Researcher Database

Hideaki Higashi
Research Center for Zoonosis Control Division of Infection and Immunity
Professor

Researcher Profile and Settings

Affiliation

  • Research Center for Zoonosis Control Division of Infection and Immunity

Job Title

  • Professor

Degree

  • (BLANK)

J-Global ID

Research Interests

  • 細菌   ヘリコバクターピロリ   感染症   癌   

Research Areas

  • Life sciences / Molecular biology

Association Memberships

  • JAPANESE SOCIETY FOR BACTERIOLOGY   日本ヘリコバクター学会   日本癌学会   

Research Activities

Published Papers

  • Hideki Sudo, Katsuhisa Yamada, Koji Iwasaki, Hideaki Higashi, Manabu Ito, Akio Minami, Norimasa Iwasaki
    PLOS ONE 8 (3) e58806  1932-6203 2013/03 [Refereed][Not invited]
     
    Background: Intervertebral disc degeneration is a significant cause of degenerative spinal diseases. Nucleus pulposus (NP) cells reportedly fail to survive in large degenerated discs with limited nutrient availability. Therefore, understanding the regulatory mechanism of the molecular response of NP cells to nutrient deprivation may reveal a new strategy to treat disc degeneration. This study aimed to identify genes related to nutrient deprivation in NP cells on a global scale in an experimental nutrient deprivation model. Methodology/Principal Findings: Rat NP cells were subjected to serum starvation. Global gene expression was profiled by microarray analysis. Confirmation of the selected genes was obtained by real-time polymerase chain reaction array analysis. Western blotting was used to confirm the expression of selected genes. Functional interactions between p21(Cip1) and caspase 3 were examined. Finally, flow cytometric analyses of NP cells were performed. Microarray analysis revealed 2922 differentially expressed probe sets with >= 1.5-fold changes in expression. Serum starvation of NP cells significantly affected the expression of several genes involved in DNA damage checkpoints of the cell cycle, including Atm, Brca1, Cdc25, Gadd45, Hus1, Ppm1D, Rad 9, Tp53, and Cyclin D1. Both p27(Kip1) and p53 protein expression was upregulated in serum-starved cells. p21(Cip1) expression remained in NP cells transfected with short interfering RNA targeting caspase 3 (caspase 3 siRNA). Both G1 arrest and apoptosis induced by serum starvation were inhibited in cells transfected with caspase 3 siRNA. Conclusions/Significance: Nutrient deprivation in NP cells results in the activation of a signaling response including DNA damage checkpoint genes regulating the cell cycle. These results provide novel possibilities to improve the success of intervertebral disc regenerative techniques.
  • Mudenda B. Hang'ombe, James C. L. Mwansa, Sergio Muwowo, Phillip Mulenga, Muzala Kapina, Eric Musenga, David Squarre, Liywali Mataa, Suzuki Y. Thomas, Hirohito Ogawa, Hirofumi Sawa, Hideaki Higashi
    TROPICAL DOCTOR 42 (3) 136 - 139 0049-4755 2012/07 [Refereed][Not invited]
     
    There has been a reduction of incidences of anthrax in the developed countries but it is still a public health problem in the developing countries where communities live in interface areas with wildlife. An outbreak of anthrax in Hippopotamus amphibious was observed in Zambia. Following the death of hippopotamuses, suspected human cases were reported. The objective of this study was to isolate and confirm Bacillus anthracis and to determine the antimicrobial susceptibility for the management of the disease. Of the specimens collected, 29.4% (95% confidence interval [CI], 11.4-56.0) were from humans, 42.1% (95% CI, 21.1-66.0) were from hippopotamuses and 20.0% (95% CI, 6.61-44.3) from the soil were found to be positive were for B. anthracis. An antimicrobial susceptibility test revealed that all the isolates were found to be sensitive to the recommended antibiotics. The disease control was achieved by case management and by explaining to the communities that they should avoid contact with animals that die from unknown causes.
  • Takeru Hayashi, Miki Senda, Hiroko Morohashi, Hideaki Higashi, Masafumi Horio, Yui Kashiba, Lisa Nagase, Daisuke Sasaya, Tomohiro Shimizu, Nagarajan Venugopalan, Hiroyuki Kumeta, Nobuo N. Noda, Fuyuhiko Inagaki, Toshiya Senda, Masanori Hatakeyama
    CELL HOST & MICROBE 12 (1) 20 - 33 1931-3128 2012/07 [Refereed][Not invited]
     
    The Helicobacter pylori type IV secretion effector CagA is a major bacterial virulence determinant and critical for gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA localizes to the inner face of the plasma membrane, where it acts as a pathogenic scaffold/hub that promiscuously recruits host proteins to potentiate oncogenic signaling. We find that CagA comprises a structured N-terminal region and an intrinsically disordered C-terminal region that directs versatile protein interactions. X-ray crystallographic analysis of the N-terminal CagA fragment (residues 1-876) revealed that the region has a structure comprised of three discrete domains. Domain I constitutes a mobile CagA N terminus, while Domain II tethers CagA to the plasma membrane by interacting with membrane phosphatidylserine. Domain III interacts intramolecularly with the intrinsically disordered C-terminal region, and this interaction potentiates the pathogenic scaffold/hub function of CagA. The present work provides a tertiary-structural basis for the pathophysiological/oncogenic action of H. pylori CagA.
  • Naoko Murata-Kamiya, Kenji Kikuchi, Takeru Hayashi, Hideaki Higashi, Masanori Hatakeyama
    CELL HOST & MICROBE 7 (5) 399 - 411 1931-3128 2010/05 [Refereed][Not invited]
     
    When delivered into gastric epithelial cells via type IV secretion, Helicobacter pylori CagA perturbs host cell signaling and thereby promotes gastric carcinogenesis. However, the mechanisms of CagA delivery, localization, and action remain poorly understood. We show that direct contact of H. pylori with epithelial cells induces externalization of the inner leaflet enriched host phospholipid, phosphatidylserine, to the outer leaflet of the host plasma membrane. CagA, which is exposed on the bacterial surface via type IV secretion, interacts with the externalized phosphatidylserine to initiate its entry into cells. CagA delivery also requires energy-dependent host cell processes distinct from known endocytic pathways. Within polarized epithelial cells, CagA is tethered to the inner leaflet of the plasma membrane through interaction with phosphatidylserine and binds the polarity-regulating host kinase PAR1/MARK to induce junctional and polarity defects. Thus, host membrane phosphatidylserine plays a key role in the delivery, localization, and pathophysiological action of CagA.
  • Acacia Lamb, Xiao-Dong Yang, Ying-Hung N. Tsang, Jiang-Dong Li, Hideaki Higashi, Masanori Hatakeyama, Richard M. Peek, Steven R. Blanke, Lin-Feng Chen
    EMBO REPORTS 10 (11) 1242 - 1249 1469-221X 2009/11 [Refereed][Not invited]
     
    Helicobacter pylori-initiated chronic gastritis is characterized by the cag pathogenicity island-dependent upregulation of proinflammatory cytokines, which is largely mediated by the transcription factor nuclear factor (NF)-kappa B. However, the cag pathogenicity island-encoded proteins and cellular signalling molecules that are involved in H. pylori-induced NF-kappa B activation and inflammatory response remain unclear. Here, we show that H. pylori virulence factor CagA and host protein transforming growth factor-beta-activated kinase 1 (TAK1) are essential for H. pylori-induced activation of NF-kappa B. CagA physically associates with TAK1 and enhances its activity and TAK1-induced NF-kappa B activation through the tumour necrosis factor receptor-associated factor 6-mediated, Lys 63-linked ubiquitination of TAK1. These findings show that polyubiquitination of TAK1 regulates the activation of NF-kappa B, which in turn is used by H. pylori CagA for the H. pylori-induced inflammatory response.
  • Huai-Sheng Lu, Yasuhiro Saito, Mayumi Umeda, Naoko Murata-Kamiya, Hong-Mei Zhang, Hideaki Higashi, Masanori Hatakeyama
    CANCER SCIENCE 99 (10) 2004 - 2011 1347-9032 2008/10 [Refereed][Not invited]
     
    Helicobacter pylori (H. pylori) cagA-positive strains are associated with gastritis, peptic ulcerations, and gastric adenocarcinoma. Upon delivery into gastric epithelial cells, the cagA-encoded CagA protein specifically binds and aberrantly activates SHP-2 oncoprotein in a manner that is dependent on CagA tyrosine phosphorylation. CagA-deregulated SHP-2 then elicits aberrant Erk activation while causing an elongated cell shape known as the hummingbird phenotype. In polarized epithelial cells, CagA also binds to PAR1b/MARK2 and inhibits the PAR1b kinase activity, thereby disrupting tight junctions and epithelial cell polarity independent of CagA tyrosine phosphorylation. We show here that the CagA-multimerization (CM) sequence that mediates interaction of CagA with PAR1b is not only essential for the CagA-triggered junctional defects but also plays an important role in induction of the hummingbird phenotype by potentiating CagA-SHP-2 complex formation. We also show that the CM sequence of CagA isolated from East Asian H. pylori (referred to as the E-CM sequence) binds PAR1b more strongly than that of CagA isolated from Western H. pylori (referred to as the W-CM sequence). Within Western CagA species, the ability to bind PAR1b is proportional to the number of W-CM sequences. Furthermore, the level of PAR1b-binding activity of CagA correlates with the magnitude of junctional defects and the degree of hummingbird phenotype induction. Our findings reveal that structural diversity in the CM sequence is an important determinant for the degree of virulence of CagA, a bacterial oncoprotein that is associated with gastric carcinogenesis. (Cancer Sci 2008; 99: 2004-2011)
  • D. Miyamoto, M. Miyamoto, A. Takahashi, Y. Yomogita, H. Higashi, S. Kondo, M. Hatakeyama
    ONCOGENE 27 (25) 3508 - 3515 0950-9232 2008/06 [Refereed][Not invited]
     
