Fumiaki Ito

Publications of Fumiaki Ito

• Anaphase DNA bridges induced by lack of RecQ5 in Drosophila syncytial embryos.

  Authors: Haruna Sakurai, Misa Okado, Fumiaki Ito, Katsumi Kawasaki

  FEBS letters. 06/2011; 585(12):1923-8.

  Drosophila melanogaster RecQ5, a member of the RecQ family, is expressed in early embryos. The loss of maternally-derived RecQ5 leads to spontaneous mitotic defects in syncytial embryos. WeDrosophila melanogaster RecQ5, a member of the RecQ family, is expressed in early embryos. The loss of maternally-derived RecQ5 leads to spontaneous mitotic defects in syncytial embryos. We demonstrate that the mitotic defects are derived from anaphase DNA bridges. Pairs of daughter nuclei that had been linked by the bridges concurrently exited from the cycle and were eliminated by Chk2-dependent centrosome inactivation. These results suggest that the lack of RecQ5 leads to spontaneous double-stranded DNA breaks (DSBs). RecQ5 may function in the resolution of anaphase DNA bridges during mitosis or in DSB repair during interphase in syncytial Drosophila embryos.

• Dual regulation of hepatocyte apoptosis by reactive oxygen species: Increases in transcriptional expression and decreases in proteasomal degradation of BimEL.

  Authors: Yasuhiro Ishihara, Kenji Takeuchi, Fumiaki Ito, Norio Shimamoto

  Journal of cellular physiology. 04/2011; 226(4):1007-16.

  Reactive oxygen species (ROS) have a fundamental role in intracellular signaling transduction. We show here that time-dependent extracellular signal-regulated kinase (ERK) activation due toReactive oxygen species (ROS) have a fundamental role in intracellular signaling transduction. We show here that time-dependent extracellular signal-regulated kinase (ERK) activation due to inactivation of protein tyrosine phosphatases was closely linked to hepatocyte apoptosis under sustained exposure to ROS, which is produced through inhibition of ROS-scavenging enzymes. We found, for the first time, that active ERK transcriptionally increased BimEL expression among seven proteins of the Bcl-2 family. Transfection of Bim siRNA inhibited BimEL expression and hepatocyte apoptosis. Although ERK activation also elicited BimEL phosphorylation and subsequent ubiquitination, exposure to ROS for 9 h decreased proteasome activity. Collectively, the amount of BimEL was elevated by its increased expression and decreased degradation, leading to apoptosis. Exposure to ROS for 6 h caused neither reduction of proteasome activity nor hepatocyte apoptosis. These results indicate that the duration of exposure to ROS determines the fate of cells, that is, survival or death, in addition to the species, amounts, and generation sites of ROS.

• Increased expression of c-Fos by extracellular signal-regulated kinase activation under sustained oxidative stress elicits BimEL upregulation and hepatocyte apoptosis.

  Authors: Yasuhiro Ishihara, Fumiaki Ito, Norio Shimamoto

  The FEBS journal. 03/2011; 278(11):1873-81.

  We previously reported that the inhibition of catalase and glutathione peroxidase activities by treatment with 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid evoked sustained increases in theWe previously reported that the inhibition of catalase and glutathione peroxidase activities by treatment with 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid evoked sustained increases in the levels of reactive oxygen species and apoptosis in rat primary hepatocytes. Apoptosis was accompanied by increased expression of BimEL, following activation of extracellular signal-regulated kinase. The aim of this study was to characterize the mechanism underlying hepatocyte apoptosis by identifying the transcription factor that induces BimEL expression. The bim promoter region was cloned into a promoterless-luc vector, and promoter activity was monitored by a luciferase assay. The luciferase activity increased in the presence of ATZ + mercaptosuccinic acid. Pretreatment with a MEK inhibitor, U0126, or an antioxidant, vitamin C, suppressed the promoter activity. Furthermore, ATZ + mercaptosuccinic acid-induced luciferase activity was attenuated by mutation of the activator protein-1 binding site in the bim promoter region. The amounts of total and phosphorylated c-Fos increased over time in the presence of ATZ + mercaptosuccinic acid, whereas the amounts of total and phosphorylated c-Jun remained unchanged. Chromatin immunoprecipitation revealed that both c-Fos and c-Jun localized to the activator protein-1-binding site in the bim promoter region. BimEL expression and hepatocyte apoptosis were suppressed by knockdown of c-Fos and c-Jun, respectively. These results indicate that increases in c-Fos following extracellular signal-regulated kinase activation are critical for BimEL upregulation and apoptosis.

