31 results on '"Miyazaki, H."'
Search Results
2. Functional analysis of the C-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a "shuttle" peptide to help secretion.
- Author
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Muto, T, Feese, M D, Shimada, Y, Kudou, Y, Okamoto, T, Ozawa, T, Tahara, T, Ohashi, H, Ogami, K, Kato, T, Miyazaki, H, and Kuroki, R
- Abstract
Thrombopoietin (TPO) is a cytokine that primarily stimulates megakaryocytopoiesis and thrombopoiesis. TPO has a unique C-terminal tail peptide of about 160 amino acids that consists mostly of hydrophilic residues and contains six N-linked sugar chains. In order to investigate the biological function of the C-terminal domain, two series of mutations were performed. One is systematic truncation from the C terminus. Another is elimination of N-glycosylation sites in the C-terminal domain by Asn to Gln mutations. After the mutant proteins were expressed by mammalian cells, it was found that the elimination of the N-linked sugar sites did not affect the biological activity, whereas truncation of the C-terminal domain resulted in elevation of in vitro activity up to 4-fold. The C-terminal peptide itself was found to inhibit the in vitro activity. Moreover, both the C-terminal truncation and the elimination of the N-glycosylation sites decreased the secretion level progressively down to (1)/(10) that of wild type, and the amount of the mutant left in the cell increased. The N-glycosylation in the C-terminal region was found to be important for secretion of TPO. Among six N-glycosylation sites in the C-terminal region, two locations, Asn-213 and Asn-234, were found to be critical for secretion, and two other locations, Asn-319 and Asn-327, did not affect the secretion.
- Published
- 2000
3. GD3 synthase gene expression in PC12 cells results in the continuous activation of TrkA and ERK1/2 and enhanced proliferation.
- Author
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Fukumoto, S, Mutoh, T, Hasegawa, T, Miyazaki, H, Okada, M, Goto, G, Furukawa, K, and Urano, T
- Abstract
A rat pheochromocytoma cell line (PC12) transfected with ganglioside GD3 synthase gene showed a marked change in the ganglioside profile and enhanced proliferation and no response of neurite extension to nerve growth factor (NGF) stimulation. In these transfectant cells, a continuous phosphorylation of TrkA and the activation of ERK1/2 without NGF treatment were observed. Proliferation inhibition experiments with kinase inhibitors such as herbimycin A, K-252a, and PD98059 revealed that the enhanced proliferation was actually due to the activation of the Ras/MEK/ERK pathway. A TrkA dimer was detected in the GD3 synthase transfectant cells regardless of NGF treatment by cross-linking and immunoblotting. The increased expression of GD1b and GT1b in these transfectant cells might induce the conformational change of TrkA to form a dimer and to be activated continuously. These results may indicate regulatory roles of gangliosides in cell proliferation under physiological and malignant processes.
- Published
- 2000
4. Increased D-type cyclin expression together with decreased cdc2 activity confers megakaryocytic differentiation of a human thrombopoietin-dependent hematopoietic cell line.
- Author
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Matsumura, I, Tanaka, H, Kawasaki, A, Odajima, J, Daino, H, Hashimoto, K, Wakao, H, Nakajima, K, Kato, T, Miyazaki, H, and Kanakura, Y
- Abstract
At the late phase of megakaryocytopoiesis, megakaryocytes undergo endomitosis, which is characterized by DNA replication without cell division. Although a number of cell cycle regulatory molecules have been identified, the precise roles of these molecules in megakaryocytic endomitosis are largely unknown. In a human interleukin-3-dependent cell line transfected with the thrombopoietin (TPO) receptor c-mpl (F-36P-mpl), either treatment with TPO or the overexpression of activated ras (Ha-Ras(G12V)) induced megakaryocytic maturation with polyploid formation. We found that TPO stimulation or Ha-Ras(G12V) expression led to up-regulation of cyclin D1, cyclin D2, and cyclin D3 expression. In addition, expression levels of cyclin A and cyclin B were reduced during the total course of both TPO- and Ha-Ras(G12V)-induced megakaryocytic differentiation, thereby leading to decreased cdc2 kinase activity. Neither the induced expression of cyclin D1, cyclin D2, or cyclin D3 nor the expression of a dominant negative form of cdc2 alone could induce megakaryocytic differentiation of F-36P-mpl cells. In contrast, overexpression of dominant negative cdc2 together with cyclin D1, cyclin D2, or cyclin D3 facilitated megakaryocytic differentiation in the absence of TPO. These results suggest that both D-type cyclin expression and decreased cdc2 kinase activity may participate in megakaryocytic differentiation.
- Published
- 2000
5. Attenuation of interleukin 2 signal in the spleen cells of complex ganglioside-lacking mice.
- Author
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Zhao, J, Furukawa, K, Fukumoto, S, Okada, M, Furugen, R, Miyazaki, H, Takamiya, K, Aizawa, S, Shiku, H, Matsuyama, T, and Furukawa, K
- Abstract
T cell development and function in complex ganglioside-lacking (GM2/GD2 synthase gene-disrupted) mice were analyzed. GM1, asialo-GM1, and GD1b were representative gangliosides expressed on T cells of the wild type mice and completely deleted on those of the mutant mice. The sizes and cell numbers of the mutant mice spleen and thymus were significantly reduced. Spleen cells from the mutant mice showed clearly reduced proliferation compared with the wild type when stimulated by interleukin 2 (IL-2) but not when treated with concanavalin A or anti-CD3 cross-linking. Expression levels of IL-2 receptor alpha, beta, and gamma were almost equivalent, and up-regulation of alpha chain after T cell activation was also similar between the mutant and wild type mice. Activation of JAK1, JAK3, and SAT5 after IL-2 treatment was reduced, and c-fos expression was delayed and reduced in the mutant spleen cells, suggesting that the IL-2 signal was attenuated in the mutant mice probably due to the modulation of IL-2 receptors by the lack of complex gangliosides.
- Published
- 1999
6. Molecular cloning of a novel alpha2,3-sialyltransferase (ST3Gal VI) that sialylates type II lactosamine structures on glycoproteins and glycolipids.
- Author
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Okajima, T, Fukumoto, S, Miyazaki, H, Ishida, H, Kiso, M, Furukawa, K, Urano, T, and Furukawa, K
- Abstract
A novel member of the human CMP-NeuAc:beta-galactoside alpha2, 3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of alpha2, 3-sialyltransferase toward Galbeta1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity, i.e. it utilized Galbeta1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.
- Published
- 1999
7. Expression cloning of mouse cDNA of CMP-NeuAc:Lactosylceramide alpha2,3-sialyltransferase, an enzyme that initiates the synthesis of gangliosides.
