Researchers Database

tago kenji

    DepartmentofBiochemistry,DivisionofStructuralBiochemistry Assistant Professor
Last Updated :2021/12/04

Researcher Information

URL

Research funding number

  • 20306111

J-Global ID

Research Interests

  • シグナル伝達   細胞情報伝達機構   癌   シグナル伝達系   がん   蛋白質   細胞・組織   薬理学   受容体   生体分子   Gタンパク質   Gタンパク質共役受容体   新規分子プローブ   G蛋白質共役受容体   細胞遊走制御   G蛋白質   創薬科学   機能抗体   オーファン受容体   モノクローナル抗体   

Research Areas

  • Life sciences / Functional biochemistry

Academic & Professional Experience

  • 2012/04 - Today  Jichi Medical UniversitySchool of Medicine, Department of BiochemistryAssistant Professor
  • 2006/06 - 2012/03  NAISTDepartment of BioscienceAssistant Professor
  • 2002/04 - 2006/05  St. Jude Children's Research HospitalDepartment of Tumor Cell BiologyResearch Associate
  • 1998/04 - 2002/03  Jichi Medical UniversitySchool of MedicineResearch Assistant

Education

  • 1993/04 - 1998/03  Tokyo Institute of Technology  生命理工学研究科
  • 1989/04 - 1993/03  Saitama University  理学部  生化学科

Association Memberships

  • THE MOLECULAR BIOLOGY SOCIETY OF JAPAN   THE JAPANESE BIOCHEMICAL SOCIETY   

Published Papers

  • Sui Sawaguchi, Mizuki Goto, Yukino Kato, Marina Tanaka, Kenji Tago, Hiroaki Oizumi, Katsuya Ohbuchi, Kazushige Mizoguchi, Yuki Miyamoto, Junji Yamauchi
    Polymers 13 (7) 2021/03 [Refereed]
     
    Pelizaeus-Merzbacher disease (PMD), also known as hypomyelinating leukodystrophy 1 (HLD1), is an X-linked recessive disease affecting in the central nervous system (CNS). The gene responsible for HLD1 encodes proteolipid protein 1 (plp1), which is the major myelin structural protein produced by oligodendroglial cells (oligodendrocytes). HLD15 is an autosomal recessive disease affecting the glutamyl-prolyl-aminoacyl-tRNA synthetase 1 (eprs1) gene, whose product, the EPRS1 protein, is a bifunctional aminoacyl-tRNA synthetase that is localized throughout cell bodies and that catalyzes the aminoacylation of glutamic acid and proline tRNA species. Here, we show that the HLD15-associated nonsense mutation of Arg339-to-Ter (R339X) localizes EPRS1 proteins as polymeric aggregates into Rab7-positive vesicle structures in mouse oligodendroglial FBD-102b cells. Wild-type proteins, in contrast, are distributed throughout the cell bodies. Expression of the R339X mutant proteins, but not the wild-type proteins, in cells induces strong signals regulating Rab7. Whereas cells expressing the wild-type proteins exhibited phenotypes with myelin web-like structures bearing processes following the induction of differentiation, cells expressing the R339X mutant proteins did not. These results indicate that HLD15-associated EPRS1 mutant proteins are localized in Rab7-positive vesicle structures where they modulate Rab7 regulatory signaling, inhibiting cell morphological differentiation. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD15.
  • Kohei Hattori, Kenji Tago, Shiori Memezawa, Arisa Ochiai, Sui Sawaguchi, Yukino Kato, Takanari Sato, Kazuma Tomizuka, Hiroaki Ooizumi, Katsuya Ohbuchi, Kazushige Mizoguchi, Yuki Miyamoto, Junji Yamauchi
    Medicines (Basel, Switzerland) 8 (2) 2021/02 [Refereed]
     
    Genetic hypomyelinating diseases are a heterogeneous group of disorders involving the white matter. One infantile hypomyelinating leukoencephalopathy is associated with the homozygous variant (Cys4-to-Ser (C4S)) of the c11orf73 gene. Methods: We observed that in mouse oligodendroglial FBD-102b cells, the C4S mutant proteins but not the wild type ones of C11orf73 are microscopically localized in the lysosome. And, they downregulate lysosome-related signaling in an immunoblotting technique. Results: The C4S mutant proteins specifically interact with Filamin A, which is known to anchor transmembrane proteins to the actin cytoskeleton; the C4S mutant proteins and Filamin A are also observed in the lysosome fraction. While parental FBD-102b cells and cells harboring the wild type constructs exhibit morphological differentiation, cells harboring C4S mutant constructs do not. It may be that morphological differentiation is inhibited because expression of these C4S mutant proteins leads to defects in the actin cytoskeletal network involving Filamin A. Conclusions: The findings that leukoencephalopathy-associated C11ORF73 mutant proteins specifically interact with Filamin A, are localized in the lysosome, and inhibit morphological differentiation shed light on the molecular and cellular pathological mechanisms that underlie infantile hypomyelinating leukoencephalopathy.
  • Yuki Uchihara, Kenji Tago, Hiroomi Tamura, Megumi Funakoshi-Tago
    Molecular oncology 15 (1) 167 - 194 2021/01 [Refereed]
     
    The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), found in anaplastic large-cell lymphoma (ALCL), localizes to the cytosol, nucleoplasm, and nucleolus. However, the relationship between its localization and transforming activity remains unclear. We herein demonstrated that NPM-ALK localized to the nucleolus by binding to nucleophosmin 1 (NPM1), a nucleolar protein that exhibits shuttling activity between the nucleolus and cytoplasm, in a manner that was dependent on its kinase activity. In the nucleolus, NPM-ALK interacted with Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2), which is involved in rRNA biosynthesis. Moreover, enforced expression of NPM-ALK induced tyrosine phosphorylation of EBP2. Knockdown of EBP2 promoted the activation of the tumor suppressor p53, leading to G0 /G1 -phase cell cycle arrest in Ba/F3 cells transformed by NPM-ALK and ALCL patient-derived Ki-JK cells, but not ALCL patient-derived SUDH-L1 cells harboring p53 gene mutation. In Ba/F3 cells transformed by NPM-ALK and Ki-JK cells, p53 activation induced by knockdown of EBP2 was significantly inhibited by Akt inhibitor GDC-0068, mTORC1 inhibitor rapamycin, and knockdown of Raptor, an essential component of mTORC1. These results suggest that the knockdown of EBP2 triggered p53 activation through the Akt-mTORC1 pathway in NPM-ALK-positive cells. Collectively, the present results revealed the critical repressive mechanism of p53 activity by EBP2 and provide a novel therapeutic strategy for the treatment of ALCL.
  • Megumi Funakoshi-Tago, Kenji Tago, Chin Li, Shingo Hokimoto, Hiroomi Tamura
    Scientific reports 10 (1) 19588 - 19588 2020/11 [Refereed]
     
    The consumption of coffee has been suggested to effectively enhance the therapeutic effects of tamoxifen against breast cancer; however, the underlying molecular mechanisms remain unclear. We herein attempted to clarify how coffee decoction exerts anti-cancer effects in cooperation with tamoxifen using the estrogen receptor α (ERα)-positive breast cancer cell line, MCF-7. The results obtained showed that coffee decoction down-regulated the expression of ERα, which was attributed to caffeine inhibiting its transcription. Coffee decoction cooperated with tamoxifen to induce cell-cycle arrest and apoptotic cell death, which may have been mediated by decreases in cyclin D1 expression and the activation of p53 tumor suppressor. The inclusion of caffeine in coffee decoction was essential, but not sufficient, to induce cell-cycle arrest and apoptotic cell death, suggesting the requirement of unknown compound(s) in coffee decoction to decrease cyclin D1 expression and activate apoptotic signaling cascades including p53. The activation of p53 through the cooperative effects of these unidentified component(s), caffeine, and tamoxifen appeared to be due to the suppression of the ERK and Akt pathways. Although the mechanisms by which the suppression of these pathways induces p53-mediated apoptotic cell death remain unclear, the combination of decaffeinated coffee, caffeine, and tamoxifen also caused cell-cycle arrest and apoptotic cell death, suggesting that unknown compound(s) present in decaffeinated coffee cooperate with caffeine and tamoxifen.
  • Megumi Funakoshi-Tago, Yusuke Nonaka, Kenji Tago, Mika Takeda, Yuma Ishihara, Ami Sakai, Mari Matsutaka, Kenji Kobata, Hiroomi Tamura
    Scientific reports 10 (1) 2584 - 2584 2020/02 [Refereed][Not invited]
     
    Coffee is a complex mixture of many bioactive compounds possessing anti-inflammatory properties. However, the mechanisms by which coffee exerts anti-inflammatory effects remains unclear and the active ingredients have not yet been identified. In this study, we found that coffee extract at more than 2.5%(v/v) significantly inhibited LPS-induced inflammatory responses in RAW264.7 cells and that anti-inflammatory activity of coffee required the roasting process. Interestingly, we identified pyrocatechol, a degradation product derived from chlorogenic acid during roasting, as the active ingredient exhibiting anti-inflammatory activity in coffee. HPLC analysis showed that 124 μM pyrocatechol was included in 100% (v/v) roasted coffee. A treatment with 5%(v/v) coffee extract and more than 2.5 μM pyrocatechol inhibited the LPS-induced activation of NF-κB and also significantly activated Nrf2, which acts as a negative regulator in LPS-induced inflammation. Furthermore, intake of 60% (v/v) coffee extract and 74.4 μM pyrocatechol, which is the concentration equal to contained in 60% (v/v) coffee, markedly inhibited the LPS-induced inflammatory responses in mice. Collectively, these results demonstrated that pyrocatechol, which was formed by the roasting of coffee green beans, is one of the ingredients contributing to the anti-inflammatory activity of coffee.
  • Yuki Uchihara, Tomoyuki Ohe, Tadahiko Mashino, Takayuki Kidokoro, Kenji Tago, Hiroomi Tamura, Megumi Funakoshi-Tago
    Pharmacological reports : PR 71 (6) 1067 - 1078 1734-1140 2019/12 [Refereed][Not invited]
     
    BACKGROUND: Inhibitors for signal transducer and activator of transcription 3 (STAT3), Stattic, BP-1-102, and LLL12 significantly induce apoptosis in transformed Ba/F3 cells expressing an oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) that induces the activation of STAT3. We found that the antioxidant reagent, N-acetyl cysteine (NAC) prevented the abilities of Stattic and BP-1-102, but not LLL12 to induce apoptosis in transformed cells expressing NPM-ALK, providing a novel problem in use of STAT3 inhibitors. We herein investigated the mechanisms how NAC prevented the effects of Sttatic and BP-1-102. METHODS: Ba/F3 cells expressing NPM-ALK and SUDHL-1 cells were treated with antioxidants such as NAC, Trolox or edaravone in combination with STAT3 inhibitors. Phosphorylation of STAT3, cell proliferation rate, cell viability, cell cycle, internucleosomal DNA fragmentation and the intracellular accumulation of reactive oxygen species (ROS) was investigated. The binding of STAT3 inhibitors and NAC was analyzed by LC-MS. RESULTS: NAC but not Trolox and edaravone diminished the abilities of Stattic and BP-1-102 to induce apoptosis in cells expressing NPM-ALK. The ROS levels in cells expressing NPM-ALK were not markedly affected by the treatments with Stattic and BP-1-102 in combination with NAC, suggesting that NAC inhibited the activity of Stattic and BP-1-102 independent of its antioxidant activity. LC-MS analysis revealed that NAC directly bound to Stattic and BP-1-102. Furthermore, these NAC adducts exhibited no cytotoxicity, and failed to affect the activity of STAT3. CONCLUSIONS: NAC antagonizes the activities of Stattic and BP-1-102, which inhibit STAT3 activation by interacting with cysteine residues in STAT3.
  • Yuki Uchihara, Reiko Komori, Kenji Tago, Hiroomi Tamura, Megumi Funakoshi-Tago
    Biochemical pharmacology 170 113666 - 113666 0006-2952 2019/12 [Refereed][Not invited]
     
    Anaplastic large cell lymphoma (ALCL) is associated with a characteristic chromosomal translocation that generates the oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Methotrexate is a commonly used chemotherapeutic drug in the treatment of multiple cancers due to its inhibition of dihydrofolate reductase (DHFR), which suppresses the synthesis of DNA. In the present study, we found that low-dose methotrexate significantly induced apoptosis in transformed Ba/F3 cells expressing NPM-ALK by inhibiting the activation of signal transducer and activator of transcription factor 3 (STAT3), a critical downstream molecule of NPM-ALK. Although methotrexate prevented the phosphorylation of STAT3, it did not affect the activity of NPM-ALK. A co-treatment with folinic acid prevented the methotrexate-induced inhibition of STAT3 activation and induction of apoptosis, suggesting that methotrexate exerts its cytotoxic effects by depleting tetrahydrofolate (THF) in transformed cells by NPM-ALK. Furthermore, methotrexate induced the down-regulation of the anti-apoptotic protein, MCL-1, DNA damage, and the activation of a p53 tumor suppressor, leading to apoptosis through the inhibition of STAT3. Methotrexate significantly induced apoptosis in ALK inhibitor-resistant cells expressing the NPM-ALK mutant harboring the point mutation, G262R, and in ALCL patient-derived NPM-ALK-positive Ki-JK cells. Collectively, these results demonstrate the potential therapeutic application of methotrexate, which inhibits the activation of STAT3, to NPM-ALK-positive ALCL.
  • Megumi Funakoshi-Tago, Rina Tsuruya, Fumihito Ueda, Aki Ishihara, Tadashi Kasahara, Hiroomi Tamura, Kenji Tago
    Cytokine 123 154753 - 154753 1043-4666 2019/11 [Refereed][Not invited]
     
    In the majority of myeloproliferative neoplasms (MPNs) patients, a point mutation, V617F has been found in Janus kinase 2 (JAK2) gene, and this JAK2 mutant provoked aberrant signaling pathway. In the current study, we found that suppressor of cytokine signaling proteins 3 (SOCS3) possessed the tumor suppressive activity against the JAK2 V617F mutant-provoked cellular transformation. The knockdown of SOCS3 increased the expression level of the JAK2 V617F mutant, which enhanced the activation of signaling mediators, including signal transducer and activator of transcription 3 and 5 (STAT3, STAT5) and extracellular signal-regulated kinase (ERK), and also increased of the proliferation rate and tumorigenesis activity of Ba/F3 cells expressing the JAK2 V617F mutant and erythropoietin receptor (EpoR). In contrast, the enforced expression of SOCS3 significantly inhibited the JAK2 V617F mutant-induced activation of downstream signaling molecules, cell proliferation, and tumorigenesis by down-regulating the expression level of the JAK2 V617F mutant. SOCS3 interacted with the JAK2V617F mutant through its SH2 domain and was phosphorylated at Tyr-204 and Tyr-221 in its SOCS box by the JAK2V617F mutant. SOCS3 mutants carrying a mutation in the SH2 domain (R71E) and a substitution at Tyr-221 (Y221F) failed to exert inhibitory effects on JAK2V617F mutant-induced cellular transformation and tumorigenesis. Collectively, these results imply that SOCS3 plays a negative role in the JAK2 V617F mutant-induced oncogenic signaling pathway through its SH2 domain and the phosphorylation of Tyr-221 in its SOCS box.
  • Kenji Tago, Megumi Funakoshi-Tago, Satoshi Ohta, Hirotoshi Kawata, Hiroshi Saitoh, Hisanaga Horie, Chihiro Aoki-Ohmura, Junji Yamauchi, Akira Tanaka, Jitsuhiro Matsugi, Ken Yanagisawa
    Molecular oncology 13 (11) 2493 - 2510 1574-7891 2019/11 [Refereed][Not invited]
     
    It is well established that nuclear factor κB (NF-κB) acts as one of the most important transcription factors for tumor initiation and progression, as it both protects cells from apoptotic/necrotic signals and accelerates angiogenesis and tumor metastasis, which is mediated via the expression of target genes. However, it has not yet been clarified how oncogenic signals accelerate the activation of NF-κB. In the current study, we utilized untransformed NIH-3T3 cells stably harboring a κB-driven luciferase gene to show that an oncogenic mutant of Ras GTPase augmented TNFα-induced NF-κB activation. Notably, enforced expression of cyclin-dependent kinase inhibitors, such as p27Kip1 and p21Cip1 , effectively canceled the accelerated activation of NF-κB, suggesting that oncogenic Ras-induced cell cycle progression is essential for the hyperactivation of NF-κB. Furthermore, we found that Ras (G12V) augmented the transcriptional activation of NF-κB, and this activation required the p38 MAP kinase. We observed that a downstream kinase of p38 MAP kinase, MSK1, was activated by Ras (G12V) and catalyzed the phosphorylation of p65/RelA at Ser-276, which is critical for its transcriptional activation. Significantly, phosphorylation of the p65/RelA subunit at Ser-276 was elevated in patient samples of colorectal cancer harboring oncogenic mutations of the K-Ras gene, and the expression levels of NF-κB target genes were drastically enhanced in several cancer tissues. These observations strongly suggest that oncogenic signal-induced acceleration of NF-κB activation is caused by activation of the p38 MAP kinase-MSK1 signaling axis and by cell cycle progression in cancer cells.
  • Yuki Uchihara, Kenji Tago, Megumi Funakoshi-Tago
    Nihon yakurigaku zasshi. Folia pharmacologica Japonica 153 (4) 147 - 154 0015-5691 2019 [Refereed][Not invited]
     
    Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) are caused by a fusion protein, BCR-ABL, which induces cellular transformation by activating the signaling molecules, STAT5 and Akt. The specific BCR-ABL inhibitors including imatinib, nilotinib, and dasatinib, are clinically utilized in the treatment with CML and ALL patients. Although these BCR-ABL inhibitors are initially successful in the treatment of leukemia, many patients develop drug resistance due to the appearance of the gatekeeper mutation of BCR-ABL, T315I. Recently, we found that taxodione, a quinone methide diterpene isolated from a conifer, Taxodium distichum, significantly induced apoptosis in human myelogenous leukemia-derived K562 cells, which is positive for the bcr-abl gene. Taxodione reduced the activities of mitochondrial respiratory chain complex III, leading to the production of reactive oxygen species (ROS). An antioxidant agent, N-acetylcysteine (NAC), canceled taxodione-induced ROS production and apoptotic cell death, suggesting that taxodione induced apoptosis through ROS accumulation. Furthermore, in K562 cells treated with taxodione, BCR-ABL, STAT5 and Akt were sequestered in mitochondrial fraction, and their localization changes decrease their abilities to stimulate cell proliferation. Strikingly, NAC canceled these taxodione-caused inhibition of BCR-ABL, STAT5 and Akt. In addition, taxodione significantly induced apoptosis in transformed Ba/F3 cells by not only BCR-ABL but also T315I-mutated BCR-ABL through the generation of ROS, suggesting that taxodione has potential as anti-tumor drug with high efficacy to overcome BCR-ABL T315I mutation-mediated resistance in leukemia cells. It's also expected that these knowledge becomes an important clue in the development of anti-cancer drugs against the broad range of tumors.
  • Yuri Urai, Minami Yamawaki, Natsumi Watanabe, Yoich Seki, Takako Morimoto, Kenji Tago, Keiichi Homma, Hiroyuki Sakagami, Yuki Miyamoto, Junji Yamauchi
    Biochemical and biophysical research communications 503 (3) 2047 - 2053 0006-291X 2018/09 [Refereed][Not invited]
     
    The intracellular molecular transport system is a basic and general cellular mechanism that is regulated by an array of signaling molecules. Sar1 small GTPases are molecules that play a key role in controlling vehicle transport between the endoplasmic reticulum (ER) and Golgi bodies. Like other small GTPases, the activities of Sar1a depend on their guanine-nucleotide-binding states, which are regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the well-known function of mammalian Sar1 in the intracellular transport system, little is known about when and how Sar1 is activated during cell morphological changes. Here we show that the C-terminal, but not the N-terminal, regions of Sec23A and Sec23B, the effector proteins of Sar1a, specifically bind to the active, GTP-bound form of Sar1a. An affinity precipitation (pull-down) assay using a recombinant C-terminal region of Sec23B reveals that Sar1a is activated following differentiation in neuronal cell lines. In neuronal N1E-115 cells, GTP-bound Sar1a is increased when cells elongate neuronal processes. Similar results are observed in morphological differentiation in oligodendroglial FBD-102b cells. Additionally, prolactin regulatory element binding (PREB), the GEF for Sar1 (Sar1 activator), increases the binding ability to the nucleotide-free form of Sar1a when morphological differentiation occurs. Nucleotide-free small GTPases preferentially interact with the cognate, active GEFs. These results provide evidence that using previously unreported pull down assays reveals that Sar1 and PREB are upregulated following the induction of morphological differentiation, suggesting the potential role of signaling through Sar1a during morphological differentiation.
  • Megumi Funakohi-Tago, Tomoki Sakata, Satoru Fujiwara, Ayaka Sakakura, Takeshi Sugai, Kenji Tago, Hiroomi Tamura
    European journal of pharmacology 834 246 - 256 0014-2999 2018/09 [Refereed][Not invited]
     
