FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2002596952> ?p ?o ?g. }

• W2002596952 endingPage "34899" @default.
• W2002596952 startingPage "34888" @default.
• W2002596952 abstract "Diacylglycerol kinase (DGK) regulates the level of the second messenger diacylglycerol and produces phosphatidic acid (PA), another signaling molecule. The Arabidopsis thaliana genome encodes seven putative diacylglycerol kinase isozymes (named AtDGK1 to -7), structurally falling into three major clusters. So far, enzymatic activity has not been reported for any plant Cluster II DGK. Here, we demonstrate that a representative of this cluster, AtDGK7, is biochemically active when expressed as a recombinant protein in Escherichia coli. AtDGK7, encoded by gene locus At4g30340, contains 374 amino acids with an apparent molecular mass of 41.2 kDa. AtDGK7 harbors an N-terminal catalytic domain, but in contrast to various characterized DGKs (including AtDGK2), it lacks a cysteine-rich domain at its N terminus, and, importantly, its C-terminal DGK accessory domain is incomplete. Recombinant AtDGK7 expressed in E. coli exhibits Michaelis-Menten type kinetics with 1,2-dioleoyl-sn-glycerol as substrate. AtDGK7 activity was affected by pH, detergents, and the DGK inhibitor R59022. We demonstrate that both AtDGK2 and AtDGK7 phosphorylate diacylglycerol molecular species that are typically found in plants, indicating that both enzymes convert physiologically relevant substrates. AtDGK7 is expressed throughout the Arabidopsis plant, but expression is strongest in flowers and young seedlings. Expression of AtDGK2 is transiently induced by wounding. R59022 at ∼80 μm inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development. Diacylglycerol kinase (DGK) regulates the level of the second messenger diacylglycerol and produces phosphatidic acid (PA), another signaling molecule. The Arabidopsis thaliana genome encodes seven putative diacylglycerol kinase isozymes (named AtDGK1 to -7), structurally falling into three major clusters. So far, enzymatic activity has not been reported for any plant Cluster II DGK. Here, we demonstrate that a representative of this cluster, AtDGK7, is biochemically active when expressed as a recombinant protein in Escherichia coli. AtDGK7, encoded by gene locus At4g30340, contains 374 amino acids with an apparent molecular mass of 41.2 kDa. AtDGK7 harbors an N-terminal catalytic domain, but in contrast to various characterized DGKs (including AtDGK2), it lacks a cysteine-rich domain at its N terminus, and, importantly, its C-terminal DGK accessory domain is incomplete. Recombinant AtDGK7 expressed in E. coli exhibits Michaelis-Menten type kinetics with 1,2-dioleoyl-sn-glycerol as substrate. AtDGK7 activity was affected by pH, detergents, and the DGK inhibitor R59022. We demonstrate that both AtDGK2 and AtDGK7 phosphorylate diacylglycerol molecular species that are typically found in plants, indicating that both enzymes convert physiologically relevant substrates. AtDGK7 is expressed throughout the Arabidopsis plant, but expression is strongest in flowers and young seedlings. Expression of AtDGK2 is transiently induced by wounding. R59022 at ∼80 μm inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development. Lipid second messengers, generated in response to diverse stimuli through the activity of lipid kinases and phospholipases, are involved in a variety of biological responses in plant cells (1Meijer H.J.G. Munnik T. Annu. Rev. Plant Biol. 2003; 54: 265-306Crossref PubMed Scopus (478) Google Scholar, 2Wang X. Curr. Opin. Plant Biol. 2004; 7: 329-336Crossref PubMed Scopus (310) Google Scholar). Diacylglycerol kinase (EC 2.7.1.107) is a lipid kinase that phosphorylates diacylglycerol (DAG) 5The abbreviations and trivial names used are: DAG, diacylglycerol; EST, expressed sequence tag; Bis-Tris, bis(2-hydroxyethyl)-imino-tris(hydroxymethyl)methane; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Na-DC, sodium deoxycholate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; 1,2-SAG, 1-stearoyl-2-arachidonoyl-sn-glycerol; 1,2-DOG, 1,2-dioleoyl-sn-glycerol; 1,2-POG, 1-palmitoyl, 2-oleoyl-sn-glycerol; 1,2-SLG, 1-stearoyl, 2-linoleoyl-sn-glycerol; 1,2-OPG, 1,2-1-oleoyl, 2-palmitoyl-sn-glycerol; PA, phosphatidic acid; CaM, calmodulin; RT, reverse transcription; R59022, 6-{2-{4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl}ethyl}-7-methyl-5H-thiazolo(3,2-α)pyrimidine-5-one; R59949, 3-{2-{4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl}ethyl}-2,3-dihydro-2-thioxo-4(1H)-quinazolinone; MPSS, massively parallel signature sequencing; TPM, transcripts/million; HPLC, high pressure liquid chromatography. 