- Author
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Fukumoto, S, Miyazaki, H, Goto, G, Urano, T, Furukawa, K, and Furukawa, K
- Abstract
Expression cloning of a cDNA for the alpha2,3-sialyltransferase (GM3 synthase) (EC 2.4.99.-) gene was performed using a GM3-lacking mouse fibroblast line L cell and anti-GM3 monoclonal antibody. Plasmids from a cDNA library generated with poly(A)+ RNA of a mouse fibrosarcoma line CMS5j and pdl3027 (polyoma T antigen) were co-transfected into L cells. The isolated cDNA clone pM3T-7 predicted a type II membrane protein with 13 amino acids of cytoplasmic domain, 17 amino acids of transmembrane region, and a large catalytic domain with 329 amino acids. Introduction of the cDNA clone into L cells resulted in the neo-synthesis of GM3 and high activity of alpha2,3-sialyltransferase. Among glycosphingolipids, only lactosylceramide showed significant activity as an acceptor, indicating that this gene product is a sialyltransferase specific for the synthesis of GM3. An amino acid sequence deduced from the cloned cDNA showed the typical sialyl motif with common features among alpha2,3-sialyltransferases. Among various mouse tissues, brain, liver, and testis showed relatively high expression of a 2.3-kilobase mRNA, whereas all tissues, more or less, expressed this gene.
- Published
- 1999
8. In vitro biosynthesis of human renin and identification of plasma inactive renin as an activation intermediate.
- Author
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Hirose, S, Kim, S, Miyazaki, H, Park, Y S, and Murakami, K
- Abstract
The biosynthesis and post-translational modifications, including proteolytic processing and core glycosylation, of the human renin precursor have been studied in vitro in a cell-free system. For this purpose, highly enriched renin mRNA was isolated from a renin-producing juxtaglomerular cell tumor and translated in rabbit reticulocyte lysate containing [35S]methionine in the presence or absence of dog pancreas microsomal membranes. Fluorographic analysis of the radioactive translation products, immunoprecipitated and then resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that the primary translation product, preprorenin (Mr = 45,000), is initially processed to glycosylated prorenin (Mr = 47,000) during or shortly after its sequestration into the lumen of the microsomal membranes. The vectorial translocation across the membrane was confirmed by the observation that the proform was resistant to digestion with trypsin while preprorenin was sensitive. Radiosequencing and the use of prorenin-specific antibodies established the cleavage points of the pre- and profragment and showed that the in vitro precursor of human renin contains a 23-residue signal peptide and a 43-residue prosegment. The post-translational modification which, despite the removal of signal peptide, resulted in an increase in apparent Mr, reflects the glycosylation as examined using Xenopus oocytes microinjected with renin mRNA in the presence of tunicamycin, an inhibitor of protein glycosylation. Four anti-peptide antibodies which specifically recognize the NH2 terminus (Pro 1), two middle parts (Pro 2A and Pro 2B), and COOH terminus (Pro 3) of the prosegment, respectively, have been raised and used to characterize plasma prorenin. Renin precursors (pre- and prorenin) synthesized in vitro or in the kidney reacted with these antibodies (anti-Pro 1, anti-Pro 2A, anti-Pro 2B, and anti-Pro 3). However, quite unexpectedly, human plasma prorenin was recognized only by anti-Pro 3, indicating that plasma prorenin is a truncated version of intact prorenin, which lacks a large portion of the NH2 terminus of the prosegment and may represent an activation intermediate. This somewhat surprising result may lead to a better understanding of the exact roles and activation mechanisms of plasma prorenin existing in a relatively large amount.
- Published
- 1985
- Full Text
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9. Expression cloning of rat cDNA encoding UDP-galactose:GD2 beta1,3-galactosyltransferase that determines the expression of GD1b/GM1/GA1.
- Author
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Miyazaki, H, Fukumoto, S, Okada, M, Hasegawa, T, and Furukawa, K
- Abstract
Using an anti-GD1b monoclonal antibody, expression cloning of a cDNA for the beta1,3-galactosyltransferase gene (EC 2.4.1.62) was performed. KF4C, mouse melanoma B16 transfected with polyoma T antigen gene, and GM2/GD2 synthase cDNA was used as a recipient cell line for the cDNA library transfection. A cDNA clone of GD3 synthase, pD3T-31 was co-transfected with a cDNA library prepared from rat brain RNA using the pcDNAI expression vector. The isolated cDNA clone pM1T-9 predicted a type II membrane protein with 4 amino acids of cytoplasmic domain, 21 amino acids of transmembrane region, and a large catalytic domain with 346 amino acids. Introduction of the cDNA clone into a mouse melanoma line B16 previously transfected with a GM2/GD2 synthase gene resulted in the neo-synthesis of GM1. Co-transfection of the cell line with pM1T-9 and a GD3 synthase cDNA resulted in the expression of GD1b as well as GM1. Moreover, introduction of pM1T-9 into L cell (lacking GM3 synthase), previously transfected with GM2/GD2 synthase gene, resulted in the definite expression of asialo-GM1. These results indicated that GD1b/GM1/GA1 synthases were identical, as previously suggested based on enzymological analysis. In Northern blots of the beta1, 3-galactosyltransferase gene with total RNA from various rat tissues, a 1.6-kilobase mRNA was strongly expressed in spleen, thymus, kidney, and testis. However, the expression level of the gene in the adult brain tissue was not especially high. On the other hand, this gene was expressed at high levels in the rat brain of embryonal day 12, and reached a peak at around birth, then fell to low level in the adult brain.
- Published
- 1997
10. Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells.
- Author
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Takeuchi, M, Takasaki, S, Miyazaki, H, Kato, T, Hoshi, S, Kochibe, N, and Kobata, A
- Abstract
The asparagine-linked sugar chains of human erythropoietin produced by recombinant Chinese hamster ovary cells and naturally occurring human urinary erythropoietin were liberated by hydrazinolysis and fractionated by paper electrophoresis, lectin affinity chromatography, and Bio-Gel P-4 column chromatography. Both erythropoietins had three asparagine-linked sugar chains in one molecule, all of which were acidic complex type. Structural analysis of them revealed that the sugar chains from both erythropoietins are quite similar except for sialyl linkage. All sugar chains of erythropoietin produced by recombinant Chinese hamster ovary cells contain only the NeuAc alpha 2—-3Gal linkage, while those of human urinary erythropoietin contain the NeuAc alpha 2—-6Gal linkage together with the NeuAc alpha 2—-3Gal linkage. The major sugar chains were of fucosylated tetraantennary complex type with and without N-acetyllactosamine repeating units in their outer chain moieties in common, and small amounts of 2,4- and 2,6-branched triantennary and biantennary sugar chains were detected. This paper proved, for the first time, that recombinant technique can produce glycoprotein hormone whose carbohydrate structures are common to the major sugar chains of the native one.
- Published
- 1988
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11. Occurrence of 9-deoxy-delta 9,delta 12-13,14-dihydroprostaglandin D2 in human urine.
