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.     

The effect of explant origin and collection season on stilbene biosynthesis in cell cultures of Vitis amurensis Rupr.

Plant cell and tissue cultures are considered as a source of valuable secondary metabolites but usually produce insufficient level of the compounds, which is the limiting factor for their application in biotechnology. We obtained 18 callus cell cultures from different organs of wild grape Vitis amurensis Rupr. collected at different seasons and analyzed stilbene accumulation in combination with calli growth parameters. This analysis showed that temporal and tissue origin of the calli affected the rate of stilbene biosynthesis. Stem-derived calli accumulated higher stilbene levels and exhibited a higher expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes than calli derived from the leaves and petioles. The highest content of stilbenes was detected in the calli initiated from grapevine stems collected in the autumn. In general, all “autumn” cell cultures contained more than 2 mg g^??1 dry wt (up to 11 mg g^??1 dry wt) and exhibited high PAL and STS genes expression in comparison with the calli initiated in the summer. The content of stilbenes in the “autumn” cell cultures were comparable to the highest stilbene contents detected in other plant sources described in the literature. Thus, selecting the most optimal explant source for cell culture establishment could be an effective approach towards developing plant cell cultures producing high stilbene levels.

     

Structure and expression profiling of a novel calcium-dependent protein kinase gene <Emphasis Type="Italic">PgCDPK1a</Emphasis> in roots,leaves, and cell cultures of <Emphasis Type="Italic">Panax ginseng</Emphasis>

Calcium-dependent protein kinases (CDPKs) are proposed to play an essential role in plant defense responses. In this study, we aimed to define the full sequence of a CDPK gene of Panax ginseng and analyze its expression in roots, leaves, and cell cultures of P. ginseng, one of the most valuable Chinese traditional medicinal herbs. We isolated the full-length cDNA of a P. ginseng CDPK gene, which was designated PgCDPK1a. PgCDPK1a shares high sequence identity at the amino acidic level with previously reported CDPK sequences for other plant species. We analyzed PgCDPK1a expression in the leaves of wild-growing P. ginseng plants, and in the roots and leaves of cultivated P. ginseng plants growing in an open experimental nursery at a natural ginseng habitat. PgCDPK1a was more actively expressed in the young leaves of cultivated P. ginseng plants than in that of wild-growing ones. Finally, we analyzed the expression of the gene in control GV and five rolC and rolB transgenic callus cultures of P. ginseng with different levels of fresh biomass accumulation, pathogen-related gene expression, and ginsenoside production. We observed a strong positive correlation between fresh biomass accumulation of P. ginseng cell cultures and expression of the PgCDPK1a gene. There was a less clear negative correlation between the expression of pathogen-related genes and the content of ginsenosides with the PgCDPK1a expression in cell cultures of P. ginseng. Perhaps, PgCDPK1a is involved in ginseng growth, as a positive regulator.     

Influence of calcium influx induced by the calcium ionophore, A23187, on resveratrol content and the expression of CDPK and STS genes in the cell cultures of Vitis amurensis

The present study examines the effect of calcium influx induced by the calcium ionophore (CI) on the biosynthesis of resveratrol and the expression of stilbene synthase (STS) and calcium-dependent protein kinase (CDPK) genes in cell cultures of Vitis amurensis, which have different levels of resveratrol production. The present study utilized the control cell culture V2 of V. amurensis, which contains no more than 0.02?% dry weight (DW) of resveratrol, in addition to rolB transgenic cell cultures VB1 and VB2, which have increased resveratrol contents (0.1–0.8?% DW). Treatment with the CI at a 1?μM concentration significantly increased STS gene expression (6 of 10 analyzed STS genes) and resveratrol production in the control V2 cell culture by fourfold; however, use of the CI at 10?μM significantly decreased resveratrol production by 2–4 fold in all cell cultures tested. In the control V2 grape cell culture, treatment with the CI increased expression of all of the CDPK genes except VaCDPK1a and VaCDPK3a. In the rolB transgenic VB2 grape cell culture treated with the CI, we detected alterations in expression of several CDPK genes, but these changes in gene expression were not significant. Our results indicated that treatment with 1?μM of the CI increased resveratrol content and production in control grape cells by selectively increasing the expression of STS genes. Conversely, the CI treatment did not significantly increase resveratrol content and production, or the expression of CDPK or STS genes in the rolB transgenic cells. Likely, untreated VB2 cells have increased concentrations of cytoplasmic calcium, and therefore, treatment with the CI did not significantly change CDPK expression. These results suggest that the rolB gene has an important role in the regulation of calcium-dependent transduction pathways in transformed cells.     