5The abbreviations and trivial names used are: DAG, diacylglycerol; EST, expressed sequence tag; Bis-Tris, bis(2-hydroxyethyl)-imino-tris(hydroxymethyl)methane; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Na-DC, sodium deoxycholate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; 1,2-SAG, 1-stearoyl-2-arachidonoyl-sn-glycerol; 1,2-DOG, 1,2-dioleoyl-sn-glycerol; 1,2-POG, 1-palmitoyl, 2-oleoyl-sn-glycerol; 1,2-SLG, 1-stearoyl, 2-linoleoyl-sn-glycerol; 1,2-OPG, 1,2-1-oleoyl, 2-palmitoyl-sn-glycerol; PA, phosphatidic acid; CaM, calmodulin; RT, reverse transcription; R59022, 6-{2-{4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl}ethyl}-7-methyl-5H-thiazolo(3,2-α)pyrimidine-5-one; R59949, 3-{2-{4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl}ethyl}-2,3-dihydro-2-thioxo-4(1H)-quinazolinone; MPSS, massively parallel signature sequencing; TPM, transcripts/million; HPLC, high pressure liquid chromatography. to yield phosphatidic acid (PA) in a reaction that uses ATP as phosphate donor. In plants, both DAG and PA may have signaling functions. DAG has been demonstrated to activate both ion pumping in patch-clamped guard cell protoplasts and opening of stomata (3Lee Y. Assmann S.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2127-2131Crossref PubMed Scopus (73) Google Scholar), whereas PA accumulates in response to different kinds of stresses and regulates the activity of several enzymes (4Munnik T. Trends Plant Sci. 2001; 6: 227-233Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar).Various PA targets have been identified in plants, including, for example, Arabidopsis phosphoinositide-dependent protein kinase 1 (5Deak M. Casamayor A. Currie R.A. Downes C.P. Alessi D.R. FEBS Lett. 1999; 451: 220-226Crossref PubMed Scopus (110) Google Scholar) and a calcium-dependent protein kinase from carrot (6Farmer P.K. Choi J.H. Biochim. Biophys. Acta. 1999; 1434: 6-17Crossref PubMed Scopus (76) Google Scholar). Recently, using PA affinity chromatography coupled to mass spectrometry, PA-binding proteins were identified in plants, including phosphoenolpyruvate carboxylase, Hsp90, 14-3-3 proteins, and others (7Testerink C. Dekker H.L. Lim Z.Y. Johns M.K. Holmes A.B. Koster C.G. Ktistakis N.T. Munnik T. Plant J. 2004; 39: 527-536Crossref PubMed Scopus (161) Google Scholar), providing new entries into the field of plant PA research. Furthermore, Zhang et al. (8Zhang Q. Qin C. Zhao J. Wang X. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9508-9513Crossref PubMed Scopus (402) Google Scholar) have demonstrated that PA interacts with ABI1 phosphatase 2C, thereby regulating abscisic acid signaling in Arabidopsis thaliana.Animal DGKs have been extensively studied, and models for their function have been elaborated (9Topham M.K. Prescott S.M. Thromb. Haemost. 2002; 88: 912-918Crossref PubMed Scopus (34) Google Scholar, 10Martelli A.M. Bortul R. Tabellini G. Bareggi R. Manzoli L. Narducci P. Cocco L. Cell Mol. Life Sci. 2002; 59: 1129-1137Crossref PubMed Scopus (45) Google Scholar). DGK activity has also been reported in several plant species, including Catharanthus roseus, tobacco, wheat, tomato, and Arabidopsis (11Heim S. Bauleke A. Wylegalla C. Wagner K.G. Plant Sci. 1987; 49: 159-165Crossref Scopus (27) Google Scholar, 12Kamada Y. Muto S. Biochim. Biophys. Acta. 1991; 1093: 72-79Crossref PubMed Scopus (41) Google Scholar, 13Wissing J.B. Wagner K.G. Plant Physiol. 1992; 98: 1148-1153Crossref PubMed Scopus (26) Google Scholar, 14Lundberg G.A. Sommarin M. Biochim. Biophys. Acta. 1992; 1123: 177-183Crossref PubMed Scopus (21) Google Scholar, 15Snedden W.A. Blumwald E. Plant J. 2000; 24: 317-326Crossref PubMed Google Scholar, 16Gómez-Merino F.C. Ornatowska M. Abdel-Haliem M.E.F. Zanor M.