- Author
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Hirata, Y, Hayashi, H, Ito, S, Kikawa, Y, Ishibashi, M, Sudo, M, Miyazaki, H, Fukushima, M, Narumiya, S, and Hayaishi, O
- Abstract
We have developed a highly sensitive and specific solid-phase enzyme immunoassay for 9-deoxy-delta 9,delta 12-dihydroprostaglandin D2 (delta 12-PGJ2) and studied the occurrence of this novel PGD2 metabolite in human urine. The assay detected delta 12-PGJ2 over the range of 2-200 pg, and the antiserum showed 2% cross-reaction with PGJ2 and less than 0.2% with other PGs. We used this assay and purified the delta 12-PGJ2-like immunoreactive substance from human urine. Purification consisted of chromatographies on a Sep-Pak C18 cartridge, a silicic acid column, reversed-phase high-performance liquid chromatography, and finally an affinity column of anti-delta 12-PGJ2 antibody. As a result, about 850 ng of delta 12-PGJ2-like immunoreactive substance were recovered from 60 liters of human urine. The purified material was identified as delta 12-PGJ2 by gas chromatography/high resolution-selected ion monitoring using the molecular ion m/z 448[M]+. and ions [M - 15]+, [M - 43]+, [M - 100]+., and [M - 143]+. The amounts of delta 12-PGJ2 in the urine from normal, volunteer men and women were 151.5 +/- 20.0 and 65.6 +/- 5.4 ng/24 h (mean +/- S.E., n = 5), respectively. The delta 12-PGJ2 amount in urine did not alter significantly during storage for at least 24 h or by the addition of authentic PGD2 to urine samples, suggesting that the delta 12-PGJ2 we determined was not derived from the decomposition of PGD2 in the urine during storage or purification. Moreover, when a single dose of PGD2 (1 mg/kg) was injected intravenously into cynomolgus monkeys, the urinary level of delta 12-PGJ2 increased 20- to 180-fold over the normal levels, whereas the delta 12-PGJ2 level decreased by 40-50% of the normal levels, following the administration of indomethacin at a dose of 1 mg/kg. These results indicate that delta 12-PGJ2 is formed naturally in the body and excreted as a urinary PGD2 metabolite.
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- 1988
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12. A 56,000-dalton renin-binding protein in hog kidney is an endogenous renin inhibitor.
- Author
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Ueno, N., primary, Miyazaki, H., additional, Hirose, S., additional, and Murakami, K., additional
- Published
- 1981
- Full Text
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13. FACS-array-based cell purification yields a specific transcriptome of striatal medium spiny neurons in a murine Huntington disease model.
- Author
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Miyazaki H, Yamanaka T, Oyama F, Kino Y, Kurosawa M, Yamada-Kurosawa M, Yamano R, Shimogori T, Hattori N, and Nukina N
- Subjects
- Animals, Corpus Striatum pathology, Disease Models, Animal, Huntingtin Protein genetics, Huntingtin Protein metabolism, Huntington Disease genetics, Huntington Disease pathology, Male, Mice, Mice, Transgenic, Corpus Striatum metabolism, Flow Cytometry, Huntington Disease metabolism, Transcriptome
- Abstract
Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin gene. Results from previous studies have suggested that transcriptional dysregulation is one of the key mechanisms underlying striatal medium spiny neuron (MSN) degeneration in HD. However, some of the critical genes involved in HD etiology or pathology could be masked in a common expression profiling assay because of contamination with non-MSN cells. To gain insight into the MSN-specific gene expression changes in presymptomatic R6/2 mice, a common HD mouse model, here we used a transgenic fluorescent protein marker of MSNs for purification via FACS before profiling gene expression with gene microarrays and compared the results of this "FACS-array" with those obtained with homogenized striatal samples (STR-array). We identified hundreds of differentially expressed genes (DEGs) and enhanced detection of MSN-specific DEGs by comparing the results of the FACS-array with those of the STR-array. The gene sets obtained included genes ubiquitously expressed in both MSNs and non-MSN cells of the brain and associated with transcriptional regulation and DNA damage responses. We proposed that the comparative gene expression approach using the FACS-array may be useful for uncovering the gene cascades affected in MSNs during HD pathogenesis., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Miyazaki et al.)
- Published
- 2020
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14. Parallel homodimer structures of the extracellular domains of the voltage-gated sodium channel β4 subunit explain its role in cell-cell adhesion.
- Author
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Shimizu H, Tosaki A, Ohsawa N, Ishizuka-Katsura Y, Shoji S, Miyazaki H, Oyama F, Terada T, Shirouzu M, Sekine SI, Nukina N, and Yokoyama S
- Subjects
- Animals, CHO Cells, Cell Adhesion, Cricetulus, Crystallography, X-Ray, Cysteine chemistry, Cystine chemistry, Dimerization, Humans, Hydrogen Bonding, Hydrophobic and Hydrophilic Interactions, Mice, Peptide Fragments chemistry, Peptide Fragments genetics, Peptide Fragments metabolism, Protein Conformation, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Multimerization, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Voltage-Gated Sodium Channel beta-4 Subunit chemistry, Voltage-Gated Sodium Channel beta-4 Subunit genetics, Models, Molecular, Voltage-Gated Sodium Channel beta-4 Subunit metabolism
- Abstract
Voltage-gated sodium channels (VGSCs) are transmembrane proteins required for the generation of action potentials in excitable cells and essential for propagating electrical impulses along nerve cells. VGSCs are complexes of a pore-forming α subunit and auxiliary β subunits, designated as β1/β1B-β4 (encoded by SCN1B-4B, respectively), which also function in cell-cell adhesion. We previously reported the structural basis for the trans homophilic interaction of the β4 subunit, which contributes to its adhesive function. Here, using crystallographic and biochemical analyses, we show that the β4 extracellular domains directly interact with each other in a parallel manner that involves an intermolecular disulfide bond between the unpaired Cys residues (Cys
58 ) in the loop connecting strands B and C and intermolecular hydrophobic and hydrogen-bonding interactions of the N-terminal segments (Ser30 -Val35 ). Under reducing conditions, an N-terminally deleted β4 mutant exhibited decreased cell adhesion compared with the wild type, indicating that the β4 cis dimer contributes to the trans homophilic interaction of β4 in cell-cell adhesion. Furthermore, this mutant exhibited increased association with the α subunit, indicating that the cis dimerization of β4 affects α-β4 complex formation. These observations provide the structural basis for the parallel dimer formation of β4 in VGSCs and reveal its mechanism in cell-cell adhesion., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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15. Reactive oxygen generated by NADPH oxidase 1 (Nox1) contributes to cell invasion by regulating matrix metalloprotease-9 production and cell migration.
