30 results on '"Mori S"'
Search Results
2. Ligand-induced ubiquitination of the platelet-derived growth factor beta-receptor plays a negative regulatory role in its mitogenic signaling.
- Author
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Mori, S., primary, Heldin, C.H., additional, and Claesson-Welsh, L., additional
- Published
- 1993
- Full Text
- View/download PDF
3. Ligand-induced polyubiquitination of the platelet-derived growth factor beta-receptor.
- Author
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Mori, S, primary, Heldin, C.H., additional, and Claesson-Welsh, L, additional
- Published
- 1992
- Full Text
- View/download PDF
4. Identification of a hydrophobic region in the carboxyl terminus of the platelet-derived growth factor beta-receptor which is important for ligand-mediated endocytosis.
- Author
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Mori, S., primary, Claesson-Welsh, L., additional, and Heldin, C.H., additional
- Published
- 1991
- Full Text
- View/download PDF
5. Transcriptional activation of beta-tropomyosin mediated by serum response factor and a novel Barx homologue, Barx1b, in smooth muscle cells.
- Author
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Nakamura, M, Nishida, W, Mori, S, Hiwada, K, Hayashi, K, and Sobue, K
- Abstract
Tropomyosin (TM) is a regulatory protein of actomyosin system. Muscle type-specific expression of TM isoforms is generated from different genes and by alternative splicing. beta-TM isoforms in chicken skeletal and smooth muscles are encoded by a single gene and transcribed from the same promoter. We previously reported a smooth muscle cell (SMC) phenotype-dependent change in beta-TM expression (Kashiwada, K., Nishida, W., Hayashi, K., Ozawa, K., Yamanaka, Y., Saga, H., Yamashita, T., Tohyama, M., Shimada, S., Sato, K., and Sobue, K. (1997) J. Biol. Chem. 272, 15396-15404), and identified beta-TM as an SMC-differentiation marker. Here, we characterized the transcriptional machinery of the beta-TM gene in SMCs. Promoter and gel mobility shift analyses revealed an obligatory role for serum response factor and its interaction with the CArG box sequence in the SMC-specific transcription of the beta-TM gene in differentiated SMCs. We further isolated a novel homologue of the Barx homeoprotein family, Barx1b, from chicken gizzard. Barx1b was exclusively localized to SMCs of the upper digestive organs and their attached arteries and to craniofacial structures. Serum response factor and Barx1b bound each other directly, coordinately transactivated the beta-TM gene in differentiated SMCs and heterologous cells, and formed a ternary complex with a CArG probe. Taken together, these results suggest that SRF and Barx1b are coordinately involved in the SMC-specific transcription of the beta-TM gene in the upper digestive organs and their attached arteries.
- Published
- 2001
6. OUT, a novel basic helix-loop-helix transcription factor with an Id-like inhibitory activity.
- Author
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Narumi, O, Mori, S, Boku, S, Tsuji, Y, Hashimoto, N, Nishikawa, S, and Yokota, Y
- Abstract
Transcription factors belonging to the basic helix-loop-helix (bHLH) family are involved in various cell differentiation processes. We report the isolation and functional characterization of a novel bHLH factor, termed OUT. OUT, structurally related to capsulin/epicardin/Pod-1 and ABF-1/musculin/MyoR, is expressed mainly in the adult mouse reproductive organs, such as the ovary, uterus, and testis, and is barely detectable in tissues of developing embryos. Physical association of OUT with the E protein was predicted from the primary structure of OUT and confirmed by co-immunoprecipitation. However, unlike other bHLH factors, this novel protein failed to bind E-box or N-box DNA sequences and inhibited DNA binding of homo- and heterodimers consisting of E12 and MyoD in gel mobility shift assays. In luciferase assays, OUT inhibited the induction of E-box-dependent transactivation by MyoD-E12 heterodimers. Deletion studies identified the domain responsible for the inhibitory action of OUT in its bHLH and C-terminal regions. Moreover, terminal differentiation of C2C12 myoblasts was inhibited by exogenous introduction of OUT. These inhibitory functions of OUT closely resemble those of the helix-loop-helix inhibitor Id proteins. Based on these findings, we propose that this novel protein functions as a negative regulator of bHLH factors through the formation of a functionally inactive heterodimeric complex.
- Published
- 2000
7. Platelet-derived growth factor activates p38 mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration.
- Author
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Matsumoto, T, Yokote, K, Tamura, K, Takemoto, M, Ueno, H, Saito, Y, and Mori, S
- Abstract
Members of the mitogen activated protein (MAP) kinase family, extracellular signal-regulated kinase, stress-activated protein kinase-1/c-Jun NH2-terminal kinase, and p38, are central elements that transduce the signal generated by growth factors, cytokines, and stressing agents. It is well known that the platelet-derived growth factor (PDGF) activates extracellular signal-regulated kinase, which leads to cellular mitogenic response. On the other hand, the role of the other MAP kinases in mediating the cellular function of PDGF remains unclear. In the present study, we have investigated the functional role of the other MAP kinases in PDGF-mediated cellular responses. We show that ligand stimulation of PDGF receptors leads to the activation of p38 but not stress-activated protein kinase-1/c-Jun NH2-terminal kinase. Experiments using a specific inhibitor of p38, SB203580, show that the activation of p38 is required for PDGF-induced cell motility responses such as cell migration and actin reorganization but not required for PDGF-stimulated DNA synthesis. Analyses of tyrosine residue-mutated PDGF receptors show that Src homology 2 domain-containing proteins including Src family kinases, phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, the Src homology 2 domain-containing phosphatase SHP-2, phospholipase C-gamma, and Crk do not play a major role in mediating the PDGF-induced activation of p38. Finally, the expression of dominant-negative Ras but not dominant-negative Rac inhibited p38 activation by PDGF, suggesting that Ras is a potent mediator in the p38 activation pathway downstream of PDGF receptors. Taken together, our present study proposes the existence of a Ras-dependent pathway for the activation of p38, which is important for cell motility responses elicited by PDGF stimulation.