-I. Mueller-Roeber B. Schnarrenberger C. Wittmann-Liebold B. Proceedings of the XIIth Congress on Genes, Gene Families, and Isozymes. Monduzzi Editore, Bologna, Italy2003: 247-250Google Scholar), and molecular data bases reveal that they are present in a number of other crop plants such as rice, maize, grape, sweet orange, and cotton. However, functional analysis of DGK genes is still fragmentary. Two DGK cDNAs, LeDGK1 and LeCBDGK, respectively, have been cloned from tomato (15Snedden W.A. Blumwald E. Plant J. 2000; 24: 317-326Crossref PubMed Google Scholar). The two enzymes are derived from the same gene via alternative splicing and are identical except for a 29-amino acid-long C-terminal extension in the case of LeCBDGK, which represents a calmodulin (CaM)-binding domain. The two proteins lack the cysteine-rich domain (CRD) present in other eukaryotic DGKs, but are active in vitro. LeCBDGK is found both in association with membranes and in soluble cell extracts. The Ca2+/CaM-dependent translocation of LeCBDGK to its membrane-associated substrate DAG might represent a means of activation in vivo, analogous to the Ca2+-dependent translocation of certain mammalian DGKs. In contrast, LeDGK1 only associates with the membrane fraction, via a Ca2+/CaM-independent mechanism, which might represent a means of encoding specificity in cellular responses by alternative splicing (15Snedden W.A. Blumwald E. Plant J. 2000; 24: 317-326Crossref PubMed Google Scholar). In A. thaliana, seven candidate genes (named AtDGK1 to -7) encode putative DGK isoforms. The AtDGK1 cDNA has been isolated and reported to be mainly expressed in roots, shoots, and leaves, but its enzyme product was not active in vitro (17Katagiri T. Mizoguchi T. Shinozaki K. Plant Mol. Biol. 1996; 30: 647-653Crossref PubMed Scopus (45) Google Scholar). We have previously cloned the AtDGK2 cDNA and demonstrated that its encoded enzyme is catalytically active. AtDGK2 transcripts are found in the whole plant except in stems and are induced by exposure to 4 °C, pointing to a role in cold signal transduction (18Gómez-Merino F.C. Brearley C.A. Ornatowska M. Abdel-Haliem M.E.F. Zanor M.I. Mueller-Roeber B. J. Biol. Chem. 2004; 279: 8230-8241Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). AtDGK1 and -2 share a similar domain organization and fall into the Cluster I of plant DGKs.Here we report the molecular cloning and characterization of AtDGK7, which encodes an enzyme structurally belonging to the Cluster II of plant DGKs. The AtDGK7 gene is transcribed throughout the plant and most prominently in flowers and young tissues. Recombinant AtDGK7 enzyme is catalytically active and, importantly, accepts DAG molecular species containing at least one saturated fatty acid as the preferred substrate. We also demonstrate that both AtDGK2 and AtDGK7 are able to phosphorylate DAG molecular species that are typically found in plants, indicating that both enzymes convert physiologically relevant substrates. The DGK inhibitor 6-{2-{4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl}ethyl}-7-methyl-5H-thiazolo(3,2-α)pyrimidine-5-one (R59022) at around 50-100 μm inhibits recombinant AtDGK2 in vitro. It also modifies root growth within the same concentration range, indicating the involvement of this enzyme in developmental processes. In contrast, AtDGK7 was found to be affected by R59022 only at concentrations above 100 μm.EXPERIMENTAL PROCEDURESGeneral—Manipulation and analysis of nucleic acids were performed according to standard molecular-biological techniques described (19Sambrook J. Russell D. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000Google Scholar). Restriction enzymes were purchased from Roche Applied Science (Mannheim, Germany) and New England Biolabs (Frankfurt am Main, Germany). Oligonucleotides were obtained from TibMolbiol (Berlin, Germany) or Eurogentec (Cologne, Germany). DNA sequencing was performed by SeqLab (Göttingen, Germany). Unless otherwise indicated, other chemicals were purchased from Roche Applied Science, Merck, or Sigma. Escherichia coli strain XL-1 Blue (Stratagene, Heidelberg, Germany) was employed for general DNA work. For sequence analyses, the tools provided by the National Center for Biotechnology Information (available on the World Wide Web at www.ncbi.nlm.nih.gov/), the ExPASy Molecular Biology Server (available on the World Wide Web at us.expasy.org/), and The Arabidopsis Information Resource (available on the World