    Hydroxytyrosol (HT) is a polyphenol contained in olives and exhibits antioxidant activity. We herein investigated the effects of HT and its derivatives, hydroxytyrosol acetate (HT-A) and hydroxytyrosol butyrate (HT-B), on the protection of neuronal cells against apoptosis induced by the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA). The pretreatment of SH-SY5Y cells with HT-B, but not HT or HT-A significantly reduced the 6-OHDA-induced generation of reactive oxygen species, activation of caspase-3, and subsequent cell death. HT-B also induced the protein expression of the transcription factor, NF-E2-related factor-2 (Nrf2) and its transcriptional activation, resulting in the up-regulated expression of heme oxygenase-1 (HO-1), which conferred neuroprotection against 6-OHDA-induced oxidative damage. Furthermore, three cysteine residues, Cys151, Cys273, and Cys288 in Kelch-like ECH-associated protein 1 (Keap1) were necessary for the HT-B-induced activation of Nrf2. Collectively, the present results demonstrated that HT-B, harboring higher fat solubility than HT and HT-A, effectively elicited adaptive responses to oxidative stress by activating the Nrf2/HO-1 axis in neuronal cells.
  • Tomomi Niino, Kenji Tago, Daisuke Yasuda, Kyoko Takahashi, Tadahiko Mashino, Hiroomi Tamura, Megumi Funakoshi-Tago
    Biochemical pharmacology 155 182 - 197 0006-2952 2018/09 [Refereed][Not invited]
     
    5-Hydroxyoxindole is a urinary metabolite of indole that exhibits antioxidant activity. In the present study, we found that a 5-hydroxyoxindole derivative (5-HI) significantly inhibited LPS-induced inflammatory effects in the murine macrophage cell line, RAW264.7. 5-HI induced the expression of the transcription factor, Nrf2, which is typically ubiquitinated by Keap1, an adaptor component of the ubiquitin E3 ligase complex, resulting in its proteasomal degradation. By utilizing Keap1-/- MEFs reconstituted with Keap1 mutants harboring substitutions in their major cysteine residues, we clarified the importance of Cys151 in Keap1 as a sensor for 5-HI in the induction of Nrf2 expression. Furthermore, 5-HI induced the activation of the MKK3/6-p38 pathway, which is required for the transcriptional activation of Nrf2. The knockdown of Nrf2 enhanced the LPS-induced expression of inflammatory mediators, including iNOS, NO, and CCL2, and effectively repressed the inhibitory effects of 5-HI on their expression. Although 5-HI and antioxidant N-acetyl cysteine (NAC) both reduced LPS-induced ROS generation, the treatment with NAC did not affect the LPS-induced expression of inflammatory mediators, suggesting that the anti-inflammatory activity of 5-HI mediated by Nrf2 is independent of redox control. Furthermore, when injected into mice with 5-HI, the expression of Nrf2 was significantly increased, and the LPS-induced mRNA expression of CXCL1, CCL2, TNFα, and IL-6 were remarkably inhibited in the kidneys, liver, and lungs, and the production of these cytokines in serum was effectively reduced. Collectively, these results suggest that 5-HI has potential in the treatment of inflammatory diseases through the activation of Nrf2.
  • 核小体に局在するNPM-ALKの結合分子の同定
    内原 脩貴, 多胡 めぐみ, 多胡 憲治, 田村 悦臣
    日本生化学会大会プログラム・講演要旨集 (公社)日本生化学会 91回 [3T12a - 216)] 2018/09
  • Yuki Uchihara, Kenji Tago, Hidetoshi Taguchi, Yuji Narukawa, Fumiyuki Kiuchi, Hiroomi Tamura, Megumi Funakoshi-Tago
    Biochemical pharmacology 154 357 - 372 0006-2952 2018/08 [Refereed][Not invited]
     
    Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) are hematopoietic malignancies caused by the constitutive activation of BCR-ABL tyrosine kinase. Although direct BCR-ABL inhibitors, such as imatinib, were initially successful in the treatment of leukemia, many patients developed drug resistance over time due to the gatekeeper mutation of BCR-ABL T315I. In the present study, we found that taxodione, a quinone methide diterpene isolated from Taxodium distichum, significantly induced apoptosis in human myelogenous leukemia-derived K562 cells, which were transformed by BCR-ABL. Taxodione reduced the activities of mitochondrial respiratory chain (MRC) complexes III and V, which appeared to induce the production of reactive oxygen species (ROS). N-acetylcysteine (NAC), an antioxidant agent, canceled taxodione-induced ROS production, reductions in MRC activities, particularly complex V, and apoptotic cell death. Furthermore, in K562 cells treated with taxodione, BCR-ABL and its major signaling molecules, such as STAT5 and Akt were sequestered in mitochondrial fraction, and their localization changes decrease their abilities to stimulate cell proliferation, suggesting that these actions seem to be a mechanism how taxodione functions as an anti-tumor drug. Strikingly, NAC canceled these taxodione-caused anti-cancer effects. Taxodione induced apoptosis in transformed Ba/F3 cells induced not only by BCR-ABL, but also T315I-mutated BCR-ABL through the generation of ROS. Collectively, the present results suggest that in the treatment of leukemia, taxodione has potential as a compound with high efficacy to overcome BCR-ABL T315I mutation-mediated resistance in leukemia cells.
  • Yuki Uchihara, Takayuki Kidokoro, Kenji Tago, Tadahiko Mashino, Hiroomi Tamura, Megumi Funakoshi-Tago
    European journal of pharmacology 825 1 - 9 0014-2999 2018/04 [Refereed][Not invited]
     
    Crizotinib is an inhibitor of anaplastic lymphoma kinase (ALK) and is of significant therapeutic benefit to patients with non-small cell lung cancer (NSCLC) harboring the EML4-ALK fusion gene. In the present study, we demonstrated that α-tocopherol, a major component of vitamin E, attenuated the effects of crizotinib independently of its anti-oxidant properties. α-Tocopherol significantly inhibited crizotinib-induced apoptosis in cells transformed by EML4-ALK. It also effectively attenuated the crizotinib-induced inhibition of EML4-ALK and its downstream molecules, STAT3 and ERK, and suppressed the inhibitory effects of crizotinib on EML4-ALK-mediated transformation in the focus formation assay. On the other hand, other members of the vitamin E family, namely, β-tocopherol, γ-tocopherol, δ-tocopherol, and α-tocotrienol, and a water-soluble analog of vitamin E, Trolox had no effects on the anti-tumor activity of crizotinib in cells transformed by EML4-ALK. Collectively, these results revealed the risk of the anti-tumor activity of crizotinib being attenuated when it is administrated in combination with vitamin E supplements containing α-tocopherol as a major component.
  • Yuki Miyamoto, Tomohiro Torii, Kenji Tago, Akito Tanoue, Shou Takashima, Junji Yamauchi
    Science advances 4 (4) eaar4471  2018/04 [Refereed][Not invited]
     
    During development of the peripheral nervous system in mammals, Schwann cells wrap their plasma membranes around neuronal axons, forming multiple myelin sheaths. A mature myelin sheath insulates axons and increases nerve conduction velocity while protecting nerve fibers from various stresses such as physical ones. Despite this functional importance, the molecular units that underlie dynamic morphological changes in formation of myelin sheaths are not sufficiently understood. Arf1 is a small guanosine triphosphate-binding protein that plays multiple roles in intracellular trafficking and related signaling, both of which are processes involved in cell morphogenesis. We demonstrate that the Arf1 guanine nucleotide exchange factor, brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1)/Arfgef1, and the effector Arf1 regulate the initiation of myelination of axons by Schwann cells. Schwann cell-specific BIG1 conditional knockout mice, which have been generated here, exhibit reduced myelin thickness and decreased localization of myelin protein zero in the myelin membrane, compared with their littermate controls. BIG1 knockout mouse nerves specifically decrease the amounts of Arf1 in the AP1 clathrin adaptor protein subunits but not the Arf1 binding to GGA1 (Golgi-localized, gamma-adaptin ear-containing, Arf-binding protein 1) transporting proteins. The amounts of Arf1 in the COPI coatomer protein subunits were comparable in the knockout mice and controls. Similar results in myelin thickness are observed in Arf1 conditional knockout mice, which have also been generated here. Thus, the BIG1 and Arf1 unit plays a key role in Schwann cell myelination, newly adding it to the list of molecular units controlling myelination.
  • Akira Kawashima, Tadayoshi Karasawa, Kenji Tago, Hiroaki Kimura, Ryo Kamata, Fumitake Usui-Kawanishi, Sachiko Watanabe, Satoshi Ohta, Megumi Funakoshi-Tago, Ken Yanagisawa, Tadashi Kasahara, Koichi Suzuki, Masafumi Takahashi
    Journal of immunology (Baltimore, Md. : 1950) 199 (10) 3614 - 3622 0022-1767 2017/11 [Refereed][Not invited]
     
    The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a molecular platform that induces caspase-1 activation and subsequent IL-1β maturation, and is implicated in inflammatory diseases; however, little is known about the negative regulation of NLRP3 inflammasome activation. In this article, we identified an E3 ligase, Ariadne homolog 2 (ARIH2), as a posttranslational negative regulator of NLRP3 inflammasome activity in macrophages. ARIH2 interacted with NLRP3 via its NACHT domain (aa 220-575) in the NLRP3 inflammasome complex. In particular, we found that while using mutants of ARIH2 and ubiquitin, the really interesting new gene 2 domain of ARIH2 was required for NLRP3 ubiquitination linked through K48 and K63. Deletion of endogenous ARIH2 using CRISPR/Cas9 genome editing inhibited NLRP3 ubiquitination and promoted NLRP3 inflammasome activation, resulting in apoptosis-associated speck-like protein containing a caspase recruitment domain oligomerization, pro-IL-1β processing, and IL-1β production. Conversely, ARIH2 overexpression promoted NLRP3 ubiquitination and inhibited NLRP3 inflammasome activation. Our findings reveal a novel mechanism of ubiquitination-dependent negative regulation of the NLRP3 inflammasome by ARIH2 and highlight ARIH2 as a potential therapeutic target for inflammatory diseases.
  • Kenji Tago, Satoshi Ohta, Masaki Kashiwada, Megumi Funakoshi-Tago, Jitsuhiro Matsugi, Shin-Ichi Tominaga, Ken Yanagisawa
    Heliyon 3 (10) e00436  2017/10 [Refereed][Not invited]
     
    The ST2 gene was originally identified as a primary responsive gene, and the expressions of its gene products are induced by stimulation with growth factors and by oncogenic stresses. In this study, we observed that oncogenic Ras mutant induced the expression of ST2 and ST2L proteins. Interestingly, the enforced expression of ST2 gene products in NIH-3T3 murine fibroblasts remarkably enhanced Ras (G12V)-induced cellular transformation. Furthermore, when the expression of ST2 gene products was silenced by RNA-interference technique, Ras (G12V)-induced cellular transformation was drastically suppressed. According to these observations, it was indicated that the oncogenic Ras-induced expression of ST2 gene products is required for the acceleration of cellular transformation, and this seems to be independent of the stimulation with IL-33, a ligand for ST2/ST2L. Interestingly, knockdown of ST2 gene products caused a reduction in Rb phosphorylation in transformed murine fibroblasts, suggesting the functional involvement of ST2 gene products in cell cycle progression during cellular transformation. Our current study strongly suggests the importance of ST2 gene products in cellular transformation, and the presence of novel mechanism how ST2 gene products affect the cellular transformation and cell proliferation.
  • Kazue Sugiyama, Kenji Tago, Sayumi Matsushita, Masashi Nishikawa, Katsuya Sato, Yoshinori Muto, Takahiro Nagase, Hiroshi Ueda
    Cellular signalling 32 115 - 123 0898-6568 2017/04 [Refereed][Not invited]
     
    PLEKHG2 is a Gβγ-dependent guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42, and has been shown to mediate signalling pathways such as actin cytoskeleton reorganization and serum response element (SRE)-dependent gene transcription. Here we show that the constitutively active mutant of the Gαs subunit significantly attenuated PLEKHG2-induced SRE-mediated gene transcription. Strikingly, we observed that the constitutive activation of endogenous Gαs by treatment with CTx caused a similar inhibitory effect on PLEKHG2-induced activation of SRE. However, both the enforced expression of the catalytic subunit β of protein kinase A and the treatment with dibutyl-cyclic AMP failed to mimic the inhibitory effect of Gαs on PLEKHG2. Furthermore, the dominant negative mutant of protein kinase A had no effect on PLEKHG2-mediated SRE activation. Performing immunoprecipitation and an in vitro pulldown assay, we found that PLEKHG2 directly interacted with the active form of the Gαs subunit in cells. The interaction between PLEKHG2 and Gαs required the N-terminal region of PLEKHG2, which includes the DH domain, a functional domain of GEF, suggesting that Gαs directly masks the DH domain of PLEKHG2. In a previous study, we reported that Gβγ accelerates PLEKHG2-mediated SRE-dependent gene transcription. Interestingly, Gαs also inhibited the hyperactivation of SRE induced by the co-expression of Gβγ and PLEKHG2; however, Gαs and Gβγ bind to different regions of PLEKHG2. This is the first report to show that PLEKHG2 is a novel effector of Gαs, and is negatively regulated by the Gαs subunit through direct interaction.
  • Fumihito Ueda, Kenji Tago, Hiroomi Tamura, Megumi Funakoshi-Tago
    The Journal of biological chemistry 292 (5) 1826 - 1846 0021-9258 2017/02 [Refereed][Not invited]
     
    The erythropoietin receptor (EpoR) regulates development of blood cells, and its full activation normally requires the cytokine erythropoietin (Epo). In the case of myeloproliferative neoplasms (MPN), Epo-independent signaling through EpoR can be caused by a point mutation, V617F, in the EpoR-interacting tyrosine kinase Janus kinase 2 (JAK2). In cells expressing the JAK2 V617F mutant, eight tyrosine residues in the intracellular domain of EpoR are phosphorylated, but the functional role of these phosphorylations in oncogenic signaling is incompletely understood. Here, to evaluate the functional consequences of the phosphorylation of these tyrosine residues, we constructed an EpoR-8YF mutant in which we substituted all eight tyrosine residues with phenylalanine. Co-expression of EpoR-8YF with the JAK2 V617F mutant failed to induce cytokine-independent cell proliferation and tumorigenesis, indicating that JAK2-mediated EpoR phosphorylation is the reason for JAK2 V617F mutant-induced oncogenic signaling. An exhaustive mutational analysis of the eight EpoR tyrosine residues indicated that three of these residues, Tyr-343, Tyr-460, and Tyr-464, are required for the JAK2 V617F mutant to exhibit its oncogenic activity. We also showed that phosphorylation at these three residues was necessary for full activation of the transcription factor STAT5, which is a critical downstream factor of JAK2 V617F-induced oncogenic signaling. In contrast, Epo stimulation could moderately stimulate the proliferation of cells expressing wild type JAK2 and EpoR-8YF, suggesting that the requirement of the phosphorylation of these three tyrosine residues seems to be specific for the oncogenic proliferation provoked by V617F mutation. Collectively, these results have revealed that phosphorylation of Tyr-343, Tyr-460, and Tyr-464 in EpoR underlies JAK2 V617F mutant-induced tumorigenesis. We propose that the targeted disruption of this pathway has therapeutic utility for managing MPN.
  • Megumi Funakoshi-Tago, Takuro Moriwaki, Fumihito Ueda, Hiroomi Tamura, Tadashi Kasahara, Kenji Tago
    Cellular signalling 31 41 - 57 0898-6568 2017/02 [Refereed][Not invited]
     
    The JAK2 V617F mutant-mediated aberrant signaling pathway is a hallmark of myeloproliferative neoplasms (MPNs). Although cytokine-inducible Src homology 2 protein (CIS) and suppressors of cytokine signaling (SOCS) are negative regulators of the JAK-STAT pathway, the functional role of CIS/SOCS family members in the JAK2 V617F mutant-induced oncogenic signaling pathway has not yet been elucidated. In this study, we found that the expression of CIS and SOCS1 was induced through the activation of signal transducer and activator of transcription 5 (STAT5) in not only the cells stimulated with Epo or IL-3 but also the cells transformed by the JAK2 V617F mutant. Cell proliferation and tumor formation in nude mice induced by the JAK2 V617F mutant were significantly enhanced when the expression of CIS was silenced using an RNA interference technique, whereas the knockdown of SOCS1 had no effect. The enforced expression of CIS caused apoptotic cell death in the transformed by JAK2 V617F mutant and drastically inhibited the JAK2 V617F mutant-induced tumor formation. CIS interacted with phosphorylated EpoR at Y401, which was critical for the activation of STAT5 and ERK. Whereas the activation of STAT5 and ERK in the transformed cells by JAK2 V617F mutant was increased by the knockdown of CIS, the enforced expression of CIS reduced the activation of these molecules. Furthermore, these anti-tumor effects of CIS required the function of SH2 domain and its tyrosine phosphorylation at Y253. We herein elucidated the mechanism by which CIS functions as a novel type of tumor suppressor in JAK2 V617F mutant-induced tumorigenesis.
  • Kenji Tago, Satoshi Ohta, Megumi Funakoshi-Tago, Chihiro Aoki-Ohmura, Jitsuhiro Matsugi, Shin-Ichi Tominaga, Ken Yanagisawa
    FEBS open bio 7 (2) 293 - 302 2211-5463 2017/02 [Refereed][Not invited]
     
    The ST2 gene was originally identified as a primary responsive gene induced by stimulation with growth factors and by oncogenic stress. The ST2 gene harbors two distinct promoters - a distal promoter and a proximal promoter. In this study, we identified a novel type of serum-responsive element in the ST2 proximal promoter using reporter gene analysis; this element includes a possible responsive element for STAT family proteins. Indeed, enforced expression of constitutively active STAT3 activated this promoter element and induced the expression of ST2 gene products. Furthermore, an oncogenic Ras (G12V) mutant also caused the expression of ST2 gene products by utilizing the proximal promoter. We also clarified that activation of the ST2 promoter by either growth stimulation or oncogenic Ras was suppressed by the inhibitors for STAT3 and ERK pathways. Our observations strongly suggest the importance of STAT family and ERK pathways for the induction of ST2 gene products by cell growth stimulation.
  • Chihiro Maki, Megumi Funakoshi-Tago, Ryohei Aoyagi, Fumihito Ueda, Masaki Kimura, Kenji Kobata, Kenji Tago, Hiroomi Tamura
    PloS one 12 (3) e0173264  1932-6203 2017 [Refereed][Not invited]
     
    Although epidemiological data have indicated that a strong negative association exists between coffee consumption and the prevalence of obesity-associated diseases, the molecular mechanisms by which coffee intake prevents obesity-associated diseases has not yet been elucidated. In this study, we found that coffee intake significantly suppressed high-fat diet (HFD)-induced metabolic alternations such as increases in body weight and the accumulation of adipose tissue, and up-regulation of glucose, free fatty acid, total cholesterol and insulin levels in the blood. We also found that coffee extract significantly inhibited adipogenesis in 3T3-L1 preadipocytes. In the early phase of adipogenesis, 3T3-L1 cells treated with coffee extract displayed the retardation of cell cycle entry into the G2/M phase called as mitotic clonal expansion (MCE). Coffee extract also inhibited the activation of CCAAT/enhancer-binding protein β (C/EBPβ) by preventing its phosphorylation by ERK. Furthermore, the coffee extract suppressed the adipogenesis-related events such as MCE and C/EBPβ activation through the down-regulation of insulin receptor substrate 1 (IRS1). The stability of the IRS1 protein was markedly decreased by the treatment with coffee extract due to proteasomal degradation. These results have revealed an anti-adipogenic function for coffee intake and identified IRS1 as a novel target for coffee extract in adipogenesis.
  • Yuki Uchihara, Fumihito Ueda, Kenji Tago, Yosuke Nakazawa, Tomoyuki Ohe, Tadahiko Mashino, Shigenobu Yokota, Tadashi Kasahara, Hiroomi Tamura, Megumi Funakoshi-Tago
    PloS one 12 (8) e0183003  1932-6203 2017 [Refereed][Not invited]
     
    Anaplastic large cell lymphomas (ALCL) are mainly characterized by harboring the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). The ALK inhibitor, crizotinib specifically induced apoptosis in Ba/F3 cells expressing NPM-ALK by inhibiting the activation of NPM-ALK and its downstream molecule, signal transducer and activator of transcription factor 3 (STAT3). We found that α-tocopherol, a major component of vitamin E, attenuated the effects of crizotinib independently of its anti-oxidant properties. Although α-tocopherol suppressed the inhibitory effects of crizotinib on the signaling axis including NPM-ALK and STAT3, it had no influence on the intake of crizotinib into cells. Crizotinib also directly inhibited the kinase activity of NPM-ALK; however, this inhibitory effect was not altered by the co-treatment with α-tocopherol. Whereas the nuclear localization of NPM-ALK was disappeared by the treatment with crizotinib, the co-treatment with α-tocopherol swept the effect of crizotinib and caused the localization of NPM-ALK in nucleus. The administration of α-tocopherol attenuated the anti-tumor activity of crizotinib against NPM-ALK-provoked tumorigenesis in vivo. Furthermore, the α-tocopherol-induced inhibition of crizotinib-caused apoptosis was also observed in NPM-ALK-positive cells derived from ALCL patients, namely, SUDHL-1 and Ki-JK. Collectively, these results not only revealed the novel mechanism underlying crizotinib-induced apoptosis in NPM-ALK-positive cells, but also suggest that the anti-tumor effects of crizotinib are attenuated when it is taken in combination with vitamin E.
  • Megumi Funakoshi-Tago, Yurika Miyagawa, Fumihito Ueda, Tadahiko Mashino, Yasuhiro Moriwaki, Kenji Tago, Tadashi Kasahara, Hiroomi Tamura
    INTERNATIONAL IMMUNOPHARMACOLOGY 40 254 - 264 1567-5769 2016/11 [Refereed][Not invited]
     