• Foreword. Target therapy for cancer: anti-cancer drugs targeting growth-factor signaling molecules.

  Authors: Fumiaki Ito

  Biological & pharmaceutical bulletin. 01/2011; 34(12):1773.

• Receptor tyrosine kinases and targeted cancer therapeutics.

  Authors: Kenji Takeuchi, Fumiaki Ito

  Biological & pharmaceutical bulletin. 01/2011; 34(12):1774-80.

  The majority of growth factor receptors are composed of extracellular, transmembrane, and cytoplasmic tyrosine kinase (TK) domains. Receptor tyrosine kinase (RTK) activation regulates many keyThe majority of growth factor receptors are composed of extracellular, transmembrane, and cytoplasmic tyrosine kinase (TK) domains. Receptor tyrosine kinase (RTK) activation regulates many key processes including cell growth and survival. However, dysregulation of RTK has been found in a wide range of cancers, and it has been shown to correlate with the development and progression of numerous cancers. Therefore, RTK has become an attractive therapeutic target. One way to effectively block signaling from RTK is inhibition of its catalytic activity with small-molecule inhibitors. Low-molecular-weight TK inhibitors (TKIs), such as imatinib, targeting tumors with mutant c-Kit, and gefitinib, targeting non-small cell lung cancer with mutant epidermal growth factor receptor (EGFR), have received marketing approval in Japan. MET, fibroblast growth factor receptor (FGFR), and insulin-like growth factor-I receptor (IGF-IR) are frequently genetically altered in advanced cancers. TKIs of these receptors have not yet appeared on the market, but many anticancer drug candidates are currently undergoing clinical trials. Most of these TKIs were designed to compete with ATP at the ATP-binding site within the TK domain. This review will focus on small-molecule TKIs targeting MET, FGFR, and IGF-IR and discuss the merits and demerits of two types of agents, i.e., those with only one or a few targets and those directed at multiple targets. Targeting agents specifically inhibiting the target kinase were previously searched for based on the hypothesis that a narrow target window might reduce unexpected side effects, but agents with multiple targets have been recently developed to overcome tumors resistant against a single-targeting agent.

• Novel aspects of epidermal growth factor receptor in relation to tumor development.

  Authors: Fumiaki Ito, Kenji Takeuchi

  The FEBS journal. 11/2009;

• EGF receptor in relation to tumor development: molecular basis of responsiveness of cancer cells to EGFR-targeting tyrosine kinase inhibitors.

  Authors: Kenji Takeuchi, Fumiaki Ito

  The FEBS journal. 11/2009;

  The function of the epidermal growth factor receptor (EGFR) is dysregulated in various types of malignancy as a result of gene amplification, mutations, or abnormally increased ligand production.The function of the epidermal growth factor receptor (EGFR) is dysregulated in various types of malignancy as a result of gene amplification, mutations, or abnormally increased ligand production. Therefore, the tyrosine kinase activity of the EGFR is a promising therapeutic target. EGFR tyrosine kinase inhibitors, such as gefitinib (Iressa), show evident anticancer effects in patients with non-small cell lung cancer. The induction of apoptosis has been considered to be the major mechanism for these gefitinib-mediated anticancer effects. Lung cancer cells harboring mutant EGFRs become dependent on them for their survival and, consequently, undergo apoptosis following the inhibition of EGFR tyrosine kinase by gefitinib. Gefitinib has been shown to inhibit cell survival and growth signaling pathways such as the extracellular signal-regulated kinase 1/2 pathway and the Akt pathway, as a consequence of the inactivation of EGFR. However, the precise downstream signaling molecules of extracellular signal-regulated kinase 1/2 and Akt have not yet been elucidated. In this minireview we have highlighted the effect of tyrosine kinase inhibitors on members of the Bcl-2 family of proteins, which are downstream signaling molecules and serve as the determinants that control apoptosis. We also discuss tyrosine kinase inhibitor-induced apoptosis via c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase.

