Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance

Recently we reported on a plasma membrane tobacco protein (designated NtCBP4) that binds calmodulin. When overexpressed in transgenic plants, NtCBP4 confers Pb2+ hypersensitivity associated with enhanced accumulation of this toxic metal. To further investigate possible modulation of Pb2+ tolerance in plants, we prepared transgenic plants that express a truncated version of this protein (designated NtCBP4DeltaC) from which its C-terminal, with the calmodulin-binding domain and part of the putative cyclic nucleotide-binding domain, was removed. In contrast to the phenotype of transgenic plants expressing the full-length gene, transgenic plants expressing the truncated gene showed improved tolerance to Pb2+, in addition to attenuated accumulation of this metal. Furthermore, disruption by T-DNA insertion mutagenesis of the Arabidopsis CNGC1 gene, which encodes a homologous protein, also conferred Pb2+ tolerance. We suggest that NtCBP4 and AtCNGC1 are components of a transport pathway responsible for Pb2+ entry into plant cells.     

Cyclic-nucleotide- and Ca2+/calmodulin-regulated channels in plants: targets for manipulating heavy-metal tolerance, and possible physiological roles

Recently we discovered a tobacco protein (designated NtCBP4) that modulates heavy-metal tolerance in transgenic plants. Structurally, NtCBP4 is similar to mammalian cyclic-nucleotide-gated non-selective cation channels containing six putative transmembrane domains, a predicted pore region, a conserved cyclic-nucleotide-binding domain, and a high-affinity calmodulin-binding site that coincides with its cyclic-nucleotide-binding domain. Transgenic tobacco expressing the plasma-membrane-localized NtCBP4 exhibit improved tolerance to Ni(2+) and hypersensitivity to Pb(2+), which are associated with a decreased accumulation of Ni(2+) and an enhanced accumulation of Pb(2+) respectively. Transgenic plants expressing a truncated version of NtCBP4, from which regulatory domains had been removed, have a different phenotype. Here we describe our approach to studying the involvement of NtCBP4 in heavy-metal tolerance and to elucidate its physiological role.     

A plant kinesin heavy chain-like protein is a calmodulin-binding protein

Calmodulin, a calcium modulated protein, regulates the activity of several proteins that control cellular functions. A cDNA encoding a unique calmodulin-binding protein, PKCBP, was isolated from a potato expression library using protein-protein interaction based screening. The cDNA encoded protein bound to biotinylated calmodulin and ³⁵S-labeled calmodulin in the presence of calcium and failed to bind in the presence of EGTA, a calcium chelator. The deduced amino acid sequence of the PKCBP has a domain of about 340 amino acids in the C-terminus that showed significant sequence similarity with the kinesin heavy chain motor domain and contained conserved ATP- and microtubule-binding sites present in the motor domain of all known kinesin heavy chains. Outside the motor domain, the PKCBP showed no sequence similarity with any of the known kinesins, but contained a globular domain in the N-terminus and a putative coiled-coil region in the middle. The calmodulin-binding region was mapped to a stretch of 64 amino acid residues in the C-terminus region of the protein. The gene is differentially expressed with the highest expression in apical buds. A homolog of PKCBP from Arabidopsis (AKCBP) showed identical structural organization indicating that kinesin heavy chains that bind to calmodulin are likely to exist in other plants. This paper presents evidence that the motor domain has microtubule stimulated ATPase activity and binds to microtubules in a nucleotide-dependent manner. The kinesin heavy chain-like calmodulin-binding protein is a new member of the kinesin superfamily as none of the known kinesin heavy chains contain a calmodulin-binding domain. The presence of a calmodulin-binding motif and a motor domain in a single polypeptide suggests regulation of kinesin heavy chain driven motor function(s) by calcium and calmodulin.     

