The myristoylated amino-terminus of an Arabidopsis calcium-dependent protein kinase mediates plasma membrane localization

Calcium-dependent protein kinases (CDPK) are a major group of calcium-stimulated kinases found in plants and some protists. Many CDPKs are membrane-associated, presumably because of lipid modifications at their amino termini. We investigated the subcellular location and myristoylation of AtCPK5, a member of the Arabidopsis CDPK family. Most AtCPK5 was associated with the plasma membrane as demonstrated by two-phase fractionation of plant microsomes and by in vivo detection of AtCPK5-GFP fusion proteins. AtCPK5 was a substrate for plant N-myristoyltransferase and myristoylation was prevented by converting the glycine at the proposed site of myristate attachment to alanine (G2A). In transgenic plants, a G2A mutation completely abolished AtCPK5 membrane association, indicating that myristoylation was essential for membrane binding. The first sixteen amino acids of AtCPK5 were sufficient to direct plasma membrane localization. In addition, differentially phosphorylated forms of AtCPK5 were detected both in planta and after expression of AtCPK5 in a cell-free plant extract. Our results demonstrate that AtCPK5 is myristoylated at its amino terminus and that myristoylation is required for membrane binding.     

Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.     

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.     

A novel yeast two-hybrid approach to identify CDPK substrates: characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein

Calcium-dependent protein kinases (CDPKs) are sensor-transducer proteins capable of decoding calcium signals in diverse phosphorylation-dependent calcium signaling networks in plants and some protists. Using a novel yeast two-hybrid (YTH) approach with constitutively active and/or catalytically inactive forms of AtCPK11 as bait, we identified AtDi19 as an AtCPK11-interacting protein. AtDi19 is a member of a small family of stress-induced genes. The interaction was confirmed using pull-down assays with in vitro translated AtCPK11 and GST-AtDi19 and localization studies in Arabidopsis protoplasts cotransfected with AtCPK11:GFP and AtDi19:DsRed2 protein fusions. We further showed that the interaction of AtDi19 is specific to both AtCPK4 and AtCPK11, whereas other closely related CPKs from Arabidopsis interacted weakly (e.g., AtCPK12) or did not interact (e.g., AtCPK26, AtCPK5 and AtCPK1) with AtDi19. Deletion analyses showed that a region containing two predicted nuclear localization signals (NLS) and a nuclear export signal (NES) of AtDi19 is essential for interaction with AtCPK11. We further demonstrated that AtDi19 is phosphorylated by AtCPK11 in a Ca(2+)-dependent manner at Thr105 and Ser107 within the AtDi19 bipartite NLS using in vitro kinase assays. Our data suggest that disruption of the autoinhibitor domain leading to the formation of a constitutively active CDPK may stabilize kinase-substrate interactions without affecting specificity.     

Induction of anthraquinone biosynthesis in Rubia cordifolia cells by heterologous expression of a calcium-dependent protein kinase gene

Calcium-dependent protein kinases (CDPKs) play an important role in plant cell responses to stress and pathogenic attack. In this study, we investigated the effect of heterologous expression of the Arabidopsis CDPK gene, AtCPK1, on anthraquinone production in transgenic Rubia cordifolia cells. AtCPK1 variants (a constitutively active, Ca(2+) -independent form and a non-active form used as a negative control) were transferred to callus cells by agrobacterial transformation. Overexpression of the constitutively active, Ca(2+) -independent form in R. cordifolia cells caused a 10-fold increase in anthraquinone content compared with non-transformed control cells, while the non-active form of AtCPK1 had no effect on anthraquinone production. Real-time PCR measurements showed that the activation of anthraquinone biosynthesis in transgenic calli correlated with the activation of isochorismate synthase gene expression. The activator effect of AtCPK1 was stable during prolonged periods of transgenic cell cultivation (more than 3 years) and the transgenic cultures exhibited high growth. Our results provide the first evidence that a CDPK gene can be used for the engineering of secondary metabolism in plant cells.     

