Biomedical subjects

Chae Oh Lim

Publications and source records attributed to Chae Oh Lim.

At least 19 recordsLinked to original sources

PubMed2006

Gene expression profiles during heat acclimation in Arabidopsis thaliana suspension-culture cells.

Thermotolerance is induced by moderated heat acclimation. Suspension cultures of heat-acclimated Arabidopsis thaliana L. (Heynh.), ecotype Columbia, show thermotolerance against lethal heat shock (9 min, 50 degrees C), as evidenced by a chlorophyll assay and fluorescein diacetate staining. To monitor the genome-wide transcriptome changes induced by heat acclimation at 37 degrees C, we constructed an A. thaliana cDNA microarray containing 7,989 unique genes, and applied it to A. thaliana suspension-culture cells harvested at various times (0.5, 1, 2.5, 6, and 16 h) during heat acclimation. Data analysis revealed 165 differentially expressed genes that were grouped into ten clusters. We compared these genes with published and publicly available microarray heat-stress-related data sets in AtGenExpress. Heat-shock proteins were strongly expressed, as previously reported, and we found several of the up-regulated genes encoded detoxification and regulatory proteins. Moreover, the transcriptional induction of DREB2 (dehydration responsive element-binding factor 2) subfamily genes and COR47/rd17 under heat stress suggested cross-talk between the signaling pathways for heat and dehydration responses.

Acclimatization↗

PubMed2005

Phosphorylation and concomitant structural changes in human 2-Cys peroxiredoxin isotype I differentially regulate its peroxidase and molecular chaperone functions.

The H2O2-catabolizing peroxidase activity of human peroxiredoxin I (hPrxI) was previously shown to be regulated by phosphorylation of Thr90. Here, we show that hPrxI forms multiple oligomers with distinct secondary structures. HPrxI is a dual function protein, since it can behave either as a peroxidase or as a molecular chaperone. The effects of phosphorylation of hPrxI on its protein structure and dual functions were determined using site-directed mutagenesis, in which the phosphorylation site was substituted with aspartate to mimic the phosphorylated status of the protein (T90D-hPrxI). Phosphorylation of the protein induces significant changes in its protein structure from low molecular weight (MW) protein species to high MW protein complexes as well as its dual functions. In contrast to the wild type (WT)- and T90A-hPrxI, the T90D-hPrxI exhibited a markedly reduced peroxidase activity, but showed about sixfold higher chaperone activity than WT-hPrxI.

Amino Acid Substitution↗

PubMed2005

Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.).

Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca(2+)-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5'-TWCG(C/T)GTKKKKTKCG-3' (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate beta-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.

Amino Acid Sequence↗

PubMed2005

Arabidopsis ubiquitin-specific protease 6 (AtUBP6) interacts with calmodulin.

Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.

Amino Acid Motifs↗

PubMed2005

WRKY group IId transcription factors interact with calmodulin.

Calmodulin (CaM) is a ubiquitous Ca(2+)-binding protein known to regulate diverse cellular functions by modulating the activity of various target proteins. We isolated a cDNA encoding AtWRKY7, a novel CaM-binding transcription factor, from an Arabidopsis expression library with horseradish peroxidase-conjugated CaM. CaM binds specifically to the Ca(2+)-dependent CaM-binding domain (CaMBD) of AtWRKY7, as shown by site-directed mutagenesis, a gel mobility shift assay, a split-ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Furthermore, we show that the CaMBD of AtWRKY7 is a conserved structural motif (C-motif) found in group IId of the WRKY protein family.

Amino Acid Sequence↗

PubMed2005

Over-expression of Chinese cabbage calreticulin 1, BrCRT1, enhances shoot and root regeneration, but retards plant growth in transgenic tobacco.

Calreticulin (CRT) is a ubiquitously expressed, high capacity Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). A cDNA encoding a calreticulin, BrCRT1 (Brassica rapa Calreticulin 1), has been isolated from Chinese cabbage (B. rapa subsp. pekinensis) flower bud. Constitutive over-expression of the BrCRT1 gene promotes robust shoot production and root formation at sub-optimal concentrations of BA/NAA, which are important factors controlling plant regeneration in tissue culture. In contrast, the suppressed BrCRT1 line exhibited a slight reduction of shoot and root regeneration. In spite of enhanced regeneration in tissue culture, the seedling and plant growth rate was inhibited in soil. The steady state level of BrCRT1 transcripts was sensitive to exogenous auxins and cytokinins, and rapidly accumulated within 30 min, and this induction required de novo protein synthesis. Together with the results of transgenic tobacco plants and mRNA analysis in Chinese cabbage, our data suggest that BrCRT1 genes may up-regulate the competency of vegetative tissue to respond to hormonal signals involved in shoot and root regeneration processes.