A tobacco plasma membrane calmodulin-binding transporter confers Ni2+ tolerance and Pb2+ hypersensitivity in transgenic plants

All organisms require a minimal amount of metal ions to sustain their metabolism, growth and development. At the same time, their intrinsic metal-uptake systems render them vulnerable to toxic levels of metals in the biosphere. Using radiolabeled recombinant calmodulin as a probe to screen a tobacco cDNA library, we have discovered a protein designated NtCBP4 (Nicotiana tabacum calmodulin-binding protein) that can modulate plant tolerance to heavy metals. Structurally, NtCBP4 is similar to vertebrate and invertebrate K+ and to non-selective cation channels, as well as to recently reported proteins from barley and Arabidopsis. Here we report on the subcellular localization of NtCBP4 and the phenotype of transgenic plants overexpressing this protein. The localization of NtCBP4 in the plasma membrane was manifested by fractionating tobacco membranes on sucrose gradients or by aqueous two-phase partitioning, and subsequently using immunodetection. Several independent transgenic lines expressing NtCBP4 had higher than normal levels of NtCBP4. These transgenic lines were indistinguishable from wild type under normal growth conditions. However, they exhibited improved tolerance to Ni2+ and hypersensitivity to Pb2+, which are associated with reduced Ni2+ accumulation and enhanced Pb2+ accumulation, respectively. To our knowledge this is the first report of a plant protein that modulates plant tolerance or accumulation of Pb2+. We propose that NtCBP4 is involved in metal uptake across the plant plasma membrane. This gene may prove useful for implementing selective ion tolerance in crops and improving phytoremediation strategies.     

A sustained increase in the intracellular Ca2+ concentration induces proteolytic cleavage of EAG2 channel

Voltage-gated EAG2 channel is abundant in the brain and enhances cancer cell growth by controlling cell volume. The channel contains a cyclic nucleotide-binding homology (CNBH) domain and multiple calmodulin-binding motifs. Here we show that a raised intracellular Ca²⁺ concentration causes proteolytic digestion of heterologously expressed and native EAG2 channels. A treatment of EAG2-expressing cells with the Ca²⁺ ionophore A23187 for 1 h reduces the full-length protein by ∼80% with a concomitant appearance of 30–35-kDa peptides. Similarly, a treatment with the Ca²⁺-ATPase inhibitor thapsigargin for 3 h removes 30–35-kDa peptides from ∼1/3 of the channel protein. Moreover, an incubation of the isolated rat brain membrane with CaCl₂ leads to the generation of fragments with similar sizes. This Ca²⁺-induced digestion is not seen with EAG1. Mutations in a C-terminal calmodulin-binding motif alter the degrees and positions of the cleavage. Truncated channels that mimic the digested proteins exhibit a reduced current density and altered channel gating. In particular, these shorter channels lack a rapid activation typical in EAG channels with more than 20-mV positive shifts in voltage dependence of activation. The truncation also eliminates the ability of EAG2 channel to reduce cell volume. These results suggest that a sustained increase in the intracellular Ca²⁺ concentration leads to proteolytic cleavage at the C-terminal cytosolic region following the CNBH domain by altering its interaction with calmodulin. The observed Ca²⁺-induced proteolytic cleavage of EAG2 channel may act as an adaptive response under physiological and/or pathological conditions.     

A novel kinesin-like protein with a calmodulin-binding domain

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with ³⁵S-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca²⁺-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCK1 is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca²⁺/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.     

Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C

We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.     

A novel calcium/calmodulin-regulated kinesin-like protein is highly conserved between monocots and dicots

