Search PubMed⌕ Search

Biomedical subjects

T Nishimoto

Publications and source records attributed to T Nishimoto.

At least 37 records · Page 2Linked to original sources
PubMed2000

A subunit of the mammalian oligosaccharyltransferase, DAD1, interacts with Mcl-1, one of the bcl-2 protein family.

DAD1 is a mammalian homologue of Saccharomyces cerevisiae Ost2p, a subunit of the oligosaccharyltransferase complex. Loss of its function induces apoptosis in hamster BHK21 cells. By means of a two-hybrid method involving DAD1 as bait, the C-terminal region of Mcl-1, one of the bcl-2 family, was isolated. Consistently, DAD1 binds well to Mcl-1 in COS cells when overexpressed. On deletion analysis, the C-terminal domain of Mcl-1 containing BH(2) (bcl-2 homologous domain) was found to be essential for the interaction with DAD1. On the other hand, the C-terminal half of DAD1 was concluded to be essential for the interaction with Mcl-1. Surprisingly, a DeltaC-DAD1 mutant lacking only 4 amino acid residues from the C-terminus did not complement the tsBN7 mutation, while it interacted well with Mcl-1. In contrast, DeltaN-DAD1 lacking 20 amino acid residues from the N-terminus still exhibited the ability to complement the tsBN7 mutation. Thus, the C-terminus of DAD1 was suggested to play an important role in N-linked glycosylation and to complement the tsBN7 mutation. Mcl-1 may be required for the inhibition of apoptotic cell death caused by a loss of DAD1.

Animals↗
PubMed1999

Identification of XDRP1; a Xenopus protein related to yeast Dsk2p binds to the N-terminus of cyclin A and inhibits its degradation.

Using the N-terminus of cyclin A1 in a two-hybrid screen as a bait, we identified a Xenopus protein, XDRP1, that contains a ubiquitin-like domain in its N-terminus and shows significant homology in its C-terminal 50 residues to Saccharomyces cerevisiae Dsk2 and Schizosaccharomyces pombe dph1. XDRP1 is a nuclear phosphoprotein in Xenopus cells, and its phosphorylation is mediated by cyclin A-dependent kinase. XDRP1 binds to both embryonic and somatic forms of cyclin A (A1 and A2) in Xenopus cells, but not to B-type cyclins. The N-terminal ubiquitin-like domain of XDRP1, but not the C-terminal Dsk2-like domain, is required for interaction with cyclin A. XDRP1 requires residues 130-160 of cyclin A1 for efficient binding, which do not include the destruction box of cyclin A. The addition of bacterially expressed XDRP1 protein to frog egg extract inhibited the Ca(2+)-induced degradation of cyclin A, but not that of cyclin B. The injection of XDRP1 protein into fertilized Xenopus eggs blocked embryonic cell division.

Amino Acid Sequence↗
PubMed1999

A new role of ran GTPase.

Ran is a G protein similar to Ras, but it has no membrane binding site. RanGEF, RCC1, is on chromatin and RanGAP, RanGAP1/Rna1p is in cytoplasm. Ran, thus, shuttles between the nucleus and the cytoplasm to complete its GTPase cycle, carrying out nucleocytoplasmic transport of macromolecules. A majority of Ran binding proteins, thus far found, are required for this process. A recently found novel Ran-binding protein, RanBPM, however, is localized in the centrosome. Subsequently, four groups reported that RanGTP, but not RanGDP, can induce microtubule self-organization in Xenopus egg extracts where no nuclear membrane is present. Thus, Ran is suggested to have a new role beyond the nucleocytoplasmic transport of macromolecules. In both microtubule assembly and nucleocytoplasmic transport, chromosomal localization of RCC1 is important to carry out the functions of RanGTPase. In this regard, a future intriguing question is how RCC1 interacts with chromatin DNA.

Animals↗
PubMed1999

Model of the ran-RCC1 interaction using biochemical and docking experiments.