• EGFR mutation up-regulates EGR1 expression through the ERK pathway.

  Authors: Mari Maegawa, Tokuzo Arao, Hideyuki Yokote, Kazuko Matsumoto, Kanae Kudo, Kaoru Tanaka, Hiroyasu Kaneda, Yoshihiko Fujita, Fumiaki Ito, Kazuto Nishio

  Anticancer research. 05/2009; 29(4):1111-7.

  BACKGROUND: DelE746_A750-type EGFR is a constitutively active type of mutation that enhances EGFR signaling. However, the changes in gene expression that occur in mutant EGFR-harboring cells has notBACKGROUND: DelE746_A750-type EGFR is a constitutively active type of mutation that enhances EGFR signaling. However, the changes in gene expression that occur in mutant EGFR-harboring cells has not been fully studied. MATERIALS AND METHODS: A gene expression analysis of HEK293 cells transfected with wild-type or mutant EGFR was performed focusing on the significant gene. RESULTS: Early growth response 1 (EGR1), a transcription factor, was the most strongly up-regulated gene in mutant EGFR-transfected cells among the genes examined. An increase in EGR1 expression in the mutant EGFR cells was confirmed using RT-PCR or immunoblotting. The expression was up-regulated by EGF stimulation and down-regulated by EGFR-tyrosine kinase inhibitor. In addition, the MEK inhibitor U0126 inhibited EGR1 expression, while the phosphatidylinositol 3-kinase inhibitor LY294002 did not. CONCLUSION: Mutant EGFR constitutively up-regulates EGR1 through the ERK pathway, and its expression is correlated with EGFR signal activation. Findings provide an insight into a target gene of mutant EGFR and further improve the understanding of the oncogenic properties of EGFR.

• Mitogen-activated protein kinase phosphatase-1 modulated JNK activation is critical for apoptosis induced by inhibitor of epidermal growth factor receptor-tyrosine kinase.

  Authors: Kenji Takeuchi, Tomohiro Shin-Ya, Kazuto Nishio, Fumiaki Ito

  The FEBS journal. 02/2009;

  Alterations resulting in enhanced epidermal growth factor receptor (EGFR) expression or function have been documented in a variety of tumors. Therefore, EGFR-tyrosine kinase is a promisingAlterations resulting in enhanced epidermal growth factor receptor (EGFR) expression or function have been documented in a variety of tumors. Therefore, EGFR-tyrosine kinase is a promising therapeutic target. Although in vitro and in vivo studies have shown the anti-tumor activity of EGFR-tyrosine kinase inhibitors against various tumor types, little is known about the mechanism by which such inhibitors effect their anti-tumor action. AG1478 is known to selectively inhibit EGFR-tyrosine kinase. In this study, we showed that AG1478 caused apoptosis and apoptosis-related reactions such as the activation of caspase 3 in human non-small cell lung cancer cell line PC-9. To investigate the signaling route by which AG1478 induced apoptosis, we examined the activation of c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase p38 in AG1478-treated PC-9 cells. JNK, but not p38, was significantly activated by AG1478 as determined by both immunoblot analysis for levels of phosphorylated JNK and an in vitro activity assay. Various types of stimuli activated JNK through phosphorylation by the dual-specificity JNK kinases, but the dual-specificity JNK kinases MKK4 and MKK7 were not activated by AG1478 treatment. However, JNK phosphatase, i.e. mitogen-activated protein kinase phosphatase-1 (MKP-1), was constitutively expressed in the PC-9 cells, and its expression level was reduced by AG1478. The inhibition of JNK activation by ectopic expression of MKP-1 or a dominant-negative form of JNK strongly suppressed AG1478-induced apoptosis. These results reveal that JNK, which is activated through the decrease in the MKP-1 level, is critical for EGFR-tyrosine kinase inhibitor-induced apoptosis.