    SHP-2 protein tyrosine phosphatase plays an important role in activation of the RAS-dependent signaling. Gain-of-function mutations in the PTPN11 gene, which encodes SHP-2, have been found in the leukemia-prone developmental disorder Noonan syndrome as well as sporadic childhood leukemias, indicating that SHP-2 is a bona. de human oncoprotein. However, the role of SHP-2 mutations in non-hematological malignancies remains obscure. Here, we screened for PTPN11 mutations in primary solid tumors and identified a 1520C > A mutation that causes threonine-507 to lysine (T507K) substitution in the phosphatase domain of SHP-2 in a case of hepatocellular carcinoma. T507K SHP-2 exhibited altered substrate specificity with slightly elevated basal phosphatase activity. Upon expression in NIH3T3 cells, T507K SHP-2 induced transformed foci, which was not observed with wild type, Noonan-specific or leukemia-specific SHP-2. Furthermore, NIH3T3 cells transformed by T507K SHP-2 showed anchorage-independent growth and developed tumors in nude mice. These results indicate that quantitative and/or qualitative alteration in phosphatase activity determines the transforming potential as well as target cell/tissue spectrum of individual SHP-2 mutants as oncoproteins. Although rare in solid tumors, the identified T507K SHP-2 represents a distinct class of SHP-2 mutants with oncogenic RAS-like transforming activity, which could contribute to the development of solid tumors.
  • Yo Kurashima, Naoko Murata-Kamiya, Kenji Kikuchi, Hideaki Higashi, Takeshi Azuma, Satoshi Kondo, Masanori Hatakeyama
    INTERNATIONAL JOURNAL OF CANCER 122 (4) 823 - 831 0020-7136 2008/02 [Refereed][Not invited]
     
    Infection with Helicobacter pylori cagA-positive strains causes gastritis and peptic ulceration and is associated with gastric adenocarcinoma. The cagA gene product CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases at the C-terminal EPIYA-repeat region. Tyrosine-phosphorylated CagA specifically binds and activates SHP-2 tyrosine phosphatase, causing cell morphological transformation known as the hummingbird phenotype. CagA also destabilizes the E-cadherin/beta-catenin complex to elicit aberrant activation of the beta-catenin signal that underlies intestinal metaplasia. Here we show that translocalization of membranous beta-catenin and subsequent activation of the beta-catenin signal by CagA requires the EPIYA-repeat region, which is characterized by structural variation between CagA of H. pylori isolated in Western countries (Western CagA) and that of East Asian H. pylori isolates (East Asian CagA), but is independent of CagA tyrosine phosphorylation. Detailed analysis using a series of Western and East Asian CagA mutants revealed that deregulation of beta-catenin requires residues 10091086 and residues 908-1012 of ABCCC Western CagA and ABD East Asian CagA, respectively, and is mediated by the 16-aminoacid CagA multimerization sequence that is conserved between the 2 geographically distinct H. pylori CagA species. Our results indicate that aberrant activation of the P-catenin signal, which promotes precancerous intestinal metaplasia, is an inherent and fundamental CagA activity that is independent of the structural polymorphism of CagA. (C) 2007 Wiley-Liss, Inc.
  • Naomi Ohnishi, Hitomi Yuasa, Shinya Tanaka, Hirofumi Sawa, Motohiro Miura, Atsushi Matsui, Hideaki Higashi, Manabu Musashi, Kazuya Lwabuchi, Misao Suzuki, Gen Yamada, Takeshi Azuma, Masanori Hatakeyama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 (3) 1003 - 1008 0027-8424 2008/01 [Refereed][Not invited]
     
    Infection with cagA-positive Helicobacter pylori is associated with gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma of B cell origin. The cagA-encoded CagA protein is delivered into gastric epithelial cells via the bacterial type IV secretion system and, upon tyrosine phosphorylation by Src family kinases, specifically binds to and aberrantly activates SHIP-2 tyrosine phosphatase, a bona fide oncoprotein in human malignancies. CagA also elicits junctional and polarity defects in epithelial cells by interacting with and inhibiting partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) independently of CagA tyrosine phosphorylation. Despite these CagA activities that contribute to neoplastic transformation, a causal link between CagA and in vivo oncogenesis remains unknown. Here, we generated transgenic mice expressing wild-type or phosphorylation-resistant CagA throughout the body or predominantly in the stomach. Wild-type CagA transgenic mice showed gastric epithelial hyperplasia and some of the mice developed gastric polyps and adenocarcinomas of the stomach and small intestine. Systemic expression of wild-type CagA further induced leukocytosis with IL-3/GM-CSF hypersensitivity and some mice developed myeloid leukemias and B cell lymphomas, the hematological malignancies also caused by gain-of-function SHIP-2 mutations. Such pathological abnormalities were not observed in transgenic mice expressing phosphorylation-resistant CagA. These results provide first direct evidence for the role of CagA as a bacterium-derived oncoprotein (bacterial oncoprotein) that acts in mammals and further indicate the importance of CagA tyrosine phosphorylation, which enables CagA to deregulate SHIP-2, in the development of H. pylori-associated neoplasms.
  • N. Murata-Kamiya, Y. Kurashima, Y. Teishikata, Y. Yamahashi, Y. Saito, H. Higashi, H. Aburatani, T. Akiyama, R. M. Peek, T. Azuma, M. Hatakeyama
    ONCOGENE 26 (32) 4617 - 4626 0950-9232 2007/07 [Refereed][Not invited]
     
    Infection with Helicobacter pylori cagA-positive strains is associated with gastric adenocarcinoma. Intestinal metaplasia is a precancerous lesion of the stomach characterized by transdifferentiation of the gastric mucosa to an intestinal phenotype. The H. pylori cagA gene product, CagA, is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Tyrosine-phosphorylated CagA specifically binds to and activates SHP-2 phosphatase, thereby inducing cell-morphological transformation. We report here that CagA physically interacts with E-cadherin independently of CagA tyrosine phosphorylation. The CagA/E-cadherin interaction impairs the complex formation between E-cadherin and beta-catenin, causing cytoplasmic and nuclear accumulation of beta-catenin. CagA-deregulated beta-catenin then transactivates beta-catenin-dependent genes such as cdx1, which encodes intestinal specific CDX1 transcription factor. In addition to b-catenin signal, CagA also transactivates p21(WAF1/Cip1), again, in a phosphorylation-independent manner. Consequently, CagA induces aberrant expression of an intestinal-differentiation marker, goblet-cell mucin MUC2, in gastric epithelial cells that have been arrested in G1 by p21(WAF1/Cip1). These results indicate that perturbation of the E-cadherin/beta-catenin complex by H. pylori CagA plays an important role in the development of intestinal metaplasia, a premalignant transdifferentiation of gastric epithelial cells from which intestinal-type gastric adenocarcinoma arises.
  • Iraj Saadat, Hideaki Higashi, Chikashi Obuse, Mayumi Umeda, Naoko Murata-Kamiya, Yasuhiro Saito, Huaisheng Lu, Naomi Ohnishi, Takeshi Azuma, Atsushi Suzuki, Shigeo Ohno, Masanori Hatakeyama
    NATURE 447 (7142) 330 - U8 0028-0836 2007/05 [Refereed][Not invited]
     
    Helicobacter pylori cagA-positive strains are associated with gastritis, ulcerations and gastric adenocarcinoma(1). CagA is delivered into gastric epithelial cells(2) and, on tyrosine phosphorylation, specifically binds and activates the SHP2 oncoprotein(3-7), thereby inducing the formation of an elongated cell shape known as the 'hummingbird' phenotype(2,3). In polarized epithelial cells, CagA also disrupts the tight junction and causes loss of apical basolateral polarity(8,9). We show here that H. pylori CagA specifically interacts with PAR1/MARK kinase, which has an essential role in epithelial cell polarity(10,11). Association of CagA inhibits PAR1 kinase activity and prevents atypical protein kinase C ( aPKC)-mediated PAR1 phosphorylation, which dissociates PAR1 from the membrane(12,13), collectively causing junctional and polarity defects. Because of the multimeric nature of PAR1 (ref. 14), PAR1 also promotes CagA multimerization, which stabilizes the CagA - SHP2 interaction(15). Furthermore, induction of the hummingbird phenotype by CagA-activated SHP2 requires simultaneous inhibition of PAR1 kinase activity by CagA. Thus, the CagA - PAR1 interaction not only elicits the junctional and polarity defects but also promotes the morphogenetic activity of CagA. Our findings revealed that PAR1 is a key target of H. pylori CagA in the disorganization of gastric epithelial architecture underlying mucosal damage, inflammation and carcinogenesis.
  • Shumei Ren, Hideaki Higashi, Huaisheng Lu, Takeshi Azuma, Masanori Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 281 (43) 32344 - 32352 0021-9258 2006/10 [Refereed][Not invited]
     
    Helicobacter pylori cagA-positive strains are associated with gastric adenocarcinoma. The cagA gene product CagA is delivered into gastric epithelial cells where it localizes to the plasma membrane and undergoes tyrosine phosphorylation at the EPIYA-repeat region, which contains the EPIYA-A segment, EPIYA-B segment, and Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D segment. In host cells, CagA specifically binds to and deregulates SHP-2 phosphatase via the tyrosine-phosphorylated EPIYA-C or EPIYA-D segment, thereby inducing an elongated cell shape known as the hummingbird phenotype. In this study, we found that CagA multimerizes in cells in a manner independent of its tyrosine phosphorylation. Using a series of CagA mutants, we identified a conserved amino acid sequence motif (FPLXRXXXVXDL-SKVG), which mediates CagA multimerization, within the EPIYA-C segment as well as in a sequence that located immediately downstream of the EPIYA-C or EPIYA-D segment. We also found that a phosphorylation-resistant CagA, which multimerizes but cannot bind SHP-2, inhibits the wild-type CagA-SHP-2 complex formation and abolishes induction of the hummingbird phenotype. Thus, SHP-2 binds to a preformed and tyrosine-phosphorylated CagA multimer via its two Src homology 2 domains. These results, in turn, indicate that CagA multimerization is a prerequisite for CagA-SHP-2 interaction and subsequent deregulation of SHP-2. The present work raises the possibility that inhibition of CagA multimerization abolishes pathophysiological activities of CagA that promote gastric carcinogenesis.
  • M Naito, T Yamazaki, R Tsutsumi, H Higashi, K Onoe, S Yamazaki, T Azuma, M Hatakeyama
    GASTROENTEROLOGY 130 (4) 1181 - 1190 0016-5085 2006/04 [Refereed][Not invited]
     
    Background & Aims: Helicobacter pylori CagA-positive strain is associated with gastric adenocarcinoma. CagA is delivered into gastric epithelial cells, where it undergoes tyrosine phosphorylation at the EPIYA sites by Src family kinases (SFKs). Owing to homologous recombination within the 3'-region of the cagA gene, 4 distinct EPIYA sites, each of which is defined by surrounding sequences, are variably assembled in both number and order among CagA proteins from different clinical H pylori isolates. Tyrosine-phosphorylated CagA specifically binds and deregulates SHP-2 via the Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D site, and C-terminal Src kinase (Csk) via the EPIYA-A or EPIYA-B site. Here we investigated the influence of EPIYA-repeat polymorphism on the CagA activity. Methods: A series of EPIYA-repeat variants of CagA were expressed in AGS gastric epithelial cells and the ability of individual CagA to bind SHP-2 or Csk was determined by the sequential immunoprecipitation and immunoblotting method. Results: CagA proteins carrying multiple EPIYA-C or EPIYA-D sites bound and deregulated SHP-2 more strongly than those having a single EPIYA-C or EPIYA-D. Furthermore, the ability of CagA to bind Csk was correlated with the number of EPIYA-A and EPIYA-B sites. Because Csk inhibits SFK, CagA with greater Csk-binding activity more strongly inhibited Src-dependent CagA phosphorylation and more effectively attenuated induction of cell elongation caused by CagA-SHP-2 interaction. Conclusions: EFIYA-repeat polymorphism of CagA greatly influences the magnitude and duration of phosphorylation-dependent CagA activity, which may determine the potential of individual CagA as a bacterial virulence factor that directs gastric carcinogenesis.
  • R Tsutsumi, A Takahashi, T Azuma, H Higashi, M Hatakeyama
    MOLECULAR AND CELLULAR BIOLOGY 26 (1) 261 - 276 0270-7306 2006/01 [Refereed][Not invited]
     