    IL-33 functions as a ligand for ST2L, which is mainly expressed in immune cells, including mast cells. IL-33 is a potent inducer of pro-inflammatory cytokines, such as IL-6, and has been implicated in the pathogenesis of allergic inflammatory diseases. Therefore, IL-33 has recently been attracting attention as a new target for the treatment of inflammatory diseases. In the present study, we demonstrated that a water-soluble bis-malonic acid fullerene derivative (C-60-dicyclopropane-1,1,1',1'-tetracarboxylic acid) markedly diminished the IL-33-induced expression of IL-6 in bone marrow-derived mast cells (BMMC). The bis-malonic acid fullerene derivative suppressed the canonical signaling steps required for NF-kappa B activation such as the degradation of Ma and nuclear translocation of NF-kappa B by directly inhibiting the IL-33-induced MK activation. Although p38 and JNK also contributed to IL-33-induced expression of IL-6, the bis-malonic acid fullerene derivative did not affect their activation. Furthermore, the bis-malonic acid fullerene derivative had no effect on the NF-kappa B activation pathway induced by TNFa and IL-1. These results suggest that the bis-malonic fullerene derivative has potential as a specific drug for the treatment of IL-33-induced inflammatory diseases by specifically inhibiting the NF-kappa B activation pathway. (C) 2016 Elsevier B.V. All rights reserved.
  • Megumi Funakoshi-Tago, Kentaro Ohsawa, Toshiyuki Ishikawa, Fumika Nakamura, Fumihito Ueda, Yuji Narukawa, Fumiyuki Kiuchi, Hiroomi Tamura, Kenji Tago, Tadashi Kasahara
    INTERNATIONAL IMMUNOPHARMACOLOGY 40 550 - 560 1567-5769 2016/11 [Refereed][Not invited]
     
    Flavonoids, particularly those derived from plants, harbor biological effects such as and-inflammation and the inhibition of cancer progression. In the present study, we investigated the effects of 10 kinds of flavonoids isolated from Nepalese propolis on the LPS signaling pathway in order to clarify their anti-inflammatory activities. Five types of flavonoids: isoliquiritigenin, chrysin, 3',4'-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, and cearoin, markedly inhibited inflammatory responses including LPS-induced NO production by suppressing the expression of iNOS mRNA and LPS-induced mRNA expression of TNF alpha and CCL2. Their inhibitory effects on LPS-induced inflammatory responses correlated with the intensities of these flavonoids to suppress the LPS-induced activation of nuclear factor kappa B (NF-kappa B), an essential transcription factor for the mRNA expression of iNOS, TNF alpha, and CCL2. Among these flavonoids, 3',4'-dihydroxy-4-methoxydalbergione and 4-methoxydalbergion markedly inhibited the LPS-induced activation of IKK, thereby abrogating the degradation of I kappa B alpha and nuclear localization of NF-kappa B. On the other hand, isoliquiritigenin, chrysin, and cearoin failed to inhibit these signaling steps, but suppressed the transcriptional activity of NF-kappa B, which caused their anti-inflammatory effects. The results of the present study revealed that these five kinds of flavonoids are the components of Nepalese propolis that exhibit anti-inflammatory activities with a different regulatory mechanism for the activation of NF-kappa B. (C) 2016 Elsevier B.V. All rights reserved.
  • Kazuhisa Watanabe, Kazuhiro Nakayama, Satoshi Ohta, Kenji Tago, Supichaya Boonvisut, Elizabeth J. Millings, Stuart G. Fischer, Charles A. LeDuc, Rudolph L. Leibel, Sadahiko Iwamoto
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 477 (4) 712 - 716 0006-291X 2016/09 [Refereed][Not invited]
     
    A diabetes susceptibility gene, immunoglobulin-like domain containing receptor 2 (Ildr2), encodes a transmembrane protein localized to the endoplasmic reticulum membrane that is closely related to hepatic lipid metabolism. The livers of ob/ob mice in which Ildr2 is transiently overexpressed are relieved of hepatic steatosis. However, the molecular mechanisms through which ILDR2 affects these changes in hepatic lipid metabolism remain unknown. This study aimed to identify ILDR2-interacting proteins to further elucidate the molecular mechanisms underlying the role of ILDR2 in lipid homeostasis. We purified ILDR2-containing protein complexes using tandem affinity purification tagging and identified ZNF70, a member of the Kruppel C2H2-type zinc finger protein family, as a novel ILDR2-interacting protein. We demonstrated that ZNF70 interacts with ZFP64 and activates HES1 transcription by binding to the HES1 promoter. In addition, HES1 gene expression is increased in ILDR2-knockdown HepG2 cells, in which ZNF70 is translocated from the cytoplasm to the nucleus, suggesting that ZNF70 migration to the nucleus after dissociating from the ILDR2-ZNF70 complex activates HES1 transcription. These results support a novel link between ILDR2 and HES1 gene expression and suggest that ILDR2 is involved in a novel pathway in hepatic steatosis. (C) 2016 Elsevier Inc. All rights reserved.
  • Satoshi Ohta, Kenji Tago, Megumi Funakoshi-Tago, Jitsuhiro Matsugi, Ken Yanagisawa
    CELLULAR SIGNALLING 28 (8) 1025 - 1036 0898-6568 2016/08 [Refereed][Not invited]
     
    A member of the interleukin-1 family, interleukin-33 (NF-HEV/IL-33), is a ligand for the receptor, ST2L and stimulates the production of Th2 cytokines. Although IL-33 localizes to the nucleus and may be involved in the regulation of transcription independent of ST2L, its functions in the nucleus currently remain unclear. We herein demonstrated that the expression of IL-33 was markedly enhanced in NIH-3T3 cells transformed by an oncogenic H-Ras mutant (H-Ras (G12V)), and the induced IL-33 was mainly located in the nuclei of these cells. The enforced expression of IL-33 accelerated H-Ras (G12V)-induced transformation in NIH-3T3 cells, and this transforming activity was markedly reduced by the knockdown of IL-33 with shRNA. We subsequently analyzed several signaling molecules regulated by Ras in order to elucidate the mechanism by which IL-33 contributes to Ras (G12V)-induced transformation. We found that the knockdown of IL-33 effectively attenuated the Ras (G12V)-induced expression of cyclin D1. However, the knockdown of IL-33 failed to affect cyclin D1 mRNA expression levels, and epoxomicin, a proteasome. inhibitor, did not cancel the IL-33 knockdown -induced down-regulation of its protein levels. We showed that Ras (G12V)-induced cyclin D1 protein synthesis was markedly suppressed by the knockdown of IL-33. Taken together, the results of the present study strongly suggest a novel role for IL-33 in cellular transformation. (C) 2016 Elsevier Inc All rights reserved.
  • Shin-ichi Tominaga, Satoshi Ohta, Kenji Tago
    Biochemistry and Biophysics Reports 5 8 - 15 2405-5808 2016/03 [Refereed][Not invited]
     
    The ST2 gene is induced in murine fibroblast cells at the start of cell proliferation. Although IL-33 has been identified as a ligand for one of the two major gene products of ST2 - namely, the transmembrane receptor form ST2L - prompting immunological research on inflammation, the roles of the ST2 gene products in cell proliferation remain to be elucidated.Using a cell proliferation assay system with NIH-3T3 cells, a normal murine fibroblast cell line, we found that treatment with recombinant ST2 caused an acceleration of cell proliferation, suggesting that ST2 acts in an autocrine/paracrine fashion. Strikingly, shRNA-induced knockdown of both ST2 gene products, ST2 and ST2L, reduced cell proliferation. This effect was effectively canceled by the expression of shRNA-resistant ST2, but not shRNA-resistant ST2L.The novel enhancement of cell proliferation by ST2 appears to involve positive feedback. Since the ST2 level is increased in various diseases involving inflammation, future investigations into the role of ST2 gene products in relation to various diseases, including malignancies, may be warranted.
  • Megumi Funakoshi-Tago, Takahiro Hattori, Fumihito Ueda, Kenji Tago, Tomoyuki Ohe, Tadahiko Mashino, Hiroomi Tamura
    Biochemistry and Biophysics Reports 5 259 - 265 2405-5808 2016/03 [Refereed][Not invited]
     
    Obesity and its associated metabolic diseases represent some of the most rapidly expanding health issues worldwide, and, thus, the development of a novel chemical compound to suppress adipogenesis is strongly expected. We herein investigated the effects of water-soluble fullerene derivatives: a bis-malonic acid derivative and three types of proline-type fullerene derivatives, on adipogenesis using NIH-3T3 cells overexpressing PPARγ. One of the proline-type fullerene derivatives (P3) harboring three carboxy groups significantly inhibited lipid accumulation and the expression of adipocyte-specific genes, such as aP2, induced by the PPARγ agonist rosiglitazone. On the other hand, the bis-malonic acid derivative (M) and the 2 other proline-type fullerene derivatives (P1, P2), which have two carboxy groups, had no effect on PPARγ-mediated lipid accumulation or the expression of aP2. P3 fullerene also inhibited lipid accumulation induced by the combined stimulation with 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin in 3T3-L1 preadipocytes. During the differentiation of 3T3-L1 cells into adipocytes, P3 fullerene did not affect the expression of C/EBPδ, C/EBPβ, or PPARγ, but markedly inhibited that of aP2 mRNA. These results suggest that P3 fullerene exhibits anti-obesity activity by preventing the activation of PPARγ.
  • Kenji Tago, Megumi Funakoshi-Tago
    Seikagaku 88 (2) 207 - 210 2189-0544 2016 [Refereed][Not invited]
  • Fumihito Ueda, Keito Iizuka, Kenji Tago, Yuji Narukawa, Fumiyuki Kiuchi, Tadashi Kasahara, Hiroomi Tamura, Megumi Funakoshi-Tago
    INTERNATIONAL IMMUNOPHARMACOLOGY 28 (2) 967 - 976 1567-5769 2015/10 [Refereed][Not invited]
     
    Leonotis nepetaefolia R. Br., also known as Klip Dagga or Lion's Ear, has traditionally been used as a folk medicine to treat inflammatory diseases such as rheumatism, bronchitis, and asthma; however, the components that exhibit its anti-inflammatory activity have not yet been identified. In the present study, we investigated the effects of three types of diterpenoids, nepetaefuran, leonotinin, and leonotin, which were isolated from L. nepetaefolia R. Br., on the LPS signaling pathway in order to elucidate the anti-inflammatory mechanism involved. Nepetaefuran more potently inhibited the LPS-induced production of NO and CCL2 than leonotinin by suppressing the expression of iNOS mRNA and CCL2 mRNA. On the other hand, leonotin failed to inhibit the production of NO and CCL2 induced by LPS. Although nepetaefuran and leonotinin had no effect on the LPS-induced degradation of I kappa B alpha or nuclear translocation of NF-kappa B p65, they markedly inhibited the transcriptional activity of NF-kappa B. Nepetaefuran and leonotinin also inhibited the transcriptional activity of the GAL4-NF-kappa B p65 fusion protein. On the other hand, nepetaefuran, leonotinin and leonotin did not affect the LPS-induced activation of MAP kinase family members such as ERK, p38, and JNK In addition, inhibitory effect of nepetaefuran and leonotinin on NF-kappa B activation is well correlated with their ability to induce activation of Nrf2 and ER stress. Taken together, these results demonstrated that nepetaefuran and leonotinin could be the components responsible for the anti-inflammatory activity of L nepetaefolia R. Br. by specifically inhibiting the LPS-induced activation of NF-kappa B. (C) 2015 Elsevier B.V. All rights reserved.
  • Shigeyuki Ohta, Sayaka Sakaguchi, Yuki Kobayashi, Norikazu Mizuno, Kenji Tago, Hiroshi Itoh
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 38 (4) 594 - 600 0918-6158 2015/04 [Refereed][Not invited]
     
    GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) and is highly expressed in parts of tumor cells. The involvement of GPR56 in tumorigenesis has been reported. We generated agonistic monoclonal antibodies against human GPR56 and analyzed the action and signaling pathway of GPR56. The antibodies inhibited cell migration through the Gq and Rho pathway in human glioma U87-MG cells. Co-immunoprecipitation analysis indicated that the interaction between the GPR56 extracellular domain and transmembrane domain was potentiated by agonistic antibodies. These results demonstrated that functional antibodies are invaluable tools for GPCR research and should open a new avenue for therapeutic treatment of tumors.
  • Shin-ichi Tominaga, Kenji Tago, Hidetoshi Tsuda, Mayumi Komine
    CYTOKINE 72 (1) 105 - 108 1043-4666 2015/03 [Refereed][Not invited]
     
    The interleukin-33 (IL-33)-ST2L signaling pathway has been shown to play important roles in the field of immunology, especially as a trigger for allergic reactions such as bronchial asthma. However, coming back to the original finding that the ST2 gene is induced during initiation of the cell cycle of fibroblastic cell lines, the possible functions of the ST2 gene products and their specific ligand, IL-33, in the field of cell growth regulation are still interesting problems to be solved. In this study, we used NIH-3T3 mouse cell line and added IL-33 before and after cell proliferation assay, which revealed the dual function of IL-33. When IL-33 was added to the confluent cells before the start of cell proliferation, it suppressed the cell growth concentration-dependently. On the other hand, if IL-33 was added after the start of cell proliferation, it enhanced the cell growth. The negative effect of IL-33 on cell proliferation is a novel finding and would provide an important clue to the roles of IL-33 and ST2/ST2L in growth regulation. (C) 2014 Elsevier Ltd. All rights reserved.
  • Megumi Funakoshi-Tago, Kazuhi Okamoto, Rika Izumi, Kenji Tago, Ken Yanagisawa, Yuji Narukawa, Fumiyuld Kiuchi, Tadashi Kasahara, Hiroomi Tamura
    INTERNATIONAL IMMUNOPHARMACOLOGY 25 (1) 189 - 198 1567-5769 2015/03 [Refereed][Not invited]
     
    Propolis has been used in folk medicine to improve health and prevent inflammatory diseases; however, the components that exhibit its anti-inflammatory activity remain unknown. We herein investigated the effects of flavonoids isolated from Nepalese propolis on the IL-33 signaling pathway to clarify the anti-inflammatory mechanism involved. Of the 8 types of flavonoids isolated from Nepalese propolis, 4 types of compounds, such as 3',4'-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, cearoin, and chrysin, markedly inhibited the IL-33-induced mRNA expression of inflammatory genes including IL-6, TNF alpha. and IL-13 in bone marrow-derived mast cells (BMMC). These four flavonoids also inhibited the IL-33-induced activation of nudear factor kappa B (NF-kappa B), which was consistent with their inhibitory effects on cytokine expression. The effects of these flavonoids are attributed to inhibition of IL-33-induced activation of IKK, which leads to the degradation of I kappa B alpha and nuclear localization of NF-kappa B. On the other hand, other flavonoids isolated from Nepalese propolis, such as isoliquiritigenin, plathymenin, 7-hydroxyflavanone, and (+)-medicarpin, had no effect on the IL-33 signaling pathway or cytokine expression. Therefore, these results indicate that 3',4'-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, cearoin, and chrysin are the substances responsible for the anti-inflammatory activity of Nepalese propolis. (C) 2015 Elsevier B.V. All rights reserved.
  • K. Tago, M. Funakoshi-Tago, H. Itoh, Y. Furukawa, J. Kikuchi, T. Kato, K. Suzuki, K. Yanagisawa
    ONCOGENE 34 (3) 310 - 318 0950-9232 2015/01 [Refereed][Not invited]
     
    Tumor suppressor protein p19(ARF) (Arf; p14(ARF) in humans) functions in both p53-dependent and -independent modes to counteract hyper-proliferative signals caused by proto-oncogene activation, but its p53-independent activities remain poorly understood. Using the tandem affinity purification-tag technique, we purified Arf-containing protein complexes and identified p68 DEAD-box protein (DDX5) as a novel interacting protein of Arf. In this study, we found that DDX5 interacts with c-Myc, and harbors essential roles for c-Myc-mediated transcription and its transforming activity. Furthermore, when c-Myc was forcibly expressed, the expression level of DDX5 protein was drastically increased through the acceleration of protein synthesis of DDX5, suggesting the presence of an oncogenic positive feedback loop including c-Myc and DDX5. Strikingly, Arf blocked the physical interaction between DDX5 and c-Myc, and drove away DDX5 from the promoter of c-Myc target genes. These observations most likely indicate the mechanism by which Arf causes p53-independent tumor-suppressive activity.
  • Tomohiro Torii, Yuki Miyamoto, Kenji Tago, Kazunori Sango, Kazuaki Nakamura, Atsushi Sanbe, Akito Tanoue, Junji Yamauchi
    The Journal of biological chemistry 289 (49) 33887 - 903 0021-9258 2014/12 [Refereed][Not invited]
     
    The mechanism of neurite growth is complicated, involving continuous cytoskeletal rearrangement and vesicular trafficking. Cytohesin-2 is a guanine nucleotide exchange factor for Arf6, an Arf family molecular switch protein, controlling cell morphological changes such as neuritogenesis. Here, we show that cytohesin-2 binds to a protein with a previously unknown function, CCDC120, which contains three coiled-coil domains, and is transported along neurites in differentiating N1E-115 cells. Transfection of the small interfering RNA (siRNA) specific for CCDC120 into cells inhibits neurite growth and Arf6 activation. When neurites start to extend, vesicles containing CCDC120 and cytohesin-2 are transported in an anterograde manner rather than a retrograde one. As neurites continue extension, anterograde vesicle transport decreases. CCDC120 knockdown inhibits cytohesin-2 localization into vesicles containing CCDC120 and diffuses cytohesin-2 in cytoplasmic regions, illustrating that CCDC120 determines cytohesin-2 localization in growing neurites. Reintroduction of the wild type CCDC120 construct into cells transfected with CCDC120 siRNA reverses blunted neurite growth and Arf6 activity, whereas the cytohesin-2-binding CC1 region-deficient CCDC120 construct does not. Thus, cytohesin-2 is transported along neurites by vesicles containing CCDC120, and it mediates neurite growth. These results suggest a mechanism by which guanine nucleotide exchange factor for Arf6 is transported to mediate neurite growth.
  • Megumi Funakoshi-Tago, Masaki Tsukada, Toshiro Watanabe, Yuka Mameda, Kenji Tago, Tomoyuki Ohe, Shigeo Nakamura, Tadahiko Mashino, Tadashi Kasahara
    INTERNATIONAL IMMUNOPHARMACOLOGY 20 (1) 258 - 263 1567-5769 2014/05 [Refereed][Not invited]
     
    JAK2 V617F mutant, a gene responsible for human myeloproliferative neoplasms (MPNs), causes not only cellular transformation but also resistance to various anti-cancer drugs. We previously reported that pyrrolidinium fullerene markedly induced the apoptosis of JAK2 V617F mutant-induced transformed cells through the reduction of apoptosis signal-regulating kinase 1 (ASK1), following inhibition of the c-Jun N-terminal kinase (INK) pathway. In the current study, we found that the replacement of the 2-hydrogen atom (-H) or N-methyl group (-CH3) by the butyl group (-C4C9) caused the more than 3-fold potent cytotoxic effects on cells transformed by the JAK2 V617F mutant. Strikingly, these chemical modification of pyrrolidinium fullerene resulted in more marked reduction of ASK1 protein and a more potent inhibitory effect on the JNK signaling cascade. On the other hand, when modified with a longer alkyl group, the derivatives lacked their cytotoxicity. These observations clearly indicate that the modification of pyrrolidinium fullerene with a suitable length of alkyl group such as butyl group enhances its apoptotic effect through inhibition of the ASK1-MKK4/7-JNK pathway. (C) 2014 Elsevier B.V. All rights reserved.
  • Yoshiyuki Inoue, Koumei Shirasuna, Hiroaki Kimura, Fumitake Usui, Akira Kawashima, Tadayoshi Karasawa, Kenji Tago, Katsuya Dezaki, Satoshi Nishimura, Junji Sagara, Tetsuo Noda, Yoichiro Iwakura, Hiroko Tsutsui, Shun'ichiro Taniguchi, Ken Yanagisawa, Toshihiko Yada, Yoshikazu Yasuda, Masafumi Takahashi
    JOURNAL OF IMMUNOLOGY 192 (9) 4342 - 4351 0022-1767 2014/05 [Refereed][Not invited]
     