• Epidermal growth factor receptor lacking C-terminal autophosphorylation sites retains signal transduction and high sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor.

  Authors: Mari Maegawa, Tokuzo Arao, Hideyuki Yokote, Kazuko Matsumoto, Kanae Kudo, Kaoru Tanaka, Hiroyasu Kaneda, Yoshihiko Fujita, Fumiaki Ito, Kazuto Nishio

  Cancer science. 02/2009;

  Constitutively active mutations of epidermal growth factor receptor (EGFR) (delE746_A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in theConstitutively active mutations of epidermal growth factor receptor (EGFR) (delE746_A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in the C-terminal region of EGFR. These pathways are thought to be important for cellular sensitivity to EGFR tyrosine kinase inhibitors (TKI). To examine the correlation between phosphorylation of the tyrosine residues in the C-terminal region of EGFR and cellular sensitivity to EGFR TKI, we used wild-type (wt) EGFR, as well as the following constructs: delE746_A750 EGFR; delE746_A750 EGFR with substitution of seven tyrosine residues to phenylalanine in the C-terminal region; and delE746_A750 EGFR with a C-terminal truncation at amino acid 980. These constructs were transfected stably into HEK293 cells and designated HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D-Tr, respectively. The HEK293/D cells were found to be 100-fold more sensitive to EGFR TKI (AG1478) than HEK293/Wt. Surprisingly, the HEK293/D7F and HEK293/D-Tr cells, transfected with EGFR lacking the C-terminal autophosphorylation sites, retained high sensitivity to EGFR TKI. In these three high-sensitivity cells, the ERK pathway was activated without ligand stimulation, which was inhibited by EGFR TKI. In addition, although EGFR in the HEK293/D7F and HEK293/D-Tr cells lacked significant tyrosine residues for EGFR signal transduction, phosphorylation of Src homology and collagen homology (Shc) was spontaneously activated in these cells. Our results indicate that tyrosine residues in the C-terminal region of EGFR are not required for cellular sensitivity to EGFR TKI, and that an as-yet-unknown signaling pathway of EGFR may exist that is independent of the C-terminal region of EGFR. (Cancer Sci 2009).

• Loss of RecQ5 leads to spontaneous mitotic defects and chromosomal aberrations in Drosophila melanogaster.

  Authors: Minoru Nakayama, Shin-Ichiroh Yamaguchi, Yoshiko Sagisu, Haruna Sakurai, Fumiaki Ito, Katsumi Kawasaki

  DNA repair. 12/2008;

  RecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation inRecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation in RecQ5, Drosophila melanogaster RecQ5 is highly expressed in early embryos, suggesting an important role for it in the DNA metabolism of the early embryo. In this present study, we generated RecQ5 mutants in D. melanogaster. Embryos lacking maternally derived RecQ5 contained irregular nuclei in early embryogenesis. These irregular nuclei emerged in nuclear cycle 11-13, lost cell-cycle markers, and were located below the surface monolayer of nuclei. By time-lapse microscopy, these irregular nuclei were observed not to divide, whereas all neighboring nuclei proceeded through normal mitotic division with synchrony. These data suggest that the irregular nuclei exited from the nuclear division cycle. This phenotype is reminiscent of the effect of X-ray irradiation on wild-type embryos and was rescued by expression of RecQ5. Thus, the maternal supply of RecQ5 is important for the nuclear cycles in syncytical embryos. Furthermore, the frequencies of spontaneous and induced chromosomal aberrations were increased in RecQ5 mutant neuroblasts. These data imply that DNA damage accumulates spontaneously in RecQ5 mutants. Therefore, endogenous genomic damage may be produced in Drosophila development, and RecQ5 would be involved in the maintenance of genomic stability by suppressing the accumulation of DNA damage.

• Growth stimulation of non-small cell lung cancer cell lines by antibody against epidermal growth factor receptor promoting formation of ErbB2/ErbB3 heterodimers.