- Author
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Shinohara M, Adachi Y, Mitsushita J, Kuwabara M, Nagasawa A, Harada S, Furuta S, Zhang Y, Seheli K, Miyazaki H, and Kamata T
- Subjects
- Animals, Antioxidants pharmacology, Caco-2 Cells, Cell Line, Cell Movement genetics, Cell Movement physiology, Epidermal Growth Factor pharmacology, Humans, I-kappa B Kinase genetics, I-kappa B Kinase metabolism, Immunoblotting, Immunoprecipitation, Matrix Metalloproteinase 2 genetics, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 9 genetics, NADH, NADPH Oxidoreductases genetics, NADH, NADPH Oxidoreductases metabolism, NADPH Oxidase 1, Onium Compounds pharmacology, Promoter Regions, Genetic genetics, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins physiology, Rats, Reverse Transcriptase Polymerase Chain Reaction, Vitamin E pharmacology, rho GTP-Binding Proteins metabolism, Cell Movement drug effects, Matrix Metalloproteinase 9 metabolism, NADH, NADPH Oxidoreductases physiology, Reactive Oxygen Species metabolism
- Abstract
A mediating role of the reactive oxygen species-generating enzyme Nox1 has been suggested for Ras oncogene transformation phenotypes including anchorage-independent cell growth, augmented angiogenesis, and tumorigenesis. However, little is known about whether Nox1 signaling regulates cell invasiveness. Here, we report that the cell invasion activity was augmented in K-Ras-transformed normal rat kidney cells and attenuated by transfection of Nox1 small interference RNAs (siRNAs) into the cells. Diphenyleneiodonium (DPI) or Nox1 siRNAs blocked up-regulation of matrix metalloprotease-9 at both protein and mRNA levels in K-Ras-transformed normal rat kidney cells. Furthermore, DPI and Nox1 siRNAs inhibited the activation of IKKalpha kinase and the degradation of IkappaB alpha, suppressing the NFkappaB-dependent matrix metalloprotease-9 promoter activity. Additionally, epidermal growth factor-stimulated migration of CaCO-2 cells was abolished by DPI and Nox1 siRNAs, indicating the requirement of Nox1 activity for the motogenic effect of epidermal growth factor. This Nox1 action was mediated by down-regulation of the Rho activity through the low molecular weight protein-tyrosine phosphatase-p190RhoGAP-dependent mechanism. Taken together, our findings define a mediating role of Nox1-generated reactive oxygen species in cell invasion processes, most notably metalloprotease production and cell motile activity.
- Published
- 2010
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16. Inhibition of Rho kinases enhances the degradation of mutant huntingtin.
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Bauer PO, Wong HK, Oyama F, Goswami A, Okuno M, Kino Y, Miyazaki H, and Nukina N
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- Amides pharmacology, Animals, Ataxin-3, Autophagy, Blotting, Western, Cell Death drug effects, Cells, Cultured, Embryo, Mammalian cytology, Embryo, Mammalian drug effects, Embryo, Mammalian metabolism, Fibroblasts cytology, Fibroblasts drug effects, Fibroblasts metabolism, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Humans, Huntingtin Protein, Mice, Nerve Tissue Proteins genetics, Neuroblastoma genetics, Neuroblastoma metabolism, Neuroblastoma pathology, Nuclear Proteins genetics, Profilins genetics, Profilins metabolism, Protein Binding, Protein Kinase C genetics, Protein Kinase C metabolism, Pyridines pharmacology, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Small Interfering pharmacology, Receptors, Androgen genetics, Receptors, Androgen metabolism, Repressor Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Enzyme Inhibitors pharmacology, Mutation genetics, Nerve Tissue Proteins metabolism, Nuclear Proteins metabolism, Peptides metabolism, rho-Associated Kinases antagonists & inhibitors
- Abstract
Huntington disease (HD) is a fatal hereditary neurodegenerative disease caused by an expansion of the polyglutamine (polyQ) stretch in huntingtin (htt). Whereas the pathological significance of the expanded polyQ has been clearly established and a tremendous effort to develop therapeutic tools for HD has been exerted, there is yet no effective cure. Whereas many molecules able to reduce the polyQ accumulation and aggregation have been identified, including several Rho kinase (ROCK) inhibitors, it remains very important to determine the mechanism of action of the potential drugs. ROCK inhibitors, including Y-27632 were reported to decrease aggregation of htt and androgen receptor (AR) through ROCK1 and protein kinase C-related protein kinase-2 (PRK-2). A downstream effector of ROCK1, actin-binding factor profilin, was shown to inhibit the mutant htt aggregation but not AR by direct interaction. We found that the anti-aggregation effect of ROCK inhibitors was not limited to the mutant htt and AR and that Y-27632 was also able to reduce the aggregation of ataxin-3 and atrophin-1 with expanded polyQ. These results suggested that in addition to the mechanism reported for htt and AR, there might also be other common mediators involved in the reduced aggregation of different polyQ proteins. In this study, we show that Y-27632 not only reduced the mutant htt aggregation by enhancing its degradation, but surprisingly was able to activate the main cellular degradation pathways, proteasome, and macroautophagy. We also show that this unique effect was mediated by ROCK1 and ROCK2.
- Published
- 2009
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17. RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells.
- Author
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Doi H, Okamura K, Bauer PO, Furukawa Y, Shimizu H, Kurosawa M, Machida Y, Miyazaki H, Mitsui K, Kuroiwa Y, and Nukina N
- Subjects
- Animals, Brain metabolism, Brain pathology, Cell Line, Disease Models, Animal, Humans, Huntingtin Protein, Huntington Disease genetics, Huntington Disease pathology, Immunohistochemistry, Intranuclear Inclusion Bodies genetics, Intranuclear Inclusion Bodies pathology, Mice, Mice, Transgenic, Nerve Tissue Proteins genetics, Neurons cytology, Neurons pathology, Nuclear Proteins genetics, RNA-Binding Protein FUS genetics, Exons, Huntington Disease metabolism, Intranuclear Inclusion Bodies metabolism, Nerve Tissue Proteins metabolism, Nuclear Proteins metabolism, Peptides metabolism, RNA-Binding Protein FUS metabolism
- Abstract
Formation of intracellular aggregates is the hallmark of polyglutamine (polyQ) diseases. We analyzed the components of purified nuclear polyQ aggregates by mass spectrometry. As a result, we found that the RNA-binding protein translocated in liposarcoma (TLS) was one of the major components of nuclear polyQ aggregate-interacting proteins in a Huntington disease cell model and was also associated with neuronal intranuclear inclusions of R6/2 mice. In vitro study revealed that TLS could directly bind to truncated N-terminal huntingtin (tNhtt) aggregates but could not bind to monomer GST-tNhtt with 18, 42, or 62Q, indicating that the tNhtt protein acquired the ability to sequester TLS after forming aggregates. Thioflavin T assay and electron microscopic study further supported the idea that TLS bound to tNhtt-42Q aggregates at the early stage of tNhtt-42Q amyloid formation. Immunohistochemistry showed that TLS was associated with neuronal intranuclear inclusions of Huntington disease human brain. Because TLS has a variety of functional roles, the sequestration of TLS to polyQ aggregates may play a role in diverse pathological changes in the brains of patients with polyQ diseases.