    Infection with cagA-positive Helicobacter pylori (H. pylori) is associated with atrophic gastritis, peptic ulcer, and gastric adenocarcinoma. The cagA gene product CagA is translocated from H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation by Src family kinases (SFKs). Tyrosine-phosphorylated CagA binds and activates SHP-2 phosphatase and the C-terminal Src kinase (Csk) while inducing an elongated cell shape termed the "hummingbird phenotype." Here we show that CagA reduces the level of focal adhesion kinase (FAK) tyrosine phosphorylation in gastric epithelial cells. The decrease in phosphorylated FAK is due to SHP-2-mediated dephosphorylation of FAK at the activating phosphorylation sites, not due to Csk-dependent inhibition of SFKs, which phosphorylate FAK. Coexpression of constitutively active FAK with CagA inhibits induction of the hummingbird phenotype, whereas expression of dominant-negative FAK elicits an elongated cell shape characteristic of the hummingbird phenotype. These results indicate that inhibition of FAK by SHP-2 plays a crucial role in the morphogenetic activity of CagA. Impaired cell adhesion and increased motility by CagA may be involved in the development of gastric lesions associated with cagA-positive H. pylori infection.
  • N Nakano, K Urasawa, Y Takagi, T Saito, S Kaneta, S Ishikawa, H Higashi, H Tsutsui, M Hatakeyama, A Kitabatake
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 338 (3) 1661 - 1667 0006-291X 2005/12 [Refereed][Not invited]
     
    Immature vascular smooth muscle cells (VSMCs) proliferate responding to extrinsic mitogens and accumulate in neointima after arterial injuries. Cell proliferation is positively regulated by cyclin/cyclin-dependent kinase (CDK) complex and negatively controlled by CDK inhibitors; CKIs Such as p27(kip1) and p57(kip2). In this Study, embryonic rat thoracic aorta VSMCs; A10 were G0/G1 arrested by serum starvation, re-stimulated with serum, and harvested every four hours. Both CKIs co-expressed in quiescent VSMCs and rapidly diminished by stimulation. Protein level of p27(kip1) was regulated by both transcription and post-transcription, but that of p57(kip2) was mainly by post-transcription. Supplemental overexpression of p57(kip2) inhibited the activations of G1 cyclin/CDKs and subsequent hyperphosphorylations of all three retinoblastoma pocket proteins as well as G1/S transition of cell cycle. Our findings suggest that the downregulations of not only p27(kip1), but also p57(kip2) responding to mitogenic stimulation, play key roles in the cell cycle progression of VSMCs. (c) 2005 Elsevier Inc. All rights reserved.
  • M Hatakeyama, H Higashi
    CANCER SCIENCE 96 (12) 835 - 843 1347-9032 2005/12 [Refereed][Not invited]
     
    Infection with cagA-positive Helicobacter pylori is associated with the development of gastric adenocarcinoma. The cagA gene product CagA is injected directly from the bacterium into the bacterium-attached gastric epithelial cells via the type-IV secretion system. Upon membrane localization and subsequent tyrosine phosphorylation by Src family kinases, CagA functions as a scaffolding adaptor and interacts with a number of host proteins that regulate cell growth, cell motility and cell polarity in both CagA phosphorylation-dependent and phosphorylation-independent manners. Of special interest is the interaction of CagA with the SHP-2 tyrosine phosphatase, gain-of-function mutations that of which have recently been found in a variety of human malignancies. The CagA-SHP-2 interaction is entirely dependent on CagA tyrosine phosphorylation and, through the complex formation, SHP-2 is catalytically activated and induces morphological transformation with elevated cell motility. Intriguingly, structural diversity of the tyrosine phosphorylation sites of CagA accounts for the differential activity of individual CagA to bind and activate SHP-2. Deregulation of SHP-2 and other intracellular signaling molecules by H. pylori CagA may predispose cells to accumulate multiple genetic and epigenetic changes involved in gastric carcinogenesis. Furthermore, the differential potential of individual CagA to disturb cellular functions indicates that H. pylori strains carrying biologically more active CagA are more virulent than those with less active CagA and are more closely associated with gastric carcinoma.
  • S Yamazaki, A Yamakawa, T Okuda, M Ohtani, H Suto, Y Ito, Y Yamazaki, Y Keida, H Higashi, M Hatakeyama, T Azuma
    JOURNAL OF CLINICAL MICROBIOLOGY 43 (8) 3906 - 3916 0095-1137 2005/08 [Refereed][Not invited]
     
    Colonization of the stomach mucosa by Helicobacter pylori is a major cause of acute and chronic gastric pathologies in humans. Several H. pylori virulence genes that may play a role in its pathogenicity have been identified. The most important determinants are vacA and cagA in the cag pathogenicity island (cagPAI) genes. In the present study, to consider the association of molecular genetics between vacA and the cagPAI regarding clinical outcome, we selected H. pylori strains with various genotypes of vacA in Japan and sequenced full-length vacA, cagA, and cagE genes. Sequencing of vacA and cagA genes revealed variable size, whereas the cagE gene was well conserved among strains. Each of the phylogenetic trees based on the deduced amino acid sequences of VacA, CagA, and CagE indicated that all three proteins were divided into two major groups, a Western group and an East Asian group, and the distributions of isolates exhibited similar patterns among the three proteins. The strains with s2 and s1a/m1a vacA genotypes and the Western-type 3' region cagA genotype were classified into the Western group, and the strains with the s1c/m1b vacA genotype and the East Asian-type 3' cagA genotype were included in the East Asian group. In addition, the prevalence of infection with the Western group strain was significantly higher in patients with peptic ulcer (90.0%, 9/10) than in patients with chronic gastritis (22.7%, 5/22) (x(2) = 12.64, P = 0.00057). These data suggest that the molecular genetics of vacA and cagPAI are associated and that the Western group with vacA and cagPAI genes is associated with peptic ulcer disease.
  • K Yokoyama, H Higashi, S Ishikawa, Y Fujii, S Kondo, H Kato, T Azuma, A Wada, T Hirayama, H Aburatani, M Hatakeyama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 (27) 9661 - 9666 0027-8424 2005/07 [Refereed][Not invited]
     
    Chronic infection with cagA-positive Helicobacter pylori is associated with the development of atrophic gastritis, peptic ulcers, and gastric adenocarcinoma. The cagA gene product CagA is injected into gastric epithelial cells, where it undergoes tyrosine phosphorylation by Src family kinases. Translocated CagA disturbs cellular functions by physically interacting with and deregulating intracellular signaling transducers through both tyrosine phosphorylation-dependent and -independent mechanisms. To gain further insights into the pathophysiological activities of CagA in gastric epithelial cells, we executed a genome-wide screening of CagA-responsive genes by using DNA microarray and identified nuclear factor of activated T cells (NFAT) transcription factors whose binding sites were overrepresented in the promoter regions of CagA-activated genes. Results of reporter assays confirmed that CagA was capable of activating NFAT in a manner independent of CagA phosphorylation. Expression of CagA in gastric epithelial cells provoked translocation of NFATc3, a member of the NFAT family, from the cytoplasm to the nucleus and activated an NFAT-regulated gene, p21(WAF1/CiP1). CagA-mediated NFAT activation was abolished by inhibiting calcineurin or phospholipase C gamma activity. Furthermore, treatment of cells with H. pylori VacA (vacuolating toxin), which inhibits NFAT activity in T lymphocytes, counteracted the ability of CagA to activate NFAT in gastric epithelial cells. These findings indicate that the two major H. pylori virulence factors inversely control NFAT activity. Considering the pleiotropic roles of NFAT in cell growth and differentiation, deregulation of NFAT, either positively or negatively, depending on the relative exposure of cells to CagA and VacA, may contribute to the various disease outcomes caused by H. pylori infection.
  • S Yamazaki, S Kato, N Matsukura, M Ohtani, Y Ito, H Suto, Y Yamazaki, A Yamakawa, S Tokudome, H Higashi, M Hatakeyama, T Azuma
    FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY 44 (3) 261 - 268 0928-8244 2005/06 [Refereed][Not invited]
     
    The CagA protein is one of the virulence factors of Helicobacter pylori, and two major subtypes of CagA have been observed, the Western and East Asian type. CagA is injected from the bacteria into gastric epithelial cells, undergoes tyrosine phosphorylation, and binds to Src homology 2 domain-containing protein-tyrosine phosphatase SHP-2. The East Asian type CagA binds to SHP-2 more strongly than the Western type CagA. Here, we tried to distinguish the CagA type by highly sensitive real-time PCR with the objective of establishing a system to detect H. pylori and CagA subtypes from gastric biopsies. We designed primers and probe sets for Western or East Asian-cagA at Western-specific or East Asian-specific sequence regions, respectively, and H. pylori 16S rRNA. We could detect the H. pylori 16S rRNA gene, Western and East Asian-cagA gene from DNA of gastric biopsies. The sensitivity and specificity for H. pylori infection was 100% in this system. In Thai patients, 87.8% (36/41) were cagA-positive; 26.8% (11/41) were Western-cagA positive and 53.7% (22/41) were East Asian-cagA positive, while 7.3% (3/41) reacted with both types of cagA. These results suggest that this real-time PCR system provides a highly sensitive assessment of CagA type as a new diagnostic tool for the pathogenicity of H. pylori infection. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
  • H Higashi, K Yokoyama, Y Fujii, S Ren, H Yuasa, Saadat, I, NM Kamiya, T Azuma, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 280 (24) 23130 - 23137 0021-9258 2005/06 [Refereed][Not invited]
     