Identification of proteins that interact with catalytically active calcium-dependent protein kinases from <Emphasis Type="Italic">Arabidopsis</Emphasis>

Calcium-dependent protein kinases (CDPKs) are essential sensor-transducers of calcium signaling pathways in plants. Functional characterization of CDPKs is of great interest because they play important roles during growth, development, and in response to a wide range of environmental stimuli. The Arabidopsis genome encodes 34 CDPKs, but very few substrates of these enzymes have been identified. In this study, we exploited the unique characteristics of CDPKs to develop an efficient approach for the discovery of CDPK-interacting proteins. High-throughput, semi-automated yeast two-hybrid interaction screens with two different cDNA libraries each containing 18 million prey clones were performed using catalytically impaired and constitutively active AtCPK4 and AtCPK11 variants as baits. The use of the constitutively active versions of the CPK baits improved the recovery of positive interacting proteins relative to the wild type kinase. Titration of interaction strength by growth under increasing concentrations of 3-aminotriazole (3-AT), a histidine analog and competitive inhibitor of the His3 gene product, confirmed these results. Possible mechanisms for this observed improvement are discussed. The reproducibility of this approach was assessed by the overlap of several interacting proteins of AtCPK4 and AtCPK11 and the recovery of several putative substrates and indicated that yeast two-hybrid screens using constitutively active and/or catalytically impaired forms of CDPK provides a useful tool to identify potential substrates of the CDPK family and potentially the entire protein kinase superfamily. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family

In plants, numerous Ca(2+)-stimulated protein kinase activities occur through calcium-dependent protein kinases (CDPKs). These novel calcium sensors are likely to be crucial mediators of responses to diverse endogenous and environmental cues. However, the precise biological function(s) of most CDPKs remains elusive. The Arabidopsis genome is predicted to encode 34 different CDPKs. In this Update, we analyze the Arabidopsis CDPK gene family and review the expression, regulation, and possible functions of plant CDPKs. By combining emerging cellular and genomic technologies with genetic and biochemical approaches, the characterization of Arabidopsis CDPKs provides a valuable opportunity to understand the plant calcium-signaling network.     

CDPK-driven changes in the intracellular ROS level and plant secondary metabolism

Heterologous expression of a constitutively active calcium-dependent protein kinase (CDPK) gene was previously shown to increase secondary metabolite production in cultured cells of Rubia cordifolia, but the critical question of how CDPK activates secondary metabolism remains to be answered. In this article, we report that the expression of the Arabidopsis CDPK gene, AtCPK1, in R. cordifolia cells caused moderate and stable elevation of intracellular reactive oxygen species (ROS) levels. In contrast, the non-active, mutated AtCPK1 gene did not cause such an effect. The active AtCPK1 also increased cell size, likely by restricting cell division. These results are consistent with the model in which constitutive expression of AtCPK1 mimics the effects of elicitors, acting on secondary metabolism via the activation of ROS production.     

Regulation of CDPK isoforms during tuber development

CDPK activities present during tuber development were analysed. A high CDPK activity was detected in the soluble fraction of early stolons and a lower one was detected in soluble and particulate fractions of induced stolons. The early and late CDPK activities displayed diverse specificity for in vitro substrates and different subcellular distribution. Western blot analysis revealed two CDPKs of 55 and 60 kDa that follow a precise spatial and temporal profile of expression. The 55 kDa protein was only detected in early-elongating stolons and the 60 kDa one was induced upon stolon swelling, correlating with early and late CDPK activities. A new member of the potato CDPK family, StCDPK3, was identified from a stolon cDNA library. Gene specific RT-PCR demonstrated that this gene is only expressed in early stolons, while the previously identified StCDPK1 is expressed upon stolon swelling. This expression profile suggests that StCDPK3 could correspond to the 55 kDa isoform while StCDPK1 could encode the 60 kDa isoform present in swelling stolons. StCDPK1 has myristoylation and palmitoylation consensus possibly involved in its dual intracellular localization. Transient expression studies with wild-type and mutated forms of StCDPK1 fused to GFP were used to show that subcellular localization of this isoform is controlled by myristoylation and palmitoylation. Altogether, our data suggest that sequential activation of StCDPK3 and StCDPK1 and the subcellular localisation of StCDPK1 might be critical regulatory steps of calcium signalling during potato tuber development.     