- Published
- 2008
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18. Nox1 redox signaling mediates oncogenic Ras-induced disruption of stress fibers and focal adhesions by down-regulating Rho.
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Shinohara M, Shang WH, Kubodera M, Harada S, Mitsushita J, Kato M, Miyazaki H, Sumimoto H, and Kamata T
- Subjects
- 3T3 Cells, Actins metabolism, Animals, Cell Line, Cell Transformation, Neoplastic, Cytoskeleton metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Mice, NADH, NADPH Oxidoreductases genetics, NADPH Oxidase 1, Oxidation-Reduction, Protein Tyrosine Phosphatases chemistry, Protein Tyrosine Phosphatases metabolism, Rats, Repressor Proteins genetics, Repressor Proteins metabolism, ras Proteins genetics, rho GTP-Binding Proteins genetics, Focal Adhesions metabolism, Genes, ras, NADH, NADPH Oxidoreductases metabolism, Signal Transduction physiology, Stress Fibers metabolism, ras Proteins metabolism, rho GTP-Binding Proteins metabolism
- Abstract
Generation of reactive oxygen species (ROS) by Ras oncogene-induced NADPH oxidase (Nox) 1 is required for Ras transformation phenotypes including anchorage-independent growth, morphological transformation, and tumorigenesity, but the signaling mechanism downstream of Nox1 remains elusive. Rho is known to be a critical regulator of actin stress fiber formation. Nonetheless, Rho was reported to no longer couple to loss of actin stress fibers in Ras-transformed Swiss3T3 cells despite the elevation of Rho activity. In this study, however, we demonstrate that Rho is inactivated in K-Ras-transformed normal rat kidney cells, and that abrogation of Nox1-generated ROS by Nox1 small interference RNAs or diphenyleneiodonium restores Rho activation, suggesting that Nox1-generated oxidants mediate down-regulation of the Rho activity. This down-regulation involves oxidative inactivation of the low molecular weight protein-tyrosine phosphatase by Nox1-generated ROS and a subsequent elevation in the tyrosine-phosphorylated active form of p190RhoGAP, the direct target of the phosphatase. Furthermore, the decreased Rho activity leads to disruption of both actin stress fibers and focal adhesions in Ras-transformed cells. As for Rac1, Rac1 also appears to participate in the down-regulation of Rho via Nox1. Our discovery defines a mediating role of Nox1-redox signaling for Ras oncogene-induced actin cytoskeletal changes.
- Published
- 2007
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19. Laminin alpha5 is required for dental epithelium growth and polarity and the development of tooth bud and shape.
- Author
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Fukumoto S, Miner JH, Ida H, Fukumoto E, Yuasa K, Miyazaki H, Hoffman MP, and Yamada Y
- Subjects
- Animals, Body Patterning, Cell Proliferation, Cells, Cultured, DNA, Complementary metabolism, Gene Expression Regulation, Neoplastic, Genes, Dominant, Immunohistochemistry, In Situ Hybridization, Integrin alpha6beta4 metabolism, Laminin metabolism, Mice, Mice, Knockout, Mice, Transgenic, Microscopy, Fluorescence, Organ Culture Techniques, Phosphatidylinositol 3-Kinases metabolism, Pseudopodia metabolism, RNA metabolism, RNA, Messenger metabolism, Signal Transduction, Time Factors, Epithelium metabolism, Gene Expression Regulation, Developmental, Laminin physiology, Tooth embryology, Tooth metabolism
- Abstract
In tooth development, the oral ectoderm and mesenchyme coordinately and reciprocally interact through the basement membrane for their growth and differentiation to form the proper shape and size of the tooth. Laminin alpha5 subunit-containing laminin-10/11 (LM-511/521) is the major laminin in the tooth germ basement membrane. Here, we have examined the role of laminin alpha5 (Lama5) in tooth development using laminin alpha5-null mouse primary dental epithelium and tooth germ organ cultures. Lama5-null mice develop a small tooth germ with defective cusp formation and have reduced proliferation of dental epithelium. Also, cell polarity and formation of the monolayer of the inner dental epithelium are disturbed. The enamel knot, a signaling center for tooth germ development, is defective, and there is a significant reduction of Shh and Fgf4 expression in the dental epithelium. In the absence of laminin alpha5, the basement membrane in the inner dental epithelium becomes discontinuous. In normal mice, integrin alpha6beta4, a receptor for laminin alpha5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin alpha6beta4 is expressed around the cell surface. In primary dental epithelium culture, laminin-10/11 promotes cell growth, spreading, and filopodia-like microspike formation. This promotion is inhibited by anti-integrin alpha6 and beta4 antibodies and by phosphatidylinositol 3-kinase inhibitors and dominant negative Rho-GTPase family proteins Cdc42 and Rac. In organ culture, anti-integrin alpha6 antibody and wortmannin reduce tooth germ size and shape. Our studies demonstrate that laminin alpha5 is required for the proliferation and polarity of basal epithelial cells and suggest that the interaction between laminin-10/11-integrin alpha6beta4 and the phosphatidylinositol 3-kinase-Cdc42/Rac pathways play an important role in determining the size and shape of tooth germ.
- Published
- 2006
- Full Text
- View/download PDF
20. beta Subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase.
- Author
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Wong HK, Sakurai T, Oyama F, Kaneko K, Wada K, Miyazaki H, Kurosawa M, De Strooper B, Saftig P, and Nukina N
- Subjects
- Alzheimer Disease metabolism, Amino Acid Sequence, Amyloid Precursor Protein Secretases, Amyloid beta-Protein Precursor chemistry, Animals, Aspartic Acid Endopeptidases, Binding Sites, Blotting, Western, Brain metabolism, Cell Line, Cell Membrane metabolism, Cells, Cultured, Detergents pharmacology, Endopeptidases metabolism, Fibroblasts metabolism, Genetic Vectors, Humans, Mice, Mice, Inbred C57BL, Mice, Knockout, Microscopy, Fluorescence, Molecular Sequence Data, Neurons metabolism, Phosphoric Monoester Hydrolases metabolism, Protein Binding, Protein Structure, Tertiary, RNA Interference, Sequence Homology, Amino Acid, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Transfection, Endopeptidases chemistry
- Abstract
Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and gamma-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that beta subunits of voltage-gated sodium channels are sequentially processed by BACE1 and gamma-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.
- Published
- 2005
- Full Text
- View/download PDF
21. Chimeric G alpha i2/G alpha 13 proteins reveal the structural requirements for the binding and activation of the RGS-like (RGL)-containing Rho guanine nucleotide exchange factors (GEFs) by G alpha 13.