    Helicobacter pylori contributes to the development of peptic ulcers and atrophic gastritis. Furthermore, H. pylori strains carrying the cagA gene are more virulent than cagA-negative strains and are associated with the development of gastric adenocarcinoma. The cagA gene product, CagA, is translocated into gastric epithelial cells and localizes to the inner surface of the plasma membrane, in which it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif. Tyrosine-phosphorylated CagA specifically binds to and activates Src homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) at the membrane, thereby inducing an elongated cell shape termed the hummingbird phenotype. Accordingly, membrane tethering of CagA is an essential prerequisite for the pathogenic activity of CagA. We show here that membrane association of CagA requires the EPIYA-containing region but is independent of EPIYA tyrosine phosphorylation. We further show that specific deletion of the EPIYA motif abolishes the ability of CagA to associate with the membrane. Conversely, reintroduction of an EPIYA sequence into a CagA mutant that lacks the EPIYA-containing region restores membrane association of CagA. Thus, the presence of a single EPIYA motif is necessary for the membrane localization of CagA. Our results indicate that the EPIYA motif has a dual function in membrane association and tyrosine phosphorylation, both of which are critically involved in the activity of CagA to deregulate intracellular signaling, and suggest that the EPIYA motif is a crucial therapeutic target of cagA-positive H. pylori infection.
  • H Ozawa, S Ashizawa, M Naito, M Yanagihara, N Ohnishi, T Maeda, Y Matsuda, Y Jo, H Higashi, A Kakita, M Hatakeyama
    ONCOGENE 23 (39) 6590 - 6602 0950-9232 2004/08 [Refereed][Not invited]
     
    The eukaryotic cell cycle is regulated by sequential activation and inactivation of cyclin-cyclin-dependent kinase (Cdk) complexes. In this work, we screened human cDNAs that can rescue yeast Saccharomyces cerevisiae from lethality caused by ectopic expression of human cyclin E and isolated a cDNA encoding ESXR1, a paired-like homeodomain-containing protein with a unique C-terminal proline-rich repeat region. In adult tissues, ESXR1 is primarily expressed in the testis. We demonstrate that ESXR1 prevents degradation of ubiquitinated cyclins in human cells. Accordingly, elevation of ESXR1 level results in accumulation of cyclin A and cyclin B1 and thereby provokes M-phase arrest. In human cells, the 65-kDa full-length ESXR1 protein is capable of proteolytically processing into N-terminal 45-kDa and C-terminal 20-kDa fragments. The C-terminal fragment, containing a proline-rich repeat region, is localized to the cytoplasm and displays the ability to inhibit cyclin degradation. In contrast, the N-terminal fragment, containing a paired-like homeodomain, is localized exclusively in the nucleus, suggesting that it plays a role in transcription. Our results indicate that proteolytic processing of ESXR1 plays a role in concerted regulation of the cell cycle and transcription in human cells.
  • T Azuma, M Ohtani, Y Yamazaki, H Higashi, M Hatakeyama
    GASTROENTEROLOGY 126 (7) 1926 - 1927 0016-5085 2004/06 [Refereed][Not invited]
  • T Azuma, A Yamakawa, S Yamazaki, M Ohtani, Y Ito, A Muramatsu, H Suto, Y Yamazaki, Y Keida, H Higashi, M Hatakeyama
    JOURNAL OF CLINICAL MICROBIOLOGY 42 (6) 2508 - 2517 0095-1137 2004/06 [Refereed][Not invited]
     
    The severity of Helicobacter pylori-related disease is correlated with the presence of a cag pathogenicity island (PAI). Genetic diversity within the cag PAI may have a modifying effect on the pathogenic potential of the infecting strain. We analyzed the complete cag PAI sequences of 11 representative Japanese strains according to their vacA genotypes and clinical effects and examined the relationship between the diversity of the cag PAI and clinical features. The cag PAI genes were divided into two major groups, a Western and a Japanese group, by phylogenetic analysis based on the entire cag PAI sequences. The predominant Japanese strains formed a Japanese cluster which was different from the cluster formed by Western strains. The diversity of the cag PAI was associated with the vacA and cagA genotypes. All strains with the s1c vacA genotype were in the Japanese cluster. In addition, all strains with the East Asian-type cagA genotype were also in the Japanese cluster. Patients infected with the Japanese-cluster strain had high-grade gastric mucosal atrophy. These results suggest that a distinct diversity of the cag PAI of H. pylori is present among Japanese strains and that this diversity may be involved in the development of atrophic gastritis and may increase the risk for gastric cancer.
  • M Higuchi, R Tsutsumi, H Higashi, M Hatakeyama
    CANCER SCIENCE 95 (5) 442 - 447 1347-9032 2004/05 [Refereed][Not invited]
     
    RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-l-thio-beta-D-galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP-2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach.
  • H Higashi, A Nakaya, R Tsutsumi, K Yokoyama, Y Fujii, S Ishikawa, M Higuchi, A Takahashi, Y Kurashima, Y Teishikata, S Tanaka, T Azuma, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (17) 17205 - 17216 0021-9258 2004/04 [Refereed][Not invited]
     
    The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma. CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity. We established AGS human gastric epithelial cells that inducibly express wildtype or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines. Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the "hummingbird" phenotype. Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes. Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA ( siRNA) abolished the CagA-mediated hummingbird phenotype. The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2. In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation. Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation. Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells. These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA. Our work therefore suggests a key role of SHP-2 in the pathological activity of H. pylori virulence factor CagA.
  • T Azuma, S Yamazaki, A Yamakawa, M Ohtani, A Muramatsu, H Suto, Y Ito, M Dojo, Y Yamazaki, M Kuriyama, Y Keida, H Higashi, M Hatakeyama
    JOURNAL OF INFECTIOUS DISEASES 189 (5) 820 - 827 0022-1899 2004/03 [Refereed][Not invited]
     
    We investigated the relationship between the diversity of Helicobacter pylori CagA protein and clinical outcome. The cagA gene was sequenced in 115 clinical isolates. The binding affinity of CagA to Src homology 2 domain containing tyrosine phosphatase (SHP-2) was examined by in vitro infection. Two major CagA subtypes were observed - the East Asian and the Western type. The grades of inflammation, activity of gastritis, and atrophy were significantly higher in patients with gastritis infected with the East Asian CagA-positive strain than in patients with gastritis infected with cagA-negative or Western CagA-positive strains. All strains isolated from patients with gastric cancer were East Asian CagA positive. East Asian CagA exhibited stronger SHP-2-binding activity than did Western CagA. These findings suggest that infection with East Asian CagA - positive H. pylori is associated with atrophic gastritis and gastric cancer and that persistent active inflammation induced by the East Asian CagA- positive strain may play a role in the pathogenesis of disease.
  • W Zhou, S Yamazaki, A Yamakawa, M Ohtani, Y Ito, Y Keida, H Higashi, M Hatakeyama, JM Si, T Azuma
    FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY 40 (1) 81 - 87 0928-8244 2004/01 [Refereed][Not invited]
     
    It has been reported that Helicobacter pylori infection with the type I strain, which expresses the VacA and CagA antigens, is associated with duodenal ulcer. We examined the diversity of vacA and cagA genes in 143 isolates obtained from patients with duodenal ulcer or chronic gastritis in East Asia (two different areas of Japan, Fukui and Okinawa, and also in Hangzhou, China) by polymerase chain reaction (PCR) and sequence analysis. Diversities of cagA and vacA genes were detected in East Asia. The prevalence of cagA-positive H. pylori was significantly different between Fukui and Okinawa (P = 0.0032). The prevalence of Western type CagA was significantly higher in Okinawa than in Fukui (P < 0.0001). However, there was no significant association between the genotype of cagA and clinical outcome. In Japan, the predominant vacA genotype was slc/mlb. In contrast, in Hangzhou, the predominant vacA genotype was s1c/m2, and they were all East Asian CagA-positive. These findings suggest that a distinct distribution of the vacA and cagA genotypes is present in East Asia, regardless of clinical outcome. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
  • S Umehara, H Higashi, N Ohnishi, M Asaka, M Hatakeyama
    ONCOGENE 22 (51) 8337 - 8342 0950-9232 2003/11 [Refereed][Not invited]
     
    Helicobacter pylori (H. pylori) is a causative agent of gastrointestinal diseases such as atrophic gastritis and gastroduodenal ulcer. Infection of cagA-positive H. pylori is also associated with gastric carcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The cagA gene product CagA is directly injected into the bacteria-attached host cells via the bacterial type IV secretion system. The translocated CagA deregulates intracellular signaling pathways and thereby initiates pathogenesis. In this work, we examined the biological effects of CagA on B cells, from which MALT lymphoma arises. Ectopic expression of CagA in interleukin 3-dependent B cells inhibited cell proliferation by suppressing the JAK-STAT signaling. CagA was also capable of preventing hydroxyurea-induced B-cell apoptosis through inhibiting p53 accumulation. In contrast to the effects of CagA in gastric epithelial cells, the observed CagA activities in B cells were independent of its tyrosine phosphorylation. Our results indicate that CagA possesses both phosphorylation-dependent and -independent activities in mammalian cells and that biological impacts of CagA depend on cell-type context. As a result of B-cell growth inhibition, CagA may diminish anti-H. pylori immune responses. Furthermore, CagA may play a role in the development of MALT lymphoma by impairing p53-dependent apoptosis.
  • T Takebayashi, H Higashi, H Sudo, H Ozawa, E Suzuki, O Shirado, H Katoh, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 (17) 14897 - 14905 0021-9258 2003/04 [Refereed][Not invited]
     