RCC1, the regulator of chromosome condensation, is the guanine nucleotide exchange factor (GEF) for the nuclear Ras-like GTP-binding protein Ran. Its structure was solved by X-ray crystallography and revealed a seven-bladed beta-propeller, one side of which was proposed to be the interaction site with Ran. To gain more insight into this interaction, alanine mutagenesis studies were performed on conserved residues on the surface of the structure. Purified mutant proteins were analysed by steady-state kinetic analysis of their GEF activities towards Ran. A number of residues were identified whose mutation affected either the KMor kcatof the overall reaction, or had no effect. Mutants were further analysed by plasmon surface resonance in order to get more information on individual steps of the complex reaction pathway. Ran-GDP was coupled to the sensor chip and reacted with RCC1 mutants to categorise them into different groups, demonstrating the usefulness of plasmon surface resonance in the study of complex multi-step kinetic processes. A docking solution of Ran-RCC1 structures in combination with sequence analysis allows prediction of the site of interaction between RCC1 and Ran and proposes a model for the Ran-RCC1 structure which corresponds to and extends the biochemical data. Three invariant residues which most severely affect the kcatof the reaction, D128, D182 and H304, are located in the centre of the Ran-RCC1 interface and interfere with switch II and the phosphate binding area. The structural model suggests that different guanine nucleotide exchange factors use a similar interaction site on their respective GTP-binding proteins, but that the molecular mechanisms for the release of nucleotides are likely to be different.

Alanine↗
PubMed1999

Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran.

The nucleotide exchange activity of RCC1, the only known nucleotide exchange factor for Ran, a Ras-like small guanosine triphosphatase, was required for microtubule aster formation with or without demembranated sperm in Xenopus egg extracts arrested in meiosis II. Consistently, in the RCC1-depleted egg extracts, Ran guanosine triphosphate (RanGTP), but not Ran guanosine diphosphate (RanGDP), induced self-organization of microtubule asters, and the process required the activity of dynein. Thus, Ran was shown to regulate formation of the microtubule network.

Animals↗
PubMed1999

Overexpression of D-type cyclins, E2F-1, SV40 large T antigen and HPV16 E7 rescue cell cycle arrest of tsBN462 cells caused by the CCG1/TAF(II)250 mutation.

tsBN462 cells, which have a point mutation in CCG1/TAF(II)250, a component of TFIID complex, arrest in G1 at the nonpermissive temperature of 39.5 degrees C. Overexpression of D-type cyclins rescued the cell cycle arrest of tsBN462 cells, suggesting that the cell cycle arrest was through Rb. Consistent with this, overexpression of E2F-1, whose function is repressed by the hypophosphorylated form of Rb, also rescued the cell cycle arrest. Moreover, expression of the viral oncoproteins SV40 large T antigen and HPV16 E7, which both bind Rb and inactivate its function, rescued the cell cycle arrest, whereas HPV16 E6 did not. Mutation of the Rb-binding motif in E7 abrogated its ability to rescue the cell cycle arrest. Expression of exogenous cyclin D1, SV40 large T antigen or CCG1/TAF(II)250 increased cyclin A expression at 39.5 degrees C. Coexpression of HPV16 E7 and adenovirus E1b19K, which blocks apoptosis, rescued the proliferation of tsBN462 cells at 38.5 degrees C. To investigate the mechanism underlying the lack of cyclin D1 expression, deletion analysis of cyclin D1 promoter was performed. The 0.15 kbp cyclin D1 core promoter region, which lacks any transcription factor binding motifs, still exhibited a temperature-sensitive phenotype in tsBN462 cells suggesting that CCG1/TAF(II)250 is critical for the function of the cyclin D1 core promoter.

Animals↗
PubMed1999

Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport.

The protein Ran is a small GTP-binding protein that binds to two types of effector inside the cell: Ran-binding proteins, which have a role in terminating export processes from the nucleus to the cytoplasm, and importin-beta-like molecules that bind cargo proteins during nuclear transport. The Ran-binding domain is a conserved sequence motif found in several proteins that participate in these transport processes. The Ran-binding protein RanBP2 contains four of these domains and constitutes a large part of the cytoplasmic fibrils that extend from the nuclear-pore complex. The structure of Ran bound to a non-hydrolysable GTP analogue (Ran x GppNHp) in complex with the first Ran-binding domain (RanBD1) of human RanBP2 reveals not only that RanBD1 has a pleckstrin-homology domain fold, but also that the switch-I region of Ran x GppNHp resembles the canonical Ras GppNHp structure and that the carboxy terminus of Ran is wrapped around RanBD1, contacting a basic patch on RanBD1 through its acidic end. This molecular 'embrace' enables RanBDs to sequester the Ran carboxy terminus, triggering the dissociation of Ran x GTP from importin-beta-related transport factors and facilitating GTP hydrolysis by the GTPase-activating protein ranGAP. Such a mechanism represents a new type of switch mechanism and regulatory protein-protein interaction for a Ras-related protein.