A chemical-genetic approach to elucidate protein kinase function <Emphasis Type="Italic">in planta</Emphasis>

The major objective in protein kinase research is the identification of the biological process, in which an individual enzyme is integrated. Protein kinase-mediated signalling is thereby often addressed by single knock-out mutation- or co-suppression-based reverse genetics approaches. If a protein kinase of interest is a member of a multi gene family, however, no obvious phenotypic alteration in the morphology or in biochemical parameters may become evident because mutant phenotypes may be compensated by functional redundancy or homeostasis. Here we establish a chemical-genetic screen combining ATP-analogue sensitive (as) kinase variants and molecular fingerprinting techniques to study members of the plant calcium-dependent protein kinase (CDPK) family in vivo. CDPKs have been implicated in fast signalling responses upon external abiotic and biotic stress stimuli. CDPKs carrying the as-mutation did not show altered phosphorylation kinetics with ATP as substrate, but were able to use ATP analogues as phosphate donors or as kinase inhibitors. For functional characterization in planta, we have substituted an Arabidopsis thaliana mutant line of AtCPK1 with the respective as-variant under the native CPK1 promoter. Seedlings of Arabidopsis wild type and AtCPK1 as-lines were treated with the ATP analogue inhibitor 1-NA-PP1 and exposed to cold stress conditions. Rapid cold-induced changes in the phosphoproteome were analysed by 2D-gel-electrophoresis and phosphoprotein staining. The comparison between wild type and AtCPK1 as-plants before and after inhibitor treatment revealed differential CPK1-dependent and cold-stress-induced phosphoprotein signals. In this study, we established the chemical-genetic approach as a tool, which allows the investigation of plant-specific classes of protein kinases in planta and which facilitates the identification of rapid changes of molecular biomarkers in kinase-mediated signalling networks.     

Structure of the regulatory apparatus of a calcium-dependent protein kinase (CDPK): a novel mode of calmodulin-target recognition

Calcium-dependent protein kinases (CDPKs) are a class of calcium-binding sensory proteins that are found in plants and certain protozoa, including the causative agent of malaria, Plasmodium falciparum. CDPKs have diverse regulatory functions, including involvement in the triggering of the lytic cycle of malarial infection. CDPKs contain an autoinhibitory junction (J) region whose calcium-dependent interaction with the tethered regulatory calmodulin-like domain (CaM-LD) activates the catalytic kinase domain. We report here the X-ray crystal structure of the J-CaM-LD region of CDPK from Arabidopsis thaliana (AtCPK1), determined to 2.0 A resolution using multiple-wavelength anomalous dispersion (MAD). The structure reveals a symmetric dimer of calcium-bound J-CaM-LD with domain-swap interactions, in which the J region of one protomer interacts extensively with the carboxy-terminal EF-hand domain (C-lobe) of the partner protomer. However, as the J-CaM-LD is monomeric in solution, the activated monomer was modelled to account for the intra-molecular recognition of the two domains. While the J-CaM-LD segment mimics certain aspects of target motif recognition by CaM other features are specific to CDPKs, in particular the combination of the strong interaction between the N and C-lobes of the CaM-LD and the exclusive use of only the C-lobe in the recognition of the covalently tethered target region. Combined with our previous observations showing that there is likely to be strong interactions between this tethered J region and the CaM-LD even at basal Ca(2+) concentrations, the new structural data indicate that the response to calcium of CDPKs is clearly unique among the CaM family.     

Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling

Evolutionary conservation of N-terminal N-myristoylation within protein families indicates significant functional impact of this lipid posttranslational modification for function. In the MYRbase study (Maurer-Stroh et al. (2004) Genome Biology 5, R21), protein families with relevance to asymmetric cell division in animals and the group of plant calcium-dependent protein kinases (CPKs) have surfaced with many predicted myristoylated members. Here, we describe experimental in vitro verification of predicted myristoylation and explore its impact on subcellular localization for these targets in vivo. Our results confirm that, indeed, Numb isoform A, Neuralized isoforms C and D from Drosophila melanogaster and two Neuralized-like homologues from Mus musculus have the capability for N-terminal myristoylation in vitro and in vivo (in fly tissue and in mouse 3T3 cells respectively) whereas other isoforms such as Neuralized A and B have not. The latter two cases are an example of different potential of various isoforms for posttranslational modifications. Additionally, the Arabidopsis thaliana CDPKs CPK6, CPK9 and CPK13 are shown to be substrates for myristoylation in vitro, which also affects their subcellular localization (in Arabidopsis protoplasts and tobacco leaves). At the same time, CPK6 and CPK13 do not appear to be substrates of a NMT1-like enzyme; the reasons for differing substrate specificities of NMT homologues in plants are derived from the evolutionary divergence of their N-myristoyl transferase sequences. As a methodical advance, we describe a fast and very sensitive technique (compared to traditional autoradiography) for in vitro testing of myristoylation based on thin layer chromatography read-out of the incorporated radioactive myristoyl anchor with subsequent Western blotting detection for protein yield determination using the same membrane.     