Wide Web at www.arabidopsis.org/) were used. Gene expression data obtained through Affymetrix Gene-Chip hybridizations were retrieved using the software tools of the Genevestigator package (available on the World Wide Web at www.genevestigator.ethz.ch), version of July 2004. Massively parallel signature sequencing data were retrieved from the Arabidopsis massively parallel signature sequencing (MPSS) data base (available on the World Wide Web at mpss.udel.edu/at/java.html), version of August 2004.Cloning of AtDGK7 cDNA—PCR amplification of the AtDGK7 cDNA was carried out using the Advantage-HF2 PCR kit (Clontech, Heidelberg, Germany) according to the manufacturer's protocol. A. thaliana (L.) Heynh. Col-0 flower cDNA was used as template. Primer sequences were as follows: AtDGK7-F, 5′-GCGGATCCTGATGGAGGAGACGCCGAGATC-3′; AtDGK7-R, 5′-GCGCTCGAGTTATATGAACCTCTTAGGAAC-3′. Reaction details were as follows: 95 °C for 3 min; 36 cycles of 95 °C for 45 s, 62 °C for 45 s, and 72 °C for 72 s; 72 °C for 10 min. PCR products were analyzed by agarose gel electrophoresis. Individual fragments were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and subcloned into pGEM-T Easy (Promega, Mannheim, Germany), generating the plasmid pG7. The entire AtDGK7 cDNA, present in plasmid pG7, had a length of 1125 bp (deposited under GenBank™ accession number AY686593).Quantitative Reverse Transcription-PCR Analysis—Total RNA was digested with DNase I, RNase Free (Roche Applied Sciences, Mannheim, Germany). The absence of genomic DNA contamination was subsequently confirmed by PCR, using primers annealing to an intron of actin 2 (At3g18780) as a control gene, or alternatively, with primers LEH-fw (5′-AACAGCAACAACAATGCAACTACTGATT-3′) and LEH-rev (5′-ACAAACAGAGACAAGAGACAAGACATGG-3′), that span an intron of the Arabidopsis late elongated hypocotyl 1 gene encoding a Myb transcription factor. In some cases, genomic PCR products appeared after 35 amplification cycles, and such samples were discarded. RNA integrity was confirmed on 1.5% (w/v) agarose gel prior to and after DNase I digestion. Reverse transcriptase reactions were performed with avian myeloblastosis virus reverse transcriptase (Promega) or Superscript II reverse transcriptase (Invitrogen). Real time RT-PCR was performed with 1 μl of a 1:2 dilution of the first strand cDNA reaction and SYBR Green reagent (Applied Biosystems, Foster City, CA) in a 20-μl volume, on a PerkinElmer Life Sciences Geneamp 7300 machine, with the following primer pairs: ACTIN2, actin-fw (5′-ATGGCTGAGGCTGATGATATTCAAC-3′) and actin-rev (5′-TACAAGGAGAGAACAGCTTGGATG-3′); polyubiqutin UBQ10, UBQ-fw (5′-ATGCAGATCTTTGTTAAGACTCTCAC-3′) and UBQ-rev (5′-ATAGTCTTTCCGGTGAGAGTCTTC-3′); AtDGK2, AtDGK2-fw (5′-AAGCAAGTCTCGGACATGCCT-3′) and AtDGK2-rev (5′-TTCGTTTGTGCCCGCCTAT-3′); AtDGK7, AtDGK7F-RT (5′-TGTGGACTTAGCATCACAGG-3′) and AtDGK7R-RT (5′-AGCTGAGAGTCTGTCAAGG-3′).Data were normalized to actin 2 or ubiquitin (UBQ10) as follows: nCT = CTgene - CTactin or CTubiquitin, where CT refers to the number of cycles at which SYBR green fluorescence (ΔRn) in a PCR reaches an arbitrary value during the exponential phase of DNA amplification, set at 0.3 in all experiments, and then compared according to the formula, Cr (change in signal log ratio) = nCTcontrol - nCsample.Wounding Experiments—Arabidopsis wild-type and transgenic plants harboring the promoter region of the AtDGK2 gene fused to the Escherichia coli GUS reporter gene (18Gómez-Merino F.C. Brearley C.A. Ornatowska M. Abdel-Haliem M.E.F. Zanor M.I. Mueller-Roeber B. J. Biol. Chem. 2004; 279: 8230-8241Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) were used to test the effect of wounding on AtDGK2 expression. Wounding of 4-week-old soil-grown plants was performed by thoroughly crushing whole shoots. Wounded and nonwounded plants were harvested at different time points and frozen in liquid nitrogen for RNA and protein extraction.Expression of Recombinant AtDGK7 in Bacteria—The coding region of the AtDGK7 cDNA, flanked by XhoI and SpeI sites, was transferred from plasmid pG7 to plasmid pET43c (Novagen, Darmstadt, Germany), yielding plasmid pET7. The pET43c vector allows the generation of fusion protein containing a NusA tag and a His6 tag (for detection and purification of target proteins; see