Manabu Negishi

Kyoto University, Kioto, Kyōto, Japan

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Publications (244)974.85 Total impact

  • Shingo Takeuchi · Hironori Katoh · Manabu Negishi
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    ABSTRACT: Eph/ephrin signaling plays essential roles in various tissue developments, such as axon guidance, angiogenesis and tissue separation. Interaction between Ephs and ephrins upon cell-cell contact results in forward (toward Eph-expressing cells) and reverse (toward ephrin-expressing cells) signaling. Although the molecular mechanisms downstream of Eph/ephrin forward signaling have been extensively studied, the functions and intracellular molecular mechanisms of Eph/ephrin reverse signaling are not fully understood. Rho GTPases are key regulators of the actin cytoskeleton to regulate cell morphology. In this study, we revealed that stimulation with the extracellular domain of EphB2 to activate Eph/ephrin reverse signaling induced axonal retraction in hippocampal neurons. The reduction of axonal length and branching by Eph/ephrin reverse signaling was blocked by inhibition of RhoA or ROCK. These results suggest that Eph/ephrin reverse signaling negatively regulates axonal outgrowth and branching through RhoA/ROCK pathway in hippocampal neurons. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
    No preview · Article · Apr 2015 · Journal of Biochemistry
  • Kohei Harada · Manabu Negishi · Hironori Katoh
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    ABSTRACT: Expression of EphA2 is up-regulated in various cancers derived from epithelial cells and correlates with the ability of a cancer cell to undergo migration and invasion. Here we have investigated the role of EphA2 in epithelial morphogenesis of MDCK cells in three-dimensional culture. We show that EphA2 is phosphorylated on serine 897 by hepatocyte growth factor (HGF) stimulation via a phosphatidylinositol 3-kinase /Akt-dependent mechanism, and that this phosphorylation is required for extension formation, the first step of tubulogenesis, in MDCK cysts. On the other hand, ligand ephrinA1 stimulation dephosphorylates EphA2 on serine 897 and suppresses the HGF-induced morphological change. Furthermore, activation of small GTPase RhoG is involved in the HGF-induced formation of extensions downstream of EphA2. These observations suggest that ligand-independent activity of EphA2 contributes to epithelial morphogenesis. © 2015. Published by The Company of Biologists Ltd.
    No preview · Article · Apr 2015 · Journal of Cell Science
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    Kentaro Umeda · Nariaki Iwasawa · Manabu Negishi · Izumi Oinuma
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    ABSTRACT: Precise wiring patterns of axons are one of the remarkable features of neuronal circuit formation, and establishment of the proper neuronal network requires control of outgrowth, branching, and guidance of axons. R-Ras is a Ras family small GTPase that has essential roles in multiple phases of axonal development. We recently identified afadin, an F-actin-binding protein, as an effector of R-Ras mediating axon branching through F-actin reorganization. Afadin comprises two isoforms, l-afadin having F-actin-binding domain and s-afadin lacking the F-actin-binding domain. Compared with l-afadin, s-afadin, the short splicing variant of l-afadin, contains RA domains but lacks the F-actin-binding domain. Neurons express both isoforms, however the function of s-afadin in brain remains unknown. Here we identify s-afadin as an endogenous inhibitor of cortical axon branching. In contrast to the abundant and constant expression of l-afadin throughout neuronal development, the expression of s-afadin is relatively low when cortical axons branch actively. Ectopic expression and knockdown of s-afadin suppresses and promotes branching, respectively. s-Afadin blocks the R-Ras-mediated membrane translocation of l-afadin and axon branching through inhibiting the binding of l-afadin to R-Ras. Thus, s-afadin acts as a dominant-negative isoform in R-Ras-afadin-regulated axon branching. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Mar 2015 · Molecular biology of the cell
  • Manabu Negishi · Izumi Oinuma
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    ABSTRACT: Semaphorins, which are a large family of secreted and membrane-bound molecules, were initially identified as neuronal axon-guidance signaling molecules but are now known as important key regulators for cell adhesion and motility in a wide range of organ systems, such as angiogenesis and immune response. The semaphorin receptors, neuropilins and plexins, are expressed in a variety of cell types, including neurons, endothelial cells, and cancer cells. Plexins are primarily receptors responsible for intracellular semaphorin signalings. Plexins possess an intrinsic GAP (glyceraldehyde-3-phosphate) activity for R-Ras subfamily GTPases, and this GAP activity is one of the crucial signals of semaphorins. In addition, plexins associate with a variety of signaling molecules, such as Rho GEFs and Rho GAP, and these associated molecules determine the characters of semaphorin signals. On the other hand, their signalings are critically modulated by their associated co-receptor molecules, including tyrosine kinase receptors. Semaphorins provide attractive and repulsive responses in a variety of cells, but associated co-receptors of plexins frequently hold the key to conversion between attraction and repulsion. In this chapter, we focus attention on the molecular signaling systems of plexins.
