Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: comprehensive analysis of the CDPKs gene family in rice

In plants, calcium acts as a universal second messenger in various signal transduction pathways. The plant-specific calcium-dependent protein kinases (CDPKs) play important roles regulating downstream components of calcium signaling. We conducted a genome-wide analysis of rice CDPKs and identified 29 CDPK genes and eight closely related kinase genes, including five CDPK-related kinases (CRKs), one calcium and calmodulin-dependent protein kinase (CCaMK) and two phosphoenolpyruvate (PEP) carboxylase kinase-related kinases (PEPRKs). The mRNA splicing sites of the rice CDPKs, CRKs and PEPRKs (but not OsCCaMK) are highly conserved, suggesting that these kinases are derived from a common ancestor. RNA gel blot analyses revealed that the majority of rice CDPK genes exhibited tissue-specific expression. Expression of OsCPK9 was elevated in seedlings infected by rice blast, indicating that this gene plays an important role in signaling in response to rice blast treatment. Our genomic and bioinformatic analyses will provide an important foundation for further functional dissection of the rice CDPK gene family.     

The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications

Plant calcium-dependent protein kinases (CDPKs) are key proteins implicated in calcium-mediated signaling pathways of a wide range of biological events in the organism. The action of each particular CDPK is strictly regulated by many mechanisms in order to ensure an accurate signal translation and the activation of the adequate response processes. In this work, we investigated the regulation of a CDPK involved in rice cold stress response, OsCPK17, to better understand its mode of action. We identified two new alternative splicing (AS) mRNA forms of OsCPK17 encoding truncated versions of the protein, missing the CDPK activation domain. We analyzed the expression patterns of all AS variants in rice tissues and examined their subcellular localization in onion epidermal cells. The results indicate that the AS of OsCPK17 putatively originates truncated forms of the protein with distinct functions, and different subcellular and tissue distributions. Additionally, we addressed the regulation of OsCPK17 by post-translational modifications in several in vitro experiments. Our analysis indicated that OsCPK17 activity depends on its structural rearrangement induced by calcium binding, and that the protein can be autophosphorylated. The identified phosphorylation sites mostly populate the OsCPK17 N-terminal domain. Exceptions are phosphosites T107 and S136 in the kinase domain and S558 in the C-terminal domain. These phosphosites seem conserved in CDPKs and may reflect a common regulatory mechanism for this protein family.     

Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation

Calcium-dependent protein kinases (CDPKs), the most abundant serine/threonine kinases in plants, are found in various subcellular localizations, which suggests that this family of kinases may be involved in multiple signal transduction pathways. A complete analysis to try to understand the molecular basis of the presence of CDPKs in various localizations in the cell has not been accomplished yet. It has been suggested that myristoylation may be responsible for membrane association of CDPKs. In this study, we used a rice CDPK, OSCPK2, which has a consensus sequence for myristoylation at the N-terminus, to address this question. We expressed wild-type OSCPK2 and various mutants in different heterologous systems to investigate the factors that affect its membrane association. The results show that OSCPK2 is myristoylated and palmitoylated and targeted to the membrane fraction. Both modifications are required, myristoylation being essential for membrane localization and palmitoylation for its full association. The fact that palmitoylation is a reversible modification may provide a mechanism for regulation of the subcellular localization. OSCPK2 is the first CDPK shown to be targeted to membranes by an src homology domain 4 (SH4) located at the N-terminus of the molecule.     

Structures of parasitic CDPK domains point to a common mechanism of activation

We recently determined the first structures of inactivated and calcium-activated calcium-dependent protein kinases (CDPKs) from Apicomplexa. Calcium binding triggered a large conformational change that constituted a new mechanism in calcium signaling and a novel EF-hand fold (CAD, for CDPK activation domain). Thus we set out to determine if this mechanism was universal to all CDPKs. We solved additional CDPK structures, including one from the species Plasmodium. We highlight the similarities in sequence and structure across apicomplexan and plant CDPKs, and strengthen our observations that this novel mechanism could be universal to canonical CDPKs. Our new structures demonstrate more detailed steps in the mechanism of calcium activation and possible key players in regulation. Residues involved in making the largest conformational change are the most conserved across Apicomplexa, leading us to propose that the mechanism is indeed conserved. CpCDPK3_CAD and PfCDPK_CAD were captured at a possible intermediate conformation, lending insight into the order of activation steps. PfCDPK3_CAD adopts an activated fold, despite having an inactive EF-hand sequence in the N-terminal lobe. We propose that for most apicomplexan CDPKs, the mode of activation will be similar to that seen in our structures, while specific regulation of the inactive and active forms will require further investigation.     