    Inflammation plays a key role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury. However, the mechanism by which hepatic I/R induces inflammatory responses remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by I/R is mediated through a multiple-protein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in hepatic I/R injury and found that hepatic I/R stimuli upregulated the inflammasome-component molecule, nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), but not apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). NLRP3(-/-) mice, but not ASC(-/-) and caspase-1(-/-) mice, had significantly less liver injury after hepatic I/R. NLRP3(-/-) mice showed reduced inflammatory responses, reactive oxygen species production, and apoptosis in I/R liver. Notably, infiltration of neutrophils, but not macrophages, was markedly inhibited in the I/R liver of NLRP3(-/-) mice. Bone marrow transplantation experiments showed that NLRP3 not only in bone marrow-derived cells, but also in non-bone marrow-derived cells contributed to liver injury after I/R. In vitro experiments revealed that keratinocyte-derived chemokine-induced activation of heterotrimeric G proteins was markedly diminished. Furthermore, NLRP3(-/-) neutrophils decreased keratinocyte-derived chemokine-induced concentrations of intracellular calcium elevation, Rac activation, and actin assembly formation, thereby resulting in impaired migration activity. Taken together, NLRP3 regulates chemokine-mediated functions and recruitment of neutrophils, and thereby contributes to hepatic I/R injury independently of inflammasomes. These findings identify a novel role of NLRP3 in the pathophysiology of hepatic I/R injury.
  • 多胡 憲治
    ファルマシア (公社)日本薬学会 50 (3) 249  0014-8601 2014/03 [Not refereed][Not invited]
  • Riris I Jenie, Motoki Nishimura, Mika Fujino, Michio Nakaya, Norikazu Mizuno, Kenji Tago, Hitoshi Kurose, Hiroshi Itoh
    Genes to Cells 18 (12) 1095 - 1106 1356-9597 2013/12 [Refereed][Not invited]
     
    Hyperactivation of Gq signaling causes cardiac hypertrophy, and β-adrenergic receptor-mediated Gs signaling is attenuated in hypertrophic cardiomyocytes. Here, we found the increase in a global ubiquitination in hypertrophic mouse heart. The activation of Gq signaling resulted in the ubiquitination of Gαs in neonatal rat cardiomyocytes, reduced Gαs expression, and suppressed cAMP response to β-adrenergic receptor stimulation. Ectopic expression of Gαq induced a similar suppression, which is due to the degradation of Gαs by a ubiquitin-proteasome pathway. Co-expression of Ric-8B, a positive regulator of Gαs, effectively canceled the Gαq-induced ubiquitination of Gαs and recovered the cAMP accumulation. In vitro, Gαq competes for the binding of Gαs to Ric-8B. These data show a new role of Ric-8B in the crosstalk of two distinct G protein signaling pathways, which are possibly involved in a part of mechanisms of chronic heart failure. © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.
  • Fumihito Ueda, Kazuya Sumi, Kenji Tago, Tadashi Kasahara, Megumi Funakoshi-Tago
    Cellular Signalling 25 (11) 2115 - 2124 0898-6568 2013/11 [Refereed][Not invited]
     
    A point mutation (V617F) of tyrosine kinase Janus kinase 2 (JAK2) is found in the majority of patients with myeloproliferative neoplasms (MPNs) and an aberrant signaling pathway induced by constitutively active JAK2 V617F mutant is a hallmark of MPNs. Cells transformed by JAK2 V617F mutant exhibited resistance to anti-cancer drugs such as cisplatin (CDDP), mitomycin C (MMC) and bleomycin (BLM). We first found that the expression of FANCC, a member of the Fanconi anemia (FA) proteins, was significantly induced by JAK2 V617F mutant through activation of signal transducers and activators of transcription 5 (STAT5). In addition, monoubiqitination and foci formation of FANCD2, which are critical for activation of the FA pathway, were increased in cells transformed by JAK2 V617F mutant, compared to cells expressing wild-type JAK2. Interestingly, knockdown of FANCC in cells expressing JAK2 V617F mutant induced not only the reduction of monoubiqitination and foci formation of FANCD2 but also the enhancement of sensitivity to DNA damage induced by CDDP and MMC but not BLM. Taken together, FANCC is most likely to be critical for resistance to DNA cross-linking drug-induced DNA damage in cells transformed by JAK2 V617F mutant. © 2013 Elsevier Inc.
  • Megumi Funakoshi-Tago, Kazuya Sumi, Tadashi Kasahara, Kenji Tago
    PLoS ONE 8 (1) e52844  1932-6203 2013/01 [Refereed][Not invited]
     
    The acquired mutation (V617F) of Janus kinase 2 (JAK2) is observed in the majority of patients with myeloproliferative neoplasms (MPNs). In the screening of genes whose expression was induced by JAK2 (V617F), we found the significant induction of c-Myc mRNA expression mediated by STAT5 activation. Interestingly, GSK-3β was inactivated in transformed Ba/F3 cells by JAK2 (V617F), and this enhanced the protein expression of c-Myc. The enforced expression of c-Myc accelerated cell proliferation but failed to inhibit apoptotic cell death caused by growth factor deprivation however, the inhibition of GSK-3β completely inhibited the apoptosis of cells expressing c-Myc. Strikingly, c-Myc T58A mutant exhibited higher proliferative activity in a growth-factor-independent manner however, this mutant failed to induce apoptosis. In addition, knockdown of c-Myc significantly inhibited the proliferation of transformed cells by JAK2 (V617F), suggesting that c-Myc plays an important role in oncogenic activity of JAK2 (V617F). Furthermore, JAK2 (V617F) induced the expression of a target gene of c-Myc, ornithine decarboxylase (ODC), known as the rate-limiting enzyme in polyamine biosynthesis. An ODC inhibitor, difluoromethylornithine (DFMO), prevented the proliferation of transformed cells by JAK2 (V617F). Importantly, administration of DFMO effectively delayed tumor formation in nude mice inoculated with transformed cells by JAK2 (V617F), resulting in prolonged survival therefore, ODC expression through c-Myc is a critical step for JAK2 (V617F)-induced transformation and DFMO could be used as effective therapy for MPNs. © 2013 Funakoshi-Tago et al.
  • Megumi Funakoshi-Tago, Tatsuaki Nagata, Kenji Tago, Masaki Tsukada, Kazuyuki Tanaka, Shigeo Nakamura, Tadahiko Mashino, Tadashi Kasahara
    CELLULAR SIGNALLING 24 (11) 2024 - 2034 0898-6568 2012/11 [Refereed][Not invited]
     
    The constitutively activated mutation (V617F) of tyrosine kinase Janus kinase 2 (JAK2) is found in the majority of patients with myeloproliferative neoplasms (MPNs). The development of a novel chemical compound to suppress JAK2 V617F mutant-induced onset of MPNs and clarification of the signaling cascade downstream of JAK2 V617F mutant will provide clues to treat MPNs. Here we found that a water-soluble pyrrolidinium fullerene derivative, C-60-bis (N, N-dimethylpyrrolidinium iodide), markedly induced apoptosis of JAK2 V617F mutant-induced transformed cells through a novel mechanism, inhibiting c-Jun N-terminal kinase (JNK) activation pathway but not generation of reactive oxygen species (ROS). Pyrrolidinium fullerene derivative significantly reduced the protein expression level of apoptosis signal-regulating kinase 1 (ASK1), one of the mitogen-activated protein kinase kinase kinases (MAPKKK), resulting in the inhibition of upstream molecules of JNK, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7). Strikingly, the knockdown of ASK1 enhanced the sensitivity to pyrrolidinium fullerene derivative-induced apoptosis, and the treatment with a JNK inhibitor, SP600125, also induced apoptosis of the transformed cells by JAK2 V617F mutant Furthermore, administration of both SP600125 and pyrrolidinium fullerene derivative markedly inhibited JAK2 V617F mutant-induced tumorigenesis in nude mice. Taking these findings together, JAK2 V617F mutant-induced JNK signaling pathway is an attractive target for MPN therapy, and pyrrolidinium fullerene derivative is now considered a candidate potent drug for MPNs. (C) 2012 Elsevier Inc. All rights reserved.
  • Hitomi Nakazawa, Tadayuki Sada, Michinori Toriyama, Kenji Tago, Tadao Sugiura, Mitsunori Fukuda, Naoyuki Inagaki
    JOURNAL OF NEUROSCIENCE 32 (37) 12712 - 12725 0270-6474 2012/09 [Refereed][Not invited]
     
    Axon outgrowth requires plasma membrane expansion, which results from post-Golgi vesicular transport and fusion. However, the molecular mechanisms regulating post-Golgi vesicular trafficking for membrane expansion and axon outgrowth remain unclear. Here, we show that Rab33a expression became upregulated during axon outgrowth of cultured rat hippocampal neurons. Rab33a was preferentially localized to the Golgi apparatus and to synaptophysin-positive vesicles that are transported along the growing axon. Previous studies showed that synaptophysin is localized to post-Golgi vesicles transported by fast axonal transport in developing neurons. Reduction of Rab33a expression by RNAi (RNA interference) inhibited the anterograde transport of synaptophysin-positive vesicles, leading to their decrease in axonal tips. Furthermore, this treatment reduced membrane fusion of synaptophysin-positive vesicles at the growth cones and inhibited axon outgrowth. Overexpression of Rab33a, on the other hand, induced excessive accumulation of synaptophysin-positive vesicles and concurrent formation of surplus axons. These data suggest that Rab33a participates in axon outgrowth by mediating anterograde axonal transport of synaptophysin-positive vesicles and their concomitant fusion at the growth cones.
  • Manami Toriyama, Norikazu Mizuno, Takashi Fukami, Tokuichi Iguchi, Michinori Toriyama, Kenji Tago, Hiroshi Itoh
    JOURNAL OF BIOLOGICAL CHEMISTRY 287 (16) 12691 - 12702 0021-9258 2012/04 [Refereed][Not invited]
     
    Doublecortin (DCX) is a microtubule-associated protein that is specifically expressed in neuronal cells. Genetic mutation of DCX causes lissencephaly disease. Although the abnormal cortical lamination in lissencephaly is thought to be attributable to neuronal cell migration defects, the regulatory mechanisms governing interactions between DCX and cytoskeleton in the migration of neuronal progenitor cells remain obscure. In this study we found that the G(s) and protein kinase A (PKA) signal elicited by pituitary adenylate cyclase-activating polypeptide promotes neuronal progenitor cells migration. Stimulation of G(s)-PKA signaling prevented microtubule bundling and induced the dissociation of DCX from microtubules in cells. PKA phosphorylated DCX at Ser-47, and the phospho-mimicking mutant DCX-S47E promoted cell migration. Activation of PKA and DCX-S47E induced lamellipodium formation. Pituitary adenylate cyclase-activating polypeptide and DCX-S47E stimulated the activation of Rac1, and DCX-S47E interacted with Asef2, a guanine nucleotide exchange factor for Rac1. Our data reveal a dual reciprocal role for DCX phosphorylation in the regulation of microtubule and actin dynamics that is indispensable for proper brain lamination.
  • Megumi Funakoshi-Tago, Kei Nakamura, Kenji Tago, Tadahiko Mashino, Tadashi Kasahara
    INTERNATIONAL IMMUNOPHARMACOLOGY 11 (9) 1150 - 1159 1567-5769 2011/09 [Refereed][Not invited]
     
    Flavonoids are widely distributed in many fruits and plants, and it has been shown that most flavonoids have anti-inflammatory activity; however, the mechanisms of how the flavonoids exhibit their anti-inflammatory activity have not been clarified. We therefore focus on flavonoids Apigenin, Luteolin and Fisetin because of their related structure. We found that these compounds significantly inhibited TNF alpha-induced NF-kappa B transcriptional activation; however, they had no effect on the degradation of I kappa B proteins and the nuclear translocation and DNA binding activity of NF-kappa B p65. Interestingly, the suppression of NF-kappa B activation by these flavonoids is due to inhibition of the transcriptional activation of NF-kappa B, since the compounds markedly inhibited the transcriptional activity of GAL4-NF-kappa B p65 fusion protein. In addition, while Apigenin and Luteolin slightly inhibited TNF alpha-induced JNK activation, they had no effect on TNF alpha-induced activation of ERK and p38. Unexpectedly, Fisetin enhanced and sustained activation of ERK and JNK but not p38 in response to TNF alpha. Strikingly, TNF alpha-induced expression of CCL2/MCP-1 and CXCL1/KC was significantly inhibited by Apigenin and Luteolin but not Fisetin. Furthermore, the administration of Apigenin and Luteolin markedly inhibited acute carrageenan-induced paw edema in mice; however, Fisetin failed to have an effect. These observations strongly suggest that the slight structural difference in flavonoids may cause a defective effect of Fisetin on these inflammatory responses, and this may be due to the differences in their direction of the effect on the activation pathways of MAP kinases. (C) 2011 Elsevier B.V. All rights reserved.
  • Kazuya Sumi, Kenji Tago, Tadashi Kasahara, Megumi Funakoshi-Tago
    FEBS LETTERS 585 (12) 1884 - 1890 0014-5793 2011/06 [Refereed][Not invited]
     
    JAK2 V617F mutant induces transformation through aberrant activation of various transcription factors. We found that the expression of Aurora kinase A (Aurka) was significantly induced by mutant JAK2 through c-Myc expression. Interestingly, mutant JAK2 enhanced resistance to cisplatin (CDDP)-induced DNA damage, and effectively suppressed apoptosis. Ectopic expression of Aurka in Ba/F3 cells exhibited similar resistance to CDDP, and this required kinase activity. Conversely, knockdown and inhibition of Aurka in cells expressing mutant JAK2 abolished the resistance to CDDP. Taken together, Aurka is most likely critical for resistance to DNA damage in cells transformed by JAK2 V617F mutant. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • Jun Kamishimoto, Kenji Tago, Tadashi Kasahara, Megumi Funakoshi-Tago
    CELLULAR SIGNALLING 23 (5) 849 - 856 0898-6568 2011/05 [Refereed][Not invited]
     
    The disruption of Janus kinase 2 (JAK2) signaling regulation by its point mutation, V617F, is involved in various myeloproliferative disorders (MPDs). JAK2 V617F mutant induced constitutive activation of Akt when erythropoietin receptor (EpoR) was coexpressed; however, the physiological role of Akt activation in MPDs has not been elucidated. LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, inhibited Akt activation and induced apoptotic cell death in cells expressing JAK2 V617F mutant and EpoR. Previously, it has been shown that the phosphorylation at Y479 in EpoR is critical for the interaction with PI3K, an upstream molecule of Akt. Hence, EpoR mutant with a point mutation of Y479F, which fails to activate Akt, is useful for addressing the role of Akt activation in JAK2 V617F mutant-induced tumorigenesis. Interestingly, under the expression of EpoR Y479F mutant, JAK2 V617F mutant failed to exhibit potent anti-apoptotic activity. In addition, JAK2 V617F mutant-induced phosphorylation of CREB and GSK-3 beta was significantly decreased in cells expressing EpoR Y479F mutant, resulting in the downregulation of Bcl-XL and Mcl-1 expression. Furthermore, compared with when nude mice were inoculated with cells expressing JAK2 V617F mutant and EpoR, the lifespan of nude mice inoculated with cells expressing JAK2 V617F mutant and EpoR Y479F mutant was effectively prolonged. Taken together, it was clarified that PI3K-Akt activation through the phosphorylation of EpoR at Y479 is required for oncogenic signaling of JAK2 V617F mutant and that targeted disruption of this pathway has therapeutic utility. (C) 2011 Elsevier Inc. All rights reserved.
  • Megumi Funakoshi-Tago, Kenji Tago, Yoshinori Sato, Shin-ichi Tominaga, Tadashi Kasahara
    CELLULAR SIGNALLING 23 (2) 363 - 370 0898-6568 2011/02 [Refereed][Not invited]
     
    IL-33, a member of the IL-1 family of cytokines, has been shown to activate NF-kappa B and MAP kinase family through the IL-1 receptor-related protein, ST2L. In this study, we found that IL-33 rapidly activated a tyrosine kinase, JAK2. Interestingly, we demonstrated the functional involvement of JAK2 in IL-33-induced I kappa B alpha degradation and NF-kappa B activation, since a JAK2 inhibitor, AG490, effectively inhibited this signaling pathway. Furthermore, IL-33 failed to induce I kappa B alpha degradation and NF-kappa B activation in JAK2-deficient MEFs expressing ST2L, compared with wild-type MEFs expressing ST2L In addition, the introduction of wild-type JAK2 but not kinase dead JAK2 mutant (K882R) restored the IL-33-induced efficient activation of NF-kappa B in JAK2-deficient MEFs expressing ST2L, resulting in the induction of IL-6, CCL2/MCP-1 and CXCL1/KC expression. On the other hand, the activation of ERK, JNK and p38 was unaffected by JAK2 inhibition and JAK2 deficiency. Thus, these data demonstrate that JAK2 plays an important role in regulating IL-33-induced NF-kappa B activation. (C) 2010 Published by Elsevier Inc.
  • Kenji Tago, Megumi Funakoshi-Tago, Masaki Sakinawa, Norikazu Mizuno, Hiroshi Itoh
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 (40) 30622 - 30633 0021-9258 2010/10 [Refereed][Not invited]
     
    NF-kappa B is an important transcription factor involved in various biological responses, including inflammation, cell differentiation, and tumorigenesis. kappa B-Ras was identified as an I kappa B-interacting small GTPase and is reported to disturb cytokine-induced NF-kappa B activation. In this study, we established that kappa B-Ras is a novel type of nuclear-cytoplasmic small GTPase that mainly binds to GTP, and its localization seemed to be regulated by its GTP/GDP-binding state. Unexpectedly, the GDP-binding form of the kappa B-Ras mutant exhibited a more potent inhibitory effect on NF-kappa B activation, and this inhibitory effect seemed to be due to suppression of the transactivation of a p65/RelA NF-kappa B subunit. kappa B-Ras suppressed phosphorylation at serine 276 on the p65/RelA subunit, resulting in decreased interaction between p65/RelA and the transcriptional coactivator p300. Interestingly, the GDP-bound kappa B-Ras mutant exhibited higher interactive affinity with p65/RelA and inhibited the phosphorylation of p65/RelA more potently than wild-type kappa B-Ras. Taken together, these findings suggest that the GDP-bound form of kappa B-Ras in cytoplasm suppresses NF-kappa B activation by inhibiting its transcriptional activation.
  • Akiyuki Nishimura, Ken Kitano, Jun Takasaki, Masatoshi Taniguchi, Norikazu Mizuno, Kenji Tago, Toshio Hakoshima, Hiroshi Itoh
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 (31) 13666 - 13671 0027-8424 2010/08 [Refereed][Not invited]
     
    Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered G(q)-selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of alpha subunit of G(q) protein (G alpha(q)) by inhibiting the GDP release from G alpha(q). X-ray crystal structure analysis of the G alpha(q)beta gamma-YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the G alpha(q). The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each G alpha subunit and an insight into the molecular mechanism of G protein activation.
  • Yusuke Nagai, Akiyuki Nishimura, Kenji Tago, Norikazu Mizuno, Hiroshi Itoh
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 (15) 11114 - 11120 0021-9258 2010/04 [Refereed][Not invited]
     
    The alpha subunit of stimulatory G protein (G alpha(s)) activates adenylyl cyclase, which catalyzes cAMP production, and regulates many physiological aspects, such as cardiac regulation and endocrine systems. Ric-8B (resistance to inhibitors of cholinesterase 8B) has been identified as the G alpha(s)-binding protein; however, its role in G(s) signaling remains obscure. In this study, we present evidence that Ric-8B specifically and positively regulates G(s) signaling by stabilizing the G alpha(s) protein. An in vitro biochemical study suggested that Ric-8B does not possess guanine nucleotide exchange factor activity. However, knockdown of Ric-8B attenuated beta-adrenergic agonist-induced cAMP accumulation, indicating that Ric-8B positively regulates G(s) signaling. Interestingly, overexpression and knockdown of Ric-8B resulted in an increase and a decrease in the G alpha(s) protein, respectively, without affecting the G alpha(s) mRNA level. We found that the G alpha(s) protein is ubiquitinated and that this ubiquitination is inhibited by Ric-8B. This Ric-8B-mediated inhibition of G alpha(s) ubiquitination requires interaction between Ric-8B and G alpha(s) because Ric-8B splicing variants, which are defective for G alpha(s) binding, failed to inhibit the ubiquitination. Taken together, these results suggest that Ric-8B plays a critical and specific role in the control of G alpha(s) protein levels by modulating G alpha(s) ubiquitination and positively regulates G(s) signaling.
  • Megumi Funakoshi-Tago, Kenji Tago, Miyuki Abe, Yoshiko Sonoda, Tadashi Kasahara
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 (8) 5296 - 5307 0021-9258 2010/02 [Refereed][Not invited]
     