    No preview · Article · Jan 2015
  • Mai Akada · Kohei Harada · Manabu Negishi · Hironori Katoh
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    ABSTRACT: Anoikis is a specific type of apoptosis induced by detachment of epithelial cells from extracellular matrix, and acquiring resistance to anoikis is an important step that enables cancer cells to metastasize. EphA2, which is overexpressed in a variety of human cancers, is phosphorylated by Akt on serine 897 and mediates ligand ephrin-independent promotion of anoikis resistance through the RhoG activator Ephexin4. EphB6 is frequently silenced in invasive and metastatic cancers; however, its role in cancer progression is poorly understood. Here we show that EphB6 interacts with EphA2 and suppresses EphA2-mediated promotion of anoikis resistance in MCF7 breast cancer cells. On the other hand, knockdown of EphB6 promotes anoikis resistance. We further show that expression of EphB6 decreases serine 897 phosphorylation of EphA2 and suppresses EphA2-Ephexin4 interaction and the RhoG activation. These findings implicate EphB6 as a negative regulator of EphA2 oncogenic signaling.
    No preview · Article · Sep 2014 · Cellular Signalling
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    ABSTRACT: Dock4 is a member of the Dock180 family proteins that mediates cancer cell migration through activation of Rac. However, the regulatory mechanism of Dock4 remains unclear. In this study, we show that the C-terminal proline-rich region of Dock4 is essential for the Dock4 mediated promotion of cell migration in MDA-MB-231 breast cancer cells. We found that a phoshoinositide-binding protein SH3YL1 interacted with the C-terminal proline-rich region of Dock4. Interaction of SH3YL1 with Dock4 promoted Dock4-mediated Rac1 activation and cell migration. Mutations in the phoshoinositide-binding domain disrupted the ability of SH3YL1 to promote Dock4-mediated cell migration. In addition, depletion of SH3YL1 in MDA-MB-231 cells suppressed cell migration. Taken together, these results provide evidence for a novel and functionally important interaction between Dock4 and SH3YL1 to promote cancer cell migration by regulating Rac1 activity.
    No preview · Article · May 2014 · Cellular Signalling
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    Shuhei Ueda · Manabu Negishi · Hironori Katoh
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    ABSTRACT: In neuronal development, dendritic spine formation is important for the establishment of excitatory synaptic connectivity and functional neural circuit. Developmental deficiency of spine formation results in multiple mental diseases. Dock4, a guanine nucleotide exchange factor (GEF) for Rac, has been recently reported as a candidate genetic risk factor of autism, dyslexia, and schizophrenia. We previously revealed that Dock4 is expressed in hippocampal neurons. However, the functions of Dock4 in hippocampal neurons and the underlying molecular mechanisms remain poorly understood. Here we show that Dock4 is highly concentrated in dendritic spines and implicated in spine formation through interaction with actin-binding protein cortactin. In cultured neurons, short hairpin RNA (shRNA)-mediated knockdown of Dock4 reduced the dendritic spine density, which was rescued by coexpression of shRNA-resistant wild-type Dock4 but not by a GEF-deficient mutant of Dock4 or a truncated mutant lacking the cortactin-binding region. On the other hand, knockdown of cortactin suppressed Dock4-mediated spine formation. Taken together, our studies revealed a novel and functionally important interaction between Dock4 and cortactin to regulate dendritic spine formation through activation of Rac.