- Author
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Vázquez-Prado J, Miyazaki H, Castellone MD, Teramoto H, and Gutkind JS
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Embryo, Mammalian, GTP-Binding Protein alpha Subunit, Gi2, GTP-Binding Protein alpha Subunits, G12-G13 genetics, GTP-Binding Protein alpha Subunits, Gi-Go genetics, Guanine Nucleotide Exchange Factors chemistry, Humans, Kidney, Mice, Models, Molecular, Molecular Sequence Data, Molecular Structure, NIH 3T3 Cells, Point Mutation, Proto-Oncogene Proteins genetics, RGS Proteins chemistry, RGS Proteins metabolism, Receptors, Glutamate chemistry, Receptors, Glutamate metabolism, Recombinant Fusion Proteins metabolism, Rho Guanine Nucleotide Exchange Factors, Structure-Activity Relationship, Transfection, GTP-Binding Protein alpha Subunits, G12-G13 chemistry, GTP-Binding Protein alpha Subunits, G12-G13 metabolism, GTP-Binding Protein alpha Subunits, Gi-Go chemistry, GTP-Binding Protein alpha Subunits, Gi-Go metabolism, Guanine Nucleotide Exchange Factors metabolism, Proto-Oncogene Proteins chemistry, Proto-Oncogene Proteins metabolism, Recombinant Fusion Proteins chemistry
- Abstract
The alpha-subunit of G proteins of the G(12/13) family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of G alpha(12/13) to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction between G alpha(13) and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of G alpha(i) proteins. Using G alpha(i2), which does not bind RGL domains, as the backbone in which G alpha(13) sequences were swapped or mutated, we observed that the switch region of G alpha(13) is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surprisingly, the switch region of G alpha(13) was not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of G alpha(13) and chimeric G alpha(i2) proteins was also necessary for Rho activation. These findings revealed the structural features by which G alpha(13) interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs.
- Published
- 2004
- Full Text
- View/download PDF
22. The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C terminus.
- Author
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Anzai N, Miyazaki H, Noshiro R, Khamdang S, Chairoungdua A, Shin HJ, Enomoto A, Sakamoto S, Hirata T, Tomita K, Kanai Y, and Endou H
- Subjects
- Biological Transport, Carrier Proteins chemistry, Carrier Proteins genetics, Cell Line, Humans, Kidney Tubules, Proximal metabolism, Membrane Proteins, Organic Anion Transporters chemistry, Organic Anion Transporters genetics, Organic Cation Transport Proteins, RNA, Messenger analysis, Uric Acid metabolism, Carrier Proteins physiology, Organic Anion Transporters physiology
- Abstract
The urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents. URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95, Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay, in vitro binding assay and surface plasmon resonance analysis (K(D) = 1.97-514 nM). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the V(max) of urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules.
- Published
- 2004
- Full Text
- View/download PDF
23. Essential role for phospholipase D2 activation downstream of ERK MAP kinase in nerve growth factor-stimulated neurite outgrowth from PC12 cells.
- Author
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Watanabe H, Yokozeki T, Yamazaki M, Miyazaki H, Sasaki T, Maehama T, Itoh K, Frohman MA, and Kanaho Y
- Subjects
- Actins metabolism, Animals, Enzyme Activation, PC12 Cells, Rats, Signal Transduction, Mitogen-Activated Protein Kinases metabolism, Nerve Growth Factor pharmacology, Neurites, Phospholipase D metabolism
- Abstract
The signaling pathway that triggers morphological differentiation of PC12 cells is mediated by extracellular signal-regulated kinase (ERK), the classic mitogen-activated protein (MAP) kinase. However, mediators of the pathway downstream of ERK have not been identified. We show here that phospholipase D2 (PLD2), which generates the pleiotropic signaling lipid phosphatidic acid (PA), links ERK activation to neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. Increased expression of wild type PLD2 (WT-PLD2) dramatically elongated neurites induced by NGF stimulation or transient expression of the active form of MAP kinase-ERK kinase (MEK-CA). The response was activity-dependent, because it was inhibited by pharmacological suppression of the PLD-mediated PA production and by expression of a lipase-deficient PLD2 mutant. Furthermore, PLD2 was activated by MEK-CA, whereas NGF-stimulated PLD2 activation and hypertrophic neurite extension were blocked by an MEK-specific inhibitor. Taken together, these results provide evidence that PLD2 functions as a downstream signaling effector of ERK in the NGF signaling pathway, which leads to neurite outgrowth by PC12 cells.
- Published
- 2004
- Full Text
- View/download PDF
24. Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF receptors and membrane fluidity in PC12 cells.
- Author
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Nishio M, Fukumoto S, Furukawa K, Ichimura A, Miyazaki H, Kusunoki S, Urano T, and Furukawa K
- Subjects
- Animals, Dimerization, Enzyme Activation drug effects, Fluorescent Antibody Technique, G(M1) Ganglioside genetics, Galactosyltransferases genetics, Ganglioside Galactosyltransferase, Gangliosides genetics, Gene Expression, Glycosphingolipids genetics, Humans, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases metabolism, Nerve Growth Factor metabolism, Nerve Growth Factor pharmacology, Neurites drug effects, Neurites physiology, PC12 Cells, Phosphorylation, Rats, Receptor, Nerve Growth Factor physiology, Receptor, trkA chemistry, Receptor, trkA metabolism, Transfection, G(M1) Ganglioside physiology, Membrane Fluidity physiology, Nerve Growth Factor antagonists & inhibitors, Receptor, Nerve Growth Factor analysis
- Abstract
Ganglioside GM1 has been considered to have a neurotrophic factor-like activity. To analyze the effects of endogenously generated GM1, the rat pheochromocytoma cell line PC12 was transfected with the GM1/GD1b/GA1 synthase gene and showed increased expression levels of GM1. To our surprise, GM1+-transfectant cells (GM1+ cells) showed no neurite formation after stimulation with nerve growth factor (NGF). Autophosphorylation of NGF receptor TrkA and activation of ERK1/2 after NGF treatment were scarcely detected in GM1+ cells. Binding of 125I-NGF to PC12 cells was almost equivalent between GM1+ cells and controls. However, dimer formation of TrkA upon NGF treatment was markedly suppressed in GM1+ cells in both cross-linking analysis with Bis(sulfosuccinimidyl)suberate 3 and 125I-NGF binding assay. The sucrose density gradient fractionation of the cell lysate revealed that TrkA primarily located in the lipid raft fraction moved to the non-raft fraction in GM1+ cells. p75NTR and Ras also moved from the raft to non-raft fraction in GM1+ cells, whereas flotillin and GM1 persistently resided in the lipid raft. TrkA kinase activity was differentially regulated when GM1 was added to the kinase assay system in vitro, suggesting suppressive/enhancing effects of GM1 on NGF signals based on the concentration. Measurement of fluorescence recovery after photobleaching revealed that the membrane fluidity was reduced in GM1+ cells. These results suggested that overexpressed GM1 suppresses the differentiation signals mediated by NGF/TrkA by modulating the properties of the lipid raft and the intracellular localization of NGF receptors and relevant signaling molecules.