    The retinoblastoma protein (pRB) and its homologues, p107 and p130, prevent cell cycle progression from G(0)/G(1) to S phase by forming complexes with E2F transcription factors. Upon phosphorylation by G, cyclin-cyclin-dependent kinase (Cdk) complexes such as cyclin D1-Cdk4/6 and cyclin E-Cdk2, they lose the ability to bind E2F, and cells are thereby allowed to progress into S phase. Functional loss of one or more of the pRB family members, as a result of genetic mutation or deregulated phosphorylation, is considered to be an essential prerequisite for cellular transformation. In this study, we found that pRB family proteins have the ability to stimulate cyclin D1 transcription by activation of the NF-kappaB transcription factor. The cyclin D1-inducing activity of pRB is abolished by adenovirus E1A oncoprotein but not by the deletion of the A-box, the B-box, or the C-terminal region of the pocket, indicating that multiple pocket sequences are independently involved in cyclin D1 activation. Intriguingly, tumor-derived pRB pocket mutants retain the cyclin D1-inducing activity. Our results reveal a novel role of pRB family proteins as potential activators of NF-kappaB and inducers of G(1) cyclin. Certain pRB pocket mutants may give rise to a cellular situation in which deregulated E2F and cyclin D1 cooperatively promote abnormal cell proliferation.
  • R Tsutsumi, H Higashi, M Higuchi, M Okada, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 (6) 3664 - 3670 0021-9258 2003/02 [Refereed][Not invited]
     
    diseases ranging from gastritis to cancer. The CagA protein is the product of the cagA gene carried among virulent H. pylori strains and is associated with severe disease outcomes, most notably gastric carcinoma. CagA is injected from the attached H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation. The phosphorylated CagA binds and activates SHP-2 phosphatase and thereby induces a growth factor-like morphological change termed the "humming-bird phenotype." In this work, we demonstrate that CagA is also capable of interacting with C-terminal Src kinase (Csk). As is the case with SHP-2, Csk selectively binds tyrosine-phosphorylated CagA via its SH2 domain. Upon complex formation, CagA stimulates Csk, which in turn inactivates the Src family of protein-tyrosine kinases. Because Src family kinases are responsible for CagA phosphorylation, an essential prerequisite of CagA.SHP-2 complex formation and subsequent induction of the hummingbird phenotype, our results indicate that CagA-Csk interaction down-regulates CagA.SHP-2 signaling by both competitively inhibiting CagA.SHP-2 complex formation and reducing levels of CagA phosphorylation. We further demonstrate that CagA.SHP-2 signaling eventually induces apoptosis in AGS cells. Our results thus indicate that CagA-Csk interaction prevents excess cell damage caused by deregulated activation of SHP-2. Attenuation of CagA activity by Csk may enable cagA-positive H. pylori to persistently infect the human stomach for decades while avoiding excess CagA toxicity to the host.
  • S Yamazaki, A Yamakawa, Y Ito, M Ohtani, H Higashi, M Hatakeyama, T Azuma
    JOURNAL OF INFECTIOUS DISEASES 187 (2) 334 - 337 0022-1899 2003/01 [Refereed][Not invited]
     
    Recent experiments have indicated that CagA of Helicobacter pylori is injected into epithelial cells via the type IV secretion system and undergoes tyrosine phosphorylation in cells and that translocated CagA binds the SRC homology 2 domain-containing tyrosine phosphatase (SHP-2). We investigated these phenomena in in vivo human gastric mucosa. Tyrosine-phosphorylated CagA and CagA-coimmunoprecipitated SHP-2 were detected in gastric mucosa from H. pylori-positive patients with atrophic gastritis and in noncancerous tissues from H. pylori-positive patients with early gastric cancer. In contrast, CagA was not detected in gastric mucosa with either intestinal metaplasia or cancer. Our results provide the first evidence that CagA is translocated into the gastric epithelial cells, receives tyrosine phosphorylation, and binds SHP-2 in in vivo human gastric mucosa. Deregulation of SHP-2 by CagA may play a role in the acquisition of a cellular-transformed phenotype at a relatively early stage of multistep gastric carcinogenesis.
  • The effects of cure of Helicobacter pylori infection on the signal transduction of gastric epithelial cells
    Azuma T, Yamazaki S, Yamakawa A, Ito Y, Ohtani M, Dojo M, Yamazaki Y, Higashi H, Hatakeyama M
    Aliment. Pharmacol. Ther. 18 39 - 44 2003 [Refereed][Not invited]
  • H Higashi, R Tsutsumi, A Fujita, S Yamazaki, M Asaka, T Azuma, M Hatakeyama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 99 (22) 14428 - 14433 0027-8424 2002/10 [Refereed][Not invited]
     
    Helicobacter pylori is a causative agent of gastritis and peptic ulcer. cagA(+) H. pylori strains are more virulent than cagA(-) strains and are associated with gastric carcinoma. The cagA gene product, CagA, is injected by the bacterium into gastric epithelial cells and subsequently undergoes tyrosine phosphorylation. The phosphorylated CagA specifically binds SHP-2 phosphatase, activates the phosphatase activity, and thereby induces morphological transformation of cells. CagA proteins of most Western H. pylori isolates have a 34-amino acid sequence that variably repeats among different strains. Here, we show that the repeat sequence contains a tyrosine phosphorylation site. CagA proteins having more repeats were found to undergo greater tyrosine phosphorylation, to exhibit increased SHP-2 binding, and to induce greater morphological changes. In contrast, predominant CagA proteins specified by H. pylori strains isolated in East Asia, where gastric carcinoma is prevalent, had a distinct tyrosine phosphorylation sequence at the region corresponding to the repeat sequence of Western CagA. This East Asian-specific sequence conferred stronger SHP-2 binding and morphologically transforming activities to Western CagA. Finally, a critical amino acid residue that determines SHP-2 binding activity among different CagA proteins was identified. Our results indicate that the potential of individual CagA to perturb host-cell functions is determined by the degree of SHP-2 binding activity, which depends in turn on the number and sequences of tyrosine phosphorylation sites. The presence of distinctly structured CagA proteins in Western and East Asian H. pylori isolates may underlie the strikingly different incidences of gastric carcinoma in these two geographic areas.
  • M Yamada, T Kondo, S Ashizawa, T Takebayashi, H Higashi, M Hatakeyama
    CYTOKINE 17 (2) 91 - 97 1043-4666 2002/01 [Refereed][Not invited]
     
    Interleukin 3 (IL-3)-dependent proliferation of haematopoietic cells is specifically inhibited by p130, a member of the pRB-family proteins. p130 interacts with the cell-cycle regulatory E2F transcription factors, notably E2F-4 and E2F-5, and affects promoters containing E2F-binding sites through two distinct mechanisms. First, upon complex formation with E2F, it blocks transcriptional activation by E2F. Second, the formed p130-E2F complex binds to E2F sites and actively represses transcription by inhibiting the activity of surrounding enhancer elements on the promoter. To pursue the relative contributions of each mechanism in the p130-mediated inhibition of IL-3-dependent cell proliferation, we employed a dominant-negative DP-1, which suppresses both E2F-dependent transactivation and the formation of active transcriptional repressors. Ectopic expression of the dominant negative DP-1 in the IL-3-dependent BaF3 lymphoid cells gave rise to an inhibition of cell proliferation, which was concomitantly associated with a decrease in levels of cyclin E, an indispensable molecule for G1 to S-phase cell-cycle progression. Our results indicate that blocking E2F-dependent transactivation, but not the formation of p130-E2F transcriptional repressor complexes, is responsible for the inhibition of IL-3-dependent cell growth by p130. (C) 2002 Elsevier Science Ltd.
  • H Higashi, R Tsutsumi, S Muto, T Sugiyama, T Azuma, M Asaka, M Hatakeyama
    SCIENCE 295 (5555) 683 - 686 0036-8075 2002/01 [Refereed][Not invited]
     
    Helicobacter pylori CagA protein is associated with severe gastritis and gastric carcinoma. CagA is injected from the attached Helicobacter pylori into host cells and undergoes tyrosine phosphorylation. Wild-type but not phosphorylation-resistant CagA induced a growth factor-like response in gastric epithelial cells. Furthermore, CagA formed a physical complex with the SRC homology 2 domain (SH2)-containing tyrosine phosphatase SHP-2 in a phosphorylation-dependent manner and stimulated the phosphatase activity. Disruption of the CagA-SHP-2 complex abolished the CagA-dependent cellular response. Conversely, the CagA effect on cells was reproduced by constitutively active SHP-2. Thus, upon translocation, CagA perturbs cellular functions by deregulating SHP-2.
  • S Ashizawa, H Nishizawa, M Yamada, H Higashi, T Kondo, H Ozawa, A Kakita, M Hatakeyama
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 (14) 11362 - 11370 0021-9258 2001/04 [Refereed][Not invited]
     
    The activity of the retinoblastoma protein pRB is regulated by phosphorylation that is mediated by G(1) cyclin-associated cyclin-dependent kinases (CDKs). Since the pRB-related pocket proteins p107 and p130 share general structures and biological functions with pRB, their activity is also considered to be regulated by phosphorylation. In this work, we generated phosphorylation-resistant p107 and p130 molecules by replacing potential cyclin CDK phosphorylation sites with non-phosphorylatable alanine residues. These phosphorylation-resistant mutants retained the ability to bind E2F and cyclin. Upon introduction into p16(INK4a)-deficient U2-OS osteosarcoma cells, in which cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant mutants, but not wild-type p107 or p130, were capable of inhibiting cell proliferation. Furthermore, when ectopically expressed in pRB-deficient SAOS-8 osteosarcoma cells, the wild-type as well as the phosphorylation-resistant pRB family proteins were capable of inducing large flat cells. The flat cell-inducing activity of the wild-type proteins, but not that of the phosphorylation-resistant mutants; was:abolished by coexpressing cyclin E. Our results indicate that the elevated cyclin D- or cyclin E-associated kinase leads to systemic inactivation of the pRB family proteins and suggest that dysregulation of the pRB kinase provokes an aberrant cell cycle in a broader range of cell types than those induced by genetic inactivation of the RB gene.
  • Kondo T, Higashi H, Nishizawa H, Ishikawa S, Ashizawa S, Yamada M, Makita Z, Koike T, Hatakeyama M
    J. Biol. Chem. 276 (20) 17559 - 17567 0021-9258 2001 [Refereed][Not invited]
  • Ras and signal transducer and activator of transcription (STAT) are essential and sufficient downstream components of Janus kinases in cell proliferation
    R Mizuguchi, S Noto, M Yamada, S Ashizawa, H Higashi, M Hatakeyama
    JAPANESE JOURNAL OF CANCER RESEARCH 91 (5) 527 - 533 0910-5050 2000/05 [Refereed][Not invited]
     
    Cytokines exert their activities in cell growth and differentiation by binding specific cell membrane receptors, Janus kinases (JAKs) are cytoplasmic protein tyrosine kinases that physically interact with intracellular domains of the cytokine receptors and they play crucial roles in transducing signals triggered by the cytokine-receptor interaction. We have previously shown that conditional activation of JAK through membrane-proximal dimerization confers cytokine-independence on interleukin-3 (IL-3)-dependent Ba/F3 lymphoid cells and that the cytokine-independent proliferation is completely inhibited by dominant negative Ras. In this work, we demonstrate that ectopic expression of a dominant negative form of Stat5, a major signal transducer and activator of transcription (STAT) expressed in Ba/F3 cells, also inhibits JAK-triggered mitogenesis. In contrast, overexpression of constitutively active Ras or conditional activation of Stat5 by chemical dimerization fails to confer cytokine-independence. However, concomitant activation of ectopic Ras and Stat5 molecules in Ba/F3 cells suffices for cell proliferation in the absence of IL-3, Our results indicate that Ras and STAT are essential and sufficient components of JAK-triggered mitogenesis. Our findings further indicate that the cytokine signal bifurcates into Ras and STAT pathways following JAK activation.
  • A Ohtoshi, T Maeda, H Higashi, S Ashizawa, M Hatakeyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 268 (2) 530 - 534 0006-291X 2000/02 [Refereed][Not invited]
     