Novagen, on the World Wide Web at www.novagen.com). Plasmid pET7 was transformed into E. coli BL21 (DE3) (Novagen). Cells were grown at 37 °C. At an A600 of 0.6, expression of NusA-His-AtDGK7 fusion protein was induced by the addition of isopropyl-β-d-thiogalactopyranoside (1 mm final concentration). Cells were harvested 4 h later by centrifugation, resuspended in lysis buffer containing 50 mm Tris-HCl, pH 8.0, 300 mm NaCl, 10 mm imidazole, and Complete Mini Protease Inhibitor Mixture (Roche Applied Science), and lysed by sonification. Recombinant AtDGK7 was purified using Ni2+-nitrilotriacetic acid-agarose (Qiagen) according to the manufacturer's protocol. All homogenates were frozen and stored at -80 °C until assayed. Protein concentration was determined according to Bradford (20Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar), using bovine serum albumin as a standard.Western Blot Analysis—Purified proteins (20 μg) produced by the pET7 plasmid or the pET43c empty vector were separated by SDS-PAGE electrophoresis (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar) using a 4% spacer and a 8% (w/v) separating polyacrylamide gel and transferred onto nitrocellulose membranes (Amersham Biosciences) by semidry electroblotting. For detection, nitrocellulose membranes were blocked overnight in TBS (Tris-buffered saline) containing 0.150 m NaCl, 0.025 m Tris/HCl, pH 7, and 5% (w/v) nonfat dry milk (Bio-Rad) prior to a 1-h incubation in anti-His tag antibody (Pierce). After three 15-min washing steps using TBST (TBS plus 0.05% Tween 20 (v/v)) to remove residual antibody, membranes were developed with an enhanced chemiluminescence Western blot detection kit (Pierce SuperSignal) and exposed to x-ray films (Kodak X-OmatAR) for 5 s to 5 min.DGK Enzymatic Assays—Diacylglycerol kinase activity was determined under standard conditions by measuring the incorporation of [γ-32P]ATP into phosphatidic acid at 25 °C as described (18Gómez-Merino F.C. Brearley C.A. Ornatowska M. Abdel-Haliem M.E.F. Zanor M.I. Mueller-Roeber B. J. Biol. Chem. 2004; 279: 8230-8241Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). To assess Michaelis-Menten kinetics, enzymatic activity of DGK was measured as a function of the sum of molar concentrations of CHAPS, Triton X-100, and DAG at a given mol fraction of DAG. Four stock solutions (A, B, C, and D) were prepared. Stock solution A contained 69.5 mm CHAPS, 30 mm Triton, 0.5 mm DAG (0.005 mm DAG); stock solution B contained 69 mm CHAPS, 30 mm Triton, 1 mm DAG (0.01 mm DAG); stock solution C contained 68 mm CHAPS, 30 mm Triton, 2 mm DAG (0.02 mm DAG); stock solution D contained 65 mm CHAPS, 30 mm Triton, 5 mm DAG (0.05 mm DAG). Stock solutions were prepared as follows: DAG, dissolved in chloroform/methanol (1:1), was placed in 7-ml Schott glass disposable reaction tubes (with screw cap; Schott, Mainz, Germany), dried under a stream of nitrogen vapor, resuspended in a solution of CHAPS and Triton X-100 dissolved in water, and sonified for 5 min in a Sonorex RK 100 sonifier (Bandelin, Berlin, Germany). Increasing volumes (from 3.6 to 35.0 μl) of the stock solution were pipetted into the glass disposable reaction tubes. The final volume of the reaction mix was 250 μl. Diacylglycerol kinase activity was determined by measuring the incorporation of [γ-32P]ATP into PA as described previously (18Gómez-Merino F.C. Brearley C.A. Ornatowska M. Abdel-Haliem M.E.F. Zanor M.I. Mueller-Roeber B. J. Biol. Chem. 2004; 279: 8230-8241Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Extraction and separation of phospholipids were performed as follows: 1 ml of chloroform/methanol (1:1) and 500 μl of 1 m KCl plus 0.2 m H3PO4 were added, and the mixture was mixed thoroughly; samples were centrifuged at 2500 rpm for 5 min in a Labofuge 200 centrifuge (Heraeus Sepatech, Osterode, Germany). The lower phase (lipids) was transferred to a new glass reaction tube and washed once with chloroform/methanol and KCl to discard the remaining radiolabeled ATP. The amount of phosphate incorporated was determined by counting the radioactivity in a liquid scintillation counter.Enzymatic activity of AtDGK7 was measured as a function of ATP concentration in mixed micelles. A solution containing 50 mm nonlabeled ATP was prepared as stock. Under standard