Base Sequence↗

PubMed2004

Direct interaction of a divergent CaM isoform and the transcription factor, MYB2, enhances salt tolerance in arabidopsis.

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of enzymes and proteins. Plants express numerous CaM isoforms that exhibit differential activation and/or inhibition of CaM-dependent enzymes in vitro. However, the specific biological functions of plant CaM are not well known. In this study, we isolated a cDNA encoding a CaM binding transcription factor, MYB2, that regulates the expression of salt- and dehydration-responsive genes in Arabidopsis. This was achieved using a salt-inducible CaM isoform (GmCaM4) as a probe from a salt-treated Arabidopsis expression library. Using domain mapping, we identified a Ca2+-dependent CaM binding domain in MYB2. The specific binding of CaM to CaM binding domain was confirmed by site-directed mutagenesis, a gel mobility shift assay, split ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Interestingly, the specific CaM isoform GmCaM4 enhances the DNA binding activity of AtMYB2, whereas this was inhibited by a closely related CaM isoform (GmCaM1). Overexpression of Gm-CaM4 in Arabidopsis up-regulates the transcription rate of AtMYB2-regulated genes, including the proline-synthesizing enzyme P5CS1 (Delta1-pyrroline-5-carboxylate synthetase-1), which confers salt tolerance by facilitating proline accumulation. Therefore, we suggest that a specific CaM isoform mediates salt-induced Ca2+ signaling through the activation of an MYB transcriptional activator, thereby resulting in salt tolerance in plants.

Arabidopsis↗

PubMed2004

Pathogenesis-related gene expression by specific calmodulin isoforms is dependent on NIM1, a key regulator of systemic acquired resistance.

Plants produce numerous calmodulin isoforms that exhibit differential gene expression patterns and sense different Ca2+ signals. This diversity results in different physiological responses to particular stimuli. Gm-CaM-4 and -5 are two divergent calmodulin isoforms from the soybean (Glycine max) that have been reported to be involved in plant disease resistance. However, little is known about the pathway by which these specific isoforms transduce the defense signal and up-regulate pathogenesis-related (PR) genes. Here we report that overexpression of GmCaM-4/-5 induces constitutive PR gene expression and enhances disease resistance in wild-type Arabidopsis, but not in the nim1 mutant of Arabidopsis. GmCaM-4/-5 also appear to activate trans-acting elements that bind to cis-acting elements in the Arabidopsis PR-1 promoter. Thus up-regulation of PR genes by these GmCaM isoforms is dependent on NIM1 (Non immunity 1) and unknown transcription factors.

Arabidopsis↗

PubMed2004

Pathogen- and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor.

The Ca(2+)-binding protein calmodulin mediates cellular Ca(2+) signals in response to a wide array of stimuli in higher eukaryotes. Plants express numerous CaM isoforms. Transcription of one soybean (Glycine max) CaM isoform, SCaM-4, is dramatically induced within 30 min of pathogen or NaCl stresses. To characterize the cis-acting element(s) of this gene, we isolated an approximately 2-kb promoter sequence of the gene. Deletion analysis of the promoter revealed that a 130-bp region located between nucleotide positions -858 and -728 is required for the stressors to induce expression of SCaM-4. A hexameric DNA sequence within this region, GAAAAA (GT-1 cis-element), was identified as a core cis-acting element for the induction of the SCaM-4 gene. The GT-1 cis-element interacts with an Arabidopsis GT-1-like transcription factor, AtGT-3b, in vitro and in a yeast selection system. Transcription of AtGT-3b is also rapidly induced within 30 min after pathogen and NaCl treatment. These results suggest that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen- and salt-induced SCaM-4 gene expression in both soybean and Arabidopsis.

Amino Acid Sequence↗

PubMed2004

Bax-induced cell death of Arabidopsis is meditated through reactive oxygen-dependent and -independent processes.