    The initiation of anaphase and exit from mitosis depend on the activation of the anaphase-promoting complex/cyclosome (APC/C), a multicomponent, ubiquitin-protein ligase, The WD-repeat protein called p55(CDC)(Cdc20) directly binds to and activates APC/C. By using yeast two-hybrid screening, we found that cyclin A, a critical cell cycle regulator in the S and G2/M phases, specifically interacts with p55(CDC). Ectopically expressed p55(CDC) and cyclin A form a stable protein complex in mammalian cells. The p55(CDC)-cyclin A interaction occurs through the region containing the WD repeats of p55(CDC) and the region between the destruction box and the cyclin box of cyclin A. In addition to the physical interaction, p55(CDC) is phosphorylated by cyclin A-associated kinase. These findings suggest that the function of p55(CDC) is mediated or regulated by its complex formation with cyclin A. (C) 2000 Academic Press.
  • Aso T, Yamazaki K, Amimoto K, Kuroiwa A, Higashi H, Matsuda Y, Kitajima S, Hatakeyama M
    J. Biol. Chem. 275 (9) 6546 - 6552 0021-9258 2000 [Refereed][Not invited]
  • Ohtoshi A, Maeda T, Higashi H, Ashizawa S, Yamada M, Hatakeyama M
    Biochem. Biophys. Res. Commun. 267 (3) 947 - 952 0006-291X 2000 [Refereed][Not invited]
  • A Mori, H Higashi, Y Hoshikawa, M Imamura, M Asaka, M Hatakeyama
    ONCOGENE 18 (46) 6209 - 6221 0950-9232 1999/11 [Refereed][Not invited]
     
    The retinoblastoma protein (pRB) and the related pocket proteins, p107 and p130, play crucial roles in mammalian cell cycle control. Recent studies indicate that these pocket proteins are also involved in cellular differentiation processes. We demonstrate in this work that the pRB-related p130 selectively accumulates during the in vitro differentiation of the myeloid progenitor cell, 32Dc13, into granulocyte in response to granulocyte-colony stimulating factor (G-CSF). This G-CSF-dependent granulocytic differentiation is blocked by the adenovirus E1A oncoprotein, which binds to and inactivates the pRB family of pocket proteins including p130. Furthermore, enforced overexpression of p130 but not PRE inhibits the myeloid cell proliferation that is concomitantly associated with granulocytic differentiation morphologically characterized by nuclear segmentation. However, simple G1-cell cycle al rest induced by cytokine deprivation or ectopic overexpression of the p27 cyclin-dependent kinase inhibitor, or inhibition of E2F activities by dominant negative DP-1 is not sufficient to trigger granulocytic differentiation. The differentiation-promoting activity of p130 in myeloid cells requires both the pocket domain and the spacer domain. Our results indicate that the pRB-related p130 plays a critical role in myeloid cell differentiation and suggest that coupling of cell cycle exit with the cellular differentiation program may be specifically achieved by p130.
  • S Hatakeyama, M Kitagawa, K Nakayama, M Shirane, M Matsumoto, K Hattori, H Higashi, H Nakano, K Okumura, K Onoe, RA Good, K Nakayama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 96 (7) 3859 - 3863 0027-8424 1999/03 [Refereed][Not invited]
     
    Activation of the transcription factor nuclear factor kappa B (NF-kappa B) is controlled by proteolysis of its inhibitory subunit (I kappa B) via the ubiquitin-proteasome pathway, Signal-induced phosphorylation of I kappa B alpha by a large multisubunit complex containing I kappa B kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/beta TrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with I kappa B alpha only when I kappa B alpha is phosphorylated, The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of I kappa B alpha in concert with I kappa B kinases, resulting in nuclear translocation of NF-kappa B. In addition, FWD1 strikingly evoked the ubiquitination of I kappa B alpha in the ira vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of I kappa B alpha. These results suggest that the substrate-specific degradation of I kappa B alpha is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated I kappa B alpha. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-kappa B through control of I kappa B protein stability.
  • T Terano, T Tanaka, Y Tamura, M Kitagawa, H Higashi, Y Saito, A Hirai
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 254 (2) 502 - 506 0006-291X 1999/01 [Refereed][Not invited]
     
    Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the form of triacylglycerol (TG) were dose dependently incorporated into phospholipid fraction of vascular smooth muscle cells (VSMC) and suppressed the proliferation of VSMC. Flow cytometric analysis demonstrated both EPA and DHA inhibited G(1)/S progression. EPA and DHA inhibited the phosphorylation of Cdk2 protein and Cdk2 kinase activity without altering the amount of cyclin E and p27(kip1) proteins and cyclin dependent kinase activating kinase activity by growth stimulation. This mechanisms remained to be clarified but this is the first report of a novel mechanisms of inhibition of DNA synthesis by EPA and DHA. (C) 1999 Academic Press.
  • Tanaka T, Tatsuno I, Noguchi Y, Uchida D, Oeda T, Narumiya S, Yasuda T, Higashi H, Kitagawa M, Nakayama K, Saito Y, Hirai A
    J. Biol. Chem. 273 26772 - 26778 1998 [Refereed][Not invited]
  • SuzukiTakahashi, I, H Higashi, E Yoshida, S Nishimura, M Kitagawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234 (2) 386 - 392 0006-291X 1997/05 [Refereed][Not invited]
     
    p16(INK4a), a protein that inhibits cyclin-dependent kinase 4 (Cdk4) and Cdk6, is deficient in many human cancers and in established lines of tumor cells. It has been reported that transfection with cDNA for p16(INK4a) inhibits the growth of cell lines that express retinoblastoma protein (pRB). However, it is unclear whether the introduction of cDNA for p16(INK4a) affects the growth of cells that express p16(INK4a) protein. Moreover, the effects of other cell-cycle regulators on the inhibition of cell growth by p16(INK4a) remain unknown. In this study, cDNA for p16(INK4a) was used to transfect human cell lines that had various status of expression of RE pathway-related proteins, such as members of the RE family proteins and Cdk-inhibitory proteins. We found that status of p107, p130, p15(INK4b), p18(INK4c) p21(Cip1), p27(Kip1) cyclin D1, and Cdk4 were not correlated with the growth-inhibitory activity of exogenous p16(INK4a). BY contrast, transfection with cDNA for p16(INK4a) had a significant effect on the growth of cells depended on the status not only of pRB but also of p16(INK4a). Although exogenous p16(INK4a) inhibited the growth of cells that expressed pRB but did not express p16(INK4a) (pRB(+)/p16(-) cells), it had little affect on either pRB(-)/p16(+) cells or pRB(-)/p16(+) cells. Moreover, transfection with cDNA for p16(INK4a) also inhibited the activity of the E2 promoter of the dehydrofolate reductase gene in the same manner that depended on the absence of p16(INK4a), as well as on the presence of pRB. These results suggest that deregulation of the RE pathway by p16(INK4a) deficiency plays a very important role in the proliferation of cells that lack p16(INK4a) protein. (C) 1997 Academic Press.
  • H Higashi, SuzukiTakahashi, I, E Yoshida, S Nishimura, M Kitagawa
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 231 (3) 743 - 750 0006-291X 1997/02 [Refereed][Not invited]
     
    p16(INK4a) is a inhibitory protein of Cyclin-dependent kinase 4 (Cdk4). p16 negatively regulates the cell cycle progression from G1 to S phase. Functional pie is absent from many human cancers, as well as from many established lines of tumor cells. However, it is not clear whether expression of p16 in p16-deficient tumor cells can suppress their anchorage-independent growth. Therefore, we introduced a cDNA for p16(INK4a) into the human glioblastoma cell line T98G, which lacks a gene for p16(INK4a). We isolated several clones that stably expressed various amounts of pie protein. The doubling time of the various clones was generally prolonged. Clones with high-level expression of p16 protein had characteristics of restricted growth, such as contact inhibition, while the parental T98G cells had no such characteristics. Furthermore, the efficiency of colony formation in soft agar was dramatically decreased in the case of cells that expressed exogenous pie. Our observations suggest that the expression of pie protein restricts the unbounded growth and the anchorage-independent growth of tumor cells. (C) 1997 Academic Press.
  • The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2
    M Kitagawa, H Higashi, HK Jung, SuzukiTakahashi, I, M Ikeda, K Tamai, J Kato, K Segawa, E Yoshida, S Nishimura, Y Taya
    EMBO JOURNAL 15 (24) 7060 - 7069 0261-4189 1996/12 [Refereed][Not invited]
     
    Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G(1)/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS(780)PIP- that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser78O in pRB was phosphorylated in the GI phase in a cell cycle-dependent manner, Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin, A/E Cdk2 phosphorylate different sites of pRB in vivo.
  • Higashi H, Suzuki-Takahashi I, Saitoh S, Segawa K, Taya Y, Okuyama A, Nishimura S, Kitagawa M
    Eur. J. Biochem. 237 (2) 460 - 467 0014-2956 1996 [Refereed][Not invited]
  • A VARIANT FORM OF CYCLIN-DEPENDENT KINASE-2 (CDK2) IN A MALIGNANTLY TRANSFORMED RAT-THYROID (FRTL-TC) CELL-LINE
    S KOTANI, T ENDO, M KITAGAWA, H HIGASHI, T ONAYA
    ONCOGENE 10 (4) 663 - 669 0950-9232 1995/02 [Refereed][Not invited]
     
    Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.
  • PHOSPHORYLATION OF E2F-1 BY CYCLIN A-CDK2
    M KITAGAWA, H HIGASHI, SUZUKITAKAHASHI, I, K SEGAWA, SK HANKS, Y TAYA, S NISHIMURA, A OKUYAMA
    ONCOGENE 10 (2) 229 - 236 0950-9232 1995/01 [Refereed][Not invited]
     
    Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-EZF-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated PRE but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the GO and early G1 phase, Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdkZ in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdkZ may modulate its activity.
  • 123The interactions of E2F with pRB and with p107 are regulated via the phosphorylation of pRB and p107 by a cyclin-dependent kinase
    Suzuki-Takahashi I, Kitagawa M, Saijo M, Higashi H, Ogino H, Matsumoto H, Taya Y, Nishimura S, Okuyama A
    Oncogene 10 1691 - 1698 1995 [Refereed][Not invited]
  • Higashi H, Suzuki-Takahashi I, Taya Y, Segawa K, Nishimura S, Kitagawa M
    Biochem. Biophys. Res. Commun. 216 (2) 520 - 525 0006-291X 1995 [Refereed][Not invited]
  • A CYCLIN-DEPENDENT KINASE INHIBITOR, BUTYROLACTONE-I, INHIBITS PHOSPHORYLATION OF RB PROTEIN AND CELL-CYCLE PROGRESSION
    M KITAGAWA, H HIGASHI, IS TAKAHASHI, T OKABE, H OGINO, Y TAYA, S NISHIMURA, A OKUYAMA
    ONCOGENE 9 (9) 2549 - 2557 0950-9232 1994/09 [Refereed][Not invited]
     
    Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [H-3]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression, tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
  • BUTYROLACTONE-I, A SELECTIVE INHIBITOR OF CDK2 AND CDC2 KINASE
    M KITAGAWA, T OKABE, H OGINO, H MATSUMOTO, SUZUKITAKAHASHI, I, T KOKUBO, H HIGASHI, S SAITOH, Y TAYA, H YASUDA, Y OHBA, S NISHIMURA, N TANAKA, A OKUYAMA
    ONCOGENE 8 (9) 2425 - 2432 0950-9232 1993/09 [Refereed][Not invited]
     
    We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.
  • CLONING AND SEQUENCING OF THE 7-ALPHA-HYDROXYSTEROID DEHYDROGENASE GENE FROM ESCHERICHIA-COLI HB101 AND CHARACTERIZATION OF THE EXPRESSED ENZYME
    T YOSHIMOTO, H HIGASHI, A KANATANI, XS LIN, H NAGAI, H OYAMA, K KURAZONO, D TSURU
    JOURNAL OF BACTERIOLOGY 173 (7) 2173 - 2179 0021-9193 1991/04 [Refereed][Not invited]
     
    The 7-alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.
  • Yoshimoto T, Oyama H, Takeshita T, Higashi H, Lin XS, Tsuru D
    J. Ferment. Bioeng. 70 (6) 370 - 375 0922-338X 1990 [Refereed][Not invited]

Books etc

  • Recent Advances in Gastrointestinal Carcinogenesis
    Higashi H (Joint workHelicobacter pylori CagA Deregulates the Cell Signaling.)
    Transworld Research Network 2006

MISC

  • H Higashi, A Nakaya, R Tsutsumi, K Yokoyama, Y Fujii, S Ishikawa, M Higuchi, A Takahashi, Y Kurashima, Y Teishikata, S Tanaka, T Azuma, M Hatakeyama  JOURNAL OF BIOLOGICAL CHEMISTRY  279-  (17)  17205  -17216  2004/04  [Not refereed][Not invited]
     
    The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma. CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity. We established AGS human gastric epithelial cells that inducibly express wildtype or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines. Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the "hummingbird" phenotype. Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes. Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA ( siRNA) abolished the CagA-mediated hummingbird phenotype. The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2. In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation. Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation. Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells. These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA. Our work therefore suggests a key role of SHP-2 in the pathological activity of H. pylori virulence factor CagA.
  • S Umehara, H Higashi, N Ohnishi, M Asaka, M Hatakeyama  ONCOGENE  22-  (51)  8337  -8342  2003/11  [Not refereed][Not invited]
     
    Helicobacter pylori (H. pylori) is a causative agent of gastrointestinal diseases such as atrophic gastritis and gastroduodenal ulcer. Infection of cagA-positive H. pylori is also associated with gastric carcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The cagA gene product CagA is directly injected into the bacteria-attached host cells via the bacterial type IV secretion system. The translocated CagA deregulates intracellular signaling pathways and thereby initiates pathogenesis. In this work, we examined the biological effects of CagA on B cells, from which MALT lymphoma arises. Ectopic expression of CagA in interleukin 3-dependent B cells inhibited cell proliferation by suppressing the JAK-STAT signaling. CagA was also capable of preventing hydroxyurea-induced B-cell apoptosis through inhibiting p53 accumulation. In contrast to the effects of CagA in gastric epithelial cells, the observed CagA activities in B cells were independent of its tyrosine phosphorylation. Our results indicate that CagA possesses both phosphorylation-dependent and -independent activities in mammalian cells and that biological impacts of CagA depend on cell-type context. As a result of B-cell growth inhibition, CagA may diminish anti-H. pylori immune responses. Furthermore, CagA may play a role in the development of MALT lymphoma by impairing p53-dependent apoptosis.
  • T Takebayashi, H Higashi, H Sudo, H Ozawa, E Suzuki, O Shirado, H Katoh, M Hatakeyama  JOURNAL OF BIOLOGICAL CHEMISTRY  278-  (17)  14897  -14905  2003/04  [Not refereed][Not invited]
     
    The retinoblastoma protein (pRB) and its homologues, p107 and p130, prevent cell cycle progression from G(0)/G(1) to S phase by forming complexes with E2F transcription factors. Upon phosphorylation by G, cyclin-cyclin-dependent kinase (Cdk) complexes such as cyclin D1-Cdk4/6 and cyclin E-Cdk2, they lose the ability to bind E2F, and cells are thereby allowed to progress into S phase. Functional loss of one or more of the pRB family members, as a result of genetic mutation or deregulated phosphorylation, is considered to be an essential prerequisite for cellular transformation. In this study, we found that pRB family proteins have the ability to stimulate cyclin D1 transcription by activation of the NF-kappaB transcription factor. The cyclin D1-inducing activity of pRB is abolished by adenovirus E1A oncoprotein but not by the deletion of the A-box, the B-box, or the C-terminal region of the pocket, indicating that multiple pocket sequences are independently involved in cyclin D1 activation. Intriguingly, tumor-derived pRB pocket mutants retain the cyclin D1-inducing activity. Our results reveal a novel role of pRB family proteins as potential activators of NF-kappaB and inducers of G(1) cyclin. Certain pRB pocket mutants may give rise to a cellular situation in which deregulated E2F and cyclin D1 cooperatively promote abnormal cell proliferation.
  • Attenuation of helicobacter pylori CagA-SHP-2 signaling by interaction between CagA and C-terminal Src kinase .
    J. Biol. Chem.  278, 3664-3370-  2003  [Not refereed][Not invited]
  • H Higashi, R Tsutsumi, A Fujita, S Yamazaki, M Asaka, T Azuma, M Hatakeyama  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA  99-  (22)  14428  -14433  2002/10  [Not refereed][Not invited]
     
    Helicobacter pylori is a causative agent of gastritis and peptic ulcer. cagA(+) H. pylori strains are more virulent than cagA(-) strains and are associated with gastric carcinoma. The cagA gene product, CagA, is injected by the bacterium into gastric epithelial cells and subsequently undergoes tyrosine phosphorylation. The phosphorylated CagA specifically binds SHP-2 phosphatase, activates the phosphatase activity, and thereby induces morphological transformation of cells. CagA proteins of most Western H. pylori isolates have a 34-amino acid sequence that variably repeats among different strains. Here, we show that the repeat sequence contains a tyrosine phosphorylation site. CagA proteins having more repeats were found to undergo greater tyrosine phosphorylation, to exhibit increased SHP-2 binding, and to induce greater morphological changes. In contrast, predominant CagA proteins specified by H. pylori strains isolated in East Asia, where gastric carcinoma is prevalent, had a distinct tyrosine phosphorylation sequence at the region corresponding to the repeat sequence of Western CagA. This East Asian-specific sequence conferred stronger SHP-2 binding and morphologically transforming activities to Western CagA. Finally, a critical amino acid residue that determines SHP-2 binding activity among different CagA proteins was identified. Our results indicate that the potential of individual CagA to perturb host-cell functions is determined by the degree of SHP-2 binding activity, which depends in turn on the number and sequences of tyrosine phosphorylation sites. The presence of distinctly structured CagA proteins in Western and East Asian H. pylori isolates may underlie the strikingly different incidences of gastric carcinoma in these two geographic areas.
  • M Yamada, T Kondo, S Ashizawa, T Takebayashi, H Higashi, M Hatakeyama  CYTOKINE  17-  (2)  91  -97  2002/01  [Not refereed][Not invited]
     
    Interleukin 3 (IL-3)-dependent proliferation of haematopoietic cells is specifically inhibited by p130, a member of the pRB-family proteins. p130 interacts with the cell-cycle regulatory E2F transcription factors, notably E2F-4 and E2F-5, and affects promoters containing E2F-binding sites through two distinct mechanisms. First, upon complex formation with E2F, it blocks transcriptional activation by E2F. Second, the formed p130-E2F complex binds to E2F sites and actively represses transcription by inhibiting the activity of surrounding enhancer elements on the promoter. To pursue the relative contributions of each mechanism in the p130-mediated inhibition of IL-3-dependent cell proliferation, we employed a dominant-negative DP-1, which suppresses both E2F-dependent transactivation and the formation of active transcriptional repressors. Ectopic expression of the dominant negative DP-1 in the IL-3-dependent BaF3 lymphoid cells gave rise to an inhibition of cell proliferation, which was concomitantly associated with a decrease in levels of cyclin E, an indispensable molecule for G1 to S-phase cell-cycle progression. Our results indicate that blocking E2F-dependent transactivation, but not the formation of p130-E2F transcriptional repressor complexes, is responsible for the inhibition of IL-3-dependent cell growth by p130. (C) 2002 Elsevier Science Ltd.
  • H Higashi, R Tsutsumi, S Muto, T Sugiyama, T Azuma, M Asaka, M Hatakeyama  SCIENCE  295-  (5555)  683  -686  2002/01  [Not refereed][Not invited]
     