Effects of 5-azacytidine induced DNA demethylation on methyltransferase gene expression and resveratrol production in cell cultures of Vitis amurensis

DNA becomes methylated in vivo through the action of a specific group of enzymes known as methyltransferases or methylases. Plants are known to possess the methyltransferases (Met), chromo methyltransferases (CMT), and domainrearranged methyltransferases (DRM) methylase families, which affect cytosine methylation within different contexts. DNA methylation has been proposed to play a role in secondary plant metabolism, but there is a lack of valid data connecting these two processes. In this study, we treated control and transformed with rolB gene from Agrobacterium rhizogenes cell cultures of Vitis amurensis with the demethylation agent 5-azacytidine (azaC). The purpose of the current investigation was to study effects of induced DNA demethylation on methyltransferase gene expression in connection to resveratrol production, a naturally occurring polyphenol that has a wide range of intriguing biological properties. Using semi-quantitative and real-time PCR, we showed that rolB gene transformation of V. amurensis cells decreased Met and CMT expression, but significantly increased DRM expression. AzaC treatment of the control and the rolB-transgenic calli significantly increased expression of all methylases (excluding Met). Following 3 months of azaC treatment, we detected significantly elevated levels of rolB gene expression in the transgenic calli. In current paper, we discuss how methylase expression may influence resveratrol biosynthesis and rolB transgene expression. Effects of azaC application are discussed.     

Chromaffin cell scinderin, a novel calcium-dependent actin filament-severing protein.

Scinderin, a novel Ca2+-activated actin filament-severing protein, has been purified to homogeneity from bovine adrenal medulla using a combination of several chromatographic procedures. The protein has an apparent mol. wt of 79,600 +/- 450 daltons, three isoforms (pIs 6.0, 6.1 and 6.2) and two Ca2+ binding sites (Kd 5.85 x 10(-7) M, Bmax 0.81 mol Ca2+/mol protein and Kd 2.85 x 10(-6) M, Bmax 1.87 mol Ca2+/mol protein). Scinderin interacts with F-actin in the presence of Ca2+ and produces a decrease in the viscosity of actin gels as a result of F-actin filament severing as demonstrated by electron microscopy. Scinderin is a structurally different protein from chromaffin cell gelsolin, another actin filament-severing protein described. Scinderin and gelsolin have different mol. wts, isoelectric points, amino acid composition and yield different peptide maps after limited proteolytic digestion by either Staphylococcus V8 protease or chymotrypsin. Moreover, scinderin antibodies do not cross-react with gelsolin and gelsolin antibodies fail to recognize scinderin. Immunofluorescence with anti-scinderin demonstrated that this protein is mainly localized in the subplasmalemma region of the chromaffin cell. Immunoblotting tests with the same antibodies indicated that scinderin is also expressed in brain and anterior as well as posterior pituitary. Presence of scinderin and gelsolin, two Ca2+-dependent actin filament-severing proteins in the same tissue, suggests the possibility of synergistic functions by the two proteins in the control of cellular actin filament networks. Alternatively, the actin filament-severing activity of the two proteins might be under the control of different transduction and modulating influences.     

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.     

Purification and characterization of a novel calcium-dependent protein kinase from soybean