  Authors: Mari Maegawa, Kenji Takeuchi, Eishi Funakoshi, Katsumi Kawasaki, Kazuto Nishio, Nobuyoshi Shimizu, Fumiaki Ito

  Molecular cancer research : MCR. 04/2007; 5(4):393-401.

  Antibodies are the most rapidly expanding class of human therapeutics, including their use in cancer therapy. Monoclonal antibodies (mAb) against epidermal growth factor (EGF) receptor (EGFR)Antibodies are the most rapidly expanding class of human therapeutics, including their use in cancer therapy. Monoclonal antibodies (mAb) against epidermal growth factor (EGF) receptor (EGFR) generated for cancer therapy block the binding of ligand to various EGFR-expressing human cancer cell lines and abolish ligand-dependent cell proliferation. In this study, we show that our mAb against EGFRs, designated as B4G7, exhibited a growth-stimulatory effect on various human cancer cell lines including PC-14, a non-small cell lung cancer cell line; although EGF exerted no growth-stimulatory activity toward these cell lines. Tyrosine phosphorylation of EGFRs occurred after treatment of PC-14 cells with B4G7 mAb, and it was completely inhibited by AG1478, a specific inhibitor of EGFR tyrosine kinase. However, this inhibitor did not affect the B4G7-stimulated cell growth, indicating that the growth stimulation by B4G7 mAb seems to be independent of the activation of EGFR tyrosine kinase. Immunoprecipitation with anti-ErbB3 antibody revealed that B4G7, but not EGF, stimulated heterodimerization between ErbB2 and ErbB3. ErbB3 was tyrosine phosphorylated in the presence of B4G7 but not in the presence of EGF. Further, the phosphorylation and B4G7-induced increase in cell growth were inhibited by AG825, a specific inhibitor of ErbB2. These results show that the ErbB2/ErbB3 dimer functions to promote cell growth in B4G7-treated cells. Changes in receptor-receptor interactions between ErbB family members after inhibition of one of its members are of potential importance in optimizing current EGFR family-directed therapies for cancer.

• Relationships of Drosophila melanogaster RECQ5/QE to cell-cycle progression and DNA damage.

  Authors: Minoru Nakayama, Sayako Maruyama, Hiroshi Kanda, Noriko Ohkita, Katsunori Nakano, Fumiaki Ito, Katsumi Kawasaki

  FEBS letters. 01/2007; 580(30):6938-42.

  Members of the RecQ family of DNA helicases are involved in the cellular response to DNA damage and are regulated in the cell-cycle. However, little is known about RecQ5, one of these members. TheMembers of the RecQ family of DNA helicases are involved in the cellular response to DNA damage and are regulated in the cell-cycle. However, little is known about RecQ5, one of these members. The level of RECQ5/QE, Drosophila melanogaster RecQ5, was increased after the exposure of cultured cells to methyl-methanesulfonate. Transgenic flies that overexpressed RECQ5/QE in their developing eye primordia showed mild roughening of the ommatidial lattice. DNA-damaging agents and the mei-41 mutation enhanced the phenotype caused by RECQ5/QE overexpression. Overexpression of RECQ5/QE perturbed the progression of the cell-cycle in response to DNA damage in the eye imaginal discs. These results suggest that RECQ5/QE interacts with components of the cell-cycle during its progression in response to DNA damage.

• Expression of m-Golsyn/Syntabulin gene during mouse brain development.

  Authors: Eishi Funakoshi, Masaki Fukui, Ayako Hamano, Hiroshi Okamoto, Chie Sugiyama, Norito Nishiyama, Kiyokazu Ogita, Takamitsu Hori, Nobuyoshi Shimizu, Fumiaki Ito

  Neuroscience letters. 09/2006; 403(3):244-9.