Recently, a novel kinesin-like protein (KCBP) that is regulated by Ca2+/calmodulin was isolated from dicot plants. A homolog of KCBP has not been reported in monocots. To determine if this motor protein is present in phylogenetically divergent flowering plants, Arabidopsis KCBP cDNA was used as a probe to screen a genomic library of maize, an evolutionarily divergent species. This screening resulted in isolation of a KCBP homolog. Comparison of the predicted amino acid sequence of the KCBP from maize (ZmKCBP), a monocot, with the previously reported KCBP sequences from dicot species showed that the amino acid sequence, domain organization, and gene structure are highly conserved between monocots and dicots. The C-terminal region of ZmKCBP, containing the motor domain and the calmodulin-binding domain, and the N-terminal tail, with a myosin tail homology region (MyTH4) and talin-like region, showed strong sequence similarity to the KCBP homolog from dicots. However, the coiled-coil region is less conserved between monocots and dicots. The ZmKCBP gene contained 22 exons and 21 introns. The location of 19 of the 21 introns of ZmKCBP is also conserved. The ZmKCBP protein is encoded by a single gene and expressed in all tissues. Affinity-purified antibody to the calmodulin-binding domain of Arabidopsis KCBP detected a protein in both the soluble and the microsomal fractions. The C-terminal region of ZmKCBP, containing the motor and calmodulin-binding domains, bound calmodulin in the presence of calcium and failed to bind in the presence of EGTA. The ZmKCBP, along with other KCBPs from dicots, was grouped into a distinct group in the C-terminal subfamily of kinesin-like proteins. These data suggest that the KCBP is ubiquitous and highly conserved in all flowering plants and the origin of KCBP predated the divergence of monocots and dicots.     

Calcium-Dependent Calmodulin Binding to Chromaffin Granule Membranes: Presence of a 65-Kilodalton Calmodulin-Binding Protein

The presence of calmodulin-binding sites on chromaffin granule membranes has been investigated. Saturable, high-affinity 125I-calmodulin-binding sites (KD = 9.8 nM; Bmax = 25 pmol/mg protein) were observed in the presence of 10(-4) M free calcium. A second, nonsaturable, calmodulin-binding activity could also be detected at 10(-7) M free calcium. No binding occurred at lower calcium levels. When chromaffin granule membranes were delipidated by solvent extraction, calmodulin binding was observed at 10(-4) M free calcium. However no binding was detected at lower calcium concentrations. Thus it appears that a calcium concentration of 10(-7) M promotes the binding of calmodulin to some solvent-soluble components of the chromaffin granule membrane. Calmodulin-binding proteins associated with the granule membrane identified by photoaffinity cross-linking. A calmodulin-binding protein complex, of molecular weight 82K, was formed in the presence of 10(-4) M free calcium. This cross-linked product was specific because it was not detected either in the absence of calcium, in the presence of nonlabeled calmodulin, or in the absence of cross-linker activation. When solvent-treated membranes were used, a second, specific, calmodulin-binding protein complex (70K) was formed. Since the apparent molecular weight of calmodulin in our electrophoresis system was 17K, these experiments suggested the presence of two calmodulin-binding proteins, of molecular weights 65K and 53K, in the chromaffin granule membrane. This result was confirmed by the use of calmodulin-affinity chromatography. When detergent-solubilized membranes were applied on the column in the presence of calcium, two polypeptides of apparent molecular weights of 65K and 53K were specifically eluted by EGTA buffers. Since detergent treatments or solvent extractions are necessary to detect the 53K calmodulin-binding protein, it is concluded that only the 65K calmodulin-binding polypeptide may play a role in the interaction between calmodulin and secretory granules in chromaffin cells.     

Modulation of the Plasma Membrane Ca2+ Pump

The plasma membrane calcium pump, which ejects Ca²⁺ from the cell, is regulated by calmodulin. In the absence of calmodulin, the pump is relatively inactive; binding of calmodulin to a specific domain stimulates its activity. Phosphorylation of the pump with protein kinase C or A may modify this regulation. Most of the regulatory functions of the enzyme are concentrated in a region at the carboxyl terminus. This region varies substantially between different isoforms of the pump, causing substantial differences in regulatory properties. The pump shares some motifs of the carboxyl terminus with otherwise unrelated proteins: The calmodulin-binding domain is a modified IQ motif (a motif which is present in myosins) and the last 3 residues of isoform 4b are a PDZ target domain. The pump is ubiquitous, with isoforms 1 and 4 of the pump being more widely distributed than 2 and 3. In some kinds of cells isoform 1 or 4 is missing, and is replaced by another isoform. Received: 26 January 1998/Revised: 6 April 1998     

Interaction of calmodulin with phospholamban and caldesmon: comparative studies by 1H-NMR spectroscopy.