    It has been well established that disruption of JAK2 signaling regulation is involved in various hematopoietic disorders; however, the detailed mechanism by which abnormal activation of JAK2 exhibits transforming activity remains to be elucidated. Here, to clarify the functional role of the erythropoietin receptor (EpoR) and its downstream transcription factor STAT5 in the abnormal activation of JAK2-induced hematopoietic diseases, we generated a stable transfectant of Ba/F3 cells expressing EpoR and analyzed the molecular mechanism of how JAK2 mutation induces cell growth disorder. JAK2 V617F mutant exhibited transforming activity when EpoR was coexpressed. According to a study utilizing several truncated mutants of EpoR, the ability of EpoR to facilitate the transforming activity of JAK2 V617F mutant required the intracellular domain to interact with STAT5. Strikingly, once the truncated EpoR (EpoR-H) was mutated on Tyr-343, the phosphorylation of which is known to be important for interaction with STAT5, JAK2 V617F mutant failed to exhibit transforming activity, suggesting that STAT5 is critical for JAK2 mutant-induced hematopoietic disorder. Furthermore, the expression of the constitutively active STAT5 mutant exhibited transforming activity in Ba/F3 cells, and short hairpin RNA-mediated knockdown of STAT5 significantly inhibited the transforming activity of JAK2 V617F mutant. Taking these observations together, STAT5 plays an essential role in EpoR-JAK2 V617F mutant-induced hematopoietic disorder. Although it remains unclear why the presence of EpoR is required to activate oncogenic signaling via the JAK2 mutant and STAT5, its interacting ability is a target for the treatment of these hematopoietic diseases.
  • Megumi Funakoshi-Tago, Saeko Tanabe, Kenji Tago, Hiroshi Itoh, Tadahiko Mashino, Yoshiko Sonoda, Tadashi Kasahara
    MOLECULAR PHARMACOLOGY 76 (4) 745 - 753 0026-895X 2009/10 [Refereed][Not invited]
     
    Glycyrrhiza inflata has been used as a traditional medicine with anti-inflammatory activity; however, its mechanism has not been fully understood. Licochalcone A is a major and biogenetically characteristic chalcone isolated from G. inflata. Here, we found that licochalcone A strongly inhibited tumor necrosis (TNF)-alpha-induced nuclear localization, DNA binding activity, and the transcriptional activity of nuclear factor-kappa B (NF-kappa B). Whereas licochalcone A had no effect on the recruitment of receptor-interacting protein 1 and I kappa B kinase beta (IKK beta) to TNF receptor I by TNF-alpha, it significantly inhibited TNF-alpha-induced I kappa B kinase complex (IKK) activation and inhibitor of nuclear factor-kappa B degradation. It is interesting that we found that the cysteine residue at position 179 of IKK beta is essential for licochalcone A-induced IKK inhibition, because licochalcone A failed to affect the kinase activity of the IKK beta (C179A) mutant. In contrast, a structurally related compound, echinatin, failed to inhibit TNF-alpha-induced IKK activation and NF-kappa B activation, suggesting that the 1,1-dimethy-2-propenyl group in licochalcone A is important for the inhibition of NF-kappa B. In addition, TNF-alpha-induced expression of inflammatory cytokines CCL2/monocyte chemotactic protein-1 and CXCL1/KC was clearly inhibited by licochalcone A but not echinatin. Taken together, licochalcone A might contribute to the potent anti-inflammatory effect of G. inflata through the inhibition of IKK activation.
  • Miyuki Abe, Megumi Funakoshi-Tago, Kenji Tago, Jun Kamishimoto, Eriko Aizu-Yokota, Yoshiko Sonoda, Tadashi Kasahara
    INTERNATIONAL IMMUNOPHARMACOLOGY 9 (7-8) 870 - 877 1567-5769 2009/07 [Refereed][Not invited]
     
    The somatic Jak2 mutation (V617F) was identified in most patients with polycythemia vera (PV). Here, we show that the activating Jak2 V617F mutant completely protected Ba/F3 cells from cytokine withdrawal-induced apoptotic cell death. Interestingly, Ba/F3 cells expressing Jak2 V617F mutant induced rapid tumorigenesis in nude mice, leading to rapid death. Whereas an injection of Ba/F3 cells expressing wild-type Jak2 had no effect, an injection of Ba/F3 cells expressing Jak2 V617F mutant promptly invaded and spread into various distinct organs, such as the liver and spleen. Strikingly, Jak2 inhibitor, AG490 potently inhibited cytokine-independent cell growth induced by the Jak2 V617F mutant. Also, treatment with AG490 effectively delayed Jak2 V617F mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo suggest that Jak2 harboring V617F mutation is a potent oncogene able to promote cell transformation and tumorigenesis. (C) 2009 Elsevier B.V. All rights reserved.
  • Asuka Nakata, Daisuke Urano, Yoshiaki Fujii-Kuriyama, Norikazu Mizuno, Kenji Tago, Hiroshi Itoh
    EMBO REPORTS 10 (6) 622 - 628 1469-221X 2009/06 [Refereed][Not invited]
     
    Aryl hydrocarbon receptor (AhR) is a transcription factor that works as a dioxin receptor and is also involved in various physiological phenomena, including development and cell proliferation. Here, we show that the G alpha(13) signal destabilizes AhR by promoting the ubiquitination of AhR. G alpha(13) interacts directly with AhR-interacting protein (AIP) and inhibits the interaction between AhR and AIP, a crucial interacting protein of AhR. Strikingly, a reporter gene assay and a quantitative reverse transcription-PCR analysis indicate that the G alpha(13) signal shows a potent inhibitory effect on the ligand-induced transcriptional activation of AhR. G alpha(13) results in the nuclear translocation of AhR in a ligand-independent manner. However, in the presence of active G alpha(13), AhR fails to form the active transcriptional complex. Taken together, we propose a new negative regulation of dioxin signalling by the G protein.
  • Jun-Ichi Furusawa, Megurmi Funakoshi-Tago, Kenji Tago, Tadahiko Mashino, Hideo Inoue, Yoshiko Sonoda, Tadashi Kasahara
    CELLULAR SIGNALLING 21 (5) 778 - 785 0898-6568 2009/05 [Refereed][Not invited]
     
    Licorice root, Glycyrrhiza inflata, has been used as a traditional medicine for the treatment of bronchial asthma and inflammation; however, the mechanism of its anti-inflammatory activity has not been clarified. Here, we investigated the effect of Licochalcone A, a major component of G. inflata, on the LPS signaling pathway. We found that Licochalcone A remarkably inhibited LPS-induced NO production, and TNF alpha expression and MCP-1 expression in both RAW264.7 cells and primary macrophages. Furthermore, when injected with Licochalcone A prior to injection of LPS, the serum level of TNF alpha and MCPA in C57BL/6 mice was clearly decreased, indicating that Licochalcone A has a potent anti-inflammatory effect both in vitro and in vivo. Strikingly, Licochalcone A significantly inhibited LPS-induced NF-kappa B transcriptional activation; however, it had no effect on not only the phosphorylation and degradation of I kappa B alpha but also nuclear translocation and DNA binding activity of NF-kappa B p65. Interestingly, Licochalcone A markedly inhibited the phosphorylation of p65 at serine 276. As a result, it reduced NF-kappa B transactivation by preventing the interaction of p65 with p300. Taken together, Licochalcone A might contribute to the potent anti-inflammatory effect of G. inflata through the unique mechanism of NF-kappa B inhibition. (C) 2009 Elsevier Inc. All rights reserved.
  • Megumi Funakoshi-Tago, Kenji Tago, Kazuya Sumi, Miyuki Abe, Eriko Aizu-Yokota, Tomoyuki Oshio, Yoshiko Sonoda, Tadashi Kasahara
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 (19) 12680 - 12690 0021-9258 2009/05 [Refereed][Not invited]
     
    JAK2 plays important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Recently the L611S point mutation in JAK2 has been identified in a child with acute lymphoblastic leukemia. Here we analyzed the mechanism by which JAK2 exhibits its oncogenicity. In BaF3 murine hematopoietic cells, L611S mutant increased the expression of antiapoptotic proteins including X chromosome-linked inhibitor of apoptosis protein, inhibitor of apoptosis protein, and Bcl-XL. We also showed that JAK2 L611S mutant protects BaF3 cells from cytokine withdrawal-induced apoptotic cell death and leads to cytokine-independent cell growth. Furthermore BaF3 cells expressing JAK2 L611S mutant gained the ability to induce tumorigenesis in nude mice. The L611S mutant also exhibited malignancy, including prompt invasion and spreading into various organs, leading to rapid lethality of the mice. Finally we showed that a specific JAK2 inhibitor, AG490, potently inhibited cytokine-independent cell growth induced by JAK2 L611S mutant via the induction of apoptotic cell death. In addition, treatment with AG490 significantly inhibited the JAK2 L611S mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo strongly suggest that L611S mutant of JAK2 harbors potent oncogenic activity, and this probably requires the antiapoptotic signaling pathway.
  • Jun-ichi Furusawa, Megumi Funakoshi-Tago, Tadahiko Mashino, Kenji Tago, Hideo Inoue, Yoshiko Sonoda, Tadashi Kasahara
    INTERNATIONAL IMMUNOPHARMACOLOGY 9 (4) 499 - 507 1567-5769 2009/04 [Refereed][Not invited]
     
    Licorice root has been used as a traditional medicine for the treatment of gastric ulcer, bronchial asthma and inflammation. Licochalcone A is a major component of Xinjiang licorice, Glycyrrhiza inflata. Previously we showed that Licochalcone A significantly inhibited LPS-induced NF-kappa B transcriptional activation by abrogating the phosphorylation of NF-kappa B p65 at serine 276. Glycyrrhiza inflata contains not only Licochalcone A but also Licochalcone B, Licochalcone C, Licochalcone D, Echinatin and Isoliquiritigenin, harboring the common structure of chalcones. No chalcones had any effect on LPS-induced I kappa B degradation, nuclear translocation and DNA binding activity of NF-kappa B p65; however, we observed that Licochalcone B and Licochalcone D significantly inhibited LPS-induced phosphorylation at serine 276 and transcriptional activation of NF-kappa B, the same as Licochalcone A. Interestingly, we also found that Licochalcone A, Licochalcone B and Licochalcone D effectively inhibited LPS-induced activation of PKA, which is required for the phosphorylation of NF-kappa B p65 at serine 276. Consequently, Licochalcone B and Licochalcone D significantly reduced the LPS-induced production of NO, TNF alpha and MCP-1. On the other hand, Licochalcone C, Echinatin and Isoliquitigenin failed to inhibit LPS-induced NF-kappa B activation. These findings suggest that the anti-inflammatory effect of Glycyrrhiza inflata is ascribable to the potent inhibition of NF-kappa B by Licochalcone A, Licochalcone B and Licochalcone D. (C) 2009 Elsevier B.V. All rights reserved.
  • Megumi Funakoshi-Tago, Noriyuki Kamada, Taeko Shimizu, Yusuke Hashiguchi, Kenji Tago, Yoshiko Sonoda, Tadashi Kasahara
    CYTOKINE 45 (2) 72 - 79 1043-4666 2009/02 [Refereed][Not invited]
     
    TNF receptor-associated factor 6 (TRAF6) is an essential adaptor protein for the Interleukin-1 (IL-1) signaling pathway; however, its role in the signaling of another proinflammatory cytokine, tumor necrosis factor alpha (TNF alpha, has not been explored. Interestingly, we observed that TNF alpha-induced expression of IL-6, CXCL1 and granulocyte macrophage colony stimulating factor (GM-CSF) were significantly enhanced in TRAF6-deficient MEFs. Compared to those observed in wild-type MEFs, TNF alpha-induced I kappa B kinase (IKK) activation and I kappa B alpha degradation were enhanced in TRAF6-deficient MEFs. Also, TNF alpha-induced DNA binding activity and transcriptional activation of nuclear factor kappaB (NF-kappa B) were also augmented in TRAF6-deficient MEFs. On the other hand, TRAF6 deficiency did not affect the TNF alpha-induced activation of mitogen-activated protein (MAP) kinases, ERK, JNK, and p38. Moreover, the reintroduction of exogenous TRAF6 into TRAF6-deficient MEFs clearly suppressed TNF alpha-induced IKK activation, NF-kappa B activation and subsequent cytokine expression. In contrast, both the deletion mutant (Delta N) and the point mutant (C70A) of TRAF6, which is defective in its ubiquitin ligase activity, failed to repress TNF alpha-induced IKK activation, NF-kappa B activation and cytokine production. Thus, these data suggest that TRAF6 negatively regulates TNF alpha-induced NF-kappa B activation through its ubiquitin ligase activity. (C) 2008 Elsevier Ltd. All rights reserved.
  • Megumi Funakoshi-Tago, Kenji Tago, Chiho Nishizawa, Kyoko Takahashi, Tadahiko Mashino, Susumu Iwata, Hideo Inoue, Yoshiko Sonoda, Tadashi Kasahara
    BIOCHEMICAL PHARMACOLOGY 76 (12) 1681 - 1693 0006-2952 2008/12 [Refereed][Not invited]
     
    Aberrant activation of Jak/Stat signaling causes a number of hematopoietic disorders and oncogenesis, and therefore the effective inhibitors of the Jak/Stat signaling pathway may be therapeutically useful. TEL-Jak2 gene fusion, which has been identified in human leukemia, encodes a chimeric protein endowed with constitutive tyrosine kinase activity. Expression of TEL-Jak2 protects Ba/F3 cells from IL-3 withdrawal-induced apoptotic cell death and leads to IL-3-independent growth. However, its mechanisms remain to be only partially understood. Here, we first found that Licochalcone A, one of the flavonoids isolated from the root of Glycyrrhiza inflate, inhibited TEL-Jak2-mediated cell proliferation and survival in the absence of IL-3. Licochalcone A failed to inhibit the activity of TEL-Jak2, however, this induced apoptosis of TEL-Jak2-transformed cells with a much lower concentration in the absence of IL-3 than in the presence of IL-3. Interestingly, Licochalcone A significantly inhibited the phosphorylation and nuclear localization of Stat3, which is essential for TEL-Jak2-induced cell transformation. These data suggest that Licochalcone A is a specific inhibitor for Stat3 and would be employed for the treatment of various diseases caused by disorders of the Jak/Stat pathway. (c) 2008 Elsevier Inc. All rights reserved.
  • Megumi Funakoshi-Tago, Kenji Tago, Tadashi Kasahara, Evan Parganas, James N. Ihle
    CELLULAR SIGNALLING 20 (11) 1995 - 2001 0898-6568 2008/11 [Refereed][Not invited]
     
    Janus kinase 2 (Jak2) has a pivotal role in erythropoietin (Epo) signaling pathway, including erythrocyte differentiation and Stat5 activation. In the course of screening for critical phosphorylation of tyrosine residues in Jak2, we identified tyrosine 913 (Y-913) as a novel and functional phosphorylation site, which negatively regulates Jak2. Phosphorylation at Y-913 rapidly occurred and was sustained for at least 120 min after Epo stimulation, in contrast to the transient phosphorylation of Y-1007/1008 in the activation loop of Jak2. Interestingly, phosphorylation defective mutation of Y-913 ((YF)-F-913) results in a significant enhancement of Epo-induced Jak2 activation, whereas phosphorylation mimic mutation of Y-913 ((YE)-E-913) completely abrogated its activation. Furthermore, Jak2 deficient fetal liver cells expressing (YF)-F-913 mutant generated many mature erythroid BFU-E and CFU-E colonies, while (YE)-E-913 mutant failed to reconstitute Jak2 deficiency. We also demonstrate, in Jak1, phosphorylation of Y-939, a corresponding tyrosine residue with Y-913, negatively regulated Jak1 signaling pathway. Accordingly, our results suggest that this tyrosine phosphorylation in JH1 domain may be involved in common negative regulation mechanism for Jak family. (c) 2008 Elsevier Inc. All rights reserved.
  • Megumi Funakoshi-Tago, Kenji Tago, Morisada Hayakawa, Shin-ichi Tominaga, Tomoyuki Ohshio, Yoshiko Sonoda, Tadashi Kasahara
    CELLULAR SIGNALLING 20 (9) 1679 - 1686 0898-6568 2008/09 [Refereed][Not invited]
     
    IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38,JNK and Nuclear factor-kappa B (NF-kappa B) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappa B in TRAM deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAM and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappa B activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity. (C) 2008 Elsevier Inc. All rights reserved.
  • Megumi Funakoshi-Tago, Taeko Shimizu, Kenji Tago, Motohiro Nakamura, Hiroshi Itoh, Yoshiko Sonoda, Tadashi Kasahara
    BIOCHEMICAL PHARMACOLOGY 76 (5) 662 - 671 0006-2952 2008/09 [Refereed][Not invited]
     
    Celecoxib is a specific inhibitor of cyclooxygenase 2 (COX2). While it has been used for the treatment of chronic inflammatory conditions, including rheumatoid arthritis, its detailed anti-inflammatory mechanism has not been clarified. Here, we found that Celecoxib potently inhibited TNF alpha-induced transcriptional activity and DNA binding activity of NF-kappa B; however, Celecoxib had no effect on TNF alpha-induced IKK activation and degradation of I kappa B alpha and I kappa B beta, suggesting that it inhibited NF-kappa B activation via suppressing downstream of IKK activation and I kappa Bs degradation. Interestingly, it was also found that Celecoxib abrogated TNF alpha-induced nuclear accumulation of the NF-kappa B p65 subunit. As a result, TNF alpha-induced expression of inflammatory cytokines, CXCL1/KC and CCL2/MCP-1, was clearly inhibited by Celecoxib. On the other hand, Celecoxib had no effect on the TNF alpha-induced nuclear translocation of c-jun and activation of ERK, JNK, p38 and Akt. Taken together, these data indicate that Celecoxib specifically inhibits TNF alpha-induced NF-kappa B activation at the level of its nuclear translocation. This negative regulation of NF-kappa B activation by Celecoxib might be an important mechanism leading to its anti-inflammatory activity. (C) 2008 Elsevier Inc. All rights reserved.
  • Daisuke Urano, Asuka Nakata, Norikazu Mizuno, Kenji Tago, Hiroshi Itoh
    CELLULAR SIGNALLING 20 (8) 1545 - 1554 0898-6568 2008/08 [Refereed][Not invited]
     
    Ptdlns(3, 4, 5)P-3-dependent Rac exchanger (P-Rex) 1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac. P-Rex1 is activated by G protein beta gamma subunits (G beta gamma), and the G beta gamma-induced activation is inhibited by cAMP-dependent protein kinase A (PKA). However, the details of regulatory mechanism of P-Rex1 remain to be clarified. In the present study, we investigated the mechanism of activation and inhibition of P-Rex1 using various truncated and alanine-substituted mutants and found that the domain-domain interaction of P-Rex1 is important for G beta gamma-induced activation and PKA-induced inhibition. Immunoprecipitation analysis showed that the second Disheveled/EGL-10/Pleckstrin (DEP) and first PSD-95/Dlg/ZO-1 (PDZ) domains of P-Rex1 associate with the inositol polyphosphate-4-phosphatase (IP4P) domain. Carboxyl-terminal truncation on the IP4P domain or mutations in the protein-binding pocket of the first PDZ domain abolished the association. Analysis of in vitro guanine nucleotide exchange assay, PAK1/2 phosphorylation, and Rac-specific actin reorganization revealed that G beta gamma could activate a complex of the P-Rex1 mutant lacking the IP4P domain and the isolated IP4P domain as well as full-length P-Rex1. Moreover, PKA phosphorylation prevented the domain-domain interaction and G beta gamma-binding. These results provide a new insight into the regulation of other Rho-family GEFs and cell responses induced by the heterotrimeric G protein. (c) 2008 Elsevier Inc. All rights reserved.
  • Tokuichi Iguchi, Kensei Sakata, Kotaro Yoshizaki, Kenji Tago, Norikazu Mizuno, Hiroshi Itoh
    JOURNAL OF BIOLOGICAL CHEMISTRY 283 (21) 14469 - 14478 0021-9258 2008/05 [Refereed][Not invited]
     
    In the developing forebrain, the migration and positioning of neural progenitor cells (NPCs) are regulated coordinately by various molecules. Mutation of these molecules, therefore, causes cortical malformation. GPR56 has been reported as a cortical malformation-related gene that is mutated in patients with bilateral frontoparietal polymicrogyria. GPR56 encodes an orphan G protein-coupled receptor, and its mutations reduce the cell surface expression. It has also been reported that the expression level of GPR56 is involved in cancer cell adhesion and metastasis. However, it remains to be clarified how GPR56 functions in brain development and which signaling pathways are activated by GPR56. In this study, we showed that GPR56 is highly expressed in NPCs and has the ability to inhibit NPC migration. We found that GPR56 coupled with G alpha(12/13) and induced Rho-dependent activation of the transcription mediated through a serum-responsive element and NF-kappa B-responsive element and actin fiber reorganization. The transcriptional activation and actin reorganization were inhibited by an RGS domain of the p115 Rho-specific guanine nucleotide exchange factor (p115 RhoGEF RGS) and dominant negative form of Rho. Moreover, we have demonstrated that a functional anti-GPR56 antibody, which has an agonistic activity, inhibited NPC migration. This inhibition was attenuated by p115 RhoGEF RGS, C3 exoenzyme, and GPR56 knockdown. These results indicate that GPR56 participates in the regulation of NPC movement through the G alpha(12/13) and Rho signaling pathway, suggesting its important role in the development of the central nervous system.
  • Yo Sugawara, Hiroko Nishii, Tomoko Takahashi, Junji Yamauchi, Norikazu Mizuno, Kenji Tago, Hiroshi Itoh
    CELLULAR SIGNALLING 19 (6) 1301 - 1308 0898-6568 2007/06 [Refereed][Not invited]
     
