes that directly binds calcium and associates with many downstream signal transducers including protein kinases. All eukaryotes encode calcium-dependent and/or calmodulin-dependent kinases, however there are distinct protein families across kingdoms. Here, we compare the activation mechanisms of calmodulin-dependent protein kinases (CaMKs), calcium- and calmodulin-dependent protein kinases (CCaMKs) and calcium-dependent protein kinases (CDPKs), noting striking similarities regarding phosphorylation in a regulatory segment known as the autoinhibitory junction. We thus propose that conserved regulation by phosphorylation underlies the activation of calcium-responsive proteins from different kingdoms. [Display omitted] • Structural comparison of AIJ in CaMKs, CCaMKs, and CDPKs. • Conserved autophosphorylation in AIJ of CaMKs, CCaMKs, and CDPKs across kingdoms. • Conserved mechanism-of-action of CaMKs, CCaMKs, and CDPKs. • Phosphorylation in the AIJ may allow for the decoding of specific calcium signatures. [ABSTRACT FROM AUTHOR]

Plant calcium (Ca2+)-dependent protein kinases (CPKs) represent the primary Ca2+-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca2+/CaMbinding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and was inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+ activation and might also allow CPK28 to remain active when Ca2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reducedsensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming. [ABSTRACT FROM AUTHOR]

AtCPK4 and AtCPK11 are Arabidopsis thaliana Ca2+-dependent protein kinase (CDPK) paralogs that have been reported to positively regulate abscisic acid (ABA) signal transduction by phosphorylating ABA-responsive transcription factor-4 (AtABF4). By contrast, RcCDPK1, their closest Ricinus communis ortholog, participates in the control of anaplerotic carbon flux in developing castor oil seeds by catalyzing inhibitory phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser451. LC-MS/MS revealed that AtCPK4 and RcCDPK1 transphosphorylated several common, conserved residues of AtABF4 and its castor ortholog, TRANSCRIPTION FACTOR RESPONSIBLE FOR ABA REGULATON. Arabidopsis atcpk4/atcpk11 mutants displayed an ABA-insensitive phenotype that corroborated the involvement of AtCPK4/11 in ABA signaling. A kinase-client assay was employed to identify additional AtCPK4/RcCDPK1 targets. Both CDPKs were separately incubated with a library of 2095 peptides representative of Arabidopsis protein phosphosites; five overlapping targets were identified including PLANT INTRACELLULAR RAS-GROUP-RELATED LEUCINE-RICH REPEAT PROTEIN-9 (AtPIRL9) and the E3-ubiquitin ligase ARABIDOPSIS TOXICOS EN LEVADURA 6 (AtATL6). AtPIRL9 and AtATL6 residues phosphorylated by AtCPK4/RcCDPK1 conformed to a CDPK recognition motif that was conserved amongst their respective orthologs. Collectively, this study provides evidence for novel AtCPK4/RcCDPK1 substrates, which may help to expand regulatory networks linked to Ca2+- and ABA-signaling, immune responses, and central carbon metabolism. • Ca2+-dependent protein kinases (CDPKs) play pivotal roles in plant cell biology. • Arabidopsis AtCPK4 and castor RcCDPK1 and are closely related CDPK orthologs. • They exhibited highly comparable site-specific phosphorylation of multiple targets. • Several novel, shared targets of AtCPK4 andRcCDPK1 were identified. • This may help to expand pathways linked to plant development and stress signaling. [ABSTRACT FROM AUTHOR]