Amino Acid Sequence↗
PubMed1999

On the mechanism of genistein-induced activation of protein kinase A-dependent Cl- conductance in cardiac myocytes.

Genistein, an inhibitor of protein tyrosine kinase (PTK), enhanced the activation of the cardiac isoform of the protein kinase A (PKA)-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- conductance in guinea-pig ventricular cells. We examined the mechanism(s) underlying this excitatory action of genistein by using patch-clamp techniques. The CFTR Cl- conductance, activated by isoproterenol (ISO, 10 nM; [Cl-] 153 mM extracellular, 21 mM intracellular; 36 degrees C), was enhanced by 20 microM genistein. Daidzein, a structural analogue of genistein with little inhibitory action on PTK, also enhanced CFTR Cl- currents. After maximal activation of the Cl- conductance by a cocktail of adenosine 3', 5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine and okadaic acid or vanadate plus forskolin in the pipette, genistein was no longer stimulatory but was rather slightly inhibitory at 100 microM. Direct exposure of myocytes to higher concentrations of genistein (50-100 microM) elicited outwardly rectifying currents with a reversal potential of -47 mV in the absence of ISO. In the presence of 50 microM H-89, a PKA inhibitor, genistein had no effect. Vanadate in the pipette at a concentration (100 microM) inhibiting phosphotyrosine phosphatases alone did not prevent the action of genistein. In contrast, no conductance was activated by tyrphostins B42 or 51 or lavendustin A, other PTK inhibitors. Genistein's stimulation of cardiac CFTR Cl- conductance appears to be independent of the PTK pathway and to be due to its direct interaction with CFTR Cl- channels.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Abnormalities of developmental cell death in Dad1-deficient mice.

BACKGROUND: Dad1, the defender against apoptotic cell death, comprises the oligosaccharyltransferase complex and is well conserved among eukaryotes. In hamster BHK21-derived tsBN7 cells, loss of Dad1 causes apoptosis which cannot be prevented by Bcl-2. RESULTS: To determine the role of Dad1 function in vivo, we prepared by gene targeting, mice harbouring a disrupted Dad1 gene. Homozygous mutants died shortly after they were implanted with the characteristic features of apoptosis. In an in vitro blastocyst culture system, Dad1-null cells displayed abnormalities which were comparable to those obtained in vivo. However, oligosaccharyltransferase activity was apparently retained even after the Dad1-null cells were destined to die. Some live-born heterozygous mutants displayed soft-tissue syndactyly. Mild thymic hypoplasia was also indicated in heterozygotes. CONCLUSION: These results suggest the involvement of the Dad1 gene in the acquisition of a common syndactyly phenotype, as well as in the control of programmed cell death during development.

Animals↗
PubMed1999

Saccharomyces cerevisiae putative G protein, Gtr1p, which forms complexes with itself and a novel protein designated as Gtr2p, negatively regulates the Ran/Gsp1p G protein cycle through Gtr2p.

Prp20p and Rna1p are GDP/GTP exchanging and GTPase-activating factors of Gsp1p, respectively, and their mutations, prp20-1 and rna1-1, can both be suppressed by Saccharomyces cerevisiae gtr1-11. We found that gtr1-11 caused a single amino acid substitution in Gtr1p, forming S20L, which is a putative GDP-bound mutant protein, while Gtr1p has been reported to bind to GTP alone. Consistently, gtr1-S20N, another putative GDP-bound mutant, suppressed both prp20-1 and rna1-1. On the other hand, gtr1-Q65L, a putative GTP-bound mutant, was inhibitory to prp20-1 and rna1-1. Thus, the role that Gtr1p plays in vivo appears to depend upon the nucleotide bound to it. Our data suggested that the GTP-bound Gtr1p, but not the GDP-bound Gtr1p, interacts with itself through its C-terminal tail. S. cerevisiae possesses a novel gene, GTR2, which is homologous to GTR1. Gtr2p interacts with itself in the presence of Gtr1p. The disruption of GTR2 suppressed prp20-1 and abolished the inhibitory effect of gtr1-Q65L on prp20-1. This finding, taken together with the fact that Gtr1p-S20L is a putative, inactive GDP-bound mutant, implies that Gtr1p negatively regulates the Ran/Gsp1p GTPase cycle through Gtr2p.