    Helicobacter pylori CagA protein is associated with severe gastritis and gastric carcinoma. CagA is injected from the attached Helicobacter pylori into host cells and undergoes tyrosine phosphorylation. Wild-type but not phosphorylation-resistant CagA induced a growth factor-like response in gastric epithelial cells. Furthermore, CagA formed a physical complex with the SRC homology 2 domain (SH2)-containing tyrosine phosphatase SHP-2 in a phosphorylation-dependent manner and stimulated the phosphatase activity. Disruption of the CagA-SHP-2 complex abolished the CagA-dependent cellular response. Conversely, the CagA effect on cells was reproduced by constitutively active SHP-2. Thus, upon translocation, CagA perturbs cellular functions by deregulating SHP-2.
  • Biochem. Biophys. Res.Commun.  268, 530-534-  2000  [Not refereed][Not invited]
  • J. Biol. Chem.  273, 26772-26778-  1998  [Not refereed][Not invited]
  • SuzukiTakahashi, I, H Higashi, E Yoshida, S Nishimura, M Kitagawa  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  234-  (2)  386  -392  1997/05  [Not refereed][Not invited]
     
    p16(INK4a), a protein that inhibits cyclin-dependent kinase 4 (Cdk4) and Cdk6, is deficient in many human cancers and in established lines of tumor cells. It has been reported that transfection with cDNA for p16(INK4a) inhibits the growth of cell lines that express retinoblastoma protein (pRB). However, it is unclear whether the introduction of cDNA for p16(INK4a) affects the growth of cells that express p16(INK4a) protein. Moreover, the effects of other cell-cycle regulators on the inhibition of cell growth by p16(INK4a) remain unknown. In this study, cDNA for p16(INK4a) was used to transfect human cell lines that had various status of expression of RE pathway-related proteins, such as members of the RE family proteins and Cdk-inhibitory proteins. We found that status of p107, p130, p15(INK4b), p18(INK4c) p21(Cip1), p27(Kip1) cyclin D1, and Cdk4 were not correlated with the growth-inhibitory activity of exogenous p16(INK4a). BY contrast, transfection with cDNA for p16(INK4a) had a significant effect on the growth of cells depended on the status not only of pRB but also of p16(INK4a). Although exogenous p16(INK4a) inhibited the growth of cells that expressed pRB but did not express p16(INK4a) (pRB(+)/p16(-) cells), it had little affect on either pRB(-)/p16(+) cells or pRB(-)/p16(+) cells. Moreover, transfection with cDNA for p16(INK4a) also inhibited the activity of the E2 promoter of the dehydrofolate reductase gene in the same manner that depended on the absence of p16(INK4a), as well as on the presence of pRB. These results suggest that deregulation of the RE pathway by p16(INK4a) deficiency plays a very important role in the proliferation of cells that lack p16(INK4a) protein. (C) 1997 Academic Press.
  • H Higashi, SuzukiTakahashi, I, E Yoshida, S Nishimura, M Kitagawa  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  231-  (3)  743  -750  1997/02  [Not refereed][Not invited]
     
    p16(INK4a) is a inhibitory protein of Cyclin-dependent kinase 4 (Cdk4). p16 negatively regulates the cell cycle progression from G1 to S phase. Functional pie is absent from many human cancers, as well as from many established lines of tumor cells. However, it is not clear whether expression of p16 in p16-deficient tumor cells can suppress their anchorage-independent growth. Therefore, we introduced a cDNA for p16(INK4a) into the human glioblastoma cell line T98G, which lacks a gene for p16(INK4a). We isolated several clones that stably expressed various amounts of pie protein. The doubling time of the various clones was generally prolonged. Clones with high-level expression of p16 protein had characteristics of restricted growth, such as contact inhibition, while the parental T98G cells had no such characteristics. Furthermore, the efficiency of colony formation in soft agar was dramatically decreased in the case of cells that expressed exogenous pie. Our observations suggest that the expression of pie protein restricts the unbounded growth and the anchorage-independent growth of tumor cells. (C) 1997 Academic Press.
  • H Higashi, SuzukiTakahashi, I, E Yoshida, S Nishimura, M Kitagawa  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS  231-  (3)  743  -750  1997/02  [Not refereed][Not invited]
     
    p16(INK4a) is a inhibitory protein of Cyclin-dependent kinase 4 (Cdk4). p16 negatively regulates the cell cycle progression from G1 to S phase. Functional pie is absent from many human cancers, as well as from many established lines of tumor cells. However, it is not clear whether expression of p16 in p16-deficient tumor cells can suppress their anchorage-independent growth. Therefore, we introduced a cDNA for p16(INK4a) into the human glioblastoma cell line T98G, which lacks a gene for p16(INK4a). We isolated several clones that stably expressed various amounts of pie protein. The doubling time of the various clones was generally prolonged. Clones with high-level expression of p16 protein had characteristics of restricted growth, such as contact inhibition, while the parental T98G cells had no such characteristics. Furthermore, the efficiency of colony formation in soft agar was dramatically decreased in the case of cells that expressed exogenous pie. Our observations suggest that the expression of pie protein restricts the unbounded growth and the anchorage-independent growth of tumor cells. (C) 1997 Academic Press.
  • The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2
    M Kitagawa, H Higashi, HK Jung, SuzukiTakahashi, I, M Ikeda, K Tamai, J Kato, K Segawa, E Yoshida, S Nishimura, Y Taya  EMBO JOURNAL  15-  (24)  7060  -7069  1996/12  [Not refereed][Not invited]
     
    Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G(1)/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS(780)PIP- that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser78O in pRB was phosphorylated in the GI phase in a cell cycle-dependent manner, Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin, A/E Cdk2 phosphorylate different sites of pRB in vivo.
  • The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2
    EMBO J  15, 7060-7069-  1996  [Not refereed][Not invited]
  • THE INTERACTIONS OF E2F WITH PRB AND WITH P107 ARE REGULATED VIA THE PHOSPHORYLATION OF QRB AND P107 BY A CYCLIN-DEPENDENT KINASE
    SUZUKITAKAHASHI, I, M KITAGAWA, M SAIJO, H HIGASHI, H OGINO, H MATSUMOTO, Y TAYA, S NISHIMURA, A OKUYAMA  ONCOGENE  10-  (9)  1691  -1698  1995/05  [Not refereed][Not invited]
     
    It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evidence for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk, Therefore,we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays, We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as web as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.
  • A VARIANT FORM OF CYCLIN-DEPENDENT KINASE-2 (CDK2) IN A MALIGNANTLY TRANSFORMED RAT-THYROID (FRTL-TC) CELL-LINE
    S KOTANI, T ENDO, M KITAGAWA, H HIGASHI, T ONAYA  ONCOGENE  10-  (4)  663  -669  1995/02  [Not refereed][Not invited]
     
    Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.
  • Biochem. Biophys. Res. Commun  216-  (2)  520  -525  1995  [Not refereed][Not invited]
  • PHOSPHORYLATION OF E2F-1 BY CYCLIN A-CDK2
    M KITAGAWA, H HIGASHI, SUZUKITAKAHASHI, I, K SEGAWA, SK HANKS, Y TAYA, S NISHIMURA, A OKUYAMA  ONCOGENE  10-  (2)  229  -236  1995/01  [Not refereed][Not invited]
     
    Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-EZF-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated PRE but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the GO and early G1 phase, Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdkZ in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdkZ may modulate its activity.
  • Biochem. Biophys. Res. Commun  216-  (2)  520  -525  1995  [Not refereed][Not invited]
  • A CYCLIN-DEPENDENT KINASE INHIBITOR, BUTYROLACTONE-I, INHIBITS PHOSPHORYLATION OF RB PROTEIN AND CELL-CYCLE PROGRESSION
    M KITAGAWA, H HIGASHI, IS TAKAHASHI, T OKABE, H OGINO, Y TAYA, S NISHIMURA, A OKUYAMA  ONCOGENE  9-  (9)  2549  -2557  1994/09  [Not refereed][Not invited]
     
    Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [H-3]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression, tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
  • A CYCLIN-DEPENDENT KINASE INHIBITOR, BUTYROLACTONE-I, INHIBITS PHOSPHORYLATION OF RB PROTEIN AND CELL-CYCLE PROGRESSION
    M KITAGAWA, H HIGASHI, IS TAKAHASHI, T OKABE, H OGINO, Y TAYA, S NISHIMURA, A OKUYAMA  ONCOGENE  9-  (9)  2549  -2557  1994/09  [Not refereed][Not invited]
     
    Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [H-3]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression, tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
  • BUTYROLACTONE-I, A SELECTIVE INHIBITOR OF CDK2 AND CDC2 KINASE
    M KITAGAWA, T OKABE, H OGINO, H MATSUMOTO, SUZUKITAKAHASHI, I, T KOKUBO, H HIGASHI, S SAITOH, Y TAYA, H YASUDA, Y OHBA, S NISHIMURA, N TANAKA, A OKUYAMA  ONCOGENE  8-  (9)  2425  -2432  1993/09  [Not refereed][Not invited]
     
    We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.
  • BUTYROLACTONE-I, A SELECTIVE INHIBITOR OF CDK2 AND CDC2 KINASE
    M KITAGAWA, T OKABE, H OGINO, H MATSUMOTO, SUZUKITAKAHASHI, I, T KOKUBO, H HIGASHI, S SAITOH, Y TAYA, H YASUDA, Y OHBA, S NISHIMURA, N TANAKA, A OKUYAMA  ONCOGENE  8-  (9)  2425  -2432  1993/09  [Not refereed][Not invited]
     
    We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.
  • CLONING AND SEQUENCING OF THE 7-ALPHA-HYDROXYSTEROID DEHYDROGENASE GENE FROM ESCHERICHIA-COLI HB101 AND CHARACTERIZATION OF THE EXPRESSED ENZYME
    T YOSHIMOTO, H HIGASHI, A KANATANI, XS LIN, H NAGAI, H OYAMA, K KURAZONO, D TSURU  JOURNAL OF BACTERIOLOGY  173-  (7)  2173  -2179  1991/04  [Not refereed][Not invited]
     
    The 7-alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.
  • CLONING AND SEQUENCING OF THE 7-ALPHA-HYDROXYSTEROID DEHYDROGENASE GENE FROM ESCHERICHIA-COLI HB101 AND CHARACTERIZATION OF THE EXPRESSED ENZYME
    T YOSHIMOTO, H HIGASHI, A KANATANI, XS LIN, H NAGAI, H OYAMA, K KURAZONO, D TSURU  JOURNAL OF BACTERIOLOGY  173-  (7)  2173  -2179  1991/04  [Not refereed][Not invited]
     
    The 7-alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1, with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method. Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence. The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778. The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed about 200-fold-higher enzyme activity than the host. The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3. The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form.
  • J. Ferment. Bioeng  70, 370-375-  1990  [Not refereed][Not invited]
  • J. Ferment. Bioeng  70-  (6)  370  -375  1990  [Not refereed][Not invited]

Awards & Honors

  • 2003 日本癌学会奨励賞
  • 2002 消化管細胞機能研究会 特別賞
  • 2002 日本ヘリコバクター学会 上原H. pylori 最優秀賞

Research Grants & Projects

  • Regulation of cell cycle

Educational Activities

Teaching Experience

  • 感染症学特別研究Ⅰ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Inter-Graduate School Classes(General Subject):Natural and Applied Sciences
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
  • 感染症学特別演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Zoonotic Science
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 人獣共通感染症、新興・再興感染症、疫学、自然宿主、媒介動物、感染環、ウイルス、細菌、原虫、寄生虫 
  • 研究倫理演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 人獣共通感染症対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 感染症学特別研究ⅡA
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 感染症学特別研究ⅡB
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 海外インターンシップA
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 海外インターンシップB
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 国際感染症学コア科目Ⅰ 人獣共通感染症制御学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 国際感染症学コア科目Ⅰ 生体防御学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 国際感染症学コア科目ⅡB 製剤開発特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学特別研究
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 専門獣医科学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 人獣共通感染症対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学特論演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 動物実験倫理特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目A 人獣共通感染症制御学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 研究倫理演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • インターンシップ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 専門獣医科学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院


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