An Arabidopsis protoplast system was developed for dissecting plant cell death in individual cells. Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces apoptotic-like cell death in Arabidopsis. Bax accumulation in Arabidopsis mesophyll protoplasts expressing murine Bax cDNA from a glucocorticoid-inducible promoter results in cytological characteristics of apoptosis, namely DNA fragmentation, increased vacuolation, and loss of plasma membrane integrity. In vivo targeting analysis monitored using jellyfish green fluorescent protein (GFP) reporter indicated full-length Bax was localized to the mitochondria, as it does in animal cells. Deletion of the carboxyl-terminal transmembrane domain of Bax completely abolished targeting to mitochondria. Bax expression was followed by reactive oxygen species (ROS) accumulation. Treatment of protoplasts with the antioxidant N -acetyl- -cysteine (NAC) during induction of Bax expression strongly suppressed Bax-mediated ROS production and the cell death phenotype. However, some population of the ROS depleted cells still induced cell death, indicating that there is a process that Bax-mediated plant cell death is independent of ROS accumulation. Accordingly, suppression of Bax-mediated plant cell death also takes place in two different processes. Over-expression of a key redox-regulator, Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) down-regulated ROS accumulation and suppressed Bax-mediated cell death and transient expression of Arabidopsis Bax inhibitor-1 (AtBI-1) substantially suppressed Bax-induced cell death without altering cellular ROS level. Taken together, our results collectively suggest that the Bax-mediated cell death and its suppression in plants is mediated by ROS-dependent and -independent processes.

Animals↗

PubMed2004

Pn-AMP1, a plant defense protein, induces actin depolarization in yeasts.

Pn-AMP1, Pharbitis nil antimicrobial peptide 1, is a small cysteine-rich peptide implicated in host-plant defense. We show here that Pn-AMP1 causes depolarization of the actin cytoskeleton in Saccharomyces cerevisiae and Candida albicans. Pn-AMP1 induces rapid depolarization of actin cables and patches within 15 min. Increased osmolarity or temperature induces transient actin depolarization and results in increased sensitivity to Pn-AMP1, while cells conditioned to these stresses show less sensitivity. Mutations in components of a cell wall integrity pathway (Wsc1p, Rom2p, Bck1p and Mpk1p), which regulate actin repolarization, result in increased sensitivity to Pn-AMP1. A genetic screen reveals that mutations in components of the alpha-1,6-mannosyltransferase complex (Mnn10p, Mnn11p and Och1p), which regulate mannosylation of cell wall proteins, confer resistance to Pn-AMP1. FITC-conjugated Pn-AMP1 localizes to the outer surface of the cell with no significant staining observed in spheroplasts. Taken together, these results indicate that cell wall proteins are determinants of resistance to Pn-AMP1, and the ability of a plant defense protein to induce actin depolarization is important for its antifungal activity.

Actins↗

PubMed2003

Regulation of the dual specificity protein phosphatase, DsPTP1, through interactions with calmodulin.

Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.

Amino Acid Motifs↗

PubMed2003

BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor.

Mitogen-activated protein kinase (MAPK) cascades are known to transduce plant defense signals, but the downstream components of the MAPK have as yet not been elucidated. Here, we report an MAPK from rice (Oryza sativa), BWMK1, and a transcription factor, OsEREBP1, phosphorylated by the kinase. The MAPK carries a TDY phosphorylation motif instead of the more common TEY motif in its kinase domain and has an unusually extended C-terminal domain that is essential to its kinase activity and translocation to the nucleus. The MAPK phosphorylates OsEREBP1 that binds to the GCC box element (AGCCGCC) of the several basic pathogenesis-related gene promoters, which in turn enhances DNA-binding activity of the factor to the cis element in vitro. Transient co-expression of the BWMK1 and OsEREBP1 in Arabidopsis protoplasts elevates the expression of the beta-glucuronidase reporter gene driven by the GCC box element. Furthermore, transgenic tobacco (Nicotiana tabacum) plants overexpressing BWMK1 expressed many pathogenesis-related genes at higher levels than wild-type plants with an enhanced resistance to pathogens. These findings suggest that MAPKs contribute to plant defense signal transduction by phosphorylating one or more transcription factors.

Active Transport, Cell Nucleus↗

PubMed2002

NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants.