- Published
- 1999
8. Intrinsic nucleoside diphosphate kinase-like activity is a novel function of the 20 S proteasome.
- Author
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Yano, M, Mori, S, and Kido, H
- Abstract
The eukaryotic 20 S proteasome is the prototype of a new family of the N-terminal nucleophil hydrolases and is composed of numerous low molecular mass subunits arranged in a stack of four rings, each containing seven different alpha- or beta-subunits. Among the beta-type subunits in the yeast proteasome, three proteolytically active ones were identified, although the functions of the other beta- and alpha-type subunits remain to be clarified. We report here that the purified 20 S proteasome exhibits intrinsic nucleoside diphosphate (NDP) kinase-like activity. The proteasome exhibited a preference for ATP and dATP as phosphate donors, and a broad specificity for NDPs, other than GDP, as phosphate acceptors, unlike conventional NDP kinase, which catalyzes the transfer of gamma-phosphate between NDPs and nucleoside triphosphates. During the transfer of gamma-phosphate, the proteasome formed acid-labile phosphohistidine as autophosphorylated intermediates, and NDP-dependent dephosphorylation of the latter then occurred. These enzymatic properties are similar to those of the molecular chaperone, Hsp70, which also exhibits intrinsic NDP kinase-like activity, instead of ATPase activity. C5 among the beta-type subunits and C8 among the alpha-type subunits were autophosphorylated during the gamma-phosphate transfer reaction and were photoaffinity labeled with 8-azido-[alpha-(32)P]ATP, suggesting that the C5 and C8 subunits of the proteasome are responsible for the NDP kinase-like activity.
- Published
- 1999
9. Functional analysis of aquaporin-1 deficient red cells. The Colton-null phenotype.
- Author
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Mathai, J C, Mori, S, Smith, B L, Preston, G M, Mohandas, N, Collins, M, van Zijl, P C, Zeidel, M L, and Agre, P
- Abstract
The aquaporin-1 (AQP1) water transport protein contains a polymorphism corresponding to the Colton red blood cell antigens. To define the fraction of membrane water permeability mediated by AQP1, red cells were obtained from human kindreds with the rare Colton-null phenotype. Homozygosity or heterozygosity for deletion of exon I in AQP1 correlated with total or partial deficiency of AQP1 protein. Homozygote red cell morphology appeared normal, but clinical laboratory studies revealed slightly reduced red cell life span in vivo; deformability studies revealed a slight reduction in membrane surface area. Diffusional water permeability (Pd) was measured under isotonic conditions by pulsed field gradient NMR. Osmotic water permeability (Pf) was measured by change in light scattering after rapid exposure of red cells to increased extracellular osmolality. AQP1 contributes approximately 64% (Pd = 1.5 x 10(-3) cm/s) of the total diffusional water permeability pathway, and lipid permeation apparently comprises approximately 23%. In contrast, AQP1 contributes > 85% (Pf = 19 x 10(-3) cm/s) of the total osmotic water permeability pathway, and lipid permeation apparently comprises only approximately 10%. The ratio of AQP1-mediated Pf to Pd predicts the length of the aqueous pore to be 36 A.
- Published
- 1996
10. Degradation process of ligand-stimulated platelet-derived growth factor beta-receptor involves ubiquitin-proteasome proteolytic pathway.
- Author
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Mori, S, Tanaka, K, Omura, S, and Saito, Y
- Abstract
The platelet-derived growth factor beta-receptor undergoes polyubiquitination as a consequence of ligand binding. We have previously reported that ligand-induced ubiquitination of the receptor plays a negative regulatory role in its mitogenic signaling possibly by promoting the efficient degradation of the ligand-activated receptor (Mori, S., Heldin, C.-H., and Claesson-Welsh, L. (1993) J. Biol. Chem. 268, 577-583). In the present study, we have examined effects of different kinds of cell-penetrating proteasome inhibitors, including substrate-related peptidyl aldehydes, Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (where Bu is butyl and Cbz is benzyloxycarbonyl) (PSI) and Cbz-Leu-Leu-norvalinal (MG115), and a Streptomyces metabolite lactacystin, on degradation of the receptor in intact cells with the aim of evaluating the role of the receptor ubiquitination in the proteasome-dependent proteolytic process. These proteasome inhibitors were found to considerably inhibit ligand-stimulated degradation of the wild-type beta-receptor; however, their inhibitory effect was not observed when the cells expressing the ubiquitination-deficient mutant beta-receptor were analyzed. These data suggest that the degradation process of the ligand-stimulated beta-receptor involves the ubiquitin-proteasome proteolytic pathway.
- Published
- 1995
11. Inhibition of Ras/Raf interaction by anti-oncogenic mutants of neurofibromin, the neurofibromatosis type 1 (NF1) gene product, in cell-free systems.
- Author
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Mori, S, Satoh, T, Koide, H, Nakafuku, M, Villafranca, E, and Kaziro, Y
- Abstract
The neurofibromatosis type 1 (NF1) gene encodes a protein, neurofibromin, containing GTPase-activating protein-related domain (GRD) that stimulates intrinsic GTPase activity of Ras protein. By screening a randomly mutagenized NF1-GRD library in Saccharomyces cerevisiae, we isolated two NF1-GRD mutants (NF201 and NF204) with single amino acid substitutions, which suppress the heat shock-sensitive phenotype of the RAS2(G19V) mutant. The NF1-GRD mutants also suppress the oncogenic Ras-induced transformation of NIH 3T3 mouse fibroblasts (Nakafuku, M., Nagamine, M., Ohtoshi, A., Tanaka, K., Toh-e, A., and Kaziro, Y. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6706-6710). In this paper, we investigated the molecular mechanism of inhibition of the transforming Ras-specific function by the NF1-GRD mutants in mammalian cells. In human embryonic kidney (HEK) 293 cells, the mutant NF1-GRDs attenuated the stimulation of mitogen-activated protein kinase by Ras(G12V), but not by platelet-derived growth factor. In cell-free systems, purified recombinant NF1-GRD mutants showed an inhibitory effect on the association of Ras.guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with Raf at several times lower concentrations than the wild type. Furthermore, it was revealed that the binding affinity of the mutant NF1-GRDs toward Ras.GTP gamma S is approximately 5-10 times higher than the wild type. These results suggest that the mutant NF1-GRDs tightly bind to an oncogenic Ras in its GTP-bound active conformation and block the interaction between Ras and its effector, Raf.
