102 results on '"Ishikawa T"'
Search Results
2. Glutathione-associated cis-diamminedichloroplatinum(II) metabolism and ATP-dependent efflux from leukemia cells. Molecular characterization of glutathione-platinum complex and its biological significance.
- Author
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Ishikawa, T, primary and Ali-Osman, F, additional
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- 1993
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3. Brefeldin A binds to glutathione S-transferase and is secreted as glutathione and cysteine conjugates by Chinese hamster ovary cells.
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Brüning, A, primary, Ishikawa, T, additional, Kneusel, R.E., additional, Matern, U, additional, Lottspeich, F, additional, and Wieland, F.T., additional
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- 1992
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4. ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt.
- Author
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Müller, M., primary, Ishikawa, T., additional, Berger, U., additional, Klünemann, C., additional, Lucka, L., additional, Schreyer, A., additional, Kannicht, C., additional, Reutter, W., additional, Kurz, G., additional, and Keppler, D., additional
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- 1991
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5. ATP-dependent primary active transport of cysteinyl leukotrienes across liver canalicular membrane. Role of the ATP-dependent transport system for glutathione S-conjugates.
- Author
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Ishikawa, T, primary, Müller, M, additional, Klünemann, C, additional, Schaub, T, additional, and Keppler, D, additional
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- 1990
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6. Activation of mammalian phosphofructokinases by ribose 1,5-bisphosphate.
- Author
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Ishikawa, E, primary, Ogushi, S, additional, Ishikawa, T, additional, and Uyeda, K, additional
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- 1990
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7. A new class of rat glutathione S-transferase Yrs-Yrs inactivating reactive sulfate esters as metabolites of carcinogenic arylmethanols.
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Hiratsuka, A, primary, Sebata, N, additional, Kawashima, K, additional, Okuda, H, additional, Ogura, K, additional, Watabe, T, additional, Satoh, K, additional, Hatayama, I, additional, Tsuchida, S, additional, and Ishikawa, T, additional
- Published
- 1990
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8. Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis.
- Author
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Singh, S K, Rozycki, J, Ortega, J, Ishikawa, T, Lo, J, Steven, A C, and Maurizi, M R
- Abstract
Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.
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- 2001
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9. Formation of Nepsilon-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.
- Author
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Kato, Y, Mori, Y, Makino, Y, Morimitsu, Y, Hiroi, S, Ishikawa, T, and Osawa, T
- Abstract
The objectives of this study were to estimate the structure of the lipid hydroperoxide-modified lysine residue and to prove the presence of the adducts in vivo. The reaction of lipid hydroperoxide toward the lysine moiety was investigated employing N-benzoyl-glycyl-L-lysine (Bz-Gly-Lys) as a model compound of Lys residues in protein and 13-hydroperoxyoctadecadienoic acid (13-HPODE) as a model of the lipid hydroperoxides. One of the products, compound X, was isolated from the reaction mixture of 13-HPODE and Bz-Gly-Lys and was then identified as N-benzoyl-glycyl-Nepsilon-(hexanonyl)lysine. To prove the formation of Nepsilon-(hexanonyl)lysine, named HEL, in protein exposed to the lipid hydroperoxide, the antibody to the synthetic hexanonyl protein was prepared and then characterized in detail. Using the anti-HEL antibody, the presence of HEL in the lipid hydroperoxide-modified proteins and oxidized LDL was confirmed. Furthermore, the positive staining by anti-HEL antibody was observed in human atherosclerotic lesions using an immunohistochemical technique. The amide-type adduct may be a useful marker for the lipid hydroperoxide-derived modification of biomolecules.
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- 1999
10. Expression of multidrug resistance protein/GS-X pump and gamma-glutamylcysteine synthetase genes is regulated by oxidative stress.
- Author
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Yamane, Y, Furuichi, M, Song, R, Van, N T, Mulcahy, R T, Ishikawa, T, and Kuo, M T
- Abstract
Expression of the MRP1 gene encoding the GS-X pump and of the gamma-GCSh gene encoding the heavy (catalytic) subunit of the gamma-glutamylcysteine synthetase is frequently elevated in many drug-resistant cell lines and can be co-induced by many cytotoxic agents. However, mechanisms that regulate the expression of these genes remain to be elucidated. We report here that like gamma-GCSh, the expression of MRP1 can be induced in cultured cells treated with pro-oxidants such as tert-butylhydroquinone, 2,3-dimethoxy-1, 4-naphthoquinone, and menadione. Intracellular reactive oxygen intermediate (ROI) levels were increased in hepatoma cells treated with tert-butylhydroquinone for 2 h as measured by flow cytometry using an ROI-specific probe, dihydrorhodamine 123. Elevated GSH levels in stably gamma-GCSh-transfected cell lines down-regulated endogenous MRP1 and gamma-GCSh expression. ROI levels in these transfected cells were lower than those in the untransfected control. In the cell lines in which depleting cellular GSH pools did not affect the expression of the MRP1 and gamma-GCSh genes, only minor increased intracellular levels of ROIs were observed. These results suggest that intracellular ROI levels play an important role in the regulation of MRP1 and gamma-GCSh expression. Our data also suggest that elevated intracellular GSH levels not only facilitate substrate transport by the MRP1/GS-X pump as previously demonstrated, but also suppress MRP1 and gamma-GCSh expression.
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- 1998
11. Molecular cloning and functional expression of rat liver glutathione-dependent dehydroascorbate reductase.
- Author
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Ishikawa, T, Casini, A F, and Nishikimi, M
- Abstract
We have isolated a cDNA clone for a novel glutathione-dependent dehydroascorbate reductase from a rat liver cDNA library in lambdagt11 by immunoscreening. The authenticity of the clone was confirmed as follows: first, the antibody that had been purified through affinity for the protein expressed by the cloned lambdagt11 phage recognized only the enzyme in a crude extract from rat liver; and second, two internal amino acid sequences of purified enzyme were identified in the protein sequence predicted from the cDNA. The predicted protein consists of 213 amino acids with a molecular weight of 24,929, which is smaller by approximately 3,000 than the value obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This discrepancy of the molecular weight was explained by post-translational modification because the recombinant protein expressed by a mammalian system (Chinese hamster ovary cells) was of the same size as rat liver enzyme but larger than the protein expressed by a bacterial system (Escherichia coli). Chinese hamster ovary cells, originally devoid of glutathione-dependent dehydroascorbate reductase activity, was made to elicit the enzyme activity (1.5 nmol/min/mg of cytosolic protein) by expression of the recombinant protein. Additionally, the cells expressing the enzyme were found to accumulate 1.7 times as much ascorbate as the parental cells after incubation with dehydroascorbate. This result points to the importance of the dehydroascorbic acid reductase in maintaining a high concentration of ascorbate in the cell.
- Published
- 1998
12. Characterization of cyclic AMP-requiring yeast mutants altered in the regulatory subunit of protein kinase.
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Uno, I, Matsumoto, K, and Ishikawa, T
- Abstract
The CYR3 mutant of yeast, Saccharomyces cerevisiae, partially accumulated unbudded cells and required cAMP for the best growth at 35 degrees C. The CYR3 mutation was partially dominant over the wild type counterpart and suppressed by the bcy1 mutation which is responsible for the deficiency of the regulatory subunit of cAMP-dependent protein kinase. The molecular weights of cAMP-dependent protein kinase and its catalytic and regulatory subunits were 160,000, 30,000, and 50,000, respectively. No significant differences in the molecular weights of cAMP-dependent protein kinase and the subunits were found between the wild type and CYR3 mutant strains. However, the cAMP-dependent protein kinase activity of CYR3 cells showed significantly higher Ka values for activation by cAMP at 35 degrees C than those of wild type and a clear difference in the electrophoretic mobility of the regulatory subunit was found between the wild type and CYR3 enzymes. The CYR3 mutation was suppressed by the IAC mutation which caused the production of a significantly high level of cAMP. The results indicate that the CYR3 phenotype was produced by a structural mutation in the CYR3 gene coding for the regulatory subunit of cAMP-dependent protein kinase in yeast.