    The heterotrimeric G protein alpha q subunit (G alpha q) mediates a variety of cell functions by activating the effector molecule phospholipase Cl. Gaq activity is regulated by G protein beta gamma subunits, G protein-coupled receptors, RGS proteins, and Ric-8. In this study, we identified the lipid raft resident proteins, flotillin-1/reggie-2 and flotillin-2/reggie-1, as G alpha q-binding proteins. The interactions of G alpha q and flotillins were independent of the nucleotide-binding state of G alpha q, and the N-terminal portion of flotillins was critical for the interaction. A short interfering RNA-mediated knockdown of flotillins, particularly flotillin-2, attenuated the UTP-induced activation of p38 mitogen-activated protein kinase (MAPK) but not that of ERK1/2. The activation of p38 MAPK was inhibited by the Src family tyrosine kinase inhibitor PP2 and the cholesterol-depleting agent methyl-beta-cyclodextrin, which is generally used for the disruption of lipid rafts. In contrast, the activation of ERK1/2 was not inhibited by these compounds. These lines of evidence suggested that a Gq-coupled receptor activates specifically p38 MAPK through lipid rafts and Src kinase activation, in which flotillins positively modulate the Gq signaling. (c) 2007 Elsevier Inc. All rights reserved.
  • W den Besten, ML Kuo, K Tago, RT Williams, CJ Sherr
    ISRAEL MEDICAL ASSOCIATION JOURNAL 8 (4) 249 - 251 1565-1088 2006/04 [Refereed][Not invited]
     
    The Ink4a-Arf locus, which encodes two distinct tumor suppressor proteins, is inactivated in many cancers. Whereas p16(Ink4a) is an inhibitor of cyclin D-dependent kinases, p19(Arf) (p14(ARF) in humans) antagonizes the E3 ubiquitin protein ligase activity of Mdm2 to activate p53. We now recognize that Arf functions in both p53-dependent and -independent modes to counteract hyper-proliferative signals originating from proto-oncogene activation, but its p53-independent activities remain poorly understood. Arf proteins are highly basic (> 20% arginine content, pl > 12) and predominantly localize within nucleoli in physical association with an abundant acidic protein, nucleophosmin (NPM/B23). When bound to NPM, Arf proteins are relatively stable with half-lives of 6-8 hours. Although mouse p19(Arf) contains only a single lysine residue and human p14(ARF) has none, both proteins are N-terminally ubiquitinated and degraded in proteasomes. Through as yet uncharacterized mechanisms, p19(Arf) induces p53-independent sumoylation of a variety of cellular target proteins with which it interacts, including both Mdm2 and NPM. A naturally occurring NPM mutant (NPMc) expressed in myeloid leukemia cells redirects both wild-type NPM and p19(Arf) to the cytoplasm, inhibits Arf-induced sumoylation, and attenuates p53 activity. Thus, ubiquitination and sumoylation can each influence Arf tumor suppressor activity.
  • K Tago, S Chiocca, CJ Sherr
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 (21) 7689 - 7694 0027-8424 2005/05 [Refereed][Not invited]
     
    The mouse p19(Arf) protein has both p53-dependent and p53-independent tumor-suppressive activities. Arf triggers sumoylation of many cellular proteins, including Mdm2 and nucleophosmin (NPM/ B23), with which p19(Arf) physically interacts in vivo, and this occurs equally well in cells expressing or lacking functional p53. In an Arf-null NIH 3T3 cell derivative (MT-Arf cells) engineered to reexpress an Arf transgene driven by a zinc-inducible metallothionein promoter, sumoylation of endogenous Mdm2 and NPM proteins was initiated as p19(Arf) was induced and was observed before p53-dependent cell cycle arrest. Predominately nucleoplasmic molecules visualized by immunofluorescence with antibodies to small ubiquitin-like modifier (SUMO) 1 localized to nucleoli as p19(Arf) accumulated there. Two Arf mutants, one of which binds to Mdm2 and NPM but is excluded from nucleoli and the other of which enters nucleoli but is handicapped in binding to Mdm2 and NPM, were defective in inducing sumoylation of these two target proteins and did not localize bulk sumoylated molecules to nucleoli. The CELO adenovirus protein, Gam1, which inhibits the SUMO activating enzyme (El) and leads to down-regulation of the SUMO conjugating enzyme (E2/Ubc9), had no overt effect on the ability of p19(Arf) to activate p53 or the p53-responsive genes encoding Mdm2 and p21(Cip1), despite the fact that Arf-induced sumoylation of Mdm2 was blocked. Reduction of Ubc9 levels with short hairpin RNAs rendered similar results. We suggest that Arf's p53-independent effects on gene expression and tumor suppression might depend on Arf-induced sumoylation.
  • M Funakoshi-Tago, K Tago, K Andoh, Y Sonoda, S Tominaga, T Kasahara
    JOURNAL OF BIOCHEMISTRY 137 (2) 189 - 197 0021-924X 2005/02 [Refereed][Not invited]
     
    Interleukin-1 (IL-1) mediates numerous host responses through the rapid activation of nuclear factor-kappa B (NF-kappa B), but the signal pathways leading to NF-kappa B activation are regulated at multiple stages. Here, we propose a novel regulatory system for IL-1-induced NF-kappa B activation by a tyrosine kinase, c-Src. The kinase activity of c-Src increases in an IL-1-dependent manner and the ectopic expression of c-Src augments IL-1-induced NF-kappa B activation, suggesting the involvement of c-Src in IL-1 signaling. However, a Src family inhibitor, PP2 failed to inhibit IL-1-induced NF-kappa B activation, and the expression of a c-Src mutant lacking kinase activity (c-Src KD) augmented IL-1-induced NF-kappa B activation as well as wild type c-Src, indicating that the tyrosine kinase activity is not required for IL-1-induced NF-kappa B activation. Furthermore, a physiological interaction between c-Src and I kappa B kinase gamma (IKK gamma) was observed, implying the involvement of c-Src in the IKK-complex. While c-Src augmented IL-1-induced IKK activation independent of its kinase activity, the region comprising amino acids 361-440 in the c-Src kinase domain are required for NF-kappa B activation. The same region of c-Src is also required for IL-1-induced IKK activation and the association with IKK gamma. Taken together, our results suggest that c-Src plays a critical role in IL-1-induced NF-kappa B activation through the IKK complex.
  • C. J. Sherr, D. Bertwistle, W. Den Besten, M. -L. Kuo, M. Sugimoto, K. Tago, R. T. Williams, F. Zindy, M. F. Roussel
    MOLECULAR APPROACHES TO CONTROLLING CANCER 70 129 - 137 0091-7451 2005 [Refereed][Not invited]
     
    The Ink4a-Arf locus encodes two closely wedded tumor suppressor proteins (p16(Ink4a) and p19(Arf)) that inhibit cell proliferation by activating Rb and p53, respectively. With few exceptions, the Arf gene is repressed during mouse embryonic development, thereby helping to limit p53 expression during organogenesis. However, in adult mice, sustained hyperproliferative signals conveyed by somatically activated oncogenes can induce Arf gene expression and trigger a p53 response that eliminates incipient cancer cells. Disruption of this tumor surveillance pathway predisposes to cancer, and inactivation of INK4a-ARF by deletion, silencing, or mutation has been frequently observed in many forms of human cancer. Although it is accepted that much of Arf's tumor-suppressive activity is mediated by p53, more recent genetic evidence has pointed to additional p53-independent functions of Arf, including its ability to inhibit gene expression by a number of other transcription factors. Surprisingly, the enforced expression of Arf in mammalian cells promotes the sumoylation of several Arf-interacting proteins, implying that Arf has an associated catalytic activity. We speculate that transcriptional down-regulation in response to Arf-induced sumoylation may account for Arf's p53-independent functions.
  • Iwahana H, Hayakawa M, Kuroiwa K, Tago K, Yanagisawa K, Noji S, Tominaga S
    Biochimica et biophysica acta 1681 (1) 1 - 14 0006-3002 2004/11 [Refereed][Not invited]
  • H Iwahana, M Hayakawa, K Kuroiwa, K Tago, K Yanagisawa, S Noji, S Tominaga
    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1681 (1) 1 - 14 0167-4781 2004/11 [Refereed][Not invited]
     
    The ST2 gene encodes receptor-like molecules that are very similar to the interleukin-1 receptors. Three types of ST2 gene products, ST2, ST2L, and ST2V, can be produced by alternative splicing. In the course of cloning the chicken ST2 and ST2L cDNAs, we identified a novel variant cDNA other than ST2, ST2L, or ST2V, and tentatively named it ST2LV. ST2LV was produced by alternative splicing that deleted the transmembrane domain of ST2L The chicken ST2 gene consisted of 13 exons and had two promoters followed by noncoding exons 1a and 1b, like the ST2 genes of human, mouse, and rat. The chicken ST2 mRNA was detected by RT-PCR as early as embryonic day 5. After that, the chicken ST2 mRNA was expressed in all examined organs, including the brain, eye, heart, lung, and liver. The chicken ST2LV mRNA was detected from embryonic day 10. The chicken ST2LV cDNA was transiently expressed in COS-7 cells. A protein of 69 kDa was detected in the culture supernatant, and the size of the protein was reduced to 53 kDa by treatment with peptide-N-glycosidase F, which suggested that ST2LV is a new soluble secreted and N-glycosylated variant of the ST2 gene product. (C) 2004 Elsevier B.V. All rights reserved.
  • M Funakoshi-Tago, Y Sonoda, S Tanaka, K Hashimoto, K Tago, S Tominaga, T Kasahara
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 (31) 29359 - 29365 0021-9258 2003/08 [Refereed][Not invited]
     
    Focal adhesion kinase (FAK) is widely involved in important cellular functions such as proliferation, migration, and survival, although its roles in immune and inflammatory responses have yet to be explored. We demonstrate a critical role for FAK in the tumor necrosis factor (TNF)-induced activation of nuclear factor (NF)-kappaB, using FAK-deficient (FAK-/-) embryonic fibroblasts. Interestingly, TNF-induced interleukin (IL)-6 production was nearly abolished in FAK -/- fibroblasts, whereas a normal level of production was obtained in FAK -/- or FAK +/+ fibroblasts. FAK deficiency did not affect the three types of mitogen-activated protein kinases, ERK, JNK, and p38. Similarly, TNF-induced activation of activator protein 1 or NF-IL-6 was not impaired in FAK -/- cells. Of note, TNF-induced NF-kappaB DNA binding activity and activation of IkappaB kinases (IKKs) were markedly impaired in FAK -/- cells, whereas the expression of TNF receptor I or other signaling molecules such as receptor-interacting protein (RIP), tumor necrosis factor receptor-associated factor 2 (TRAF2), IKKalpha, IKKbeta, and IKKgamma was unchanged. Also, TNF-induced association of FAK with RIP and subsequent association of RIP with TRAF2 were not observed, resulting in a failure of RIP to recruit the IKK complex in FAK -/- cells. The reintroduction of wild type FAK into FAK -/- cells restored the interaction of RIP with TRAF2 and the IKK complex and allowed recovery of NF-kappaB activation and subsequent IL-6 production. Thus, we propose a novel role for FAK in the NF-kappaB activation pathway leading to the production of cytokines.
  • M Funakoshi-Tago, K Tago, Y Sonoda, S Tominaga, T Kasahara
    EUROPEAN JOURNAL OF BIOCHEMISTRY 270 (6) 1257 - 1268 0014-2956 2003/03 [Refereed][Not invited]
     
    Interleukin-1 (IL-1) induces multiple genes via activation of transcription factors that include NF-kappaB and activator protein-1 (AP-1). We found that IL-1-mediated c-Src activation was required for AP-1 activation, but not for NF-kappaB activation and also revealed that c-Src-induced AP-1 activation was enhanced synergistically by the coexpression of TNF receptor associated factor 6 (TRAF6). In addition, c-Src interacts with TRAF6 in response to IL-1 and this interaction is required for c-Src activity. However, neither dominant negative mutants of TRAF6 (TRAF6 DN) nor kinase-dead mutant of c-Src (c-Src KD) counteracted each-induced AP-1 activation, suggesting no hierarchy between these two molecules. During the TRAF6 and c-Src-induced AP-1 activation, phosphatidylinositol 3 (PI3)-kinase, its downstream signaling molecule, Akt and c-Jun N-terminal kinase (JNK) were significantly activated and inhibition of these kinase activities down-regulated AP-1 activation through the suppression of c-fos expression. Furthermore, TRAF6 and c-Src-induced JNK activation was significantly inhibited by PI3-kinase inhibitor or a dominant negative mutant of Akt (Akt DN). Taken together, our results demonstrate that c-Src and TRAF6 are key mediators of IL-1-induced AP-1 activation and provide evidence of cross talk between c-Src and TRAF6 molecules through PI3 kinase-Akt-JNK pathways.
  • K Yanagisawa, K Tago, M Hayakawa, M Ohki, H Iwahana, S Tominaga
    BIOCHEMICAL JOURNAL 370 (Pt 1) 159 - 166 0264-6021 2003/02 [Refereed][Not invited]
     
    Interleukin-1 (IL-1)-receptor-associated kinase (IRAK) is an indispensable signalling molecule for host-defence responses initiated by a variety of ligands that bind to members of the Toll/IL-1 receptor family. Here we report a novel splice variant of mouse IRAK-1, IRAK-1-S, which is generated by utilizing a new splicing acceptor site within exon 12. IRAK-1-S cDNA is shorter than the originally reported IRAK-1 (IRAK-1-W)cDNA by 271 nucleotides, and the subsequent frameshift causes a premature termination of translation after 23 amino acids, which are unique to the IRAK-1-S protein. To elucidate the physiological function of IRAK-1-S, we overexpressed it in 293T cells and studied the effects on the IL-1 signalling cascade. As it lacks the C-terminal region of IRAK-1-W that has been reported to contain the TRAF6 (tumour necrosis factor receptor-associated factor 6) binding domain, IRAK-1-S was unable to bind TRAF6 protein, which is a proposed downstream signalling molecule. However, IRAK-I-S overexpressed in 293T cells induced constitutive activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK) independent of stimulation by IL-1, as did IRAK-1-W. To clarify the mechanism of NF-kappaB activation by IRAK-1-S in the absence of binding to TRAF6, we demonstrated that IRAK-1-S binds to IRAK-1-W through its death domain; the findings suggested that overexpressed IRAK-1-S may bind endogenous IRAK-1-W and activate TRAF6 through IRAK-1-W. These results also indicate that this novel variant may play roles in the activation of NF-kappaB and JNK by IL-1 and other ligands whose signal transduction is dependent on IRAK-I under physiological conditions.
  • T Kasahara, M Funakoshi-Tago, Y Sonoda, K Tago, S Tominaga
    PROCEEDINGS OF THE INTERNATIONAL CYTOKINE SOCIETY ANNUAL MEETING 85 - 88 2003 [Refereed][Not invited]
     
    We demonstrated a critical role for FAK in the TNF-induced activation of NF-kappaB, using FAK-deficient (FAK-/-) embryonic fibroblasts. TNF-induced IL-6 production was impaired in FAK-/- cells, compared with FAK+/- or FAK+/+ cells. TNF-induced NF-kappaB activity and activation of IkappaB-kinases (IKKs) were also impaired in FAK-/- cells. In addition, TNF-induced association of FAK with RIP and subsequent association of RIP with TRAF2 were not observed, resulting in a failure of RIP to recruit the IKK complex in. FAK-/- cells. Reintroduction of wild type FAK restored the interaction of RIP with TRAF2 and the IKK complex, and recovered NF-kappaB activation and subsequent IL-6 production. We propose a novel role for FAK in the NF-kappaB activation pathway leading to the production of cytokines.
  • K Tago, M Funakoshi, H Mano, K Yanagisawa, M Hayakawa, K Kuroiwa, H Iwahana, T Kasahara, S Tominaga
    EUROPEAN JOURNAL OF BIOCHEMISTRY 268 (24) 6526 - 6533 0014-2956 2001/12 [Refereed][Not invited]
     
    Interleukin 1 (IL-1) is known to activate the signal transduction machinery, including the transcription factor, nuclear factor kappa B (NF-kappaB). The activation mechanism of NF-kappaB has been studied intensively, while the negative regulatory mechanisms of NF-kappaB remain to be clarified. In the present study, we found that genistein, a tyrosine kinase inhibitor, augmented IL-1 alpha -dependent NF-kappaB activation, suggesting the presence of a tyrosine kinase mediating a suppression signal on NF-kappaB. As determined by luciferase reporter gene assay using kappaB-responsive element, genistein enhanced IL-1 alpha -induced NF-kappaB activation. Although genistein failed to increase luciferase activity at 1 and 3 h after IL-1 alpha stimulation, it induced prolonged activation beginning at 6 h after the initial stimulation. We next examined whether genistein augmented the DNA-binding activity of NF-kappaB, using electrophoretic mobility shift assay. In the case of the control experiment, the binding of NF- kappaB to the kappaB-responsive element peaked at 30 min after IL-1 alpha stimulation, and decreased thereafter. In contrast, treatment with genistein maintained the maximum binding activity for at least 2 h after stimulation. Moreover, genistein enhanced the IL-1 alpha -dependent degradation of I kappaB alpha. Taken together, our results indicate that genistein augments I kappaB degradation, resulting in continuous NF-kappaB activation. This suggests the possibility that tyrosine kinase negatively regulates NF-kappaB.
  • K Tago, T Noda, M Hayakawa, H Iwahana, K Yanagisawa, T Yashiro, S Tominaga
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 285 (5) 1377 - 1383 0006-291X 2001/08 [Refereed][Not invited]
     
    The human ST2 gene has been known to encode three splice variants; namely, a soluble secreted form of ST2, a transmembrane form of ST2L, and ST2V of undetermined localization. Therefore, analysis of tissue distribution and subcellular localization of ST2V is important to elucidate functional relationships among the three splice variants of the human ST2 gene. RT-PCR procedure revealed that ST2V is predominantly expressed in the stomach, small intestine, and colon. Transfection of ST2V cDNA into COS7 cells in the presence of [S-35] methionine and cystein produced radiolabeled 40 kDa protein, which is recognized by specific monoclonal antibody against human ST2. Subcellular fractionation analysis showed that ST2V protein was distributed in the insoluble fraction of the cell lysate. Finally, ST2V protein was detected on the plasma membrane of COS7 cells, which had been transfected with ST2V cDNA, by confocal laser microscopic analysis. These findings taken together, indicate that ST2V protein localizes on the plasma membrane, suggesting its possible role in modification of the ST2L-signaling pathways. (C) 2001 Academic Press.
  • K Oshikawa, K Kuroiwa, K Tago, H Iwahana, K Yanagisawa, S Ohno, SI Tominaga, Y Sugiyama
    AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 164 (2) 277 - 281 1073-449X 2001/07 [Refereed][Not invited]
     
    Previous studies have reported that ST2 is preferentially expressed on Th2 cells and plays a critical part in controlling airway inflammation in murine models of asthma. However, the clinical role of ST2 in patients with bronchial asthma remains unclear. In our study, we examined 56 patients with atopic asthma in a nonattack phase and 200 nonatopic normal volunteers for healthy control, and analyzed the relationship of their serum ST2 levels to asthma severity, pulmonary function, and laboratory data. Of the 56 patients with atopic asthma, 30 exhibited asthmatic exacerbation, and their serum ST2 levels were also analyzed. The serum ST2 levels were low, but a statistical difference was found between patients with nonattack asthma and the healthy control group (p < 0.05). We also found a differential rise of serum ST2 level that correlates well with the severity of asthma exacerbation. Furthermore, the serum ST2 levels during asthma exacerbation statistically correlated with the percentage of predicted peak expiratory flow (r = -0.634, p = 0.004) and Pa-CO2 (r = 0.516, p = 0.003). These results suggest that soluble human ST2 protein in sera may be related to Th2-mediated allergic inflammation inducing acute exacerbation in patients with atopic asthma.
  • M Funakoshi, K Tago, Y Sonoda, S Tominaga, T Kasahara
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 283 (1) 248 - 254 0006-291X 2001/04 [Refereed][Not invited]
     
    Interleukin-1 (IL-1) mediates numerous host responses through rapid activation of nuclear factor-kappaB (NF-kappaB), but signal pathways leading to the NF-kappaB activation appear to be complicated and multiplex. We propose a novel regulatory system for NF-kappaB activation by the extracellular signal-related kinase (ERK) pathway. In a human glioblastoma cell line, T98G, IL-1-induced NF-kappaB activation was significantly augmented by the pretreatment of a specific MEK inhibitor, PD98059. In contrast, ectopic expression of a constitutive activated form of Raf (v-Raf) reduced IL-1-induced NF-kappaB activation, and this inhibition was completely reversed by PD98059. Interestingly, PD98059 sustained IL-1-induced NF-kappaB DNA binding activity by an eletrophoretic mobility shift assay and also I kappaB alpha degradation presumably by augmenting and sustaining the proteasome activation. Concomitantly, two NF-kappaB dependent genes, A20 and I kappaB alpha expression were prolonged with PD98059. These data suggested that MEK-ERK pathway exerts a regulatory effect on NF-kappaB activation, providing a novel insight on the role of MEK-ERK pathway. (C) 2001 Academic Press.
  • M Funakoshi, Y Sonoda, K Tago, S Tominaga, T Kasahara
    INTERNATIONAL IMMUNOPHARMACOLOGY 1 (3) 595 - 604 1567-5769 2001/03 [Refereed][Not invited]
     