- Published
- 1995
12. Structural determinants in the platelet-derived growth factor alpha-receptor implicated in modulation of chemotaxis.
- Author
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Yokote, K, Mori, S, Siegbahn, A, Rönnstrand, L, Wernstedt, C, Heldin, C H, and Claesson-Welsh, L
- Abstract
Activation of the platelet-derived growth factor (PDGF) beta-receptor leads to cell growth and chemotaxis. The PDGF alpha-receptor also mediates a mitogenic signal, but fails to induce cell migration in certain cell types. To examine this difference in signal transduction, a series of point-mutated PDGF alpha-receptors were analyzed. Porcine aortic endothelial cells expressing mutant PDGF alpha-receptors, in which tyrosine residues 768, 993, or 1018 were changed to phenylalanine residues migrated toward PDGF, whereas wild-type alpha-receptors and mutant alpha-receptors changed at tyrosine residues 720, 944, or 988 failed to migrate. All mutant receptors were mitogenically active and their capacity to activate phosphatidylinositol 3'-kinase and phospholipase C-gamma was not different from that of the wild-type receptor. Tyr-768 was found to be phosphorylated in PDGF-stimulated cells; in the Y768F mutant, there was a considerable increase in phosphorylation of Ser-767. Tyr-993 was not phosphorylated, but mutation of this tyrosine residue to a phenylalanine residue resulted in increased efficiency of phosphorylation on Tyr-988. Tyr-1018 is known to be an autophosphorylation site. Phosphorylated Tyr-768 and Tyr-1018 may bind signal transduction molecules involved in negative modulation of the chemotactic signaling capacity, whereas phosphorylated Tyr-988 may mediate increased chemotaxis. Thus our data indicate that the PDGF alpha-receptor has an intrinsic ability to transduce a chemotactic signal, and that this signal is counteracted by overriding negative signals.
- Published
- 1996
13. trans-Activation by the hnRNP K protein involves an increase in RNA synthesis from the reporter genes.
- Author
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Lee, M H, Mori, S, and Raychaudhuri, P
- Abstract
The function of many of the pre-mRNA-binding proteins in mRNA biogenesis is unclear. We have analyzed the biochemical function of the hnRNP K protein by using a mouse cDNA clone. A previous study indicated that the expression of hnRNP K activates c-myc promoter in transient transfection assays. We show that the expression of hnRNP K results in a trans-activation of a variety of RNA polymerase II promoters. The trans-activation function depends on the sequences of hnRNP K that are also necessary for RNA binding. However, the RNA binding motifs are not sufficient for trans-activation. We could identify a mutant that bound RNA in vitro but was impaired in its ability to trans-activate the reporter genes. The trans-activation was not a result of the stabilization of the reporter mRNA, because hnRNP K increased the steady-state level of the reporter mRNA without altering its decay rate. By doing nuclear run-on assays, we provide evidence that the hnRNP K protein trans-activates the reporter genes by increasing the level of transcription.
- Published
- 1996
14. Neutron crystallography and quantum chemical analysis of bilin reductase PcyA mutants reveal substrate and catalytic residue protonation states.
- Author
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Joutsuka T, Nanasawa R, Igarashi K, Horie K, Sugishima M, Hagiwara Y, Wada K, Fukuyama K, Yano N, Mori S, Ostermann A, Kusaka K, and Unno M
- Subjects
- Biliverdine chemistry, Catalysis, Crystallography, Mutation, Bile Pigments biosynthesis, Bile Pigments chemistry, Oxidoreductases genetics, Oxidoreductases chemistry
- Abstract
PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH
+ ) in the wildtype (WT) PcyA-BV complex, and a nearby catalytic residue Asp105 was found to have two conformations (protonated and deprotonated). Semiempirical calculations have suggested that the protonation states of BV are reflected in the absorption spectrum of the WT PcyA-BV complex. In the previously determined absorption spectra of the PcyA D105N and I86D mutants, complexed with BV, a peak at 730 nm, observed in the WT, disappeared and increased, respectively. Here, we performed neutron crystallography and quantum chemical analysis of the D105N-BV and I86D-BV complexes to determine the protonation states of BV and the surrounding residues and study the correlation between the absorption spectra and protonation states around BV. Neutron structures elucidated that BV in the D105N mutant is in a neutral state, whereas that in the I86D mutant is dominantly in a protonated state. Glu76 and His88 showed different hydrogen bonding with surrounding residues compared with WT PcyA, further explaining why D105N and I86D have much lower activities for phycocyanobilin synthesis than the WT PcyA. Our quantum mechanics/molecular mechanics calculations of the absorption spectra showed that the spectral change in D105N arises from Glu76 deprotonation, consistent with the neutron structure. Collectively, our findings reveal more mechanistic details of bilin pigment biosynthesis., 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
- 2023
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- View/download PDF
15. Intracellular and extracellular ATP coordinately regulate the inverse correlation between osteoclast survival and bone resorption.
- Author
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Miyazaki T, Iwasawa M, Nakashima T, Mori S, Shigemoto K, Nakamura H, Katagiri H, Takayanagi H, and Tanaka S
- Subjects
- Animals, Apoptosis genetics, Blotting, Western, Body Size genetics, Cell Survival, Cells, Cultured, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Down-Regulation drug effects, Extracellular Space metabolism, Female, High Mobility Group Proteins genetics, High Mobility Group Proteins metabolism, Intracellular Space metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Mitochondria genetics, Mitochondria metabolism, RANK Ligand pharmacology, RNA Interference, Receptors, Purinergic P2X7 genetics, Receptors, Purinergic P2X7 metabolism, bcl-X Protein genetics, bcl-X Protein metabolism, Adenosine Triphosphate metabolism, Aging, Bone Resorption metabolism, Osteoclasts metabolism
- Abstract
Osteoclasts, highly differentiated bone-resorbing cells of hematopoietic origin, have two conflicting tendencies: a lower capacity to survive and a higher capacity to execute energy-consuming activities such as bone resorption. Here, we report that when compared with their precursors, mature mitochondria-rich osteoclasts have lower levels of intracellular ATP, which is associated with receptor activator of nuclear factor κ-B ligand (RANKL)-induced Bcl-x(L) down-regulation. Severe ATP depletion, caused by disrupting mitochondrial transcription factor A (Tfam) gene, leads to increased bone-resorbing activity despite accelerated apoptosis. Although AMP-activated protein kinase (AMPK) activation by ATP depletion is not involved in the regulation of osteoclast function, the release of ATP from intracellular stores negatively regulates bone-resorbing activity through an autocrine/paracrine feedback loop by altering cytoskeletal structures. Furthermore, osteoclasts derived from aged mice exhibit reduced mitochondrial DNA (mtDNA) and intracellular ATP levels with increased bone-resorbing activity, implicating the possible involvement of age-related mitochondrial dysfunction in osteoporosis. Thus, our study provides evidence for a mechanism underlying the control of cellular functions by reciprocal changes in intracellular and extracellular ATP, which regulate the negative correlation between osteoclast survival and bone resorption.
- Published
- 2012
- Full Text
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16. Rice OsYSL15 is an iron-regulated iron(III)-deoxymugineic acid transporter expressed in the roots and is essential for iron uptake in early growth of the seedlings.