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- 1982
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13. Amino acid substitutions and alteration in cation specificity in the melibiose carrier of Escherichia coli.
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Kawakami, T, Akizawa, Y, Ishikawa, T, Shimamoto, T, Tsuda, M, and Tsuchiya, T
- Abstract
We isolated mutants of Escherichia coli which showed Li+-resistant growth on melibiose. The melibiose carrier of the mutants lost the ability to couple to H+, whereas it retained the ability to couple to Na+. The mutated gene, melB, of the mutants was cloned, and the nucleotide sequence was determined. The nucleotide replacements caused the following substitutions of amino acid residues in the melibiose carrier: Pro-142 with Ser, Leu-232 with Phe, or Ala-236 with Thr or Val. These amino acid residues are located in slightly hydrophobic regions of the melibiose carrier. The results provide strong support for the idea that such regions or their vicinities which contain those amino acid residues play an important role in H+ (or Li+) recognition or H+ (or Li+) transport by the melibiose carrier.
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- 1988
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14. A negative vitamin D response DNA element in the human parathyroid hormone-related peptide gene binds to vitamin D receptor along with Ku antigen to mediate negative gene regulation by vitamin D.
- Author
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Nishishita, T, Okazaki, T, Ishikawa, T, Igarashi, T, Hata, K, Ogata, E, and Fujita, T
- Abstract
We found that the human parathyroid hormone-related peptide (hPTHrP) gene contained a DNA element (nVDREhPTHrP) homologous to a negative vitamin D response element in the human parathyroid hormone gene. It bound to vitamin D receptor (VDR) but not retinoic acid Xalpha receptor (RXRalpha) in the human T cell line MT2 cells. VDR binding to this element was confirmed by the Southwestern assay combined with immunodepletion using anti-VDR monoclonal antibody, and this binding activity was repressed by 1,25-dihydroxyvitamin D3. Such a repression was reversed by acid phosphatase treatment, suggesting that 1,25-dihydroxyvitamin D3 phosphorylates VDR to weaken its binding activity to nVDREhPTHrP. In electrophoretic mobility shift assay, we found anti-Ku antigen antibody specifically supershifted the MT2 nuclear proteinnVDREhPTHrP complex. The nVDREhPTHrP-bearing reporter plasmid produced vitamin D-dependent inhibition of the reporter activity in MT2 cells, which was markedly masked by the introduction of the Ku antigen expression vector in the antisense orientation. On the other hand, such a procedure did not perturb the vitamin D response element-mediated gene stimulation by vitamin D. These results indicate that nVDREhPTHrP interacts with Ku antigen in addition to VDR to mediate gene suppression by vitamin D.
- Published
- 1998
15. Coordinated induction of MRP/GS-X pump and gamma-glutamylcysteine synthetase by heavy metals in human leukemia cells.
- Author
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Ishikawa, T, Bao, J J, Yamane, Y, Akimaru, K, Frindrich, K, Wright, C D, and Kuo, M T
- Abstract
We recently reported that GS-X pump activity, as assessed by ATP-dependent transport of the glutathione-platinum complex and leukotriene C4, and intracellular glutathione (GSH) levels were remarkably enhanced in cis-diamminedichloroplatinum(II) (cisplatin)-resistant human leukemia HL-60 cells (Ishikawa, T., Wright, C. D., and Ishizuka, H. (1994) J. Biol. Chem. 269, 29085-29093). Now, using Northern hybridization and RNase protection assay, we provide evidence that the multidrug resistance-associated protein (MRP) gene, which encodes a human GS-X pump, is expressed at higher levels in cisplatin-resistant (HL-60/R-CP) cells than in sensitive cells, whereas amplification of the MRP gene is not detected by Southern hybridization. Culturing HL-60/R-CP cells in cisplatin-free medium resulted in reduced MRP mRNA levels, but these levels could be induced to rise within 30 h by cisplatin and heavy metals such as arsenite, cadmium, and zinc. The increased levels of MRP mRNA were closely related with enhanced activities of ATP-dependent transport of leukotriene C4 (LTC4) in plasma membrane vesicles. The glutathione-platinum (GS-Pt) complex, but not cisplatin, inhibited ATP-dependent LTC4 transport, suggesting that the MRP/GS-X pump transports both LTC4 and the GS-Pt complex. Expression of gamma-glutamylcysteine synthetase in the cisplatin-resistant cells was also co-induced within 24 h in response to cisplatin exposure, resulting in a significant increase in cellular GSH level. The resistant cells exposed to cisplatin were cross-resistant to melphalan, chlorambucil, arsenite, and cadmium. These observations suggest that elevated expression of the MRP/GS-X pump and increased GSH biosynthesis together may be important factors in the cellular metabolism and disposition of cisplatin, alkylating agents, and heavy metals.
- Published
- 1996
16. ATP/Mg2+-dependent Cardiac Transport System for Glutathione S-Conjugates
- Author
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Ishikawa, T
- Abstract
In the present study, the transport of glutathione S-conjugate across rat heart sarcolemma has directly been proved to be an ATP-dependent process. Incubation of sarcolemma vesicles with S-(2,4-dinitrophenyl)glutathione (DNP-SG) in the presence of ATP resulted in a substantial uptake of DNP-SG into the vesicles; Mg2+was required for ATP-stimulated transport. The rate of glutathione S-conjugate uptake was saturated with respect to ATP and DNP-SG concentrations with apparent Kmvalues of 30 µMfor ATP and 20 µMfor DNP-SG. However, other nucleoside triphosphates, viz. GTP, UTP, CTP, and TTP, did not stimulate the transport effectively. The ATP-stimulated DNP-SG uptake was not affected by ouabain, EGTA, or by valinomycin-induced K+-diffusion potential, suggesting that Na+,K+- and Ca2+-ATPase activities as well as the membrane potential are not involved in the transport mechanism. ATP could not be replaced by ADP, AMP, or by ATP analogues, adenosine 5′-(β,γ-methylene)triphosphate and adenosine 5′-(β,γ-imino)triphosphate. From these observations, it is proposed that hydrolysis of γ-phosphate of ATP is essential for the transport mechanism.
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- 1989
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17. Identification of S100b protein as copper-binding protein and its suppression of copper-induced cell damage.
- Author
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Nishikawa, T, Lee, I S, Shiraishi, N, Ishikawa, T, Ohta, Y, and Nishikimi, M
- Abstract
We have isolated from bovine brain a protein with a high capacity to inhibit the copper ion-catalyzed oxidation of L-ascorbate and identified it as S100b protein, an EF-hand calcium-binding protein, by sequencing its proteolytic peptides. Copper binding studies showed that this protein has four copper-binding sites per dimeric protein molecule with a dissociation constant of 0.46 microM and that in the presence of L-ascorbate, copper ions bind to a total of six binding sites with a great increase in affinity. Furthermore, we examined whether S100b protein can prevent copper-induced cell damage. Bovine S100b protein was found to suppress dose-dependently the hemolysis of mouse erythrocytes induced by CuCl2. We transformed Escherichia coli cells with pGEX-5X-3 vector containing a cDNA for rat S100b protein, so that this protein could be expressed as a fusion protein with glutathione S-transferase. The transformed cells were demonstrated to be markedly resistant to a treatment with CuCl2 plus H2O2 as compared with the control cells expressing glutathione S-transferase alone. These results indicate that S100b protein does suppress oxidative cell damage by sequestering copper ions.