    Interleukin-1 (IL-1) is a central regulator of the immune acid inflammatory responses by which various inflammatory genes are induced. Although IL-1 signaling is known to involve PI3-kinase. p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK), the crosstalk of their kinases on the IL-1-mediated signal transduction is not clear. We used two specific inhibitors. SB203580 which selectively inhibits p38 MAP kinase and LY294002 which inhibits PI3-kinase. respectively, to explore the involvement of these kinases in the IL-1-induced NF-kappaB activation, using a human glioblastoma cell line, T98G. Two kinase inhibitors decreased IL-1-induced IL-8 mRNA and protein levels markedly. IL-1 caused phosphorylation of p38 MAP kinase with concomitant recruitment of PIS-kinase to IL-1 receptor I (IL-IRI) and its activation. In this content, pretreatment of LY294002, but not SB203580. inhibited IL-1-induced NF-kappaB activation significantly. While IL-1 induced-AP-1 activation was moderate. both LY294002 and S8103580 suppressed IL-1-induced AP-1 activation. These observations were prominent particularly in the TRAF6 transfection system, in which overexpression of wild type TRAF6 augmented the IL-1 mediated NF-kappaB and AP-1 activation, while dominant negative TRAF6 construct (Delta TRAF6) suppressed these activation. Namely. LY294002, inhibited TRAF6-mediated IL-1-induced NF-kappaB and AP-1 activation markedly. while SB203580 inhibited TRAF6-induced AP-1 activation but not NF-kappaB activation. Above results indicated that both PI3-kinase and p38 MAP kinase: are differentially involved in IL-1-induced NF-kappaB and AP-1 activation. (C) 2001 Elsevier Science B.V. All rights reserved.
  • HJ Li, K Tago, KC Lo, K Kuroiwa, T Arai, H Iwahana, S Tominaga, K Yanagisawa
    GENOMICS 67 (3) 284 - 290 0888-7543 2000/08 [Refereed][Not invited]
     
    The ST2 gene is a member of the IL-1 receptor family and is hypothesized to be involved in helper T cell function, but its functional ligand and physiological pole remain unknown. We have cloned the human ST2L cDNA that encodes a distinct type of membrane-bound ST2 protein. The predicted 556-amino-acid sequence showed 67% identity to the mouse STSL protein. The human ST2 gene (IL1RL1) contains 13 exons and spans 40 kb in length. Its exon-intron organization was elucidated from a registered human genomic sequence derived from chromosome 2q, which contains three other genes belonging to the IL-1 receptor family in an approximately 202-kb genomic region. The tissue distribution of ST2 expression was examined by RT-PCR, and the soluble form (ST2, IL1RL1-a) and STSL (IL1RL1-b) appear to be expressed differentially. We also established stable transfectants of a human glioblastoma cell line, T98G, that express human STSL constitutively, and we confirmed cell-surface expression of human STSL protein on the transfectants. (C) 2000 Academic Press.
  • K Kuroiwa, HJ Li, K Tago, H Iwahana, K Yanagisawa, N Komatsu, K Oshikawa, Y Sugiyama, T Arai, SI Tominaga
    HYBRIDOMA 19 (2) 151 - 159 0272-457X 2000/04 [Refereed][Not invited]
     
    The human ST2 gene can be specifically induced by growth stimulation in fibroblastic cells, and can also be induced by antigen stimulation in Th2 cells. The gene encodes a soluble secreted protein, ST2, and a transmembrane protein, ST2L, which are closely related to the interleukin-l receptor. To gain insight into the biological roles of the ST2 gene, three monoclonal antibodies (MAbs) against human ST2 gene products were obtained. To obtain these antibodies, immunization was carried out using two different immunogens: purified soluble human ST2 protein (hST2), and COS7 cells, which express the extracellular portion of human ST2L, 2A5 and FB9 MAbs were derived from the immunization with soluble hST2, and HB12 was derived from the COS7 cell immunization. All three antibodies were shown to detect native forms of the human ST2 gene products by immunoprecipitation, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). In the competitive ELISA using biotinylated and nonlabelled MAbs, neither FB9 nor HB12 affected the binding of 2A5 to ST2 gene products. Based on this result, we constructed a sandwich ELISA system using 2A5 and FB9 to measure the concentration of soluble hST2 in sera. The ELISA, combined with the flow cytometry using these antibodies, will be a useful tool for elucidating the functions of human ST2 gene products in individuals.
  • H Uchida, H Tanaka, Y Kitoh, K Yanagisawa, H To, K Tago, K Mizuta, A Fujimura, S Tominaga, K Hashizume, E Kobayashi
    TRANSPLANTATION PROCEEDINGS 32 (2) 255 - 256 0041-1345 2000/03 [Refereed][Not invited]
  • H Ohto, S Kamada, K Tago, S Tominaga, H Ozaki, S Sato, K Kawakami
    MOLECULAR AND CELLULAR BIOLOGY 19 (10) 6815 - 6824 0270-7306 1999/10 [Refereed][Not invited]
     
    Drosophila sine oculis and eyes absent genes synergize in compound-eye formation, The murine homologues of these genes, Six and Eya, respectively, show overlapping expression patterns during development. We hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites, A clear synergistic activation of these promoters was observed in certain combinations of Six and Eya, To investigate the molecular basis for the cooperation, we first examined the intracellular distribution of Six and Eya proteins in transfected COS7 cells, Coexpression of Six2, Six4, or Six5 induced nuclear translocation of Eya1, Eya2, and Eya3, which were otherwise distributed in the cytoplasm, In contrast, coexpression of Six3 did not result in nuclear localization of any Eya proteins. Six and Eya proteins were coimmunoprecipitated from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, were necessary and sufficient for the nuclear translocation of Eya, In contrast, the Eya domain, a conserved domain among Eya proteins, was not sufficient for the translocation, A specific interaction between the Six domain and homeodomain of Six4 and Eya2 was observed by yeast two-hybrid analysis. Our results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes.
  • S Tominaga, K Kuroiwa, K Tago, H Iwahana, K Yanagisawa, N Komatsu
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 264 (1) 14 - 18 0006-291X 1999/10 [Refereed][Not invited]
     
    A novel variant cDNA from the human ST2 gene other than ST2 or ST2L was identified and tentatively named ST2V. Alternative splicing inserts a new exon which leads to a change in the C-terminal portion of ST2, causing it to gain a hydrophobic tail instead of losing the third immunoglobulin-like domain. ST2V is expressed in human leukemic cell line UT-7 and its sublines UT-7/GM, UT-7/EPO, and UT-7/TPO, in addition to human helper T cell line 5C10. The amount of ST2V mRNA is greatly diminished when UT-7/GM cells are induced to differentiate into either erythroblastic or megakaryoblastic phenotypes. The possible roles of the ST2V in growth and differentiation are intriguing. (C) 1999 Academic Press.
  • H Iwahana, K Yanagisawa, A Ito-Kosaka, K Kuroiwa, K Tago, N Komatsu, R Katashima, M Itakura, S Tominaga
    EUROPEAN JOURNAL OF BIOCHEMISTRY 264 (2) 397 - 406 0014-2956 1999/09 [Refereed][Not invited]
     
    The ST2 gene encodes receptor-like molecules that are very similar to the type I interleukin-1 receptor. Two distinct types of the ST2 gene products, ST2 (a soluble secreted form) and ST2L (a transmembrane form) are produced by alternative splicing. Here we demonstrate that the human ST2 gene has two alternative promoters followed by distinct noncoding first exons, which are located more than 8 kb apart and are spliced to the common exon 2 containing the translation initiation site. Within 1001 bp upstream of the transcription initiation site of the cloned distal promoter, there are four GATA-1. The main promoter used for the expression of the ST2 gene in UT-7, a human leukaemic cell line, is distinct from that in TM12, a human fibroblastic cell line. Although UT-7 cells use both distal and proximal promoters, the distal promoter is used dominantly for expression of both ST2 and ST2L mRNA. On the other hand, almost all transcription in TM12 cells starts from the proximal promoter. These results contrast with those of former studies on the rat system, in which ST2 and ST2L mRNA were generated by use of the proximal and distal promoters, respectively. Furthermore, UT-7 cells use multiple transcription initiation sites in both the proximal and distal promoters, whereas the transcription of the ST2 gene in TM12 cells starts at a unique site. Intriguingly, these results suggest that ST2 and ST2L proteins have distinct functions in different cells within different biological systems, such as those of growth control, differentiation and immunological responses.
  • K Tago, Y Kaziro, T Satoh
    JOURNAL OF BIOCHEMISTRY 123 (4) 659 - 667 0021-924X 1998/04 [Refereed][Not invited]
     
    Stimulation of the interleukin (IL)-3 receptor provokes rapid activation of the Ras pathway in various hematopoietic cell lines, Also, a wide range of G-protein-coupled receptors induce Ras activation following ligand stimulation, In this report, we investigate the mechanism underlying Ras activation upon stimulation of these two types of receptors in hematopoietic cells, Thrombin, a G-protein-coupled receptor ligand, was found to stimulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) in IL-3-dependent BaF3 cells, suggesting a significant function of thrombin receptor-mediated signaling, We show that the Ras-guanine nucleotide exchange factor mSos is indispensable for activation of the Ras pathway in IL-3- or thrombin-stimulated BaF3 cells, The activation of Ras in response to IL-3 as defined by accumulation of the GTP-bound form was impaired by conditional overexpression of a dominant-negative mutant of mSos (Delta mSos1), Furthermore, following induction of Delta mSos1, IL-3 enhancement of the kinase activities of c-Raf-1, ERK2, and JNK1 downstream of Ras was almost completely blocked, Similarly, thrombin-induced Ras-dependent ERK2 activation was diminished by Delta mSos1, However, the tyrosine phosphorylation pattern of cellular substrates upon thrombin stimulation was entirely different from the pattern of IL-3-induced tyrosine phosphorylation, Collectively, these results provide evidence that mSos plays a crucial role in both IL-3 and thrombin activation of the Ras pathway in hematopoietic cells, although molecules (including tyrosine kinases) mediating the signal to mSos are likely to be different between the two types of receptors.
  • K Takahashi, K Tago, H Okano, Y Ohya, T Katada, Y Kanaho
    JOURNAL OF IMMUNOLOGY 156 (3) 1229 - 1234 0022-1767 1996/02 [Refereed][Not invited]
     
    Activation of the membrane-bound phospholipase D (PLD) requires cytosolic factor(s), and ADP-ribosylation factor (ARF) has been identified as a cytosolic PLD activator. In the present study, we demonstrate that calmodulin (CaM) and ARF are both involved in PLD activation in rabbit peritoneal neutrophils. The PLD activity of streptolysin O-permeabilized, cytosol-depleted rabbit neutrophils was significantly enhanced when the permeabilized cells were reconstituted with bovine brain cytosol in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), whereas there was little activation of the enzyme in the absence of cytosol. The GTP gamma S-stimulated PLD activity in the presence of cytosol was augmented on increasing the concentration of free Ca2+. The PLD activity stimulated by GTP gamma S and Ca2+ in this system was inhibited by the calmodulin inhibitor W-7. These findings suggest that CaM plays a role as a cytosolic PLD activator. Moreover, highly purified CaM alone, as well as partially purified ARF alone, promoted a slight stimulation of the PLD activity in permeabilized neutrophils. Interestingly, ARF-stimulated PLD activity was augmented by CaM in the presence of GTP gamma S and Ca2+. This augmentation was again inhibited by W-7, as well as by the structurally unrelated CaM inhibitor trifluoperazine. These data imply that CaM stimulates the PLD activity of rabbit neutrophils in concert with ARF.
  • H KURIBARA, K TAGO, T YOKOZEKI, T SASAKI, Y TAKAI, N MORII, S NARUMIYA, T KATADA, Y KANAHO
    JOURNAL OF BIOLOGICAL CHEMISTRY 270 (43) 25667 - 25671 0021-9258 1995/10 [Refereed][Not invited]
     
    An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), was partially purified from bovine brain cytosol by a series of chromatographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 exoenzyme ADP-ribosyltransferase, which strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially purified PLD-activating factor with both C3 exoenzyme and NAD significantly inhibited the PLD-stimulating activity. These results suggest that rhoA p21 is, at least in part, responsible for the PLD-stimulating activity in the preparation. Recombinant isoprenylated rhoA p21 expressed in and purified from Sf9 cells activated rat brain PLD in a concentration- and GTP gamma S (guanosine 5'-O-(3-thiotriphosphate))-dependent manner. In contrast, recombinant non-isoprenylated rhoA pal (fused to glutathione S-transferase) expressed in Escherichia coli failed to activate the PLD. This difference cannot be explained by a lower affinity of non-isoprenylated rhoA p21 for GTP gamma S, as the rates of [S-35]GTP gamma S binding were very similar for both recombinant preparations and the GTP gamma S-bound form of non-isoprenylated rhoA p21 did not induce PLD activation. Interestingly, recombinant isoprenylated rhoA p21 and ARF synergistically activated rat brain PLD; a similar pattern was seen with the partially purified PLD-activating factor. The synergistic activation was inhibited by C3 exoenzyme-catalyzed ADP-ribosylation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner. Inhibition correlated with the extent of ADP-ribosylation. These findings suggest that rhoA pal regulates rat brain PLD in concert with ARF, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.