- Author
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Inoue H, Kobayashi T, Nozoye T, Takahashi M, Kakei Y, Suzuki K, Nakazono M, Nakanishi H, Mori S, and Nishizawa NK
- Subjects
- Animals, Azetidinecarboxylic Acid analogs & derivatives, Azetidinecarboxylic Acid metabolism, Base Sequence, Cell Membrane genetics, Female, Gene Expression Regulation, Plant, Genetic Complementation Test, Iron Deficiencies, Molecular Sequence Data, Mutation, Oocytes cytology, Organ Specificity physiology, Organic Anion Transporters genetics, Oryza growth & development, Plant Proteins genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Xenopus laevis, Cell Membrane metabolism, Iron metabolism, Organic Anion Transporters biosynthesis, Oryza metabolism, Plant Proteins biosynthesis
- Abstract
Graminaceous plants take up iron through YS1 (yellow stripe 1) and YS1-like (YSL) transporters using iron-chelating compounds known as mugineic acid family phytosiderophores. We examined the expression of 18 rice (Oryza sativa L.) YSL genes (OsYSL1-18) in the epidermis/exodermis, cortex, and stele of rice roots. Expression of OsYSL15 in root epidermis and stele was induced by iron deficiency and showed daily fluctuation. OsYSL15 restored a yeast mutant defective in iron uptake when supplied with iron(III)-deoxymugineic acid and transported iron(III)-deoxymugineic acid in Xenopus laevis oocytes. An OsYSL15-green fluorescent protein fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. OsYSL15 promoter-beta-glucuronidase analysis revealed that OsYSL15 expression in roots was dominant in the epidermis/exodermis and phloem cells under conditions of iron deficiency and was detected only in phloem under iron sufficiency. These results strongly suggest that OsYSL15 is the dominant iron(III)-deoxymugineic acid transporter responsible for iron uptake from the rhizosphere and is also responsible for phloem transport of iron. OsYSL15 was also expressed in flowers, developing seeds, and in the embryonic scutellar epithelial cells during seed germination. OsYSL15 knockdown seedlings showed severe arrest in germination and early growth and were rescued by high iron supply. These results demonstrate that rice OsYSL15 plays a crucial role in iron homeostasis during the early stages of growth.
- Published
- 2009
- Full Text
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17. Direct binding of integrin alphavbeta3 to FGF1 plays a role in FGF1 signaling.
- Author
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Mori S, Wu CY, Yamaji S, Saegusa J, Shi B, Ma Z, Kuwabara Y, Lam KS, Isseroff RR, Takada YK, and Takada Y
- Subjects
- Animals, Binding Sites, CHO Cells, Cell Proliferation, Cricetinae, Cricetulus, Fibroblast Growth Factors metabolism, Humans, Models, Biological, Mutation, Protein Binding, Signal Transduction, Surface Plasmon Resonance, Fibroblast Growth Factor 1 chemistry, Integrin alphaVbeta3 chemistry
- Abstract
Integrins play a role in fibroblast growth factor (FGF) signaling through cross-talk with FGF receptors (FGFRs), but the mechanism underlying the cross-talk is unknown. We discovered that FGF1 directly bound to soluble and cell-surface integrin alphavbeta3 (K(D) about 1 microm). Antagonists to alphavbeta3 (monoclonal antibody 7E3 and cyclic RGDfV) blocked this interaction. alphavbeta3 was the predominant, if not the only, integrin that bound to FGF1, because FGF1 bound only weakly to several beta1 integrins tested. We presented evidence that the CYDMKTTC sequence (the specificity loop) within the ligand-binding site of beta3 plays a role in FGF1 binding. We found that the integrin-binding site of FGF1 overlaps with the heparin-binding site but is distinct from the FGFR-binding site using docking simulation and mutagenesis. We identified an FGF1 mutant (R50E) that was defective in integrin binding but still bound to heparin and FGFR. R50E was defective in inducing DNA synthesis, cell proliferation, cell migration, and chemotaxis, suggesting that the direct integrin binding to FGF1 is critical for FGF signaling. Nevertheless, R50E induced phosphorylation of FGFR1 and FRS2alpha and activation of AKT and ERK1/2. These results suggest that the defect in R50E in FGF signaling is not in the initial activation of FGF signaling pathway components, but in the later steps in FGF signaling. We propose that R50E is a useful tool to identify the role of integrins in FGF signaling.
- Published
- 2008
- Full Text
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18. A novel NAC transcription factor, IDEF2, that recognizes the iron deficiency-responsive element 2 regulates the genes involved in iron homeostasis in plants.
- Author
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Ogo Y, Kobayashi T, Nakanishi Itai R, Nakanishi H, Kakei Y, Takahashi M, Toki S, Mori S, and Nishizawa NK
- Subjects
- Base Sequence, Binding Sites, Cation Transport Proteins genetics, Cation Transport Proteins metabolism, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Oryza genetics, Phylogeny, RNA Interference, Response Elements, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Transcription Factors genetics, Gene Expression Regulation, Plant, Homeostasis, Iron metabolism, Iron Deficiencies, Oryza metabolism, Transcription Factors metabolism
- Abstract
Iron is essential for most living organisms, and thus iron deficiency poses a major abiotic stress in crop production. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified a novel transcription factor of rice and barley, IDEF2, which specifically binds to the iron deficiency-responsive cis-acting element 2 (IDE2) by yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets experiment revealed that IDEF2 predominantly recognized CA(A/C)G(T/C)(T/C/A)(T/C/A) within IDE2 as the core-binding site. IDEF2 transcripts are constitutively present in rice roots and leaves. Repression of the function of IDEF2 by the RNA interference (RNAi) technique and chimeric repressor gene-silencing technology (CRES-T) caused aberrant iron homeostasis in rice. Several genes up-regulated by iron deficiency, including the Fe(II)-nicotianamine transporter gene OsYSL2, were less induced by iron deficiency in the RNAi rice of IDEF2, suggesting that IDEF2 is involved in the regulation of these genes. Many genes with repressed expression in IDEF2 RNAi rice possessed the IDEF2-binding core sites in their promoters, and the flanking sequences were also highly homologous to IDE2. IDEF2 bound to OsYSL2 promoter region containing the binding core site, suggesting direct regulation of OsYSL2 expression. These results reveal novel cis-element/trans-factor interactions functionally associated with iron homeostasis.
- Published
- 2008
- Full Text
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19. Perturbation of the activity of replication origin by meiosis-specific transcription.