Molecular evolution of calmodulin-like domain protein kinases (CDPKs) in plants and protists

Many genes for calmodulin-like domain protein kinases (CDPKs) have been identified in plants and Alveolate protists. To study the molecular evolution of the CDPK gene family, we performed a phylogenetic analysis of CDPK genomic sequences. Analysis of introns supports the phylogenetic analysis; CDPK genes with similar intron/exon structure are grouped together on the phylogenetic tree. Conserved introns support a monophyletic origin for plant CDPKs, CDPK-related kinases, and phosphoenolpyruvate carboxylase kinases. Plant CDPKs divide into two major branches. Plant CDPK genes on one branch share common intron positions with protist CDPK genes. The introns shared between protist and plant CDPKs presumably originated before the divergence of plants from Alveolates. Additionally, the calmodulin-like domains of protist CDPKs have intron positions in common with animal and fungal calmodulin genes. These results, together with the presence of a highly conserved phase zero intron located precisely at the beginning of the calmodulin-like domain, suggest that the ancestral CDPK gene could have originated from the fusion of protein kinase and calmodulin genes facilitated by recombination of ancient introns. Received: 11 July 2000 / Accepted: 18 April 2001     

<Emphasis Type="Italic">St</Emphasis>CDPK2 expression and activity reveal a highly responsive potato calcium-dependent protein kinase involved in light signalling

Calcium-dependent protein kinases (CDPKs) are essential calcium sensors. In this work, we have studied StCDPK2 isoform from potato both at gene and protein level. StCdpk2 genomic sequence contains eight exons and seven introns, as was observed for StCdpk1. There is one copy of the gene per genome located in chromosome 7. StCDPK2 encodes an active CDPK of 515 aminoacids, with an apparent MW of 57 kDa, which presents myristoylation and palmitoylation consensus in its N-terminus. StCDPK2 is highly expressed in leaves and green sprouts; enhanced expression was detected under light treatment, which corresponds well with light responsive cis-acting elements found in its promoter sequence. Antibodies against the recombinant StCDPK2::6xHis protein detected this isoform in soluble and particulate fractions from leaves. StCDPK2 autophosphorylation and kinase activity are both calcium dependent reaching half maximal activation at 0.6 μM calcium. The active kinase is autophosphorylated on serine and tyrosine residues and its activity is negatively modulated by phosphatidic acid (PA). Our results reveal StCDPK2 as a signalling element involved in plant growth and development and show that its activity is tightly regulated.     

Domain analysis of a groundnut calcium-dependent protein kinase: nuclear localization sequence in the junction domain is coupled with nonconsensus calcium binding domains

The signature of calcium-dependent protein kinases (CDPKs) is a C-terminal calmodulin-like domain (CaMLD) with four consensus calcium-binding sites. A junction domain (JD) joins the kinase with CaMLD and interacts with them through its autoinhibitory and CaMLD binding subdomains, respectively. We noted several CDPKs additionally have a bipartite nuclear localization signal (NLS) sequence as a subdomain in their JD, and this feature is obligatorily coupled with the absence of consensus calcium-binding sites in their respective CaMLDs. These predicted features are substantiated by undertaking investigations on a CDPK (gi:67479988) isolated from cultured groundnut (Arachis hypogea) cells. This kinase can bind 3.1 mol of Ca(2+) under saturating conditions with a considerably high K(d) of 392 mum as compared with its canonical counterparts. CD spectroscopic analysis, however, indicates the intramolecular structural changes accompanied with calcium binding to be similar to canonical CDPKs. Attesting to the presence of NLS in the JD, the endogenous kinase is localized in the nucleus of osmotically stressed Arachis cells, and in vitro binding assays indicate the NLS in the JD to interact with nuclear transport factors of the importin family. Homology modeling also indicates the feasibility of interaction of importins with the NLS present in the JD of such CDPKs in their activated form. The possible significance of obligatory coupling between the presence of NLS in the junction domain and atypical calcium binding properties of these CDPKs is discussed in the light of the known mechanisms of activation of these kinases.     