NDP kinases (NDPKs) are multifunctional proteins that regulate a variety of eukaryotic cellular activities, including cell proliferation, development, and differentiation. However, much less is known about the functional significance of NDPKs in plants. We show here that NDPK is associated with H(2)O(2)-mediated mitogen-activated protein kinase signaling in plants. H(2)O(2) stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Proteins from transgenic plants overexpressing AtNDPK2 showed high levels of autophosphorylation and NDPK activity, and they have lower levels of reactive oxygen species (ROS) than wild-type plants. Mutants lacking AtNDPK2 had higher levels of ROS than wild type. H(2)O(2) treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6, two H(2)O(2)-activated A. thaliana mitogen-activated protein kinases. In the absence of H(2)O(2) treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the myelin basic protein phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a previously uncharacterized regulatory role in H(2)O(2)-mediated MAPK signaling in plants.

Arabidopsis↗

PubMed2002

GSH-dependent peroxidase activity of the rice (Oryza sativa) glutaredoxin, a thioltransferase.

Glutaredoxin (Grx) is a 12-kDa thioltransferase that reduces disulfide bonds of other proteins and maintains the redox potential of cells. In addition to its oxidoreductase activity, we report here that a rice Grx (OsGrx) can also function as a GSH-dependent peroxidase. Because of this antioxidant activity, OsGrx protects glutamine synthetase from oxidative damage. Individually replacing the conserved Cys residues in OsGrx with Ser shows that Cys(23), but not Cys(26), is essential for the thioltransferase and GSH-dependent peroxidase activities. Kinetic characterization of OsGrx reveals that the maximal catalytic efficiency (V(max)/K(m)) is obtained with cumene hydroperoxide rather than H(2)O(2) or t-butyl hydroperoxide.

Antioxidants↗

PubMed2002

Partial purification and properties of a phosphatidylinositol 4,5-bisphosphate hydrolyzing phospholipase C from the soluble fraction of soybean sprouts.

Three soluble enzyme fractions (F-I, F-II, and F-III) that hydrolyze phophoinositides were separated from soybean sprouts by using Matrex green gel column chromatography. Among the three phosphatidylinositol (PI)-specific phopholipsase C (PLC) enzymes, only the third fraction (F-III) was able to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) as substrates. The F-I and F-II fractions only showed enzymatic activities for PI and PIP. The PIP2-hydrolyzing PLC protein, F-III, was partially purified using the chromatographic steps of the Matrex green gel, phenyl Toyopearl, Matrex orange gel, Mono S cation exchange, and superose 6 gel filtration columns. The molecular weight of the F-III protein was estimated to be about 64 kDa on SDS-PAGE. The protein showed immunocross-reactivity with a polyclonal antibody that was prepared against the X and Y motifs of animal PLC enzymes, the conserved catalytic domains. Ca2+ ion critically affected the PIP2-hydrolyzing PLC activity of the F-III protein, representing maximal activity at 10 microM Ca2+ concentration. The PIP2-hydrolyzing PLC activity of the protein was also significantly increased by sodium deoxycholate (SDC) from 0.05 to 0.08%. However, the activity was greatly reduced above the concentration, and no activity was detected at 0.3% SDC. In addition, the protein exhibited maximal PIP2-hydrolyzing PLC activity at pH, in the range of 6.5-7.5.

Antibodies↗

PubMed2002

Carnitine uptake by AGP2 in yeast Saccharomyces cerevisiae is dependent on Hog1 MAP kinase pathway.

The AGP2 gene encodes a plasma membrane carnitine transporter in S. cerevisiae. Here, we report the identification of AGP2 as an osmotic stress response gene. AGP2 was isolated from mTn3 tagged mutants that contained in-frame fusions with lacZ. The expression of AGP2 was down-regulated by osmotic stresses, including NaCl, sorbitol, and KCI. We also found that carnitine uptake was inhibited by NaCl. In the ssk1delta stelldelta double-mutant strain, the expression of AGP2 and the uptake of carnitine were greatly reduced compared to the wild-type strain. Furthermore, carnitine uptake was inhibited by the constitutive expression of PBS2, which encodes a MAPKK that activates Hog1. We concluded, therefore, that the HOG pathway plays an important role in the regulation of carnitine uptake in S. cerevisiae.

Amino Acid Transport Systems↗

PubMed2002

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library.

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.

Amino Acid Motifs↗