- Author
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Mori S and Shirahige K
- Subjects
- Cell Cycle Proteins genetics, Chromosomes, Fungal genetics, DNA-Binding Proteins genetics, Open Reading Frames genetics, Repressor Proteins genetics, S Phase physiology, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae Proteins genetics, Transcription Factors genetics, Transcription, Genetic physiology, Chromosomes, Fungal metabolism, DNA Replication physiology, DNA, Fungal biosynthesis, Meiosis physiology, Replication Origin physiology, Saccharomyces cerevisiae physiology
- Abstract
We have determined the activity of all ARSs on the Saccharomyces cerevisiae chromosome VI as chromosomal replication origins in premeiotic S-phase by neutral/neutral two-dimensional gel electrophoresis. The comparison of origin activity of each origin in mitotic and premeiotic S-phase showed that one of the most efficient origins in mitotic S-phase, ARS605, was completely inhibited in premeiotic S-phase. ARS605 is located within the open reading frame of MSH4 gene that is transcribed specifically during an early stage of meiosis. Systematic analysis of relationships between MSH4 transcription and ARS605 origin activity revealed that transcription of MSH4 inhibited the ARS605 origin activity by removing origin recognition complex from ARS605. Deletion of UME6, a transcription factor responsible for repressing MSH4 during mitotic S-phase, resulted in inactivation of ARS605 in mitosis. Our finding is the first demonstration that the transcriptional regulation on the replication origin activity is related to changes in cell physiology. These results may provide insights into changes in replication origin activity in embryonic cell cycle during early developmental stages.
- Published
- 2007
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20. Cloning and characterization of deoxymugineic acid synthase genes from graminaceous plants.
- Author
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Bashir K, Inoue H, Nagasaka S, Takahashi M, Nakanishi H, Mori S, and Nishizawa NK
- Subjects
- Amino Acid Sequence, Azetidinecarboxylic Acid analogs & derivatives, Azetidinecarboxylic Acid metabolism, Hordeum genetics, Hydrogen-Ion Concentration, Immunohistochemistry, Iron metabolism, Iron Deficiencies, Mixed Function Oxygenases chemistry, Mixed Function Oxygenases isolation & purification, Mixed Function Oxygenases metabolism, Molecular Sequence Data, Oryza genetics, Phylogeny, Plant Roots genetics, Plant Roots metabolism, Plants, Genetically Modified enzymology, Plants, Genetically Modified genetics, Plasmids, Promoter Regions, Genetic, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Siderophores biosynthesis, Triticum genetics, Zea mays genetics, Cloning, Molecular, Genes, Plant, Hordeum enzymology, Mixed Function Oxygenases genetics, Oryza enzymology, Triticum enzymology, Zea mays enzymology
- Abstract
Graminaceous plants have evolved a unique mechanism to acquire iron through the secretion of a family of small molecules, called mugineic acid family phytosiderophores (MAs). All MAs are synthesized from l-Met, sharing the same pathway from l-Met to 2'-deoxymugineic acid (DMA). DMA is synthesized through the reduction of a 3''-keto intermediate by deoxymugineic acid synthase (DMAS). We have isolated DMAS genes from rice (OsDMAS1), barley (HvDMAS1), wheat (TaD-MAS1), and maize (ZmDMAS1). Their nucleotide sequences indicate that OsDMAS1 encodes a predicted polypeptide of 318 amino acids, whereas the other three orthologs all encode predicted polypeptides of 314 amino acids and are highly homologous (82-97.5%) to each other. The DMAS proteins belong to the aldo-keto reductase superfamily 4 (AKR4) but do not fall within the existing subfamilies of AKR4 and appear to constitute a new subfamily within the AKR4 group. All of the proteins showed DMA synthesis activity in vitro. Their enzymatic activities were highest at pH 8-9, consistent with the hypothesis that DMA is synthesized in subcellular vesicles. Northern blot analysis revealed that the expression of each of the above DMAS genes is up-regulated under iron-deficient conditions in root tissue, and that of the genes OsDMAS1 and TaDMAS1 is up-regulated in shoot tissue. OsDMAS1 promoter-GUS analysis in iron-sufficient roots showed that its expression is restricted to cells participating in long distance transport and that it is highly up-regulated in the entire root under iron-deficient conditions. In shoot tissue, OsDMAS1 promoter drove expression in vascular bundles specifically under iron-deficient conditions.
- Published
- 2006
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21. Non-cytotoxic cobra cardiotoxin A5 binds to alpha(v)beta3 integrin and inhibits bone resorption. Identification of cardiotoxins as non-RGD integrin-binding proteins of the Ly-6 family.
- Author
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Wu PL, Lee SC, Chuang CC, Mori S, Akakura N, Wu WG, and Takada Y
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Cattle, Cell Adhesion, Cell Differentiation, Cell Line, Cell Proliferation, Cobra Cardiotoxin Proteins metabolism, Dose-Response Relationship, Drug, Elapidae, Endothelial Cells metabolism, Glycoproteins chemistry, Inflammation, Integrin alphaVbeta3 metabolism, Integrins chemistry, Integrins metabolism, Ligands, Mice, Microscopy, Electron, Scanning, Molecular Sequence Data, Necrosis, Neoplasms metabolism, Osteoclasts metabolism, Protein Binding, Protein Structure, Secondary, Pulmonary Artery metabolism, Recombinant Proteins chemistry, Sequence Homology, Amino Acid, Structure-Activity Relationship, Time Factors, Bone Resorption, Cobra Cardiotoxin Proteins chemistry, Integrin alphaVbeta3 chemistry, Oligopeptides chemistry
- Abstract
Severe tissue necrosis with a retarded wound healing process is a major symptom of a cobra snakebite. Cardiotoxins (CTXs) are major components of cobra venoms that belong to the Ly-6 protein family and are implicated in tissue damage. The interaction of the major CTX from Taiwan cobra, i.e. CTX A3, with sulfatides in the cell membrane has recently been shown to induce pore formation and cell internalization and to be responsible for cytotoxicity in cardiomyocytes (Wang, C.-H., Liu, J.-H., Lee, S.-C., Hsiao, C.-D., and Wu, W.-g. (2006) J. Biol. Chem. 281, 656-667). We show here that one of the non-cytotoxic CTXs, i.e. CTX A5 or cardiotoxin-like basic polypeptide, from Taiwan cobra specifically bound to alpha(v)beta3 integrin and inhibited bone resorption activity. We found that both membrane-bound and recombinant soluble alpha(v)beta3 integrins bound specifically to CTX A5 in a dose-dependent manner. Surface plasmon resonance analysis showed that human soluble alpha(v)beta3 bound to CTX A5 with an apparent affinity of approximately 0.3 microM. Calf pulmonary artery endothelial cells, which constitutively express alpha(v)beta3, showed a CTX A5 binding profile similar to that of membrane-bound and soluble alpha(v)beta3 integrins, suggesting that endothelial cells are a potential target for CTX action. We tested whether CTX A5 inhibits osteoclast differentiation and bone resorption, a process known to be involved in alpha(v)beta3 binding and inhibited by RGD-containing peptides. We demonstrate that CTX A5 inhibited both activities at a micromolar range by binding to murine alpha(v)beta3 integrin in osteoclasts and that CTX A5 co-localized with beta3 integrin. Finally, after comparing the integrin binding affinity among CTX homologs, we propose that the amino acid residues near the two loops of CTX A5 are involved in integrin binding. These results identify CTX A5 as a non-RGD integrin-binding protein with therapeutic potential as an integrin antagonist.