CDPK-mediated signalling pathways: specificity and cross-talk

Plants are constantly exposed to environmental changes and have to integrate a variety of biotic and abiotic stress stimuli. Calcium-dependent protein kinases (CDPKs) are implicated as important sensors of Ca2+ flux in plants in response to these stresses. CDPKs are encoded by multigene families, and expression levels of these genes are spatially and temporally controlled throughout development. In addition, a subset of CDPK genes responds to external stimuli. Biochemical evidence supports the idea that CDPKs are involved in signal transduction during stress conditions. Furthermore, loss-of-function and gain-of-function studies revealed that signalling pathways leading to cold, salt, drought or pathogen resistance are mediated by specific CDPK isoforms     

Molecular cloning of Plasmodium vivax calcium-dependent protein kinase 4

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.     

VfCPK1, a gene encoding calcium-dependent protein kinase from Vicia faba, is induced by drought and abscisic acid

Calcium, one of the most ubiquitous second messengers, has been shown to be involved in a wide variety of responses in plants. Calcium-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca(2+)-regulated serine/threonine protein kinase in plants and play an important role in plant calcium signal transduction. CDPKs are encoded by a large multigene family in many plants, which has been showed so far; however, the precise role of each specific CDPK is still largely unknown. A novel CDPK gene designated as VfCPK1 was cloned from epidermal peels of broad bean (Vicia faba L.) leaves using the rapid amplification of cDNA ends (RACE)-PCR technique and its expression was studied in detail. The VfCPK1 cDNA is 1783 bp long and contains an open reading frame of 1482 bp encoding 493 amino acids. VfCPK1 contains all conserved regions found in CDPKs and shows a high level of sequence similarity to many other plant CDPKs. VfCPK1 was highly expressed in leaves, especially in leaf epidermal peels of broad bean in mRNA and protein levels. Expressions of VfCPK1 at both the mRNA and protein levels were increased in leaves treated with abscisic acid or subjected to drought stress. Potential roles of VfCPK1 in epidermal peels are discussed. The nucleotide sequence data reported here were deposited in the GenBank database under accession number AY753552.     

A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK.

Calcium-Dependent Protein Kinases (CDPKs) in higher plants contain a C-terminal calmodulin-like regulatory domain. Little is known regarding physiological CDPK targets. Both kinase activity and multiple Ca2+-dependent signaling pathways have been implicated in the control of stomatal guard cell movements. To determine whether CDPK or other protein kinases could have a role in guard cell signaling, purified and recombinant kinases were applied to Vicia faba guard cell vacuoles during patch-clamp experiments. CDPK activated novel vacuolar chloride (VCL) and malate conductances in guard cells. Activation was dependent on both Ca2+ and ATP. Furthermore, VCL activation occurred in the absence of Ca2+ using a Ca2+-independent, constitutively active, CDPK* mutant. Protein kinase A showed weaker activation (22% as compared with CDPK). Current reversals in whole vacuole recordings shifted with the Nernst potential for Cl-and vanished in glutamate. Single channel recordings showed a CDPK-activated 34 +/- 5 pS Cl- channel. VCL channels were activated at physiological potentials enabling Cl- uptake into vacuoles. VCL channels may provide a previously unidentified, but necessary, pathway for anion uptake into vacuoles required for stomatal opening. CDPK-activated VCL currents were also observed in red beet vacuoles suggesting that these channels may provide a more general mechanism for kinase-dependent anion uptake.     