conditions, each enzymatic reaction contained a final concentration of 1 mm nonlabeled ATP and ∼5 mCi of 32P-labeled ATP (18Gómez-Merino F.C. Brearley C.A. Ornatowska M. Abdel-Haliem M.E.F. Zanor M.I. Mueller-Roeber B. J. Biol. Chem. 2004; 279: 8230-8241Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). To assess the effect of varying ATP on the enzymatic reaction, four different ATP final concentrations were tested: 0.10, 1.0, 2.0, and 5.0 mm, respectively. The same amount of radioactive ATP (5 mCi) was added to the assays, and the difference in specific radioactivities obtained were taken into account for calculating the enzyme activities in the individual reactions. For this experiment, the sum of the molar concentrations of CHAPS, Triton X-100, and DAG was 7.20 mm (containing 4.90 mm CHAPS, 2.20 mm Triton, and 0.10 mm DAG).The effect of the DGK inhibitor R59022 or 3-{2-{4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl}ethyl}-2,3-dihydro-2-thioxo-4(1H)-quinazolinone (R59949) on Arabidopsis AtDGK2 and AtDGK7 enzymes was tested at different concentrations of DAG and CHAPS as indicated above. The reaction mix was preincubated for 10 min with DGK inhibitor before the reaction was started by the addition of ATP.PA standard was obtained from Sigma (product code P 9511). Hartmann Analytic (Braunschweig, Germany) provided [γ-32P]ATP (15 TBq/mmol). Lipids (1,2-SAG, 1,2-DOG, and cardiolipin), salts (MgCl2, LiCl, and NaCl) and detergents (Na-DC and CHAPS) were purchased from Sigma. Serva (Heidelberg, Germany) provided Triton X-100. DAG molecular species 1,2-POG, 1,2-SLG, and 1,2-OPG were purchased from Larodan Fine Chemicals AB (Malmö, Sweden). DGK inhibitors R59022 and R59949 were purchased from Calbiochem.HPLC Analysis of the Reaction Products of DGK Assay—The products of diacylglycerol kinase assays were obtained by two-phase partitioning. The organic phase was reduced to dryness under a stream of nitrogen gas, and the glycerolipids were deacylated (22Hawkins P.T. Stephens L. Downes C.P. Biochem. J. 1986; 238: 507-516Crossref PubMed Scopus (112) Google Scholar). An aliquot of the water-soluble products of deacylation was mixed with l-[U-14C]glycerol 3-phosphate (Amersham Biosciences; specific activity >100 mCi/mmol) and resolved by anion exchange high performance liquid chromatography with a gradient of (NH4)2HPO4 (23Brearley C.A. Hanke D.E. Biochem. J. 1992; 283: 255-260Crossref PubMed Scopus (35) Google Scholar). The column eluate was collected in 1-ml fractions, and 4 ml of Ecoscint A scintillation fluid (National Diagnostics, Atlanta, GA) was added. Radioactivity was estimated by dual label scintillation counting in a Wallac 1409 DSA (Turku, Finland) scintillation counter.Effect of R59022 on Plant Growth and Development—A. thaliana (L.) Heynh. seeds (ecotype C24) were surface-sterilized with 70% ethanol for 5 min and sodium hypochlorite solution (5.0% NaClO4 plus 0.05% Tween 20) for 5 min, followed by several washes with sterile water. Subsequently, seeds were sown on MS (Murashige Skoog) plates containing 0.8% agar (square Petri dishes, 100 × 100 × 14 mm; NUNC, Wiesbaden, Germany). The DGK inhibitor R59022 was dissolved in dimethyl formamide. Dimethyl formamide (without R59022) was used as control. The seeds were imbibed at 4 °C in the dark for 1 day. Plates were then placed vertically in a growth chamber under long day conditions (16 h of light, 8 h of dark). Lengths of primary roots from the shoot/root transition zone to the root tip were measured. To analyze the effects of the inhibitor R59022 on plant growth and development, 35-100 μm R59022 was added to MS medium containing 0.8% agar.RESULTSAtDGK7 cDNA and the Deduced Primary Structure of Its Encoded Protein—In the Arabidopsis genome, seven genes encode putative DGK isoforms that fall into three major clusters. AtDGK2, a member of Cluster I, is a catalytically active enzyme. The AtDGK2 gene is located on chromosome V, and its expression is detected in various tissues of the Arabidopsis plant, including leaves and roots, as previously shown (18Gómez-Merino F.C. Brearley C.A. Ornatowska M. Abdel-Haliem M.E.F. Zanor M.I. Mueller-Roeber B. J. Biol. Chem. 2004; 279: 8230-8241Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar).To investigate whether members of other clusters