- Published
- 1997
18. A novel Go-mediated phototransduction cascade in scallop visual cells.
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Kojima, D, Terakita, A, Ishikawa, T, Tsukahara, Y, Maeda, A, and Shichida, Y
- Abstract
Scallop retinas contain ciliary photoreceptor cells that respond to light by hyperpolarization like vertebrate rods and cones, but the response is generated by a different phototransduction cascade from those of rods and cones. To elucidate the cascade, we investigated a visual pigment and a G-protein functioning in the hyperpolarizing cell. Sequencing of cDNAs and in situ hybridization experiments showed that the hyperpolarizing cells express a novel subtype of visual pigment, which showed significant differences in amino acid sequence from other visual pigments. Cloning cDNA genes of G-protein and immunohistochemical analysis revealed the presence of an alpha subunit of a Go type G-protein, 83% identical in amino acid sequence to mammalian Go(alpha) in the nervous system, in the photoreceptive region of the cells. The results demonstrate that a novel, Go-mediated, phototransduction cascade is present in the hyperpolarizing cells. The phototransduction cascade in the scallop hyperpolarizing cell provides an alternative system to investigate Go-mediated transduction pathways in the nervous system. Molecular phylogenetic analysis strongly suggests that the Go-mediated phototransduction system emerged before the divergence of animals into vertebrate and invertebrate in the course of evolution.
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- 1997
19. Hyperosmolarity-induced gene stimulation is mediated by the negative calcium responsive element.
- Author
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Okazaki, T, Ishikawa, T, Nishimori, S, Igarashi, T, Hata, K, and Fujita, T
- Abstract
The negative calcium responsive elements of the parathyroid hormone gene bind to a specific set of nuclear proteins in an extracellular calcium (Ca2+e)-dependent manner. We have found that one of the negative calcium responsive elements, named oligo B, is found in the 5'-flanking region of such vasoactive genes as the vasopressin and atrial natriuretic polypeptide genes. Furthermore, the oligo B-like sequence in the former gene is conserved throughout evolution. Because expression of some of these vasoactive genes is altered by external stimuli which change cell volume, we examined whether oligo B is involved in gene regulation by hyperosmolarity. Here, we demonstrate that the binding between oligo B and its binding nuclear proteins including a redox factor 1 was reduced by hyperosmolarity generated by sodium chloride but not by urea. Such attenuated binding was reversed by dephosphorylating nuclear proteins by a potato acid phosphatase, suggesting that NaCl treatment elicited phosphorylation of these nuclear proteins to weaken their binding activity to oligo B. Furthermore, these nuclear events led to hyperosmolarity-mediated transcriptional stimulation of the genes bearing this DNA element in the cultured cells.
- Published
- 1997
20. Identification of a human type II receptor for bone morphogenetic protein-4 that forms differential heteromeric complexes with bone morphogenetic protein type I receptors.
- Author
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Nohno, T, Ishikawa, T, Saito, T, Hosokawa, K, Noji, S, Wolsing, D H, and Rosenbaum, J S
- Abstract
Bone morphogenetic proteins (BMPs) comprise the largest subfamily of TGF-beta-related ligands and are known to bind to type I and type II receptor serine/threonine kinases. Although several mammalian BMP type I receptors have been identified, the mammalian BMP type II receptors have remained elusive. We have isolated a cDNA encoding a novel transmembrane serine/threonine kinase from human skin fibroblasts which we demonstrate here to be a type II receptor that binds BMP-4. This receptor (BRK-3) is distantly related to other known type II receptors and is distinguished from them by an extremely long carboxyl-terminal sequence following the intracellular kinase domain. The BRK-3 gene is widely expressed in a variety of adult tissues. When expressed alone in COS cells, BRK-3 specifically binds BMP-4, but cross-linking of BMP-4 to BRK-3 is undetectable in the absence of either the BRK-1 or BRK-2 BMP type I receptors. Cotransfection of BRK-2 with BRK-3 greatly enhanced affinity labeling of BMP-4 to the type I receptor, in contrast to the affinity labeling pattern observed with the BRK-1 + BRK-3 heteromeric complex. Furthermore, a subpopulation of super-high affinity binding sites is formed in COS cells upon cotransfection only of BRK-2 + BRK-3, suggesting that the different heteromeric BMP receptor complexes have different signaling potential.
- Published
- 1995
21. Characterization of a cyclic nucleotide phosphodiesterase-deficient mutant in yeast.
- Author
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Uno, I, Matsumoto, K, and Ishikawa, T
- Abstract
A mutation (pde1) was detected by suppressor activity on the CYR3 mutation which caused cAMP requirement for growth at 35 degrees C by the alteration of cAMP-dependent protein kinase. The pde1 mutant produced a significantly reduced level of cyclic nucleotide phosphodiesterase activity when assayed with 500 microM cAMP. Two cyclic nucleotide phosphodiesterases, I and II, were identified. Approximate molecular weights of these enzymes were 60,000 and 110,000, and the apparent Km values were 100 and 0.4 microM, respectively. The pde1 mutant was deficient in phosphodiesterase I activity. The cells carrying the pde1 mutation accumulated several times over the intracellular cAMP found in wild type cells. Phosphodiesterase I was not essential for growth of yeast cells, but controlled the intracellular cAMP levels in wild type and various mutant strains.
- Published
- 1983
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22. Regulation of NAD-dependent glutamate dehydrogenase by protein kinases in Saccharomyces cerevisiae.
- Author
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Uno, I, Matsumoto, K, Adachi, K, and Ishikawa, T
- Abstract
The active NAD-dependent glutamate dehydrogenase of wild type yeast cells fractionated by DEAE-Sephacel chromatography was inactivated in vitro by the addition of either the cAMP-dependent or cAMP-independent protein kinases obtained from wild type cells. cAMP-dependent inhibition of glutamate dehydrogenase activity was not observed in the crude extract of bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase. The cAMP-dependent protein kinase of CYR3 mutant cells, which has a high K alpha value for cAMP in the phosphorylation reaction, required a high cAMP concentration for the inactivation of NAD-dependent glutamate dehydrogenase. An increased inactivation of partially purified active NAD-dependent glutamate dehydrogenase (Mr = 450,000) was observed to correlate with increased phosphorylation of a protein subunit (Mr = 100,000) of glutamate dehydrogenase. The phosphorylated protein was labeled by an NADH analog, 5'-p-fluorosulfonyl[14C]benzoyladenosine. Activation and dephosphorylation of inactive NAD-dependent glutamate dehydrogenase fractions were observed in vitro by treatment with bovine alkaline phosphatase or crude yeast cell extracts. These results suggested that the conversion of the active form of NAD-dependent glutamate dehydrogenase to an inactive form is regulated by phosphorylation through cAMP-dependent and cAMP-independent protein kinases.
- Published
- 1984
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23. Characterization of cyclic AMP-requiring yeast mutants altered in the catalytic subunit of protein kinase.
- Author
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Uno, I, Matsumoto, K, Adachi, K, and Ishikawa, T
- Abstract
The cyr2 mutant of yeast, Saccharomyces cerevisiae, required cAMP for growth at 35 degrees C. The cyr2 mutation was suppressed by the bcy1 mutation which resulted in deficiency of the regulatory subunit of cAMP-dependent protein kinase. The DEAE-Sephacel elution profile of cyr2 cAMP-dependent protein kinase was markedly different from that observed for the wild-type enzyme. With histone as substrate, the cAMP-dependent protein kinase activity of cyr2 cells showed 100-fold greater Ka value for activation by cAMP at 35 degrees C than that of the wild-type cells, while the Kd value for cAMP of the mutant enzyme was not altered. The electrophoretic character, molecular weight, and pI value of the regulatory subunit of the mutant enzyme were the same as those of the wild-type enzyme. When histone, trehalase, and glutamate dehydrogenase were used as substrate, the free catalytic subunit of the mutant enzyme showed a markedly decreased affinity for ATP and was more thermolabile compared to that of the wild-type enzyme. The results indicated that the cyr2 phenotype was produced by a structural mutation in the cyr2 gene coding for the catalytic subunit of cAMP-dependent protein kinase in yeast.
- Published
- 1984
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24. Genetic and biochemical evidence that trehalase is a substrate of cAMP-dependent protein kinase in yeast.
- Author
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Uno, I, Matsumoto, K, Adachi, K, and Ishikawa, T
- Abstract
In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.