Genome-wide identification, classification, and expression analysis of CDPK and its closely related gene families in poplar (Populus trichocarpa)

Calcium-dependent protein kinases (CDPKs) are Ca²⁺-binding proteins known to play crucial roles in Ca²⁺ signal transduction pathways which have been identified throughout plant kingdom and in certain types of protists. Genome-wide analysis of CDPKs have been carried out in Arabidopsis, rice and wheat, and quite a few of CDPKs were proved to play crucial roles in plant stress responsive signature pathways. In this study, a comprehensive analysis of Populus CDPK and its closely related gene families was performed, including phylogeny, chromosome locations, gene structures, and expression profiles. Thirty Populus CDPK genes and twenty closely related kinase genes were identified, which were phylogenetically clustered into eight distinct subfamilies and predominately distributed across fifteen linkage groups (LG). Genomic organization analyses indicated that purifying selection has played a pivotal role in the retention and maintenance of Populus CDPK gene family. Furthermore, microarray analysis showed that a number of Populus CDPK and its closely related genes differentially expressed across disparate tissues and under various stresses. The expression profiles of paralogous pairs were also investigated to reveal their evolution fates. In addition, quantitative real-time RT-PCR was performed on nine selected CDPK genes to confirm their responses to drought stress treatment. These observations may lay the foundation for future functional analysis of Populus CDPK and its closely related gene families to unravel their biological roles.     

The Arabidopsis calcium-dependent protein kinase, CPK6, functions as a positive regulator of methyl jasmonate signaling in guard cells

Previous studies have demonstrated that methyl jasmonate (MeJA) induces stomatal closure dependent on change of cytosolic free calcium concentration in guard cells. However, these molecular mechanisms of intracellular Ca(2+) signal perception remain unknown. Calcium-dependent protein kinases (CDPKs) function as Ca(2+) signal transducers in various plant physiological processes. It has been reported that four Arabidopsis (Arabidopsis thaliana) CDPKs, CPK3, CPK6, CPK4, and CPK11, are involved in abscisic acid signaling in guard cells. It is also known that there is an interaction between MeJA and abscisic acid signaling in guard cells. In this study, we examined the roles of these CDPKs in MeJA signaling in guard cells using Arabidopsis mutants disrupted in the CDPK genes. Disruption of the CPK6 gene impaired MeJA-induced stomatal closure, but disruption of the other CDPK genes did not. Despite the broad expression pattern of CPK6, we did not find other remarkable MeJA-insensitive phenotypes in the cpk6-1 mutant. The whole-cell patch-clamp analysis revealed that MeJA activation of nonselective Ca(2+)-permeable cation channels is impaired in the cpk6-1 mutant. Consistent with this result, MeJA-induced transient cytosolic free calcium concentration increments were reduced in the cpk6-1 mutant. MeJA failed to activate slow-type anion channels in the cpk6-1 guard cells. Production of early signal components, reactive oxygen species and nitric oxide, in guard cells was elicited by MeJA in the cpk6-1 mutant as in the wild type. These results provide genetic evidence that CPK6 has a different role from CPK3 and functions as a positive regulator of MeJA signaling in Arabidopsis guard cells.     

The Toxoplasma gondii calcium‐dependent protein kinase 7 is involved in early steps of parasite division and is crucial for parasite survival

Apicomplexan parasites express various calcium‐dependent protein kinases (CDPKs), and some of them play essential roles in invasion and egress. Five of the six CDPKs conserved in most Apicomplexa have been studied at the molecular and cellular levels in Plasmodium species and/or in Toxoplasma gondii parasites, but the function of CDPK7 was so far uncharacterized. In T. gondii, during intracellular replication, two parasites are formed within a mother cell through a unique process called endodyogeny. Here we demonstrate that the knock‐down of CDPK7 protein in T. gondii results in pronounced defects in parasite division and a major growth deficiency, while it is dispensable for motility, egress and microneme exocytosis. In cdpk7‐depleted parasites, the overall DNA content was not impaired, but the polarity of daughter cells budding and the fate of several subcellular structures or proteins involved in cell division were affected, such as the centrosomes and the kinetochore. Overall, our data suggest that CDPK7 is crucial for proper maintenance of centrosome integrity required for the initiation of endodyogeny. Our findings provide a first insight into the probable role of calcium‐dependent signalling in parasite multiplication, in addition to its more widely explored role in invasion and egress.