拟南芥钙依赖蛋白激酶参与植物激素信号转导

在植物信号通路中,涉及到钙应答的蛋白激酶大多是钙依赖蛋白激酶。钙依赖蛋白激酶作为钙信号转导因子,参与了包括激素信号转导途径在内的很多传递过程。本工作在前人研究的基础上,对拟南芥AtCPK30基因的功能进行了深入的研究。RT-PCR分析结果表明:AtCPK30在植物根中的表达量很高,其在幼苗中的转录水平分别受ABA、IAA、2,4-D、GA_3和6-BA等激素的诱导调节。AtCPK30基因过表达的转基因株系幼苗的主根比野生型的长,同时发现转基因植株幼苗的根在缺钙的MS培养基上生长较野生型植株长,表明缺钙对转基因幼苗影响较小。用ABA、IAA、GA_3和BA处理时,转基因植株幼苗的根对激素更敏感。当野生型和转基因植株生长在含有生长素抑制剂NPA的MS培养基上时,NPA对转基因植株侧根的抑制比对野生型弱。GFP-CPK30融合蛋白的亚细胞定位研究结果表明:CPK30蛋白定位在细胞壁和细胞膜上。这些研究结果说明了AtCPK30作为钙信号转导因子,参与了多种激素调节植物根生长的过程。     

AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis

The calcium-dependent protein kinase (CDPK) family is needed in plant signaling during various physiological pathways. The Arabidopsis AtCPK6 gene belongs to the subclass of stress-inducible CDPKs, which is stimulated by salt and osmotic stress. To elucidate the physiological function of AtCPK6, transgenic Arabidopsis plants under the control of double CaMV 35S promoter were obtained. AtCPK6 over-expressing plants showed enhanced tolerance to salt/drought stresses. The elevated tolerance of the AtCPK6 over-expressing plants was confirmed by the change of proline and malondialdehyde (MDA). Real-time PCR analyses revealed that the expression levels of several stress-regulated genes were altered in AtCPK6 over-expressing plants. However, cpk6 mutant displayed no obvious difference with control. These results are likely to indicate that AtCPK6 is functionally redundant and a positive regulator involved in the tolerance to salt/drought stress in Arabidopsis.     

CLONING OF A cDNA ENCODING A 66-kDa Ca2+-DEPENDENT PROTEIN KINASE (CDPK) FROM DUNALIELLA TERTIOLECTA (CHLOROPHYTA)

A cDNA clone encoding a Ca²+-dependent protein kinase (DtCPK1) with a calculated molecular mass of 65,746 Da was isolated by sequential immuno- and hybridization-screening from a cDNA library of the halotolerant green alga, Dunaliella tertiolecta Butcher (Chlorophyceae). Primary structure analysis of DtCPK1 revealed a long variable domain preceding a catalytic domain, an autoinhibitory junction domain, and a C-terminal calmodulin-like domain containing 4 EF-hand motifs. Database searches showed that DtCPK1 has a high similarity to CCK1 , a CDPK from the green alga, Chlamydomonas eugamentos Moewus . The N-terminal long variable domain of DtCPK1 contains neither the N-myristoylation motif, which is found in many CDPKs, nor the PEST motif, which is associated with rapid protein turnover and found in one CDPK subfamily. However, a putative Ca²+-dependent lipid binding domain that might be responsible for the association of cytosolic DtCPK1 with the cell membrane was identified in the variable domain. Three CDPKs, with molecular masses of 62, 54, and 47 kDa respectively, were observed in an in-gel protein kinase assay of D. tertiolecta cells extract. No change in the activities of these CDPKs were observed for up to 30 min after D. tertiolecta cells had been subjected to a hypoosmotic shock. An antibody raised against a CDPK purified from D. tertiolecta and used to isolate the DtCPK1 cDNA clone cross-reacted strongly with the 62-kDa CDPK but weakly with the 54-kDa CDPK in a Western blot, indicating that the 62-kDa CDPK is identical to DtCPK1. There was no change in the intensity of these bands after hypoosmotic shock, implying that the cellular level of the enzyme protein is not associated with hypoosmotic shock. These results indicate that CDPK is activated only by the increase in cytosolic-free Ca²+ concentration in vivo .