- Published
- 1983
- Full Text
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25. Bioactive 6-nitronorepinephrine identified in mammalian brain.
- Author
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Shintani, F, Kinoshita, T, Kanba, S, Ishikawa, T, Suzuki, E, Sasakawa, N, Kato, R, Asai, M, and Nakaki, T
- Abstract
Norepinephrine (NE) (von Euler, U. S. (1972) in Catecholamines (Blaschko, H., and Muscholl, E., eds.) pp. 186-230, Springer-Verlag, Berlin) and nitric oxide (NO.) function as neurotransmitters in the nervous system. We have shown that NE levels in the rat hypothalamic paraventricular nucleus (Shintani, F., Kato, R., Kinoshita, N., Kanba, S., Asai, M., and Nakaki, T.(1995) Proceedings of the Satellite Symposium, 4th IBRO World Congress on Neuroscience, Otsu, 1995) diminish in the presence of NO.. This observation prompted us to explore the possibility of an in vivo interaction between NE and NO. or NO.-related molecules. In fact, nitration of NE has been shown to occur in vitro (d'Ischia, M., and Costantini, C. (1995) Bioorg. Med. Chem. 3, 923-927). We now report the identification of 6-nitronorepinephrine in the mammalian brain. Amounts of 6-nitronorepinephrine in the rat brain were attenuated by intraperitoneal administration of an inhibitor of nitric oxide synthase, NG-nitro-L-arginine methyl ester (L-NAME). This was reversed by coadministration of L-arginine, suggesting that nitric oxide synthase participated in the formation of 6-nitronorepinephrine. Moreover, we found that 6-nitronorepinephrine inhibits the activity of catechol O-methyltransferase, as well as NE transport into rat synaptosomes. A rat brain microdialysis experiment showed that perfusion of 6-nitronorepinephrine into the rat paraventricular nucleus significantly elevated NE while decreasing 3-methoxy-4-hydroxyphenylglycol and that L-NAME administered intraperitoneally decreased NE and increased 3-methoxy-4-hydroxyphenylglycol. These observations suggest that 6-nitronorepinephrine generated in nuclei containing both adrenergic and nitrergic neurons inhibits NE inactivation. We propose that 6-nitronorepinephrine is a potential signal molecule linking the actions of NE and NO..
- Published
- 1996
26. gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family.
- Author
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Kuroki, K, Eng, F, Ishikawa, T, Turck, C, Harada, F, and Ganem, D
- Abstract
Duck hepatitis B virus particles bearing the L and S envelope proteins bind a cellular glycoprotein of 180 kDa (gp180) with high affinity and specificity. Binding is mediated by the pre-S region of the L protein and is blocked by neutralizing but not by non-neutralizing monoclonal antibodies to the virus. These and other properties have suggested that gp180 may be a component of the viral entry machinery. Here we report the purification of gp180 from duck liver and the isolation and characterization of cDNA encoding it. DNA sequence analysis of this cDNA indicates that gp180 is a novel member of the basic carboxypeptidase gene family.
- Published
- 1995
27. Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase.
- Author
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Saitoh, M, Ishikawa, T, Matsushima, S, Naka, M, and Hidaka, H
- Abstract
Systematically synthesized derivatives of ML-9, 1-(5-chloronaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine, were found to inhibit both Ca2+-calmodulin-dependent and -independent smooth muscle myosin light chain kinases with a similar concentration dependence, and their inhibitions were of the competitive type with respect to ATP. Moreover, ML-9 as well as ATP or ADP exhibited an effective protection to inactivation of smooth muscle myosin light chain kinase by the nucleotide affinity label 5′-p-fluorosulfonylbenzoyladenosine, suggesting that ML-9 binds at or near the ATP-binding site on the kinase molecule. These derivatives, which were structurally unrelated to ATP and exhibited more hydrophobic properties detected by reverse-phase high-performance liquid chromatography, exhibited more potent inhibition toward smooth muscle myosin light chain kinase, indicating that the hydrophobic properties of these derivatives positively correlated well with their potencies of inhibiting the catalytic activity for the enzyme. These findings suggest that the ATP-binding site at the active center of smooth muscle myosin light chain kinase is located in a hydrophobic environment. The potent vaso-relaxing effect of ML-9 on rabbit vascular strips and on saponin-treated skinned smooth muscle cells was discussed in relation to the in vivo inhibition by this drug of smooth muscle myosin light chain kinase.
- Published
- 1987
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28. Isolation and properties of a mutant of Escherichia coli possessing defective Na+/H+ antiporter.
- Author
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Ishikawa, T, Hama, H, Tsuda, M, and Tsuchiya, T
- Abstract
A mutant of Escherichia coli with defective Na+/H+ antiporter was isolated. The rationale for its isolation was that cells possessing defective Na+/H+ antiporter, which is essential for establishment of a Na+ gradient, could not grow with a carbon source that was taken up with Na+. The mutant had no appreciable Na+/H+ antiporter activity, but its K+/H+ antiporter and Ca2+/H+ antiporter activities were normal. Judging from the reversion frequency, the defect seems to be due to a single mutation. The mutant could not grow at alkaline pH. Therefore, the Na+/H+ antiporter, but not the K+/H+ antiporter or the Ca2+/H+ antiporter, seems to be responsible for pH regulation in alkaline medium. This mutant will be useful for cloning the Na+/H+ antiporter gene and for detection of Na+-substrate cotransport systems.
- Published
- 1987
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29. Cardiac transport of glutathione disulfide and S-conjugate. Studies with isolated perfused rat heart during hydroperoxide metabolism.
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Ishikawa, T and Sies, H
- Abstract
The relationship between the intracellular GSSG level and the rate of GSSG release was studied in the isolated perfused heart. GSSG and GSH were released from the heart into the effluent perfusion fluid at rates of 110 and 370 pmol X min-1 X g of heart-1, respectively, much lower than for similar conditions in liver. Perfusion with t-butyl hydroperoxide led to an increase in GSSG release associated with an elevated level of intracellular GSSG. Saturation kinetics was found for the rate of GSSG release with respect to the intracellular GSSG level with an apparent Km value of 30 nmol X g of heart-1 and a maximal rate of 7.5 nmol X min-1 X g of heart-1. The maximal rate of GSSG release was temperature-sensitive with a temperature coefficient of 1.8. When 1-chloro-2,4-dinitrobenzene was infused during perfusion with t-butyl hydroperoxide, mutual competition was found in the release of GSSG and the glutathione-S-conjugate, S-(2,4-dinitrophenyl)-glutathione. The maximal rate for the glutathione-S-conjugate transport was estimated to be 40 nmol X min-1 X g of heart-1. These results are consistent with the existence of a transport system for GSSG in rat heart, its capacity being substantially lower than that for the S-conjugate in heart and that for GSSG in liver. Thus, the sensitivity of heart to oxidative stress may be explained, in part, by a low capacity of GSSG export.
- Published
- 1984
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30. Activation of Phosphatidylinositol Kinase and Phosphatidylinositol-4-phosphate Kinase by cAMP in Saccharomyces cerevisiae
- Author
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Kato, H, Uno, I, Ishikawa, T, and Takenawa, T
- Abstract
In Saccharomyces cerevisiae, cAMP-dependent phosphorylation plays an essential role at the start of the cell cycle. It has also recently been demonstrated that the breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is a requisite process for cell proliferation (Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988) Nature333, 188–190). To clarify the relationship between the cAMP- and inositol phospholipid-mediated signal transduction systems, alterations in the inositol phospholipid metabolism of cAMP mutants were examined.
- Published
- 1989
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31. Role of cardiac glutathione transferase and of the glutathione S-conjugate export system in biotransformation of 4-hydroxynonenal in the heart.