MISC

  • ST2Lの新規結合タンパク質IFITM3はST2Lのリソソーム分解を介してIL-33シグナルを抑制する
    多胡 憲治, 多胡 めぐみ, 太田 聡, 大村 千尋, 松儀 実広, 富永 眞一, 柳澤 健  日本生化学会大会プログラム・講演要旨集  92回-  [1T07a  -02]  2019/09  [Not refereed][Not invited]
  • 核小体に局在するNPM-ALKの機能解析
    内原 脩貴, 多胡 めぐみ, 多胡 憲治, 田村 悦臣  日本生化学会大会プログラム・講演要旨集  92回-  [3T18a  -01]  2019/09  [Not refereed][Not invited]
  • NPM-ALK発現細胞におけるメトトレキサートによるアポトーシス誘導機構
    内原 脩貴, 小森 麗子, 大藏 晃, 多胡 めぐみ, 多胡 憲治, 田村 悦臣  日本薬学会年会要旨集  139年会-  (3)  51  -51  2019/03  [Not refereed][Not invited]
  • MethotrexateによるBCR-ABL陽性白血病細胞のアポトーシス誘導機構の解析
    伊勢田 昂成, 迫 ゆうか, 内原 脩貴, 多胡 憲治, 多胡 めぐみ, 田村 悦臣  日本薬学会年会要旨集  139年会-  (3)  128  -128  2019/03  [Not refereed][Not invited]
  • MethotrexateによるJAK-STAT経路の阻害機構の解析
    大藏 晃, 内原 脩貴, 多胡 憲治, 多胡 めぐみ, 田村 悦臣  日本薬学会年会要旨集  139年会-  (3)  128  -128  2019/03  [Not refereed][Not invited]
  • 抗炎症作用を示すコーヒー含有成分の同定
    豊嶋 直樹, 松高 茉里, 古旗 賢二, 多胡 憲治, 多胡 めぐみ, 田村 悦臣  日本薬学会年会要旨集  139年会-  (3)  216  -216  2019/03  [Not refereed][Not invited]
  • がん化型Ras変異体はp38-MSK1/2経路を介してNF-κB活性化を増強する
    多胡 憲治, 多胡 めぐみ, 太田 聡, 河田 浩敏, 堀江 久永, 齊藤 博司, 山内 淳司, 田中 亨, 松儀 実広, 柳澤 健  日本生化学会大会プログラム・講演要旨集  91回-  [3T12a  -05(3P  2018/09  [Not refereed][Not invited]
  • 核小体に局在するNPM-ALKの結合分子の同定
    内原 脩貴, 多胡 めぐみ, 多胡 憲治, 田村 悦臣  日本生化学会大会プログラム・講演要旨集  91回-  [3T12a  -09(3P  2018/09  [Not refereed][Not invited]
  • Ras変異体が誘導する発がんシグナルにおける受容体型チロシンキナーゼMer(MerTK)の機能解析
    太田 聡, 多胡 憲治, 松儀 実広, 柳澤 健  生命科学系学会合同年次大会  2017年度-  [2P  -0448]  2017/12  [Not refereed][Not invited]
  • NPM-ALKの細胞内局在制御機構の解析
    内原 脩貴, 多胡 めぐみ, 多胡 憲治, 田村 悦臣  生命科学系学会合同年次大会  2017年度-  [2P  -0460]  2017/12  [Not refereed][Not invited]
  • KRasタンパク質のC末端側における新規の点変異は特異な生化学的特性と細胞がん化能を示す
    多胡 憲治, 多胡 めぐみ, 太田 聡, 大村 千尋, 松儀 実広, 柳澤 健  生命科学系学会合同年次大会  2017年度-  [4LT19  -02(3P  2017/12  [Not refereed][Not invited]
  • 【TSLPとIL-33に関する新知見】 細胞のがん化におけるIL-33前駆体の新しい機能とその分子機構に関する新たな知見
    太田 聡, 多胡 憲治, 柳澤 健  臨床免疫・アレルギー科  67-  (3)  275  -281  2017/03  [Not refereed][Not invited]
  • がん化型Ras変異体はNF-κBの転写活性化を促進する
    多胡 憲治, 多胡 めぐみ, 太田 聡, 河田 浩敏, 堀江 久永, 山内 淳司, 田中 亨, 松儀 実広, 柳澤 健  日本生化学会大会プログラム・講演要旨集  89回-  [2T10  -07(2P  2016/09  [Not refereed][Not invited]
  • 慢性骨髄増殖性腫瘍に対するメトトレキサートの新しい作用機序
    内原 脩貴, 四元 佑樹, 上田 史仁, 多胡 憲治, 多胡 めぐみ, 田村 悦臣  日本生化学会大会プログラム・講演要旨集  89回-  [2P  -241]  2016/09  [Not refereed][Not invited]
  • 多胡 憲治, 多胡 めぐみ  生化学  88-  (2)  207  -210  2016/04  [Not refereed][Not invited]
  • ParvifloronEによるTNFαシグナル伝達経路の抑制機構の解明
    小室 友紀, 野村 さやか, 多胡 憲治, 成川 佑次, 木内 文之, 多胡 めぐみ, 田村 悦臣  日本薬学会年会要旨集  136年会-  (3)  125  -125  2016/03  [Not refereed][Not invited]
  • コーヒー豆抽出液が示す抗炎症作用の検討
    谷尾 真由美, 野中 勇佑, 武田 峰佳, 八木 利恭, 古旗 賢二, 多胡 憲治, 多胡 めぐみ, 田村 悦臣  日本薬学会年会要旨集  136年会-  (3)  125  -125  2016/03  [Not refereed][Not invited]
  • メトトレキサートによるJAK2V617F変異体発現細胞のアポトーシス誘導機構
    四元 佑樹, 内原 脩貴, 上田 史仁, 多胡 憲治, 多胡 めぐみ, 田村 悦臣  日本薬学会年会要旨集  136年会-  (3)  131  -131  2016/03  [Not refereed][Not invited]
  • IL-33前駆体は、がん化型Ras変異体が誘導する形質転換とサイクリンD1のタンパク質合成に必須の役割を担う
    太田 聡, 多胡 憲治, 多胡 めぐみ, 松儀 実広, 柳澤 健  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  88回・38回-  [1P0110]  -[1P0110]  2015/12  [Not refereed][Not invited]
  • Gαsユビキチン修飾を制御する分子機構の解析
    鯉森 貴行, 西村 基喜, 竹田 浩之, 多胡 憲治, 小林 哲夫, 澤崎 達也, 伊東 広  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  88回・38回-  [2T21  -03(2P0180)]  2015/12  [Not refereed][Not invited]
  • 新規IL-33シグナル調節蛋白質IFITM3の同定
    多胡 憲治, 多胡 めぐみ, 太田 聡, 松儀 実広, 柳澤 健  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  88回・38回-  [2T21p  -04(2P0198)]  2015/12  [Not refereed][Not invited]
  • 慢性骨髄増殖性腫瘍の原因遺伝子産物JAK2V617F変異体が誘導する形質転換におけるDDX5の役割
    安積 尊, 上田 史仁, 多胡 憲治, 柳澤 健, 多胡 めぐみ, 田村 悦臣  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  88回・38回-  [3P0187]  -[3P0187]  2015/12  [Not refereed][Not invited]
  • 脂肪細胞分化におけるRNA helicase DDX5の役割
    近藤 駿介, 野間 瞭太, 上田 史仁, 多胡 めぐみ, 多胡 憲治, 柳澤 健, 中澤 洋介, 田村 悦臣  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  88回・38回-  [3P0192]  -[3P0192]  2015/12  [Not refereed][Not invited]
  • Rho特異的活性化因子PLEKHG2のGαsとの相互作用による制御機構
    杉山 和恵, 多胡 憲治, 武藤 吉徳, 長瀬 隆弘, 上田 浩  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  88回・38回-  [3P0197]  -[3P0197]  2015/12  [Not refereed][Not invited]
  • がん化型Ras変異体はNF-κBの過剰な活性化を引き起こす
    多胡 憲治, 多胡 めぐみ, 太田 聡, 松儀 実広, 柳澤 健  日本生化学会大会プログラム・講演要旨集  87回-  [4T14a  -14]  2014/10  [Not refereed][Not invited]
  • 脂肪細胞分化におけるRNA helicase DDX5の役割
    野間 瞭太, 多胡 めぐみ, 多胡 憲治, 柳沢 健, 田村 悦臣  日本生化学会大会プログラム・講演要旨集  87回-  [4P  -306]  2014/10  [Not refereed][Not invited]
  • 心筋細胞のGタンパク質シグナルにおけるRic-8Bの機能(Involvement of Ric-8B in Gs suppression by Gq signal in cardiac myocytes)
    Jenie Riris I, 仲矢 道雄, 水野 憲一, 多胡 憲治, 黒瀬 等, 伊東 広  日本生化学会大会プログラム・講演要旨集  86回-  1T19p  -13  2013/09  [Not refereed][Not invited]
  • がん化型Ras変異体が誘起する発がんシグナルはTNFαによるNF-κBの活性化を増強する(Oncogenic signal caused by Ras(G12V) augments the TNFα-induced activation of NF-κB)
    多胡 憲治, 多胡 めぐみ, 太田 聡, 松儀 実広, 柳澤 健  日本生化学会大会プログラム・講演要旨集  86回-  3T13p  -11  2013/09  [Not refereed][Not invited]
  • 慢性骨髄増殖性腫瘍由来JAK2変異体のがん化シグナルにおけるc-Myc-ODC経路の役割
    多胡 めぐみ, 鷲見 和也, 多胡 憲治, 笠原 忠  日本生化学会大会プログラム・講演要旨集  85回-  3P  -586  2012/12  [Not refereed][Not invited]
  • 低分子量G蛋白質κB-RasはRas(G12V)による発がんシグナルに関与する
    多胡 憲治, 多胡 めぐみ, 柳澤 健, 杉山 直之, 冨田 勝, 水野 憲一, 伊東 広  日本生化学会大会プログラム・講演要旨集  85回-  3P  -614  2012/12  [Not refereed][Not invited]
  • T. Kasahara, M. Funakoshi-Tago, K. Tago  CYTOKINE  59-  (3)  539  -539  2012/09  [Not refereed][Not invited]
  • 太田聡, 多胡憲治, 多胡めぐみ, 松儀実広, 柳澤健  日本分子生物学会年会プログラム・要旨集(Web)  35th-  3P-0461 (WEB ONLY)  2012  [Not refereed][Not invited]
  • 微小管結合タンパク質doublecortinのPKAによるリン酸化を介した新規アクチン骨格制御
    鳥山 真奈美, 猪口 徳一, 深見 岳史, 多胡 憲治, 水野 憲一, 伊東 広  日本生化学会大会プログラム・講演要旨集  84回-  4P  -0342  2011/09  [Not refereed][Not invited]
  • Gタンパク質シグナルにより制御されるSUMO化とその分子機構の解析
    多胡 憲治, 多胡 めぐみ, Chiocca Susanna, 水野 憲一, 伊東 広  日本生化学会大会プログラム・講演要旨集  84回-  4T10p  -7  2011/09  [Not refereed][Not invited]
  • IL-33/ST2シグナル伝達系におけるJAK2の役割について
    笠原 忠, 多胡 めぐみ, 上四元 純, 村上 はるか, 多胡 憲治  日本薬学会年会要旨集  131年会-  (3)  156  -156  2011/03  [Not refereed][Not invited]
  • M. Toriyama, N. Mizuno, T. Iguchi, M. Toriyama, T. Fukami, K. Tago, H. Itoh  MOLECULAR BIOLOGY OF THE CELL  22-  2011  [Not refereed][Not invited]
  • H. Nakazawa, T. Sada, M. Toriyama, K. Tago, M. Fukuda, N. Inagaki  MOLECULAR BIOLOGY OF THE CELL  22-  2011  [Not refereed][Not invited]
  • 大脳皮質形成における神経前駆細胞移動のGタンパク質シグナルによる多重制御機構
    水野 憲一, 鳥山 真奈美, 多胡 憲治, 伊東 広  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  83回・33回-  1T9  -3  2010/12  [Not refereed][Not invited]
  • 三量体Gタンパク質Gαsのユビキチン化はRic-8Bとの結合により抑制される
    永井 裕介, 西村 明幸, 多胡 憲治, 水野 憲一, 伊東 広  日本生化学会大会プログラム・講演要旨集  82回-  4T4a  -8  2009/09  [Not refereed][Not invited]
  • Gタンパク質シグナルによるdoublecortinのリン酸化と細胞遊走の解析
    吉田 真奈美, 水野 憲一, 多胡 憲治, 伊東 広  日本生化学会大会プログラム・講演要旨集  82回-  2P  -690  2009/09  [Not refereed][Not invited]
  • 猪口 徳一, 坂田 賢成, 吉崎 幸太郎, 多胡 憲治, 水野 憲一, 伊東 広  解剖学雑誌  84-  (2)  55  -55  2009/06  [Not refereed][Not invited]
  • 活性酸素産生を司るRac-GEFであるP-Rex1のPKAによる抑制機構
    浦野 大輔, 中田 飛鳥, 水野 憲一, 多胡 憲治, 伊東 広  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  81回・31回-  1T14  -3  2008/11  [Not refereed][Not invited]
  • TNF-αシグナル伝達におけるcelecoxibの作用について
    清水 妙子, 多胡 めぐみ, 園田 よし子, 中村 元洋, 多胡 憲治, 伊東 広, 笠原 忠  日本薬学会年会要旨集  128年会-  (3)  81  -81  2008/03  [Not refereed][Not invited]
  • Licochalcone AのTNFα誘導性NF-κBの活性化に及ぼす影響
    田辺 沙詠子, 多胡 めぐみ, 園田 よし子, 笠原 忠, 岩田 進, 中村 元洋, 多胡 憲治, 伊東 広  日本薬学会年会要旨集  128年会-  (3)  83  -83  2008/03  [Not refereed][Not invited]
  • 永野孝典, 猪口徳一, 多胡憲治, 水野憲一, 伊東広  生化学  2P-1086  2008  [Not refereed][Not invited]
  • 非定型低分子量GTPase κB-Rasの動的細胞内局在と発癌シグナルへの関与可能性(Dynamic subcellular localization of atypical small GTPase kappaB-Ras and its possible contribution to oncogenic signals)
    多胡 憲治, 多胡 めぐみ, 水野 憲一, 笠原 忠, 富永 眞一, 伊東 広  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  80回・30回-  3T15  -4  2007/11  [Not refereed][Not invited]
  • GPR56はG12/13とRhoを介して神経前駆細胞の遊走を制御する(GPR56 regulates neural progenitor cell migration through G12/13 and Rho)
    猪口 徳一, 坂田 賢成, 吉崎 幸太郎, 多胡 憲治, 水野 憲一, 伊東 広  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  80回・30回-  3T15  -10  2007/11  [Not refereed][Not invited]
  • IL-1シグナル伝達経路におけるc-Srcの役割
    多胡 めぐみ, 多胡 憲治, 園田 よし子, 富永 眞一, 笠原 忠  生化学  74-  (8)  947  -947  2002/08  [Not refereed][Not invited]
  • K Yanagisawa, K Tago, S Tominaga  FASEB JOURNAL  16-  (5)  A1179  -A1180  2002/03  [Not refereed][Not invited]
  • IL-1受容体関連ST2遺伝子産物の細胞内情報伝達系の解析
    多胡 憲治  自治医科大学紀要  24-  124  -124  2001/12  [Not refereed][Not invited]
  • IRAK-1の選択的スプライシングバリアントの発現と機能
    柳沢 健, 多胡 憲治, 早川 盛禎, 岩花 弘之, 富永 眞一  生化学  73-  (8)  731  -731  2001/08  [Not refereed][Not invited]
  • マウスT細胞におけるST2遺伝子の発現調節
    早川 盛禎, 柳沢 健, 多胡 憲治, 岩花 弘之, 富永 眞一  生化学  73-  (8)  706  -706  2001/08  [Not refereed][Not invited]
  • 大腸癌由来細胞株におけるST2遺伝子の発現とその制御
    多胡 憲治, 早川 盛禎, 柳澤 健, 岩花 弘之, 富永 眞一  生化学  73-  (8)  707  -707  2001/08  [Not refereed][Not invited]
  • NF-κB抑制因子κB-Ras2の細胞内局在の解析
    船越 めぐみ, 多胡 憲治, 園田 よし子, 富永 眞一, 笠原 忠  生化学  73-  (8)  865  -865  2001/08  [Not refereed][Not invited]
  • ニワトリST2遺伝子のゲノム構造とその発現について
    岩花 弘之, 早川 盛禎, 多胡 憲治, 柳沢 健, 富永 眞一  生化学  73-  (8)  1056  -1056  2001/08  [Not refereed][Not invited]
  • T Kasahara, M Funakoshi, Y Sonoda, K Tago  JOURNAL OF LEUKOCYTE BIOLOGY  34  -34  2001  [Not refereed][Not invited]
  • NF-κB活性調節機構におけるMAPキナーゼの役割
    船越 めぐみ, 多胡 憲治, 園田 よし子, 富永 眞一, 笠原 忠  生化学  72-  (8)  1030  -1030  2000/08  [Not refereed][Not invited]
  • ST2の新規splicing variant,ST2Vは細胞膜表面に局在する
    多胡 憲治, 黒岩 憲二, 李 紅杰, 柳沢 健, 岩花 弘之, 富永 眞一  生化学  72-  (8)  1030  -1030  2000/08  [Not refereed][Not invited]
  • ST2LV:ST2遺伝子産物の新しいバリアントについて
    岩花 弘之, 早川 盛禎, 柳沢 健, 多胡 憲治, 黒岩 憲二, 芳賀 靖, 増沢 紀男, 野地 澄晴, 冨永 眞一  生化学  72-  (8)  1031  -1031  2000/08  [Not refereed][Not invited]
  • H Iwahana, K Yanagisawa, K Kuroiwa, K Tago, K Haga, T Masuzawa, S Noji, S Tominaga  FASEB JOURNAL  14-  (6)  A1081  -A1081  2000/04  [Not refereed][Not invited]
  • 増殖刺激時のBALB/c-3T3細胞における血管内皮細胞増殖因子のprimary responseによる発現
    柳沢 健, 黒岩 憲二, 多胡 憲治, 岩花 弘之, 内田 広夫, 富永 眞一  自治医科大学紀要  22-  87  -94  1999/12  [Not refereed][Not invited]
     
    マウス線維芽細胞BALB/c-3T3を用い,VEGFの発現について検討を行った.VEGFのmRNAは対数増殖期のBALB/c-3T3細胞では発現されているが,細胞がG0期に導入されるとmRNAの発現量は減少した.又,G0期の細胞に血清による増殖刺激を与えると,VEGFのmRNAの発現が強く誘導され,この誘導はサイクロヘキシミド存在下でも観察された.このことよりVEGF mRNAの血清刺激による発現は,他のタンパク質合成を介さない,primary responseの様式によることが判明した.VEGFのタンパク質レベルについても検討を行うと,血清刺激後,mRNAの変化と平行して分泌の増加することが明らかとなった
  • 黒岩憲二, 新井孝夫, 小松則夫, LI H, 柳沢健, 岩花弘之, 多胡憲治, 富永真一  日本分子生物学会年会プログラム・講演要旨集  22nd-  687  1999/11  [Not refereed][Not invited]
  • ニワトリST2遺伝子の胎生期における発現
    岩花 弘之, 柳沢 健, 多胡 憲治, 黒岩 憲二, 芳賀 靖, 増沢 紀男, 野地 澄晴, 富永 眞一  生化学  71-  (8)  942  -942  1999/08  [Not refereed][Not invited]
  • SixとEyaとの協同作用が標的遺伝子の活性化に必要である
    大戸 裕美, 鎌田 さやか, 多胡 憲治, 富永 真一, 尾崎 秀徳, 佐藤 滋, 川上 潔  生化学  71-  (8)  949  -949  1999/08  [Not refereed][Not invited]
  • ヒト膜在型ST2 cDNAのクローニングと組織における発現
    李 紅杰, 柳沢 健, 黒岩 憲二, 多胡 憲治, 岩花 弘之, 新井 孝夫, 富永 眞一  生化学  71-  (8)  1058  -1058  1999/08  [Not refereed][Not invited]
  • IL-1刺激によるヒトグリオーマ細胞株T98GでのIL-8産生誘導におけるTRAF6の関与について
    船越 めぐみ, 多胡 憲治, 富永 真一, 園田 よし子, 笠原 忠  生化学  71-  (8)  1073  -1073  1999/08  [Not refereed][Not invited]
  • マウス線維芽細胞BALB/c-3T3におけるVEGFの発現様式
    柳沢 健, 多胡 憲治, 岩花 弘之, 富永 眞一  生化学  70-  (8)  977  -977  1998/08  [Not refereed][Not invited]
  • IL-3情報伝達系におけるSosタンパク質の機能の解析
    多胡 憲治  生化学  68-  (7)  828  -828  1996/07  [Not refereed][Not invited]
  • スタウロスポリンとマストパランの三量体G蛋白質への作用様式の比較
    多胡 憲治  生化学  65-  (8)  833  -833  1993/08  [Not refereed][Not invited]

Awards & Honors

  • 2012/01 公益財団法人武田科学振興財団 ライフサイエンス研究奨励助成
     発がんシグナルと細胞死を制御する新規GTP結合蛋白質の機能解析 
    受賞者: 多胡憲治

Research Grants & Projects

  • Analysis of nuclear signaling complexes of novel Ras family and their activities to induce cellular transformation
    Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2017/04 -2020/03 
    Author : 多胡 憲治
     
    これまで、低分子量Gタンパク質Rasが誘導する細胞増殖シグナル、発がんシグナルにおいて、NKiRasが関与することを見出し、その分子機構について解析を行って来た。特に相互作用分子としてTRB3、DDB1、NONO、SFPQが同定された。このうち、特にTRB3は新規のがん抑制遺伝子産物として機能し、一方でDDB1は形質転換に対して促進的に機能することも明らかになっている。2018年度は、がん化型Ras変異体が誘導する各種シグナル伝達系に着目し、NKiRasの関与について検討した。NKiRasのノックダウンはRas (G12V)による細胞形質転換を抑制するが、ERKおよびJNKなどのシグナルには影響を及ぼさなかった。mTORC1の活性化はNKiRasのノックダウンにより抑制されたが、その分子機構は現在まで明らかになっていない。一方で、NKiRasに依存したRasシグナル標的遺伝子として、細胞の1次繊毛や中心体に局在し、細胞増殖などに深く関与するCEP290、尿素輸送体Slc14A1が見出されている。リポーター遺伝子などを用いた解析を計画しているが、現在まで新規の知見は得られていない。一方で、古典的Rasのシグナル伝達系についても並行して解析を進めた。その結果、新規のがん化型変異体A146TがERK、Aktに依存しない新規の発がんシグナルを活性化している可能性が見出された。またRas (G12V)変異体はNF-kappaBの活性を増強する新しいメカニズムを見出した。大腸がんの病変部にてp65/RelAサブユニットのSer276のリン酸化が亢進して居ることを見出した。このSer276のリン酸化はRasにより活性化されたp38-MSK1/2経路によるものであることが明らかになった。
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2014/04 -2017/03 
    Author : Tago Kenji
     
    It is well established that genetic mutations of Ras genes are involved in many cases of tumorigenesis. In this study, we found that a member of Ras family, kappaB-Ras contributes to the cellular transformation mediated the activation of protein kinase mTORC1. Furthermore, we purified the protein complexes including kappaB-Ras, and found that TRB3 which is identified as an interacting protein of kappaB-Ras functions as novel type of tumor suppressor. Although detailed mechanism is still unclear, it is clarified that TRB3 suppresses the oncogenic signals mediated the SUMO-conjugation of kappaB-Ras.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2014/04 -2017/03 
    Author : Yanagisawa Ken
     
    We investigated the mechanism of inhibitory effect of soluble ST2 protein, which is the extracellular domain of IL-33 receptor (ST2L protein), against the LPS signal transduction. ST2 protein did not inhibit the binding of LPS with LPS receptor. Each component of LPS receptor complex, TLR 4, CD14 and MD2, did not show the binding with ST2 protein, and each combination of two components of above also failed to bind with ST2, which suggests higher structure of LPS receptor complex is required for the binding with ST2. Furthermore, We found novel proteins such as IFITM3 which bind with ST2L protein, and IL-33 is indispensable for the cellular transformation by Ras independent of ST2 protein, and IL-33 promotes the translation of cyclin D.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2013/04 -2016/03 
    Author : Tominaga Shin-ichi, TAGO KENJI
     
    The ST2 gene was originally identified as a gene induced during the initiation of cell proliferation. Since the products had a sequence very similar to the interleukin 1 receptor and IL-33 was found as a ligand inducing inflammatory responses, the major concern has been immunological aspect. In the present study, coming back to the original findings, we investigated the roles of IL-33 and ST2 in cell proliferation of NIH-3T3 fibroblastic cell line. IL-33 had dual functions, namely, a suppressive effect on the growth initiation of quiescent cells, and a growth-promoting effect on cycling cells. On the other hand, contrary to the initial hypothesis, soluble ST2 had a growth-promoting effect. The relationship between IL-33 and ST2 in terms of growth regulation remains to be elucidated in future.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2011 -2013 
    Author : TAGO Kenji
     
    kappaB-Ras is a nuclear-cytoplasmic GTPase that mainly present as GTP-bound form with no stimulating conditions, and its cellular localization is regulated by its bound guanine nucleotides. Here, we found that kappaB-Ras is essential for Ras (G12V)-induced tumorigenesis, although kappaB-Ras itself lacks the oncogenic activities. To clarify the mechanism, by which kappaB-Ras is involved in Ras (G12V)-caused tumorigenesis, we purified the protein complexes including kappaB-Ras2, and identified novel interacting proteins of kappaB-Ras, such as TRB3 and DDB1. TRB3 exhibited the tumor suppressive activity against Ras (G12V). Furthermore, TRB3 induced SUMOylation of kappaB-Ras, and this seems to cause the inhibition of Ras (G12V)-induced transformation. These observations suggest that kappaB-Ras harbors the critical roles in tumorigenesis induced by oncogenic Ras, and this is most likely to be regulated by novel tumor suppressor TRB3 through the SUMOylation of kappaB-Ras.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2009 -2011 
    Author : ITOH Hiroshi, MIZINO Norikazu, TAGO Kenji
     
    Adhesion G protein-coupled receptors(GPCRs) have a long N-terminal extracellular domain and a seven-transmembrane domain. Most of adhesion GPCRs are orphan receptors, and the activation and signal transduction mechanisms remain to be clarified. GPR56 and Latrophilin1 belong to adhesion GPCRs. We succeed in preparing the monoclonal antibody against human GPR56, which inhibits the glioma cell migration, and have constructed several mutants. Analysis using mutants showed that an extracellular domain of Latrophilin1 has an ability to inhibit the activation of Latrophilin1 transmembarane domain as well as the GPR56 transmembrane activation.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2008 -2009 
    Author : TAGO Kenji
     
    We found that κB-Ras is a novel type of nuclear-cytoplasmic small GTPase that mainly binds to GTP, and its localization seemed to be regulated by its GTP/GDP-binding state. Unexpectedly, the GDP-binding form of κB-Ras mutant exhibited a more potent inhibitory effect on NF-κB activation, and this inhibitory effect seemed to be due to suppression of the transactivation of a p65/RelA NF-κB subunit.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas
    Date (from‐to) : 2005 -2009 
    Author : ITOH Hiroshi, MIZUNO Norikazu, TAGO Kenji
     
    In this study, we found that orphan G protein-coupled receptor, GPR56, negatively regulates neural progenitor cell migration via G12/13 and Rho pathway, and the antibody which recognizes the orphan receptor is an useful tool for analyzing the function of the receptor. Moreover, structural basis for the specific inhibition of G proteins by YM-254890 has been revealed by biochemical and X-ray crystal structure analysis. Ubiquitination of the stimulatory G protein ・ subunit and its inhibition by Ric-8B was demonstrated. Crosstalk between G protein signaling and dioxin signaling was found by identifying AIP as a novel G protein interacting protein.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2007 -2008 
    Author : ITOH Horoshi, MIZINO Norikazu, TAGO Kenji
     
    Gタンパク質共役受容体(G protein-coupled receptor, GPCR)の中には、そのリガンドが不明のオーファン受容体が未だ数多く存在する. Adhesion GPCR に属するGPR56もオーファン受容体であり、ヒト大脳皮質形成および細胞のがん化に関与することが示唆されているが、その機能やシグナル伝達はほとんど不明であり、その解析を行った. その結果、アゴニスト様に働く抗GPR56抗体の作成に成功し、その抗体を用いてGPR56がGタンパク質のG12/13からRhoを介する経路を活性化して、神経前駆細胞の遊走に対して抑制的に働くことを明らかとした.


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