- Published
- 2006
- Full Text
- View/download PDF
22. Molecular conversion of NAD kinase to NADH kinase through single amino acid residue substitution.
- Author
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Mori S, Kawai S, Shi F, Mikami B, and Murata K
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphorylation, Phosphotransferases (Alcohol Group Acceptor) chemistry, Phosphotransferases (Alcohol Group Acceptor) genetics, Protein Conformation, Sequence Homology, Amino Acid, Amino Acids metabolism, Phosphotransferases (Alcohol Group Acceptor) metabolism
- Abstract
NAD kinase phosphorylates NAD+ to form NADP+ and is strictly specific to NAD+, whereas NADH kinase phosphorylates both NAD+ and NADH, thereby showing relaxed substrate specificity. Based on their primary and tertiary structures, the difference in the substrate specificities between NAD and NADH kinases was proposed to be caused by one aligned residue: Gly or polar amino acid (Gln or Thr) in five NADH kinases and a charged amino acid (Arg) in two NAD kinases. The substitution of Arg with Gly in the two NAD kinases relaxed the substrate specificity (i.e. converted the NAD kinases to NADH kinases). The substitution of Arg in one NAD kinase with polar amino acids also relaxed the substrate specificity, whereas substitution with charged and hydrophobic amino acids did not show a similar result. In contrast, the substitution of Gly with Arg in one NADH kinase failed to convert it to NAD kinase. These results suggest that a charged or hydrophobic amino acid residue in the position of interest is crucial for strict specificity of NAD kinases to NAD+, whereas Gly or polar amino acid residue is not the sole determinant for the relaxed substrate specificity of NADH kinases. The significance of the conservation of the residue at the position in 207 NAD kinase homologues is also discussed.
- Published
- 2005
- Full Text
- View/download PDF
23. Crystal structure of bacterial inorganic polyphosphate/ATP-glucomannokinase. Insights into kinase evolution.
- Author
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Mukai T, Kawai S, Mori S, Mikami B, and Murata K
- Subjects
- Amino Acid Sequence, Catalytic Domain, Crystallography, X-Ray, Molecular Sequence Data, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Alignment, Arthrobacter enzymology, Evolution, Molecular, Phosphotransferases (Alcohol Group Acceptor) chemistry
- Abstract
Inorganic polyphosphate (poly(P)) is a biological high energy compound presumed to be an ancient energy carrier preceding ATP. Several poly(P)-dependent kinases that use poly(P) as a phosphoryl donor are known to function in bacteria, but crystal structures of these kinases have not been solved. Here we present the crystal structure of bacterial poly(P)/ATP-glucomannokinase, belonging to Gram-positive bacterial glucokinase, complexed with 1 glucose molecule and 2 phosphate molecules at 1.8 A resolution, being the first among poly(P)-dependent kinases and bacterial glucokinases. The poly(P)/ATP-glucomannokinase structure enabled us to understand the structural relationship of bacterial glucokinase to eucaryotic hexokinase and ADP-glucokinase, which has remained a matter of debate. These comparisons also enabled us to propose putative binding sites for phosphoryl groups for ATP and especially for poly(P) and to obtain insights into the evolution of kinase, particularly from primordial poly(P)-specific to ubiquitous ATP-specific proteins.
- Published
- 2004
- Full Text
- View/download PDF
24. ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands.
- Author
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Tanaka M, Nanba D, Mori S, Shiba F, Ishiguro H, Yoshino K, Matsuura N, and Higashiyama S
- Subjects
- ADAM Proteins, ADAM10 Protein, ADAM12 Protein, Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport, Amino Acid Sequence, Amyloid Precursor Protein Secretases, Binding Sites, Carrier Proteins genetics, Carrier Proteins metabolism, Cell Line, Cytoskeletal Proteins, Disintegrins metabolism, Humans, Ligands, Molecular Sequence Data, Muscle Proteins genetics, Muscle Proteins metabolism, Protein Binding, RNA, Messenger analysis, Tissue Distribution, Two-Hybrid System Techniques, Carrier Proteins physiology, ErbB Receptors metabolism, Membrane Proteins metabolism, Metalloendopeptidases metabolism, Muscle Proteins physiology
- Abstract
A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.
- Published
- 2004
- Full Text
- View/download PDF
25. ZmYS1 functions as a proton-coupled symporter for phytosiderophore- and nicotianamine-chelated metals.
- Author
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Schaaf G, Ludewig U, Erenoglu BE, Mori S, Kitahara T, and von Wirén N
- Subjects
- Animals, Azetidinecarboxylic Acid metabolism, Ferritins metabolism, Ion Transport, Xenopus laevis, Azetidinecarboxylic Acid analogs & derivatives, Carrier Proteins metabolism, Membrane Proteins metabolism, Membrane Transport Proteins, Metals metabolism, Plant Proteins metabolism, Symporters metabolism, Zea mays metabolism
- Abstract
Among higher plants graminaceous species have the unique ability to efficiently acquire iron from alkaline soils with low iron solubility by secreting phytosiderophores, which are hexadentate metal chelators with high affinity for Fe(III). Iron(III)-phytosiderophores are subsequently taken up by roots via YS1 transporters, that belong to the OPT oligopeptide transporter family. Despite its physiological importance at alkaline pH, uptake of Fe-phytosiderophores into roots of wild-type maize plants was greater at acidic pH and sensitive to the proton uncoupler CCCP. To access the mechanism of Fe-phytosiderophore acquisition, ZmYS1 was expressed in an iron uptake-defective yeast mutant and in Xenopus oocytes, where ZmYS1-dependent Fe-phytosiderophore transport was stimulated at acidic pH and sensitive to CCCP. Electrophysiological analysis in oocytes demonstrated that Fephytosiderophore transport depends on proton cotransport and on the membrane potential, which allows ZmYS1-mediated transport even at alkaline pH. We further investigated substrate specificity and observed that ZmYS1 complemented the growth defect of the zinc uptake-defective yeast mutant zap1 and transported various phytosiderophore-bound metals into oocytes, including zinc, copper, nickel, and, at a lower rate, also manganese and cadmium. Unexpectedly, ZmYS1 also transported Ni(II), Fe(II), and Fe(III) complexes with nicotianamine, a structural analog of phytosiderophores, which has been shown to act as an intracellular metal chelator in all higher plants. Our results show that ZmYS1 encodes a proton-coupled broad-range metal-phytosiderophore transporter that additionally transports Fe- and Ni-nicotianamine. These biochemical properties indicate a novel role of YS1 transporters for heavy metal homeostasis in plants.