In order to identify comparative aspects of the interaction of calmodulin with its target proteins, proton magnetic-resonance studies of complex formation between calmodulin and defined segments of phospholamban and caldesmon have been undertaken. Residues 3-15 in the cytoplasmic region of phospholamban, an integral membrane protein of cardiac sarcoplasmic reticulum believed to regulate the calcium pumping ATPase, are shown to contribute to interaction with calmodulin. Using wheat germ calmodulin specifically modified with a spin-label to provide the spectral means for spatial localisation, these residues of phospholamban were correlated with binding in the vicinity of the probe attached to Cys-27 in the N-terminal domain of calmodulin. This interaction, relevant to the mechanism of calmodulin-dependent phosphorylation of phospholamban that relieves its inhibitory influence on the calcium pump, provides a useful model system for comparative study of the properties of calmodulin-binding domains. We contrast here a calmodulin-binding segment in the C-terminal region of caldesmon localised by 1H-NMR study of the interface(s) between the two proteins. These observations are discussed in the context of other calmodulin-binding sequences.     

Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn(2+). The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.     

CHRK1, a chitinase-related receptor-like kinase in tobacco

A cDNA encoding a chitinase-related receptor-like kinase, designated CHRK1, was isolated from tobacco (Nicotiana tabacum). The C-terminal kinase domain (KD) of CHRK1 contained all of the conserved amino acids of serine/threonine protein kinases. The putative extracellular domain was closely related to the class V chitinase of tobacco and to microbial chitinases. CHRK1 mRNA accumulation was strongly stimulated by infection with fungal pathogen and tobacco mosaic virus. Amino acid-sequence analysis revealed that the chitinase-like domain of CHRK1 lacked the essential glutamic acid residue required for chitinase activity. The recombinant chitinase-like domain did not show any catalytic activity for either oligomeric or polymeric chitin substrates. The recombinant KD of CHRK1 exhibited autophosphorylation, but the mutant KD with a mutation in the essential ATP-binding site did not, suggesting that CHRK1 encoded a functional kinase. CHRK1 was detected in membrane fractions of tobacco BY2 cells. Furthermore, CHRK1-GFP fusion protein was localized in plasma membranes when it was expressed in animal cells. This is the first report of a new type of receptor-like kinase containing a chitinase-like sequence in the putative extracellular domain.     

Hypertrophic cardiomyopathy-causing Asp175asn and Glu180gly Tpm1 mutations shift tropomyosin strands further towards the open position during the ATPase cycle

Yeast calmodulin known to be ubiquitylated in vivo in a Ca²⁺ dependent manner has long remained an orphan substrate. Here we identify Saccharomyces cerevisiae Asr1p as an ubiquitin E3 ligase for yeast calmodulin, a protein involved in calcium signaling. A short region within Asr1p-C harboring two putative calmodulin-binding motifs is sufficient and necessary for interaction with calmodulin. The interaction is direct, occurs in vivo and depends on physiological concentrations of Ca²⁺. A minimal set of purified proteins including Asr1p E3 ligase was sufficient for in vitro ubiquitylation of calmodulin, a reaction that required a functional Asr1p Ring domain. We propose a role of the Asr1p E3 ligase activity in coping with stress.     