- Author
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Ishikawa, T, Esterbauer, H, and Sies, H
- Abstract
There is a remarkable difference in the isozyme pattern between cardiac and hepatic glutathione S-transferases in rat (Ishikawa, T., and Sies, H. (1984) FEBS Lett. 169, 156-160), and one near-neutral isozyme (pI = 6.9) of the cardiac glutathione S-transferases was found to have a significantly high activity toward 4-hydroxynonenal. The isozyme was inhibited by the resulting glutathione S-conjugate of 4-hydroxynonenal competitively with GSH and noncompetitively with 4-hydroxynonenal. The kinetic parameters estimated for the isozyme were: kcat = 460 mol X min-1 X mol enzyme-1, Km = 50 microM for 4-hydroxynonenal, Ki = 85 microM. When the heart was perfused with 4-hydroxynonenal, a marked decrease was observed in the intracellular GSH level, accompanied by an increase of glutathione S-conjugate of 4-hydroxynonenal in the heart. The rate of the conjugation reaction was more than 30 times the rate of the spontaneous reaction, the half-life of 4-hydroxynonenal in the heart being less than 4 s. The glutathione S-conjugate of 4-hydroxynonenal was released from the heart into the perfusion medium. Saturation kinetics were observed for the release with respect to the intracellular level of the S-conjugate (Vmax = 12 nmol X min-1 X g heart-1), and there was a competition by the S-conjugate for GSSG release. The release of the glutathione S-conjugate is considered as a carrier-mediated process and to be important not only in interorgan glutathione metabolism but also in diminishing the inhibitory effect of the S-conjugate on glutathione S-transferases and glutathione reductase.
- Published
- 1986
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32. A kinetic study on the diffusion-coupled reaction of a basic horseradish peroxidase adsorbed on the carboxymethylcellulose membrane.
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Ishikawa, T., primary, Tamura, M., additional, and Yamazaki, I., additional
- Published
- 1980
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33. Flavivirus recruits the valosin-containing protein-NPL4 complex to induce stress granule disassembly for efficient viral genome replication.
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Arakawa M, Tabata K, Ishida K, Kobayashi M, Arai A, Ishikawa T, Suzuki R, Takeuchi H, Tripathi LP, Mizuguchi K, and Morita E
- Subjects
- Genome, Viral, Humans, RNA, Viral genetics, Encephalitis Virus, Japanese genetics, Encephalitis Virus, Japanese metabolism, Encephalitis Virus, Japanese physiology, Flavivirus genetics, Flavivirus metabolism, Flavivirus physiology, Nuclear Proteins metabolism, Stress Granules genetics, Stress Granules metabolism, Valosin Containing Protein metabolism, Viral Nonstructural Proteins genetics, Viral Nonstructural Proteins metabolism, Virus Replication physiology
- Abstract
Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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34. Matrix metalloproteinase-7 induces homotypic tumor cell aggregation via proteolytic cleavage of the membrane-bound Kunitz-type inhibitor HAI-1.
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Ishikawa T, Kimura Y, Hirano H, and Higashi S
- Subjects
- Amino Acid Substitution, Animals, CHO Cells, Carcinoma metabolism, Carcinoma pathology, Cell Aggregation, Cell Line, Tumor, Cholesterol Esters chemistry, Cholesterol Esters metabolism, Colonic Neoplasms metabolism, Colonic Neoplasms pathology, Cricetulus, Humans, Matrix Metalloproteinase 7 chemistry, Matrix Metalloproteinase 7 genetics, Mutation, Neoplasm Proteins antagonists & inhibitors, Neoplasm Proteins chemistry, Neoplasm Proteins genetics, Peptide Fragments antagonists & inhibitors, Peptide Fragments chemistry, Peptide Fragments genetics, Protein Interaction Domains and Motifs, Proteinase Inhibitory Proteins, Secretory antagonists & inhibitors, Proteinase Inhibitory Proteins, Secretory chemistry, Proteinase Inhibitory Proteins, Secretory genetics, Proteolysis, RNA Interference, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Solubility, Substrate Specificity, Carcinoma enzymology, Colonic Neoplasms enzymology, Matrix Metalloproteinase 7 metabolism, Neoplasm Proteins metabolism, Peptide Fragments metabolism, Proteinase Inhibitory Proteins, Secretory metabolism
- Abstract
Matrix metalloproteinase-7 (MMP-7) plays important roles in tumor progression and metastasis. Our previous studies have demonstrated that MMP-7 binds to colon cancer cells via cell surface-bound cholesterol sulfate and induces significant cell aggregation by cleaving cell-surface protein(s). These aggregated cells exhibit a dramatically enhanced metastatic potential. However, the molecular mechanism inducing this cell-cell adhesion through the proteolytic action of MMP-7 remained to be clarified. Here, we explored MMP-7 substrates on the cell surface; the proteins on the cell surface were first biotinylated, and a labeled protein fragment specifically released from the cells after MMP-7 treatment was analyzed using LC-MS/MS. We found that hepatocyte growth factor activator inhibitor type 1 (HAI-1), a membrane-bound Kunitz-type serine protease inhibitor, is an MMP-7 substrate. We also found that the cell-bound MMP-7 cleaves HAI-1 mainly between Gly
451 and Leu452 and thereby releases the extracellular region as soluble HAI-1 (sHAI-1). We further demonstrated that this sHAI-1 can induce cancer cell aggregation and determined that the HAI-1 region corresponding to amino acids 141-249, which does not include the serine protease inhibitor domain, has the cell aggregation-inducing activity. Interestingly, a cell-surface cholesterol sulfate-independent proteolytic action of MMP-7 is critical for the sHAI-1-mediated induction of cell aggregation, whereas cholesterol sulfate is needed for the MMP-7-catalyzed generation of sHAI-1. Considering that MMP-7-induced cancer cell aggregation is an important mechanism in cancer metastasis, we propose that sHAI-1 is an essential component of MMP-7-induced stimulation of cancer metastasis and may therefore represent a suitable target for antimetastatic therapeutic strategies., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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35. Low-density lipoprotein receptor-related protein 1 (LRP1)-dependent cell signaling promotes axonal regeneration.
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Yoon C, Van Niekerk EA, Henry K, Ishikawa T, Orita S, Tuszynski MH, and Campana WM
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- Animals, Axons metabolism, Female, Ganglia, Spinal metabolism, Ligands, Neurites metabolism, Neuronal Plasticity, Neurons metabolism, Rats, Rats, Inbred F344, Regeneration, Spinal Cord Injuries pathology, Transcriptional Activation, Low Density Lipoprotein Receptor-Related Protein-1 metabolism, Nerve Regeneration, Receptor, trkC metabolism, Signal Transduction
- Abstract
Low-density lipoprotein receptors (LRPs) are present extensively on cells outside of the nervous system and classically exert roles in lipoprotein metabolism. It has been reported recently that LRP1 activation could phosphorylate the neurotrophin receptor TrkA in PC12 cells and increase neurite outgrowth from developing cerebellar granule cells. These intriguing findings led us to explore the hypothesis that LRP1 activation would activate canonical neurotrophic factor signaling in adult neurons and promote axonal regeneration after spinal cord injury. We now find that treatment of adult rat dorsal root ganglion neurons in vitro with LRP1 agonists (the receptor binding domain of α-2-macroglobulin or the hemopexin domain of matrix metalloproteinase 9) induces TrkC, Akt, and ERK activation; significantly increases neurite outgrowth (p < 0.01); and overcomes myelin inhibition (p < 0.05). These effects require Src family kinase activation, a classic LRP1-mediated Trk transactivator. Moreover, intrathecal infusions of LRP1 agonists significantly enhance sensory axonal sprouting and regeneration after spinal cord injury in rats compared with control-infused animals (p < 0.05). A significant role is established for lipoprotein receptors in sprouting and regeneration after CNS injury, identifying a novel class of therapeutic targets to explore for traumatic neurological disorders.
- Published
- 2013
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36. Revisiting the supramolecular organization of photosystem II in Chlamydomonas reinhardtii.