- Published
- 2004
- Full Text
- View/download PDF
26. PACSIN3 binds ADAM12/meltrin alpha and up-regulates ectodomain shedding of heparin-binding epidermal growth factor-like growth factor.
- Author
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Mori S, Tanaka M, Nanba D, Nishiwaki E, Ishiguro H, Higashiyama S, and Matsuura N
- Subjects
- ADAM Proteins, ADAM12 Protein, Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Angiotensin II chemistry, Cell Line, Cell Membrane metabolism, Cytoplasm metabolism, Cytoskeletal Proteins, Epidermal Growth Factor metabolism, Gene Deletion, Genetic Vectors, Glutathione Transferase metabolism, Heparin-binding EGF-like Growth Factor, Humans, Immunoblotting, Intercellular Signaling Peptides and Proteins, Intracellular Signaling Peptides and Proteins, Membrane Proteins metabolism, Metalloendopeptidases metabolism, Microscopy, Confocal, Molecular Sequence Data, Muscle Proteins metabolism, Phosphoproteins metabolism, Precipitin Tests, Proline chemistry, Protein Binding, Protein Structure, Tertiary, RNA, Small Interfering metabolism, Recombinant Fusion Proteins metabolism, Tetradecanoylphorbol Acetate, Transcriptional Activation, Transfection, Two-Hybrid System Techniques, Up-Regulation, Epidermal Growth Factor chemistry, Membrane Proteins chemistry, Metalloendopeptidases chemistry, Muscle Proteins chemistry, Phosphoproteins physiology
- Abstract
A disintegrin and metalloprotease 12 (ADAM12/meltrin alpha) is a key enzyme implicated in the ectodomain shedding of membrane-anchored heparin-binding epidermal growth factor (EGF)-like growth factor (proHB-EGF)-dependent epidermal growth factor receptor (EGFR) transactivation. However, the activation mechanisms of ADAM12 are obscure. To determine how ADAM12 is activated, we screened proteins that bind to the cytoplasmic domain of ADAM12 using a yeast two-hybrid system and identified a protein called PACSIN3 that contains a Src homology 3 domain. An analysis of interactions between ADAM12 and PACSIN3 using glutathione S-transferase fusion protein revealed that a proline-rich region (amino acid residues 829-840) of ADAM12 was required to bind PACSIN3. Furthermore, co-immunoprecipitation and co-localization analyses of ADAM12 and PACSIN3 proteins also revealed their interaction in mammalian cells expressing both of them. The overexpression of PACSIN3 in HT1080 cells enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced proHB-EGF shedding. Furthermore, knockdown of endogenous PACSIN3 by small interfering RNA in HT1080 cells significantly attenuated the shedding of proHB-EGF induced by TPA and angiotensin II. Our data indicate that PACSIN3 has a novel function as an up-regulator in the signaling of proHB-EGF shedding induced by TPA and angiotensin II.
- Published
- 2003
- Full Text
- View/download PDF
27. Methionine aminopeptidase 2 is a new target for the metastasis-associated protein, S100A4.
- Author
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Endo H, Takenaga K, Kanno T, Satoh H, and Mori S
- Subjects
- Amino Acid Sequence, Aminopeptidases antagonists & inhibitors, Angiogenesis Inhibitors pharmacology, Animals, Calcium metabolism, Calmodulin metabolism, Cyclohexanes, Endothelium, Vascular drug effects, Endothelium, Vascular metabolism, Fatty Acids, Unsaturated pharmacology, Fibroblast Growth Factors pharmacology, Humans, Metalloendopeptidases antagonists & inhibitors, Methionine metabolism, Mice, Molecular Sequence Data, S100 Calcium-Binding Protein A4, Sesquiterpenes pharmacology, Spodoptera, Tumor Cells, Cultured, Aminopeptidases metabolism, Metalloendopeptidases metabolism, Neoplasm Metastasis, S100 Proteins metabolism
- Abstract
S100A4 is an EF-hand type calcium-binding protein that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins. Here we report that S100A4 physically interacts with methionine aminopeptidase 2 (MetAP2), the primary target for potent angiogenesis inhibitors, fumagillin and ovalicin. Using a yeast two-hybrid screen, S100A4 was found to interact with the N-terminal half of MetAP2. In vitro pull-down assays showed that S100A4 associates with MetAP2 in a calcium-dependent manner. In addition, the binding site of S100A4 was found located within the region between amino acid residues 170 and 229 of MetAP2. In vivo interaction of S100A4 with MetAP2 was verified by co-immunoprecipitation analysis. Immunofluorescent staining revealed that S100A4 and MetAP2 were co-localized in both quiescent and basic fibroblast growth factor-treated murine endothelial MSS31 cells, in the latter of which a significant change of intracellular distribution of both proteins was observed. Although the binding of S100A4 did not affect the in vitro methionine aminopeptidase activity of MetAP2, the cytochemical observation suggests a possible involvement of S100A4 in the regulation of MetAP2 activity through changing its localization, thereby modulating the N-terminal methionine processing of nascent substrates. These results may offer an essential clue for understanding the functional role of S100A4 in regulating endothelial cell growth and tumor metastasis.
- Published
- 2002
- Full Text
- View/download PDF
28. A triad of serum response factor and the GATA and NK families governs the transcription of smooth and cardiac muscle genes.