Amino Acid Sequence↗
PubMed1999

Disruption of the YRB2 gene retards nuclear protein export, causing a profound mitotic delay, and can be rescued by overexpression of XPO1/CRM1.

Disruption of the YRB2 gene encoding a nuclear Ran-binding protein homologous to Yrb1p/RanBP1 makes Saccharomyces cerevisiae cold sensitive for colony-formation, but not for growth in liquid medium. Schizosaccharomyces pombe Hba1p, which is homologous to Saccharomyces cerevisiae Yrb2p, rescued the cold sensitivity of Deltayrb2 cells. When released from an alpha factor block, Deltayrb2 cells underwent a prolonged delay at the short spindle stage of mitosis with a normal level of Clb/p34(CDC28) kinase activity, but there was no chromosome loss, this being consistent with the finding that Deltayrb2 was synthetic lethal with neither Deltamad1 nor Deltamad3. The cold sensitive colony-formation of Deltayrb2 cells was rescued by both XPO1/CRM1 and GSP1, but not CDC5, carried on a multicopy vector. XPO1/CRM1 rescued Deltayrb2 even in a single copy. Consistent with such a tight functional interaction, Xpo1p/Crm1p directly bound to Yrb2p, but not Yrb1p, and Deltayrb2 cells were found to have a defect in nuclear export signal (NES)-dependent nuclear protein export. From these results together, the ability of Xpo1/Crm1p to export NES-proteins is suggested to be enhanced by both Yrb2p and Gsp1p, and thereby disruption of YRB2 retards nuclear protein export, resulting in the mitotic delay.

Amino Acid Sequence↗
PubMed1999

Identification of essential amino acid residues of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue.

Amino Acid Sequence↗
PubMed1998

A protein required for nuclear-protein import, Mog1p, directly interacts with GTP-Gsp1p, the Saccharomyces cerevisiae ran homologue.

We previously isolated 25 temperature-sensitive gsp1 alleles of Saccharomyces cerevisiae Ran homologue, each of which possesses amino acid changes that differ from each other. We report here isolation of three multicopy suppressors-PDE2, NTF2, and a gene designated MOG1-all of which rescued a growth defect of these gsp1 strains. The gsp1 suppression occurred even in the absence of GSP2, another S. cerevisiae GSP1-like gene. Previously, NTF2 was reported to suppress gsp1 but not PDE2. Mog1p, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W. Both MOG1 and NTF2 suppressed a series of gsp1 alleles with similar efficiency, and both suppressed gsp1 even with a single gene dose. Consistent with the high efficiency of gsp1 suppression, Mog1p directly bound to GTP, but not to GDP-Gsp1p. The disruption of MOG1 made yeast temperature-sensitive for growth. Deltamog1, which was suppressed by overexpression of NTF2, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export. Thus, Mog1p, which was localized in the nucleus, is a Gsp1p-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p. Furthermore, the finding that PDE2 suppressed both gsp1 and rna1-1 indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway.

Amino Acid Sequence↗
PubMed1998

When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin.

A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2.

The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins [1]. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins [1]. p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2) [2]. Ran-binding protein 2 (p340(RanBP2)) is a nuclear pore protein [3] [4], and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran [2]. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier [5-7], and that SUMO-1 conjugation promotes RanGAP1's interaction with RanBP2 [2,5,6]. Here we show that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. We also show that p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts.

Animals↗
PubMed1998

Nuclear protein import, but not mRNA export, is defective in all Saccharomyces cerevisiae mutants that produce temperature-sensitive forms of the Ran GTPase homologue Gsp1p.

A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not in mRNA export. Following release from alpha-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37 degrees C, the nonpermissive temperature. While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this, we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions with effectors that were independent of each other, and are differentially affected by mutation.

Amino Acid Sequence↗