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Tokutsu R, Kato N, Bui KH, Ishikawa T, and Minagawa J
- Subjects
- Detergents chemistry, Glucosides chemistry, Photosystem II Protein Complex metabolism, Protein Structure, Quaternary, Chlamydomonas reinhardtii enzymology, Photosystem II Protein Complex chemistry, Thylakoids enzymology
- Abstract
Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-β-D-maltoside (β-DM) or n-dodecyl-α-D-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the β-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.
- Published
- 2012
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37. H2O2-triggered retrograde signaling from chloroplasts to nucleus plays specific role in response to stress.
- Author
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Maruta T, Noshi M, Tanouchi A, Tamoi M, Yabuta Y, Yoshimura K, Ishikawa T, and Shigeoka S
- Subjects
- Acclimatization, Antioxidants metabolism, Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Ascorbate Peroxidases genetics, Ascorbate Peroxidases metabolism, Cell Nucleus metabolism, Chloroplasts metabolism, Cold Temperature, Disease Resistance genetics, Gene Expression Profiling, Gene Expression Regulation, Plant radiation effects, Gene Knockdown Techniques, Genes, Plant, Light, Oligonucleotide Array Sequence Analysis, Oxidation-Reduction, Plant Leaves enzymology, Plant Leaves metabolism, Plants, Genetically Modified, RNA Interference, Real-Time Polymerase Chain Reaction, Salicylic Acid metabolism, Salicylic Acid pharmacology, Thylakoid Membrane Proteins genetics, Thylakoid Membrane Proteins metabolism, Trans-Activators genetics, Trans-Activators metabolism, Transcription, Genetic, Arabidopsis physiology, Cell Nucleus physiology, Chloroplasts physiology, Hydrogen Peroxide metabolism, Signal Transduction, Stress, Physiological
- Abstract
Recent findings have suggested that reactive oxygen species (ROS) are important signaling molecules for regulating plant responses to abiotic and biotic stress and that there exist source- and kind-specific pathways for ROS signaling. In plant cells, a major source of ROS is chloroplasts, in which thylakoid membrane-bound ascorbate peroxidase (tAPX) plays a role in the regulation of H(2)O(2) levels. Here, to clarify the signaling function of H(2)O(2) derived from the chloroplast, we created a conditional system for producing H(2)O(2) in the organelle by chemical-dependent tAPX silencing using estrogen-inducible RNAi. When the expression of tAPX was silenced in leaves, levels of oxidized protein in chloroplasts increased in the absence of stress. Microarray analysis revealed that tAPX silencing affects the expression of a large set of genes, some of which are involved in the response to chilling and pathogens. In response to tAPX silencing, the transcript levels of C-repeat/DRE binding factor (CBF1), a central regulator for cold acclimation, was suppressed, resulting in a high sensitivity of tAPX-silenced plants to cold. Furthermore, tAPX silencing enhanced the levels of salicylic acid (SA) and the response to SA. Interestingly, we found that tAPX silencing-responsive genes were up- or down-regulated by high light (HL) and that tAPX silencing had a negative effect on expression of ROS-responsive genes under HL, suggesting synergistic and antagonistic roles of chloroplastic H(2)O(2) in HL response. These findings provide a new insight into the role of H(2)O(2)-triggered retrograde signaling from chloroplasts in the response to stress in planta.
- Published
- 2012
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38. Structural analysis of the mechanism of inhibition and allosteric activation of the kinase domain of HER2 protein.
- Author
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Aertgeerts K, Skene R, Yano J, Sang BC, Zou H, Snell G, Jennings A, Iwamoto K, Habuka N, Hirokawa A, Ishikawa T, Tanaka T, Miki H, Ohta Y, and Sogabe S
- Subjects
- Allosteric Regulation drug effects, Allosteric Regulation genetics, Antineoplastic Agents chemistry, Antineoplastic Agents therapeutic use, Crystallography, X-Ray, Drug Design, Enzyme Activation drug effects, Enzyme Activation genetics, ErbB Receptors chemistry, ErbB Receptors genetics, ErbB Receptors metabolism, Humans, Neoplasms drug therapy, Neoplasms enzymology, Neoplasms genetics, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors therapeutic use, Protein Structure, Tertiary, Receptor, ErbB-2 antagonists & inhibitors, Receptor, ErbB-2 genetics, Receptor, ErbB-2 metabolism, Receptor, ErbB-4, Structure-Activity Relationship, Receptor, ErbB-2 chemistry
- Abstract
Aberrant signaling of ErbB family members human epidermal growth factor 2 (HER2) and epidermal growth factor receptor (EGFR) is implicated in many human cancers, and HER2 expression is predictive of human disease recurrence and prognosis. Small molecule kinase inhibitors of EGFR and of both HER2 and EGFR have received approval for the treatment of cancer. We present the first high resolution crystal structure of the kinase domain of HER2 in complex with a selective inhibitor to understand protein activation, inhibition, and function at the molecular level. HER2 kinase domain crystallizes as a dimer and suggests evidence for an allosteric mechanism of activation comparable with previously reported activation mechanisms for EGFR and HER4. A unique Gly-rich region in HER2 following the α-helix C is responsible for increased conformational flexibility within the active site and could explain the low intrinsic catalytic activity previously reported for HER2. In addition, we solved the crystal structure of the kinase domain of EGFR in complex with a HER2/EGFR dual inhibitor (TAK-285). Comparison with previously reported inactive and active EGFR kinase domain structures gave insight into the mechanism of HER2 and EGFR inhibition and may help guide the design and development of new cancer drugs with improved potency and selectivity.
- Published
- 2011
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39. Heart-specific small subunit of myosin light chain phosphatase activates rho-associated kinase and regulates phosphorylation of myosin phosphatase target subunit 1.
- Author
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Shichi D, Arimura T, Ishikawa T, and Kimura A
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Cytoskeletal Proteins chemistry, Hot Temperature, Humans, Membrane Proteins chemistry, Microfilament Proteins chemistry, Molecular Sequence Data, Myosins, Phosphorylation, Two-Hybrid System Techniques, Myocardium metabolism, Myosin-Light-Chain Phosphatase metabolism, rho-Associated Kinases metabolism
- Abstract
Phosphorylation of myosin regulatory light chain (MLC) plays a regulatory role in muscle contraction, and the level of MLC phosphorylation is balanced by MLC kinase and MLC phosphatase (MLCP). MLCP consists of a catalytic subunit, a large subunit (MYPT1 or MYPT2), and a small subunit. MLCP activity is regulated by phosphorylation of MYPTs, whereas the role of small subunit in the regulation remains unknown. We previously characterized a human heart-specific small subunit (hHS-M(21)) that increased the sensitivity to Ca(2+) in muscle contraction. In this study, we investigated the role of hHS-M(21) in the regulation of MLCP phosphorylation. Two isoforms of hHS-M(21), hHS-M(21)A and hHS-M(21)B, preferentially bound the C-terminal one-third region of MYPT1 and MYPT2, respectively. Amino acid substitutions at a phosphorylation site of MYPT1, Ser-852, impaired the binding of MYPT1 and hHS-M(21). The hHS-M(21) increased the phosphorylation level of MYPT1 at Thr-696, which was attenuated by Rho-associated kinase (ROCK) inhibitors and small interfering RNAs for ROCK. In addition, hHS-M(21) bound ROCK and enhanced the ROCK activity. These findings suggest that hHS-M(21) is a heart-specific effector of ROCK and plays a regulatory role in the MYPT1 phosphorylation at Thr-696 by ROCK.
- Published
- 2010
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40. Molecular details of Bax activation, oligomerization, and membrane insertion.
- Author
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Bleicken S, Classen M, Padmavathi PV, Ishikawa T, Zeth K, Steinhoff HJ, and Bordignon E
- Subjects
- Animals, Electron Spin Resonance Spectroscopy, Mice, Models, Chemical, Protein Conformation, Proto-Oncogene Proteins c-bcl-2 chemistry, BH3 Interacting Domain Death Agonist Protein metabolism, Mitochondrial Membranes metabolism, Protein Multimerization, Proto-Oncogene Proteins c-bcl-2 metabolism
- Abstract
Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices alpha5 and alpha6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.