- Author
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Nishida W, Nakamura M, Mori S, Takahashi M, Ohkawa Y, Tadokoro S, Yoshida K, Hiwada K, Hayashi K, and Sobue K
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Calmodulin-Binding Proteins biosynthesis, Cell Nucleus metabolism, Chickens, DNA-Binding Proteins biosynthesis, DNA-Binding Proteins metabolism, Deoxyribonuclease I metabolism, GATA6 Transcription Factor, Integrin alpha1, Mice, Molecular Sequence Data, Mutation, Plasmids metabolism, Promoter Regions, Genetic, Protein Binding, Protein Structure, Tertiary, RNA, Complementary metabolism, RNA, Messenger metabolism, Rats, Transcription Factors metabolism, Transcriptional Activation, Antigens, CD biosynthesis, Homeodomain Proteins biosynthesis, Microfilament Proteins biosynthesis, Muscle Proteins biosynthesis, Muscle, Smooth metabolism, Myocardium metabolism, Serum Response Factor chemistry, Serum Response Factor metabolism, Transcription Factors biosynthesis, Transcription, Genetic
- Abstract
Serum response factor and the (CC(A/T)(6)GG) (CArG) box interact to promote the transcription of c-fos and muscle genes; this tissue-specific activity may require co-regulators for serum response factor. The alpha(1) integrin promoter contains two cis-elements besides the CArG box: a TAAT sequence, a consensus binding site for homeoproteins, and a GATA-binding box. As a candidate TAAT-binding factor, we cloned an NK family homeobox gene, Nkx-3.2, which is expressed mainly in smooth muscle tissues and skeletal structures. Nkx-3.2, serum response factor, and GATA-6 were co-expressed only in the medial smooth muscle layer of arteries. These three transcription factors formed a complex with their corresponding cis-elements and cooperatively transactivated smooth muscle genes, including alpha(1) integrin, SM22alpha, and caldesmon. Cardiac muscle-specific members of the NK and GATA families exist, and the triad of Nkx-2.5, serum response factor, and GATA-4 also transactivated the cardiac atrial natriuretic factor gene, which contains a CArG-like box, a GATA-binding box, and an NK-binding element. Our findings demonstrate that smooth and cardiac muscle have a shared transcriptional machinery and that the GATA and NK families confer muscle specificity on the serum response factor/CArG interaction.
- Published
- 2002
- Full Text
- View/download PDF
29. The cytoplasmic shuttling and subsequent degradation of p27Kip1 mediated by Jab1/CSN5 and the COP9 signalosome complex.
- Author
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Tomoda K, Kubota Y, Arata Y, Mori S, Maeda M, Tanaka T, Yoshida M, Yoneda-Kato N, and Kato JY
- Subjects
- 3T3 Cells, Amino Acid Motifs, Amino Acid Sequence, Animals, COP9 Signalosome Complex, COS Cells, Cell Nucleus metabolism, Cyclin-Dependent Kinase Inhibitor p27, Hydrolysis, Intracellular Signaling Peptides and Proteins, Mice, Molecular Sequence Data, Peptide Hydrolases, Protein Binding, Protein Transport, Sequence Homology, Amino Acid, Cell Cycle Proteins metabolism, Cytoplasm metabolism, DNA-Binding Proteins physiology, Signal Transduction, Transcription Factors physiology, Tumor Suppressor Proteins metabolism
- Abstract
The fifth component of the COP9 signalosome complex, Jab1/CSN5, directly binds to and induces specific down-regulation of the cyclin-dependent kinase inhibitor p27 (p27(Kip1)). Nuclear-cytoplasmic translocation plays an important role because leptomycin B (LMB), a chemical inhibitor of CRM1-dependent nuclear export, prevents p27 degradation mediated by Jab1/CSN5. Here we show that Jab1/CSN5 functions as an adaptor between p27 and CRM1 to induce nuclear export and subsequent degradation. Jab1/CSN5, but not p27, contains a typical leucine-rich nuclear export signal (NES) sequence conserved among different species, through which CRM1 bound to Jab1/CSN5 in an LMB-sensitive manner. Alteration of conserved leucine residues to alanine within Jab1/CSN5-NES abolished the interaction with CRM1 in vitro and impaired LMB-sensitive nuclear export and the ability to induce p27 breakdown in cultured cells. A Jab1/CSN5 truncation mutant lacking NES reversed p27 down-regulation induced by the full-length Jab1/CSN5, indicating that this mutant functions as a dominant negative (DN-Jab1). Introduction of DN-Jab1 into proliferating fibroblasts increased the level of p27 protein, thereby inducing growth arrest of the cells. Random mutagenesis analysis revealed that specific aspartic acid, leucine, and asparagine residues contained in the Jab1/CSN5-binding domain of p27 were required for interaction with Jab1/CSN5 and for down-regulation of p27. Glycerol gradient and cell fractionation experiments showed that at least two different forms of Jab1/CSN5-containing complexes existed within the cell. One is the conventional 450-kDa COP9 signalosome (CSN) complex located in the nucleus, and the other is much smaller (around 100-kDa), containing only a subset of CSN components (CSN4-8 but not CSN1-3), and mainly located in the cytoplasm. Treatment of cells with LMB greatly reduced the level of the smaller complex, suggesting that it originated from the CSN complex by nuclear export. Besides Jab1/CSN5, CSN3, -6, -7, and -8 were capable of inducing p27 down-regulation, when ectopically expressed. These results indicate that cytoplasmic shuttling regulated by Jab1/CSN5 and other CSN components may be a new pathway to control the intracellular abundance of the key cell cycle regulator.
- Published
- 2002
- Full Text
- View/download PDF
30. A tyrosine residue in the juxtamembrane segment of the platelet-derived growth factor beta-receptor is critical for ligand-mediated endocytosis.
- Author
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Mori S, Rönnstrand L, Claesson-Welsh L, and Heldin CH
- Subjects
- Amino Acid Sequence, Animals, Aorta, Becaplermin, Clone Cells, Humans, Iodine Radioisotopes, Kinetics, Molecular Sequence Data, Mutagenesis, Site-Directed, Proto-Oncogene Proteins c-sis, Receptor, IGF Type 2 chemistry, Receptors, Platelet-Derived Growth Factor biosynthesis, Receptors, Platelet-Derived Growth Factor chemistry, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Swine, Transfection, Endocytosis, Endothelium, Vascular metabolism, Platelet-Derived Growth Factor metabolism, Receptors, Platelet-Derived Growth Factor metabolism, Tyrosine
- Abstract
The importance of tyrosine residues in ligand-mediated endocytosis of the platelet-derived growth factor beta-receptor was analyzed using a series of tyrosine residue-mutated beta-receptors, which together cover all of the tyrosine residues in the juxtamembrane segment, the kinase insert, and the carboxyl-terminal tail; also certain of the tyrosine residues within the first and second parts of the kinase domain were examined. Of all of these tyrosine residues, only Tyr-579 seemed to be important for internalization, since mutation of this residue resulted in substantial reduction in the rate of ligand-induced receptor internalization (approximately 60% of the wild-type level). Replacement of Tyr-579 by either an aromatic (Phe) or a nonaromatic (Asp) residue reduced the efficiency of the mutant receptors in internalization to the same extent, suggesting that the role of Tyr-579 in the beta-receptor is different from that of the previously described tyrosine-based internalization motifs, which were first determined for the low density lipoprotein receptor. Tyr-579 has been found to be an autophosphorylation site in the beta-receptor. Moreover, the internalization rate of a kinase negative receptor mutant was not altered by the additional mutation of Tyr-579. Thus, it is likely that phosphorylation of Tyr-579 is important for ligand-induced internalization of the beta-receptor.
- Published
- 1994
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