- Published
- 2004
- Full Text
- View/download PDF
25. Roles for the MH2 domain of Smad7 in the specific inhibition of transforming growth factor-beta superfamily signaling.
- Author
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Mochizuki T, Miyazaki H, Hara T, Furuya T, Imamura T, Watabe T, and Miyazono K
- Subjects
- Amino Acid Sequence, Animals, Binding Sites genetics, Bone Morphogenetic Proteins metabolism, COS Cells, Cell Line, DNA-Binding Proteins genetics, Humans, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Signal Transduction, Smad7 Protein, Static Electricity, Trans-Activators genetics, Xenopus Proteins genetics, DNA-Binding Proteins chemistry, DNA-Binding Proteins metabolism, Trans-Activators chemistry, Trans-Activators metabolism, Transforming Growth Factor beta metabolism, Xenopus Proteins chemistry, Xenopus Proteins metabolism
- Abstract
Signals by cytokines of the transforming growth factor-beta (TGF-beta) superfamily are negatively regulated by inhibitory Smads (I-Smads). Smad7 inhibits signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 inhibits TGF-beta signals less effectively. I-Smads have amino-terminal N domains and carboxyl-terminal Mad homology 2 (MH2) domains. The N domains are essential for specific inhibition of TGF-beta signaling by Smad7, whereas the MH2 domains of I-Smads are involved in the inhibition of TGF-beta superfamily signals through interaction with type I receptors. Here, we have identified four basic amino acid residues (Lys-312, Lys-316, Lys-401, and Arg-409) in the basic surface of the Smad7 MH2 domain that play important roles in interaction with type I receptors. Mutations of the four basic amino acid residues to acidic residues (K312E, K316E, K401E, and R409E) abolished the interaction of Smad7 with TGF-beta type I receptors, inhibition of Smad2 phosphorylation and transcriptional responses induced by TGF-beta, and induction of target genes of endogenous activin/Nodal signals in Xenopus early embryos. The K401E and R409E mutants of Smad7 were also unable to interact with BMP type I receptors (BMPR-I), repress the Smad5 phosphorylation and transcription induced by BMP, and effectively inhibit endogenous BMP signals in Xenopus early embryos. However, the K312E and K316E mutants were able to interact with BMPR-I and retained the ability to inhibit BMP signaling. Thus, the MH2 domain of Smad7 plays important roles in specific inhibition of TGF-beta superfamily signals through differential interaction with type I receptors.
- Published
- 2004
- Full Text
- View/download PDF
26. Gem GTPase and tau: morphological changes induced by gem GTPase in cho cells are antagonized by tau.
- Author
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Oyama F, Kotliarova S, Harada A, Ito M, Miyazaki H, Ueyama Y, Hirokawa N, Nukina N, and Ihara Y
- Subjects
- Amino Acid Sequence, Animals, Brain metabolism, CHO Cells, Cricetinae, Cricetulus, Gene Expression, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Microtubule-Associated Proteins chemistry, Microtubule-Associated Proteins metabolism, Molecular Sequence Data, RNA, Messenger biosynthesis, RNA, Messenger genetics, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins genetics, Recombinant Proteins metabolism, Transfection, Up-Regulation, ras Proteins genetics, tau Proteins deficiency, tau Proteins genetics, ras Proteins antagonists & inhibitors, ras Proteins metabolism, tau Proteins physiology
- Abstract
A series of observations have indicated that tau, one of the major microtubule-associated proteins, is involved in neuronal cell morphogenesis and axonal maintenance. Tau is also the major component of paired helical filaments found in brains affected by Alzheimer's disease. To explore an as yet unidentified role of tau in vivo, approximately 11,000 mRNAs were profiled from tau-deficient mouse brains and compared with those from control brains at the same ages. The expression of Gem GTPase, a small GTP-binding protein of the ras superfamily, was significantly increased in the brains of tau-deficient mice at 8 weeks of age. Because Gem GTPase is a negative regulator of the Rho-Rho kinase pathway for cytoskeletal organization, this protein was transiently overexpressed in Chinese hamster ovary cells that do not express tau. Overexpression of Gem GTPase induced a marked elongation of Chinese hamster ovary cells, and simultaneous expression of tau eliminated this effect, although tau did not bind directly to Gem GTPase. This anti-elongation activity of tau was attributed to its microtubule-binding domain, and homologous domains of microtubule-associated proteins 2 and 4 exhibited similar antagonistic activities. Taken together, the present results indicate that the level of Gem GTPase and its cell elongation activity are modulated by tau and suggest that tau may be involved in a Gem GTPase-mediated signal transduction pathway.
- Published
- 2004
- Full Text
- View/download PDF
27. Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation.
- Author
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Aoki N, Ueno S, Mano H, Yamasaki S, Shiota M, Miyazaki H, Yamaguchi-Aoki Y, Matsuda T, and Ullrich A
- Subjects
- Amino Acid Sequence, Animals, B-Lymphocytes metabolism, COS Cells, Cell Line, DNA, Complementary metabolism, Glutathione Transferase metabolism, Humans, Molecular Sequence Data, Mutation, Phosphorylation, Plasmids metabolism, Precipitin Tests, Promoter Regions, Genetic, Protein Binding, Protein Phosphatase 1, Protein Structure, Tertiary, Protein Tyrosine Phosphatases, Non-Receptor, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins c-fos metabolism, Sequence Homology, Amino Acid, Signal Transduction, Transfection, Tyrosine metabolism, Gene Expression Regulation, Protein Tyrosine Phosphatases chemistry, Protein-Tyrosine Kinases chemistry, Tyrosine chemistry
- Abstract
PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.
- Published
- 2004
- Full Text
- View/download PDF
28. Potent activation of RhoA by Galpha q and Gq-coupled receptors.
- Author
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Chikumi H, Vázquez-Prado J, Servitja JM, Miyazaki H, and Gutkind JS
- Subjects
- Cell Line, GTP-Binding Protein alpha Subunits, Gq-G11, Gene Deletion, Genes, Reporter, Humans, Mutation, Plasmids metabolism, Precipitin Tests, Protein Binding, Signal Transduction, Time Factors, Transfection, Heterotrimeric GTP-Binding Proteins metabolism, Receptors, Cell Surface metabolism, rhoA GTP-Binding Protein metabolism
- Abstract
Heterotrimeric G proteins of the G(i), G(s), and G(q) family control a wide array of physiological functions primarily by regulating the activity of key intracellular second messenger-generating systems. alpha subunits of the G(12) family, Galpha(12) and Galpha(13), however, can promote cellular responses that are independent of conventional second messengers but that result from the activation of small GTP-binding proteins of the Rho family and their downstream targets. These findings led to the identification of a novel family of guanine-nucleotide exchange factors (GEFs) that provides a direct link between Galpha(12/13) and Rho stimulation. Recent observations suggest that many cellular responses elicited by Galpha(q) and its coupled receptors also require the functional activity of Rho. However, available evidence suggests that Galpha(q) may act on pathways downstream from Rho rather than by promoting Rho activation. These seemingly conflicting observations and the recent development of sensitive assays to assess the in vivo levels of active Rho prompted us to ask whether Galpha(q) and its coupled receptors can stimulate endogenous Rho. Here we show that the expression of activated forms of Galpha(q) and the stimulation of G(q)-coupled receptors or chimeric Galpha(q) molecules that respond to G(i)-linked receptors can promote a robust activation of endogenous Rho in HEK-293T cells. Interestingly, this response was not prevented by molecules interfering with the ability of Galpha(13) to stimulate its linked RhoGEFs, together suggesting the existence of a novel molecular mechanism by which Galpha(q) and the large family of G(q)-coupled receptors can regulate the activity of Rho and its downstream signaling pathways.