  We recently isolated the cDNA for the mouse Golsyn/Syntabulin (m-Golsyn/Syntabulin) gene and mapped it to mouse chromosome 15B3.2 syntenic with human chromosome 8q23, on which a locus responsible forWe recently isolated the cDNA for the mouse Golsyn/Syntabulin (m-Golsyn/Syntabulin) gene and mapped it to mouse chromosome 15B3.2 syntenic with human chromosome 8q23, on which a locus responsible for primary open-angle glaucoma had been located. In the present study, we examined the expression of m-Golsyn/Syntabulin protein in various regions of mouse brain and its developmental changes by use of anti-GOLSYN antibody. m-Golsyn/Syntabulin protein was detected in various brain regions at embryonic day 14 and throughout the postnatal stages. Furthermore, as the histogenesis and maturation of brain proceeded, strong expression of the protein became detectable in cells of the choroid plexus, piriform cortex, pyramidal cell layer, and Purkinje cell layer. In situ hybridization analysis of the mouse brain revealed that localization of the m-Golsyn/Syntabulin transcript was very similar to that of m-Golsyn/Syntabulin protein, confirming the high-level expression of the m-Golsyn/Syntabulin gene in the specific brain regions. High-level expression of m-Golsyn/Syntabulin protein was also observed in the ocular tissues including the ciliary body, which is known as a site for the production of aqueous humor. These results may indicate a significant role for this protein in neuronal cells and other types of cells such as those of the choroid plexus and ciliary body.

• Role of transcription factor activator protein 1 (AP1) in epidermal growth factor-mediated protection against apoptosis induced by a DNA-damaging agent.

  Authors: Kenji Takeuchi, Yu-Ichiro Motoda, Fumiaki Ito

  The FEBS journal. 08/2006; 273(16):3743-55.

  We investigated the survival signals of epidermal growth factor (EGF) in human gastric adenocarcinoma cell line TMK-1. Treatment of TMK-1 cells with adriamycin (ADR) caused apoptosis andWe investigated the survival signals of epidermal growth factor (EGF) in human gastric adenocarcinoma cell line TMK-1. Treatment of TMK-1 cells with adriamycin (ADR) caused apoptosis and apoptosis-related reactions such as the release of cytochrome c from mitochondria and the activation of caspase 9. However, EGF treatment greatly reduced the ADR-induced apoptosis as well as these reactions. We previously reported that hepatocyte growth factor transmitted protective signals against ADR-induced apoptosis by causing activation of the phosphatidylinositol-3'-OH kinase (PtdIns3-K)/Akt signaling pathway in human epithelial cell line MKN74 [Takeuchi K & Ito F (2004) J Biol Chem279, 892-900]. However, PtdIns3-K/Akt signaling did not mediate the antiapoptotic action of EGF in TMK-1 cells. EGF increased the expression of the Bcl-X(L) protein, an antiapoptotic member of the Bcl-2 family, but not that of other anti (Bcl-2) or proapoptotic (Bad and Bax) protein members. Expression of the c-Fos and c-Jun, components of activator protein 1 (AP1), which are known to regulate bcl-X(L) gene transcription, were increased in response to EGF. Pretreatment of the cells with PD98059, an inhibitor of MAP kinase kinase, inhibited the EGF-induced c-Fos and c-Jun expression, AP1 DNA binding, Bcl-X(L) expression, and the resistance against ADR-induced apoptosis, suggesting that EGF transmitted the antiapoptotic signal in such a way that it activated AP1 via a MAP kinase signaling pathway. TMK-1 cells stably transfected with TAM67, c-Jun dominant-negative mutant, did not display EGF-induced Bcl-X(L) expression or resistance against ADR-induced apoptosis. These results indicate that AP1-mediated upregulation of Bcl-X(L) expression is critical for protection of TMK-1 cells against ADR-induced apoptosis.

• Molecular cloning of the m-Golsyn gene and its expression in the mouse brain.

  Authors: Eishi Funakoshi, Ayako Hamano, Masaki Fukui, Norito Nishiyama, Kiyokazu Ogita, Nobuyoshi Shimizu, Fumiaki Ito

  Gene expression. 02/2006; 13(1):27-40.