Elucidation of a minimal immunoreactive site of vertebrate calmodulin

The heptapeptide AsnTyrGluGluPheValGlnNH₂ corresponding to residues 137–143 of vertebrate calmodulin is as immunoreactive as the entire 148-residue protein. A reproducible and rapid procedure for producing antisera against vertebrate calmodulin has been previously described (L. J. Van Eldik and D. M. Watterson (1981) J. Biol. Chem.256, 4205–4210). Most of the antisera elicited by this method react with a major immunoreactive region (residues 127–144) in the COOH-terminal domain of vertebrate calmodulin. In this report, the minimum segment of calmodulin required for reactivity with an antiserum that readily distinguishes various types of calmodulins is defined. These studies demonstrate that a linear segment of seven amino acid residues shows a competition curve in radioimmunoassay resembling the competition curve of intact calmodulin. This heptapeptide is the smallest calmodulin segment and the only sevenresidue segment in the 135–145 region that shows quantitative immunoreactivity with the anti-calmodulin serum. These data demonstrate that this heptapeptide is a major immunoreactive site of calmodulin. However, when this immunoreactive site heptapeptide is conjugated to a carrier and injected into rabbits, it does not elicit antisera that react with the native protein. These studies demonstrate that quantitative immunoreactivity of antisera produced in animals can be found in small peptide segments and that, for calmodulin, the requirements for production of anti-peptide antibodies that react with the native protein molecule are not as simple as surface exposure of the peptide region.     

Formation of cyclic imide-like structures upon the treatment of calmodulin and a calmodulin peptide with heat

Protein cyclic imide is the putative intermediate in the formation of sites of carboxyl-methylation in eukaryotic proteins. Conditions known to induce the formation of a cyclic imide in model peptides have been applied to a protein, calmodulin. Heating of calmodulin in the dry state at 100 degrees C for 24 h after lyophilization from a pH 2.0 or pH 6.0 solution produces derivatives with altered chromatographic properties in anion-exchange HPLC. At pH 6.0, complete activity of calmodulin was retained. Analysis with Fourier transform infrared (FTIR)-photoacoustic spectroscopy demonstrated the presence of a new structure in the calmodulin molecule consistent with modification of carboxylic acid groups. The conversion of calmodulin is dependent upon the absence of Ca2+ (the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid). A peptide analogous to the calcium binding regions of calmodulin, Asp-Lys-Asp-Gly-Asn-Gly-Thr-Ile-Thr-Thr-Lys-Glu, is also converted, upon heating, to chromatographically different forms in reversed-phase chromatography. This process is also dependent upon the absence of calcium. Sequence analysis of the peptide derivatives reveals a second amino terminus, implicating peptide bond hydrolysis in the product. A dipeptide, Asp-Gly, known to form a cyclic imide structure under similar conditions is also hydrolyzed during sequence analysis consistent with cleavage occurring at the position of the cyclic imide structure. Asp3 is suggested to be the site of cyclic imide formation in the calmodulin peptide. The presence of a cyclic imide structure is also confirmed by the application of FTIR-photoacoustic spectroscopy. These data suggest that cyclic imide formation in calmodulin has been induced, possibly at one, or more, of the calcium binding loops of the protein. These modification reactions may provide a basis for future investigations of cyclic imide formation in proteins.     

The location of calmodulin in the pea plasma membrane

Plasma membrane has been prepared from pea seedlings in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Calmodulin has been detected in these plasma membrane preparations using calcium overlay techniques, immunoblots, quantitation with antibodies raised against spinach calmodulin, phosphodiesterase activation, mobility shift, and heat stability. EGTA-stable calmodulin represents 0.5-1% of the total plasma membrane protein, and it is the only detectable calcium-binding protein in plasma membrane isolated under these conditions. The anti-spinach calmodulin reacts only with the N-terminal region of spinach calmodulin representing residues 1-106. The positioning of EGTA-stable calmodulin in the plasma membrane has been probed with trypsin and anti-spinach calmodulin. The data suggest that the calmodulin N-terminal region representing residues 1-106 projects from the membrane and could be available for binding other proteins. Calcium-dependent calmodulin binding to the plasma membrane has also been detected. Calcium-dependent calmodulin-binding proteins have been characterized using calmodulin overlay methods. The exposure of calmodulin-binding domains of most of these proteins from the plasma membrane is further suggested by their reaction with azidoiodinated calmodulin.