- Published
- 2002
- Full Text
- View/download PDF
29. Phosphatidylinositol 4-phosphate 5-kinase is essential for ROCK-mediated neurite remodeling.
- Author
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Yamazaki M, Miyazaki H, Watanabe H, Sasaki T, Maehama T, Frohman MA, and Kanaho Y
- Subjects
- Amides pharmacology, Animals, Green Fluorescent Proteins, Intracellular Signaling Peptides and Proteins, Luminescent Proteins metabolism, Lysophospholipids metabolism, Mice, Microscopy, Fluorescence, Myosins metabolism, Plasmids metabolism, Pyridines pharmacology, Recombinant Fusion Proteins metabolism, Signal Transduction, Time Factors, Transfection, rho-Associated Kinases, Neurons metabolism, Phosphotransferases (Alcohol Group Acceptor) physiology, Protein Serine-Threonine Kinases metabolism, Protein Serine-Threonine Kinases physiology
- Abstract
Phosphatidylinositol 4-phosphate 5-kinase (PIP-5kin) regulates actin cytoskeletal reorganization through its product phosphatidylinositol 4,5-bisphosphate. In the present study we demonstrate that PIP-5kin is essential for neurite remodeling, which is regulated by actin cytoskeletal reorganization in neuroblastoma N1E-115 cells. Overexpression of wild-type mouse PIP-5kin-alpha inhibits the neurite formation that is normally stimulated by serum depletion, whereas a lipid kinase-defective mutant of PIP-5kin-alpha, D266A, triggers neurite extension even in the presence of serum and blocks lysophosphatidic acid-induced neurite retraction. These results phenocopy those previously reported for the small GTPase RhoA and its effector p160 Rho-associated coiled coil-forming protein kinase (ROCK). However, the ROCK-specific inhibitor Y-27632 failed to block the inhibition by PIP-5kin-alpha of neurite extension, whereas D266A did block the neurite retraction induced by overexpression of ROCK. These results, taken together, suggest that PIP-5kin-alpha functions as a downstream effector for RhoA/ROCK to couple lysophosphatidic acid signaling to neurite retraction presumably through its product phosphatidylinositol 4,5-bisphosphate.
- Published
- 2002
- Full Text
- View/download PDF
30. Overexpression of ganglioside GM1 results in the dispersion of platelet-derived growth factor receptor from glycolipid-enriched microdomains and in the suppression of cell growth signals.
- Author
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Mitsuda T, Furukawa K, Fukumoto S, Miyazaki H, Urano T, and Furukawa K
- Subjects
- 3T3 Cells, Animals, Cell Division, Dimerization, Mice, Mitogen-Activated Protein Kinases metabolism, Phosphorylation, Transfection, G(M1) Ganglioside metabolism, Glycolipids metabolism, Receptors, Platelet-Derived Growth Factor metabolism
- Abstract
To investigate the molecular mechanisms of gangliosides for the regulation of cell proliferation, Swiss 3T3 cells were transfected with GM2/GD2 synthase and GM1 synthase cDNAs, resulting in the establishment of GM1-expressing (GM1(+)) lines. Compared with the vector control (GM1(-)) cell lines, GM1(+) cells exhibited reduced cell proliferation by stimulation with platelet-derived growth factor (PDGF). In accordance with the reduced cell growth, GM1(+) cells showed earlier decreases in the phosphorylation levels of PDGF receptor and less activation of MAP kinases than GM1(-) cells. To analyze the effects of GM1 expression on the PDGF/PDGF receptor (PDGFR) signals, the glycolipid-enriched microdomain (GEM) was isolated and the following results were obtained. (i) PDGFR predominantly distributed in the non-GEM fraction in GM1(+) cells, while it was present in both GEM and non-GEM fractions in GM1(-) cells. (ii) Activation of PDGFR as detected by anti-phosphotyrosine antibody occurred almost in parallel with existing amounts of PDGFR in each fraction. (iii) GM1 binds with PDGFR in GEM fractions. These findings suggested that GM1 regulates signals via PDGF/PDGFR by controlling the distribution of PDGFR in- and outside of GEM, and also interacting with PDGFR in the GEM fraction as a functional constituent of the microdomain.
- Published
- 2002
- Full Text
- View/download PDF
31. b-series Ganglioside deficiency exhibits no definite changes in the neurogenesis and the sensitivity to Fas-mediated apoptosis but impairs regeneration of the lesioned hypoglossal nerve.
- Author
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Okada M, Itoh Mi M, Haraguchi M, Okajima T, Inoue M, Oishi H, Matsuda Y, Iwamoto T, Kawano T, Fukumoto S, Miyazaki H, Furukawa K, Aizawa S, and Furukawa K
- Subjects
- Animals, Cells, Cultured, Gangliosides deficiency, Hypoglossal Nerve pathology, Mice, Mice, Knockout, Sialyltransferases genetics, Apoptosis physiology, Gangliosides physiology, Hypoglossal Nerve physiology, Nerve Regeneration physiology, Sialyltransferases physiology, fas Receptor physiology
- Abstract
The polymorphic carbohydrate structures of gangliosides play regulatory roles. In particular, b-series gangliosides, all of which contain alpha-2,8 sialic acids, have been considered to be critical in various biological events such as adhesion, toxin binding, neurite extension, cell growth, and apoptosis. To clarify the physiological functions of b-series gangliosides in vivo, we have established a gene knockout mouse of GD3 synthase. Although all b-series structures were deleted in the mutant mice, they showed an almost complete nervous tissue morphology with no apparent abnormal behavior. Moreover, no differences in Fas-mediated apoptotic reaction of lymphocytes between wild type and the mutant mice were detected. However, the mutant mice exhibited clearly reduced regeneration of axotomized hypoglossal nerves compared with the wild type, suggesting that b-series gangliosides are more important in the repair rather than in the differentiation of the nervous system and apoptotic process induced via Fas.
- Published
- 2002
- Full Text
- View/download PDF
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