A novel calcium-dependent protein kinase (CDPK) previously reported to be activated by the direct binding of Ca2+, and requiring neither calmodulin nor phospholipids for activity [Harmon, A.C., Putnam-Evans, C.L., & Cormier, M.J. (1987) Plant Physiol. 83, 830-837], was purified to greater than 95% homogeneity from suspension-cultured soybean cells (Glycine max, L. Wayne). Purification was achieved by chromatography on DEAE-cellulose, phenyl-Sepharose, Sephadex G-100, and Blue Sepharose. The purified enzyme (native molecular mass = 52,200 Da) resolved into two immunologically related protein bands of 52 and 55 kDa on 10% SDS gels. Enzyme activity was stimulated 40-100-fold by micromolar amounts of free calcium (K0.5 = 1.5 microM free calcium) and was dependent upon millimolar Mg2+. CDPK phosphorylated lysine-rich histone III-S and chicken gizzard myosin light chains but did not phosphorylate arginine-rich histone, phosvitin, casein, protamine, or Kemptide. Phosphorylation of histone III-S, but not autophosphorylation, was inhibited by KCl. CDPK displayed a broad pH optimum (pH 7-9), and kinetic studies revealed a Km for Mg2(+)-ATP of 8 microM and a Vmax of 1.7 mumol min-1 mg-1 with histone III-S (Km = 0.13 mg/mL) as substrate. Unlike many other protein kinases, CDPK was able to utilize Mg2(+)-GTP, in addition to Mg2(+)-ATP, as phosphate donor. The enzyme phosphorylated histone III-S exclusively on serine; however, CDPK autophosphorylated on both serine and threonine residues. These properties demonstrate that CDPK belongs to a new class of protein kinase.     

Resveratrol content and expression of phenylalanine ammonia-lyase and stilbene synthase genes in rolC transgenic cell cultures of Vitis amurensis

Resveratrol, a naturally occurring polyphenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of Vitis amurensis Rupr. with the oncogene rolC of Agrobacterium rhizogenes increased resveratrol production in the two transformed callus cultures 3.7 and 11.9 times. The rolC-transformed calli were capable of producing 0.099% and 0.144% dry weight of resveratrol. We characterized phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in the two rolC transgenic callus cultures of V. amurensis. In the rolC transgenic culture with higher resveratrol content, expression of VaPAL3, VaSTS3, VaSTS4, VaSTS5, VaSTS6, VaSTS8, VaSTS9, and VaSTS10 was increased; while in the rolC culture with lower resveratrol content, expression of VaPAL3 and VaSTS9 was increased. We suggest that transformation of V. amurensis calli with the rolС gene induced resveratrol accumulation via selective enhancement of expression of individual PAL and STS genes involved in resveratrol biosynthesis. We compared the data on PAL and STS gene expression in rolC transgenic calli with the previously obtained results for rolB transgenic calli of V. amurensis. We propose that the transformation of V. amurensis with the rolC and rolB genes of A. rhizogenes increased resveratrol accumulation through different regulatory pathways.     

The effect of stress hormones,UV-C,and stilbene precursors on calmodulin (CaM) and calmodulin-like gene (CML) expression in Vitis amurensis Rupr

Plant Cell, Tissue and Organ Culture (PCTOC) - Urtica dioica L. (Urticaceae), popularly known as nettle, is a medicinal plant used by the textile, food and pharmaceutical industry. The aim of the...     

Molecular cloning, overexpression, and characterization of autophosphorylation in calcium-dependent protein kinase 1 (CDPK1) from Cicer arietinum

In plants, calcium-dependent protein kinases (CDPKs) are key intermediates in calcium-mediated signaling that couple changes in Ca²⁺ levels to a specific response. In the present study, we report the high-level soluble expression of calcium-dependent protein kinase1 from Cicer arietinum (CaCDPK1) in Escherichia coli. The expression of soluble CaCDPK1 was temperature dependent with a yield of 3–4 mg/l of bacterial culture. CaCDPK1 expressed as histidine-tag fusion protein was purified using Ni–NTA affinity chromatography till homogeneity. The recombinant CaCDPK1 protein exhibited both calcium-dependent autophosphorylation and substrate phosphorylation activities with a V _max and K _m value of 13.2 nmol/min/mg and 34.3 μM, respectively, for histone III-S as substrate. Maximum autophosphorylation was seen only in the presence of calcium. Optimum temperature for autophosphorylation was found to be 37 °C. The recombinant protein showed optimum pH range of 6–9. The role of autophosphorylation in substrate phosphorylation was investigated using histone III-S as exogenous substrate. Our results show that autophosphorylation happens before substrate phosphorylation and it happens via intra-molecular mechanism as the activity linearly depends on enzyme concentrations. Autophosphorylation enhances the kinase activity and reduces the lag phase of activation, and CaCDPK1 can utilize both ATP and GTP as phosphodonor but ATP is preferred than GTP.