  The mouse ortholog of the human GOLSYN gene, termed the m-Golsyn gene, was isolated and mapped to the region on mouse chromosome 15B3.2 syntenic with human chromosome 8q23. Three mRNA species (typeThe mouse ortholog of the human GOLSYN gene, termed the m-Golsyn gene, was isolated and mapped to the region on mouse chromosome 15B3.2 syntenic with human chromosome 8q23. Three mRNA species (type la, 1b, and type 2) were produced by use of alternative transcription initiation points and alternative splicing events. The type 1 mRNAs were expressed only in the brain, whereas the type 2 was detected in various tissues. m-Golsyn protein was expressed in various tissues including the brain. Immunohistochemical study of m-Golsyn protein showed its prominent expression in the neuronal cells in various regions of the brain and strong expression in the choroid plexus ependymal cells lining the ventricles. m-Golsyn protein was found to be homologous to syntaphilin, a regulator of synaptic vesicle exocytosis. These results indicate that the m-Golsyn protein may play an important role in intracellular protein transport in neuronal cells of the brain.

• Molecular cloning and characterization of gene for Golgi-localized syntaphilin-related protein on human chromosome 8q23.

  Authors: Eishi Funakoshi, Kin-Ya Nakagawa, Ayako Hamano, Takamitsu Hori, Atsushi Shimizu, Shuichi Asakawa, Nobuyoshi Shimizu, Fumiaki Ito

  Gene. 02/2005; 344:259-71.

  Loci for several human genetic diseases including glaucoma have been mapped to q23 region on chromosome 8. We carried out homology search analysis of the genomic sequence of a bacterial artificialLoci for several human genetic diseases including glaucoma have been mapped to q23 region on chromosome 8. We carried out homology search analysis of the genomic sequence of a bacterial artificial chromosome (BAC) clone, KB1590E11, on 8q23 region, and mapped a previously described cDNA, KIAA1472, to this BAC clone. In this study, we determined the complete genomic structure of the KIAA1472 gene and its expression in various tissues and cell lines. Four mRNA species (types 1a, 1b, 1c, and 2) were produced from this gene by use of alternative transcription start sites and alternative-splicing events. These mRNAs were expressed in various tissues, except for type 1a, which was found only in the brain. Further, type 1 mRNA could be translated into two protein isoforms with different N-terminal sequences; and type 2 mRNA, into another type of isoform. All three of these KIAA1472 gene products were localized in Golgi apparatus and contained a C-terminal hydrophobic segment characteristic of a transmembrane domain, thus indicating them to be Golgi membrane-bound proteins. Furthermore, these proteins were homologous to syntaphilin, a molecule involved in guiding vesicular transport. These results indicate that KIAA1472 gene products may play an important role in vesicular traffic in various tissues including the brain.

• Roles of the HSP70-subunit in a eukaryotic multi-site-specific endonuclease, Endo.SceI: autophosphorylation and heat stability.

  Authors: Katsumi Kawasaki, Takehiko Shibata, Fumiaki Ito

  Bioscience, biotechnology, and biochemistry. 01/2005; 68(12):2557-64.

  The 70 kDa heat shock proteins (HSP70) are a family of molecular chaperones that bind transiently to unfolded proteins in an ATP/ADP dependent manner. Endo.SceI comprises a unique example forThe 70 kDa heat shock proteins (HSP70) are a family of molecular chaperones that bind transiently to unfolded proteins in an ATP/ADP dependent manner. Endo.SceI comprises a unique example for mitochondrial HSP70, which exists in a stable complex with a nucleolytic subunit as a multi-site specific DNase. The HSP70-subunit in Endo.SceI was autophosphorylated by ATP in vitro. The autophosphorylation was higher in the Endo.SceI complex form than in the free form. Although the autophosphorylation had no significant effect on the endonucleolytic activity of Endo.SceI, the factors favoring autophosphorylation protected the endonucleolytic activity of Endo.SceI against heat inactivation. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), and ADP not only protected the endonucleolytic activity against heat inactivation in the presence of Ca(2+) ions, but also reduced the labeling of the HSP70-subunit by [gamma-(32)P]ATP in Endo.SceI. These findings suggest that the HSP70-subunit shields Endo.SceI from heat inactivation through ATP/ADP binding.