- Published
- 2010
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41. Novel mechanisms of trafficking defect caused by KCNQ1 mutations found in long QT syndrome.
- Author
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Sato A, Arimura T, Makita N, Ishikawa T, Aizawa Y, Ushinohama H, Aizawa Y, and Kimura A
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Biological Transport physiology, Cell Line, Child, DNA Mutational Analysis, Female, Humans, KCNQ1 Potassium Channel metabolism, Male, Molecular Sequence Data, Patch-Clamp Techniques, Pedigree, Sequence Alignment, KCNQ1 Potassium Channel genetics, Long QT Syndrome genetics, Mutation
- Abstract
Long QT syndrome (LQTS) is a hereditary arrhythmia caused by mutations in genes for cardiac ion channels, including a potassium channel, KvLQT1. Inheritance of LQTS is usually autosomal-dominant, but autosomal-recessive inheritance can be observed in patients with LQTS accompanied by hearing loss. In this study, we investigated the functional alterations caused by KCNQ1 mutations, a deletion (delV595) and a frameshift (P631fs/19), which were identified in compound heterozygous state in two patients with autosomal-recessive LQTS not accompanied by hearing loss. Functional analyses showed that both mutations impaired cell surface expression due to trafficking defects. The mutations severely affected outward potassium currents without apparent dominant negative effects. It was found that delV595 impaired subunit binding, whereas P631fs/19 was retained in endoplasmic reticulum due to the newly added 19-amino acid sequence containing two retention motifs (R(633)GR and R(646)LR). This is the first report of novel mechanisms for trafficking abnormality of cardiac ion channels, providing us new insights into the molecular mechanisms of LQTS.
- Published
- 2009
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42. Loss of calmodulin binding to Bax inhibitor-1 affects Pseudomonas-mediated hypersensitive response-associated cell death in Arabidopsis thaliana.
- Author
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Kawai-Yamada M, Hori Z, Ogawa T, Ihara-Ohori Y, Tamura K, Nagano M, Ishikawa T, and Uchimiya H
- Subjects
- Amino Acid Sequence, Arabidopsis anatomy & histology, Arabidopsis Proteins genetics, Calmodulin classification, Calmodulin genetics, Membrane Proteins genetics, Molecular Sequence Data, Phylogeny, Plants, Genetically Modified, Protein Binding, Arabidopsis microbiology, Arabidopsis physiology, Arabidopsis Proteins metabolism, Calmodulin metabolism, Cell Death physiology, Membrane Proteins metabolism, Pseudomonas syringae physiology
- Abstract
Bax inhibitor-1 (BI-1) is a cell death suppressor protein conserved across a variety of organisms. The Arabidopsis atbi1-1 plant is a mutant in which the C-terminal 6 amino acids of the expressed BI-1 protein have been replaced by T-DNA insertion. This mutant BI-1 protein (AtBI-CM) produced in Escherichia coli can no longer bind to calmodulin. A promoter-reporter assay demonstrated compartmentalized expression of BI-1 during hypersensitive response, introduced by the inoculation of Pseudomonas syringae possessing the avrRTP2 gene, Pst(avrRPT2). In addition, both BI-1 knockdown plants and atbi1-1 showed increased sensitivity to Pst(avrRPT2)-induced cell death. The results indicated that the loss of calmodulin binding reduces the cell death suppressor activity of BI-1 in planta.
- Published
- 2009
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43. The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase.
- Author
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Ishikawa T, Nishikawa H, Gao Y, Sawa Y, Shibata H, Yabuta Y, Maruta T, and Shigeoka S
- Subjects
- Algal Proteins genetics, Amino Acid Sequence, Animals, Ascorbic Acid genetics, Carboxylic Ester Hydrolases genetics, Escherichia coli enzymology, Escherichia coli genetics, Euglena gracilis genetics, Molecular Sequence Data, Protozoan Proteins genetics, Rats, Recombinant Proteins genetics, Recombinant Proteins metabolism, Algal Proteins metabolism, Ascorbic Acid biosynthesis, Carboxylic Ester Hydrolases metabolism, Euglena gracilis enzymology, Hexuronic Acids metabolism, Protozoan Proteins metabolism, Sugar Acids metabolism
- Abstract
We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/L-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent K(m) values for L-galactonate and L-galactono-1,4-lactone were 1.55 +/- 0.3 and 1.67 +/- 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.
- Published
- 2008
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44. Arabidopsis phosphomannose isomerase 1, but not phosphomannose isomerase 2, is essential for ascorbic acid biosynthesis.
- Author
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Maruta T, Yonemitsu M, Yabuta Y, Tamoi M, Ishikawa T, and Shigeoka S
- Subjects
- Dose-Response Relationship, Drug, Glycolysis, Hexokinase chemistry, Kinetics, Mannose chemistry, Mannose-6-Phosphate Isomerase metabolism, Models, Biological, Phosphorylation, Plant Leaves enzymology, Plant Proteins metabolism, Plants, Genetically Modified, RNA Interference, Recombinant Proteins chemistry, Arabidopsis enzymology, Ascorbic Acid metabolism, Mannose-6-Phosphate Isomerase physiology
- Abstract
We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between D-fructose 6-phosphate and D-mannose 6-phosphate (Man-6P). The apparent K(m) and V(max) values for Man-6P of purified recombinant PMI1 were 41.3+/-4.2 microm and 1.89 micromol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372+/-13 microm and 22.5 micromol/min/mg protein, respectively. Both PMI1 and PMI2 were inhibited by incubation with EDTA, Zn(2+), Cd(2+), and L-ascorbic acid (AsA). Arabidopsis PMI1 protein was constitutively expressed in both vegetative and reproductive organs under normal growth conditions, whereas the PMI2 protein was not expressed in any organs under light. The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, whereas the induction of PMI2 expression and a decrease in the AsA level were observed under long term darkness. PMI1 showed a diurnal expression pattern in parallel with the total PMI activity and the total AsA content in leaves. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown PMI1 plants, whereas the complete inhibition of PMI2 expression did not affect the total AsA levels in leaves of knock-out PMI2 plants. Consequently, this study improves our understanding of the molecular and functional properties of Arabidopsis PMI isoenzymes and provides genetic evidence of the involvement of PMI1, but not PMI2, in the biosynthesis of AsA in Arabidopsis plants.
- Published
- 2008
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45. DNA damage-induced ubiquitylation of RFC2 subunit of replication factor C complex.
- Author
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Tomida J, Masuda Y, Hiroaki H, Ishikawa T, Song I, Tsurimoto T, Tateishi S, Shiomi T, Kamei Y, Kim J, Kamiya K, Vaziri C, Ohmori H, and Todo T
- Subjects
- Alkylating Agents pharmacology, Amino Acid Substitution, Cell Line, DNA Repair drug effects, DNA Repair physiology, DNA Replication drug effects, DNA Replication physiology, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Humans, Hydrogen Peroxide pharmacology, Ligases genetics, Ligases metabolism, Multiprotein Complexes genetics, Mutation, Missense, Oxidants pharmacology, Phosphorylation drug effects, Proliferating Cell Nuclear Antigen genetics, Proliferating Cell Nuclear Antigen metabolism, Protein Processing, Post-Translational drug effects, Replication Protein C genetics, Ubiquitin-Conjugating Enzymes genetics, Ubiquitin-Conjugating Enzymes metabolism, Ubiquitin-Protein Ligases, Ubiquitination drug effects, DNA Damage physiology, Multiprotein Complexes metabolism, Protein Processing, Post-Translational physiology, Replication Protein C metabolism, Ubiquitination physiology
- Abstract
Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.
- Published
- 2008
- Full Text
- View/download PDF
46. Intramolecular disulfide bond is a critical check point determining degradative fates of ATP-binding cassette (ABC) transporter ABCG2 protein.