also encode functional DGKs, we concentrated here on AtDGK7 (encoded by gene locus At4g30340), representing a potential DGK of Cluster II. The AtDGK7 gene is located on chromosome IV, and data base analysis revealed that AtDGK7 is transcribed. In total, four AtDGK7 expressed sequence tags (ESTs; GenBank™ accession numbers AV798976, AV827682, CF773882, and Z26229) and two cDNA sequences (AF360174 and AY113915) obtained through the full-length cDNA cloning projects at RIKEN and SALK can be retrieved from the NCBI data base (available on the World Wide Web at www.ncbi.nlm.nih.gov). Because of previous data base annotation ambiguities (reported in Ref. 18Gómez-Merino F.C. Brearley C.A. Ornatowska M. Abdel-Haliem M.E.F. Zanor M.I. Mueller-Roeber B. J. Biol. Chem. 2004; 279: 8230-8241Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), we reanalyzed the AtDGK7 sequence in detail here (see Fig. 1A). RIKEN ESTs AV827682 and AV798976 were both derived from clone RAFL 09-18-D16 and represent the 5′ and 3′ regions, respectively, of the AF360174 sequence, which in total encompasses 1807 base pairs. The EST Z26229 sequence was obtained from the Versailles EST collection and, like EST AV798976, corresponds to the 3′ part of the AF360174 full-length cDNA. The EST CF773882 covers the central part of the transcript. Finally, cDNA AY113915, generated at SALK, has a length of 1156 base pairs. The available data can be summarized as follows. (i) The AF360174 and AY113915 cDNAs code for the same protein, AtDGK7, which has a deduced length of 374 amino acids, a predicted molecular mass of 41.2 kDa, and an isoelectric point of 7.73. This prediction fully corresponds to the most recent TIGR annotation (version 5.0) for AtDGK7 (available on the World Wide Web at www.tigr.org). (ii) The AF360174 cDNA contains a 5′-untranslated region at positions 1-126, the AtDGK7 open reading frame at positions 127-1251, and an unusually long 3′-untranslated region at positions 1252-1807. The presence of this long untranslated region in the AtDGK7 transcript was confirmed by the independently generated EST Z26229. The generation of the long transcript is further in full accordance with data from recent Arabidopsis MPSS analyses (24Meyers B.C. Lee D.K. Vu T.H. Tej S.S. Edberg S.B. Matvienko M. Tindell L.D. Plant Physiol. 2004; 135: 801-813Crossref PubMed Scopus (98) Google Scholar) (available on the World Wide Web at mpss.udel.edu/at/java.html). A 17-bp MPSS signature sequence corresponding to the very 3′-end of the AtDGK7 gene was found (GATCTATGTTGAGCTTT on the Crick DNA strand). (iii) The nucleotide sequences of the two cDNAs are completely identical in the overlapping region (which encompassed the complete AtDGK7 open reading frame and part of the 3′-untranslated region).Protein sequence analysis reveals that AtDGK7 has a conserved catalytic domain (DGKc; Pfam accession number PF00781) (Fig. 1, B and C), located at amino acid residues 94-240, which contains a presumed ATP-binding site with a GXGXXG consensus sequence. Although the DGKc of AtDGK7 is ∼40% identical to that of AtDGK2, the DGK accessory domain (DGKa; Pfam accession number PF00609) is incomplete in AtDGK7 (Fig. 1B). In most AtDGK isoforms, the DGKa domain encompasses ∼160 amino acid residues, but in AtDGK7, it includes only 52 residues (Asn290-Asn341). Furthermore, AtDGK7, like all other Cluster II and also Cluster III enzymes, lacks the N-terminal diacylglycerol/phorbol ester-binding domains (InterPro accession number IPR002219) as well as the upstream basic region and the extended cysteine-rich domain (extCRD-like domain) present in AtDGK2 (18Gómez-Merino F.C. Brearley C.A. Ornatowska M. Abdel-Haliem M.E.F. Zanor M.I. Mueller-Roeber B. J. Biol. Chem. 2004; 279: 8230-8241Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar).AtDGK7 Transcripts Are Detectable thr" @default.
• W2002596952 created "2016-06-24" @default.
• W2002596952 creator A5014730874 @default.
• W2002596952 creator A5016778149 @default.
• W2002596952 creator A5022096672 @default.
• W2002596952 creator A5025051810 @default.
• W2002596952 creator A5025481183 @default.
• W2002596952 creator A5068578571 @default.
• W2002596952 creator A5091543613 @default.
• W2002596952 date "2005-10-01" @default.
• W2002596952 modified "2023-10-10" @default.