• Overexpression of the human MNB/DYRK1A gene induces formation of multinucleate cells through overduplication of the centrosome.

  Authors: Eishi Funakoshi, Takamitsu Hori, Tokuko Haraguchi, Yasushi Hiraoka, Jun Kudoh, Nobuyoshi Shimizu, Fumiaki Ito

  BMC cell biology. 10/2003; 4:12.

  BACKGROUND: Previously we cloned the human MNB/DYRK1A gene from the "Down syndrome critical region" on chromosome 21. This gene encodes a dual specificity protein kinase that catalyzes itsBACKGROUND: Previously we cloned the human MNB/DYRK1A gene from the "Down syndrome critical region" on chromosome 21. This gene encodes a dual specificity protein kinase that catalyzes its autophosphorylation on serine/threonine and tyrosine residues. But, the functions of the MNB/DYRK1A gene in cellular processes are unknown. RESULTS: In this study, we examined HeLa cells transfected with cDNA encoding a green fluorescent protein (GFP)-MNB/DYRK1A fusion protein and found 2 patterns of expression: In one group of transfected cells, GFP-MNB/DYRK1A was localized as dots within the nucleus; and in the other group, it was overexpressed and had accumulated all over the nucleus. In the cells overexpressing GFP-MNB/DYRK1A, multinucleation was clearly observed; whereas in those with the nuclear dots, such aberrant nuclei were not found. Furthermore, in the latter cells, essential processes such as mitosis and cytokinesis occurred normally. Multinucleation was dependent on the kinase activity of MNB/DYRK1A, because it was not observed in cells overexpressing kinase activity-negative mutants, GFP-MNB/DYRK1A (K179R) and GFP-MNB/DYRK1A (Y310F/Y312F). Immunostaining of GFP-MNB/DYRK1A-overexpressing cells with specific antibodies against alpha- and gamma-tubulin revealed that multiple copies of centrosomes and aberrant multipolar spindles were generated in these cells. CONCLUSIONS: These results indicate that overexpression of MNB/DYRK1A induces multinucleation in HeLa cells through overduplication of the centrosome during interphase and production of aberrant spindles and missegregation of chromosomes during mitosis.

• Transmembrane delivery of polypeptide hormones bypassing the intrinsic cell surface receptors: a conjugate of insulin with α2-macroglobulin (α2M) recognizing both insulin and α2M receptors and its biological activity in relation to endocytic pathways

  Authors: Fumiaki Ito, Sachiko Ito, Nobuyoshi Shimizu

  Molecular and Cellular Endocrinology.

  125I-labeled insulin has been cross-linked to α2-macroglobulin (α2M) via a disulfide bond. The resulting insulin-α2M conjugate carried 2.2 insulin moieties per mole of α2M and was able to deliver125I-labeled insulin has been cross-linked to α2-macroglobulin (α2M) via a disulfide bond. The resulting insulin-α2M conjugate carried 2.2 insulin moieties per mole of α2M and was able to deliver insulin into rat hepatoma cells H35 and HTC. The insulin delivery was mediated predominantly through α2M receptors and 2 h after binding it was found in the lyposomal fractions in the form of conjugate. When the conjugate was applied to rat hepatoma cells it stimulated activity of tyrosine aminotransferase (TAT) with a potency one-half that of native insulin. Hepatoma cells which were treated with conjugate in the presence of bacitracin were also stimulated for TAT activity. Since bacitracin completely inhibited the α2M binding to its receptors, but inhibited conjugate binding by only 80%, this stimulation must have resulted from the remaining binding of conjugate. These results indicate that the insulin-α2M conjugate was biologically active if it bound to insulin receptors, but that the conjugate bound and internalized through α2M receptors did not act as a mediator for TAT activation. Our results using Percoll density gradients indicate a difference in intracellular processing between insulin, α2M and the conjugate. Mechanisms of action of the conjugate are discussed in relation to the receptor-mediated endocytic pathways.