- Author
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Wakabayashi K, Nakagawa H, Tamura A, Koshiba S, Hoshijima K, Komada M, and Ishikawa T
- Subjects
- ATP Binding Cassette Transporter, Subfamily G, Member 2, ATP-Binding Cassette Transporters chemistry, Adenosine Triphosphate chemistry, Cell Line, Cysteine chemistry, Dimerization, Dose-Response Relationship, Drug, Humans, Leupeptins pharmacology, Lysosomes chemistry, Neoplasm Proteins chemistry, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors, Protein Binding, Protein Denaturation, Ubiquitin chemistry, Xenobiotics, ATP-Binding Cassette Transporters physiology, Disulfides chemistry, Neoplasm Proteins physiology
- Abstract
Human ABCG2 belongs to the ATP-binding cassette (ABC) transporter family and plays an important role in various biological reactions, such as xenobiotic elimination and homeostasis of protoporphyrin. We previously reported that ABCG2 exists in the plasma membrane as a homodimer bound via a disulfide bond at Cys-603. In the present study, we examined the importance of an intramolecular disulfide bond for stability of the ABCG2 protein. Substitution of either Cys-592 or Cys-608 located in the extracellular loop to glycine resulted in a significant decrease in protein levels of ABCG2 when expressed in Flp-In-293 cells. Interestingly, the protein levels of those ABCG2 variants were remarkably enhanced by treatment with the proteasome inhibitor MG132. Concomitantly, increases in ubiquitinated forms of those variant proteins were detected by immunoprecipitation. In contrast, neither the protein level nor the ubiquitinated state of the ABCG2 wild-type (WT) was affected by MG132 treatment. Ubiquitin-mediated protein degradation is suggested to be involved in degradation of misfolded ABCG2 proteins lacking the intramolecular disulfide bond. On the other hand, the protein level of ABCG2 WT increased more than 4-fold when cells were treated with bafilomycin A(1), which inhibits lysosomal degradation, whereas the C592G or C608G variant was little affected by the same treatment. These results strongly suggest that two distinct pathways exist for protein degradation of ABCG2 WT and mutants lacking the intramolecular disulfide bond. Namely, the WT ABCG2 is degraded in lysosomes, and the misfolded ABCG2 lacking intramolecular disulfide bond undergoes ubiquitin-mediated protein degradation in proteasomes.
- Published
- 2007
- Full Text
- View/download PDF
47. Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption.
- Author
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Soga T, Baran R, Suematsu M, Ueno Y, Ikeda S, Sakurakawa T, Kakazu Y, Ishikawa T, Robert M, Nishioka T, and Tomita M
- Subjects
- Animals, Dipeptides metabolism, Electrophoresis, Capillary, Gene Expression Profiling, Glutathione Synthase metabolism, Male, Metabolism, Mice, Mice, Inbred C57BL, Models, Biological, Glutathione metabolism, Liver metabolism, Oligopeptides metabolism, Oxidative Stress
- Abstract
Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with gamma-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Our method can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers.
- Published
- 2006
- Full Text
- View/download PDF
48. Regulation of the epithelial Na+ channel by cytosolic ATP.
- Author
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Ishikawa T, Jiang C, Stutts MJ, Marunaka Y, and Rotin D
- Subjects
- 3T3 Cells, Adenosine Monophosphate metabolism, Amino Acid Motifs, Animals, Cell Line, Dogs, Dose-Response Relationship, Drug, Electrophysiology, Epithelial Sodium Channels, Humans, Hydrolysis, Lithium chemistry, Mice, Patch-Clamp Techniques, Rats, Sodium Channels metabolism, Time Factors, Adenosine Triphosphate metabolism, Cytosol metabolism, Gene Expression Regulation, Sodium metabolism, Sodium Channels chemistry
- Abstract
The epithelial Na+ channel (ENaC), composed of three subunits (alphabetagamma), is expressed in various Na(+)-absorbing epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. By using patch clamp techniques, we have examined the effect of cytosolic ATP on the activity of the rat alphabetagammaENaC (rENaC) stably expressed in NIH-3T3 cells and in Madin-Darby canine kidney epithelial cells. The inward whole-cell current attributable to rENaC activity ran down when these cells were dialyzed with an ATP-free pipette solution in the conventional whole-cell voltage-clamping technique. This run down was prevented by 2 mM ATP (but not by AMP or ADP) in the pipette solution or by the poorly or non-hydrolyzable analogues of ATP (adenosine 5'-O-(thiotriphosphate) and adenosine 5'-(beta,gamma-imino)triphosphate) in both cell lines, suggesting that protection from run down was mediated through non-hydrolytic nucleotide binding. Accordingly, we demonstrate binding of ATP (but not AMP) to alpharENaC expressed in Madin-Darby canine kidney cells, which was inhibited upon mutation of the two putative nucleotide-binding motifs of alpharENaC. Single channel analyses indicated that the run down of currents observed in the whole-cell recording was attributable to run down of channel activity, defined as NPo (the product of the number of channels and open probability). We propose that this novel ATP regulation of ENaC may be, at least in part, involved in the fine-tuning of ENaC activity under physiologic and pathophysiologic conditions.
- Published
- 2003
- Full Text
- View/download PDF
49. Functional and structural analyses of cryptochrome. Vertebrate CRY regions responsible for interaction with the CLOCK:BMAL1 heterodimer and its nuclear localization.
- Author
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Hirayama J, Nakamura H, Ishikawa T, Kobayashi Y, and Todo T
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Biological Clocks, Cryptochromes, DNA Primers, Escherichia coli metabolism, Flavoproteins metabolism, Mice, Models, Molecular, Molecular Sequence Data, Protein Conformation, Receptors, G-Protein-Coupled, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Repressor Proteins metabolism, Restriction Mapping, Sequence Alignment, Sequence Homology, Amino Acid, Transcription, Genetic, Zebrafish, Drosophila Proteins, Eye Proteins, Flavoproteins chemistry, Flavoproteins genetics, Photoreceptor Cells, Invertebrate
- Abstract
Mouse mCRY1 and zebrafish zCRY1a and zCRY3 belong to the DNA photolyase/Cryptochrome family. mCRY1 and zCRY1a repress CLOCK:BMAL1-mediated transcription, whereas zCRY3 does not. Reciprocal chimeras between zCRY1a and zCRY3 were generated to determine the zCRY1a regions responsible for nuclear translocation, interaction with the CLOCK:BMAL1 heterodimer, and repression of CLOCK:BMAL1-mediated transcription. Three regions, RD-2a-(126-196), RD-1-(197-263), and RD-2b-(264-293), were identified. Proteins in this family consist of an N-terminal alpha/beta domain and a C-terminal helical domain connected by an interdomain loop. RD-2a is within this loop, RD-1 is at the N-terminal 50 amino acids, and RD-2b at the following 31 amino acid residues of the helical domain. Either RD-2a or RD-1 is required for interaction with the CLOCK: BMAL1 heterodimer, and either RD-1 or RD-2b is required for the nuclear translocation of CRY. Both of these functions are prerequisites for the transcriptional repressor activity. The functional nuclear localizing signal in the RD-2b region also was identified. The sequence is well conserved among repressor-type CRYs, including mCRY1. Mutations in the nuclear localizing signal of mCRY1 reduce the extent of its nuclear localization. These findings show that both nuclear localization and interaction with the CLOCK:BMAL heterodimer are essential for transcriptional repression by CRY.
- Published
- 2003
- Full Text
- View/download PDF
50. Identification of a cis element for tissue-specific alternative splicing of chloroplast ascorbate peroxidase pre-mRNA in higher plants.
- Author
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Yoshimura K, Yabuta Y, Ishikawa T, and Shigeoka S
- Subjects
- Ascorbate Peroxidases, Base Sequence, DNA, Plant, Molecular Sequence Data, Mutagenesis, Site-Directed, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Nucleic Acid, Alternative Splicing, Chloroplasts enzymology, Peroxidases genetics, Plants enzymology, RNA Precursors genetics, RNA, Messenger genetics
- Abstract
Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leaf in vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.
- Published
- 2002
- Full Text
- View/download PDF
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