• W2002596952 title "Arabidopsis AtDGK7, the Smallest Member of Plant Diacylglycerol Kinases (DGKs), Displays Unique Biochemical Features and Saturates at Low Substrate Concentration" @default.
• W2002596952 cites W1490605885 @default.
• W2002596952 cites W1539503448 @default.
• W2002596952 cites W1552494303 @default.
• W2002596952 cites W1568470731 @default.
• W2002596952 cites W1577385687 @default.
• W2002596952 cites W1590152235 @default.
• W2002596952 cites W1593810032 @default.
• W2002596952 cites W1963793114 @default.
• W2002596952 cites W1963833854 @default.
• W2002596952 cites W1970605680 @default.
• W2002596952 cites W1974910268 @default.
• W2002596952 cites W1975210463 @default.
• W2002596952 cites W1977552304 @default.
• W2002596952 cites W1986273917 @default.
• W2002596952 cites W1986582487 @default.
• W2002596952 cites W1994489278 @default.
• W2002596952 cites W1997655256 @default.
• W2002596952 cites W2004488776 @default.
• W2002596952 cites W2011124981 @default.
• W2002596952 cites W2022958525 @default.
• W2002596952 cites W2024556021 @default.
• W2002596952 cites W2049534556 @default.
• W2002596952 cites W2052818515 @default.
• W2002596952 cites W2076716764 @default.
• W2002596952 cites W2079166554 @default.
• W2002596952 cites W2085313009 @default.
• W2002596952 cites W2096451658 @default.
• W2002596952 cites W2100837269 @default.
• W2002596952 cites W2102724568 @default.
• W2002596952 cites W2111624352 @default.
• W2002596952 cites W2120068626 @default.
• W2002596952 cites W2120237563 @default.
• W2002596952 cites W2127029436 @default.
• W2002596952 cites W2146660725 @default.
• W2002596952 cites W2151626778 @default.
• W2002596952 cites W2155797625 @default.
• W2002596952 cites W2159847655 @default.
• W2002596952 cites W2166586026 @default.
• W2002596952 cites W2168659200 @default.
• W2002596952 cites W2186209348 @default.
• W2002596952 cites W2255612709 @default.
• W2002596952 cites W22940201 @default.
• W2002596952 cites W4232965715 @default.
• W2002596952 cites W4293247451 @default.
• W2002596952 cites W44861545 @default.
• W2002596952 doi "https://doi.org/10.1074/jbc.m506859200" @default.
• W2002596952 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16081412" @default.
• W2002596952 hasPublicationYear "2005" @default.
• W2002596952 type Work @default.
• W2002596952 sameAs 2002596952 @default.
• W2002596952 citedByCount "65" @default.
• W2002596952 countsByYear W20025969522012 @default.
• W2002596952 countsByYear W20025969522013 @default.
• W2002596952 countsByYear W20025969522014 @default.
• W2002596952 countsByYear W20025969522015 @default.
• W2002596952 countsByYear W20025969522017 @default.
• W2002596952 countsByYear W20025969522018 @default.
• W2002596952 countsByYear W20025969522019 @default.
• W2002596952 countsByYear W20025969522020 @default.
• W2002596952 countsByYear W20025969522021 @default.
• W2002596952 countsByYear W20025969522022 @default.
• W2002596952 countsByYear W20025969522023 @default.
• W2002596952 crossrefType "journal-article" @default.
• W2002596952 hasAuthorship W2002596952A5014730874 @default.
• W2002596952 hasAuthorship W2002596952A5016778149 @default.
• W2002596952 hasAuthorship W2002596952A5022096672 @default.
• W2002596952 hasAuthorship W2002596952A5025051810 @default.
• W2002596952 hasAuthorship W2002596952A5025481183 @default.
• W2002596952 hasAuthorship W2002596952A5068578571 @default.
• W2002596952 hasAuthorship W2002596952A5091543613 @default.
• W2002596952 hasBestOaLocation W20025969521 @default.
• W2002596952 hasConcept C104317684 @default.
• W2002596952 hasConcept C121332964 @default.
• W2002596952 hasConcept C12554922 @default.
• W2002596952 hasConcept C143065580 @default.
• W2002596952 hasConcept C181199279 @default.
• W2002596952 hasConcept C184235292 @default.
• W2002596952 hasConcept C185592680 @default.
• W2002596952 hasConcept C18903297 @default.
• W2002596952 hasConcept C192562407 @default.
• W2002596952 hasConcept C195794163 @default.
• W2002596952 hasConcept C2775988993 @default.
• W2002596952 hasConcept C2777289219 @default.
• W2002596952 hasConcept C2779491563 @default.
• W2002596952 hasConcept C2994592520 @default.
• W2002596952 hasConcept C36020004 @default.

• next