    Preview · Article · Mar 2013 · Molecular biology of the cell
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    ABSTRACT: EphA2 is activated through phosphorylation on serine 897 (S897) by Akt to promote cancer cell motility and invasion, independently of stimulation by ephrin, its ligand. Here we show that S897 phosphorylation of EphA2 strengthens the interaction between EphA2 and Ephexin4, a guanine nucleotide exchange factor for the small GTPase RhoG. S897A mutation of EphA2 abolished the EphA2/Ephexin4-mediated RhoG activation, promotion of cell migration, and resistance to anoikis. Our results suggest that S897-phosphorylated EphA2 recruits Ephexin4 to promote cell migration and anoikis resistance, providing a molecular link between S897 phosphorylation of EphA2 and tumor progression.
    Preview · Article · Jan 2013 · FEBS Open Bio
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    Full-text · Dataset · Jan 2013
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    Gen-Ichi Tasaka · Manabu Negishi · Izumi Oinuma
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    ABSTRACT: Semaphorins have been identified as repulsive guidance molecules in the developing nervous system. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 induces repulsion in axon and dendrites by functioning as a GTPase-activating protein (GAP) for R-Ras and M-Ras, respectively. In axons, Sema4D stimulation induces growth cone collapse, and downregulation of R-Ras activity by Plexin-B1-mediated GAP activity is required for the action. Axonal R-Ras GAP activity downregulates phosphatidylinositol 3-kinase signaling pathway, and thereby induces inactivation of a microtubule assembly promoter protein, CRMP-2. However, in contrast to the well studied roles of semaphorins and plexins in axonal guidance, signaling molecules linking M-Ras GAP to dendritic cytoskeleton remain obscure. Here we identified an Ena/VASP ligand, Lamellipodin (Lpd), as a novel effector of M-Ras in dendrites. Lpd was expressed in F-actin-rich distal dendritic processes and was required for both basal and M-Ras-mediated dendrite development. Subcellular fractionation showed M-Ras-dependent membrane translocation of Lpd, which was suppressed by Sema4D. Furthermore, the Ena/VASP-binding region within Lpd was required for dendrite development, and its membrane targeting was sufficient to overcome the Sema4D-mediated reduction of dendritic outgrowth and disappearance of F-actin from distal dendrites. Furthermore, in utero electroporation experiments also indicated that regulation of the M-Ras-Lpd system by the GAP activity of Plexin is involved in the normal development of cortical dendrites in vivo. Overall, our study sheds light on how repulsive guidance molecules regulate actin cytoskeleton in dendrites, revealing a novel mechanism that the M-Ras-Lpd system regulates actin-based dendrite remodeling by Sema/Plexin in rats or mice of either sex.
    Full-text · Article · Jun 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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    Nariaki Iwasawa · Manabu Negishi · Izumi Oinuma
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    ABSTRACT: Regulation of axon growth, guidance, and branching is essential for constructing a correct neuronal network. R-Ras, a Ras-family small GTPase, has essential roles in axon formation and guidance. During axon formation, R-Ras activates a series of phosphatidylinositol 3-kinase signaling, inducing activation of a microtubule-assembly promoter-collapsin response mediator protein-2. However, signaling molecules linking R-Ras to actin cytoskeleton-regulating axonal morphology remain obscure. Here we identify afadin, an actin-binding protein harboring Ras association (RA) domains, as an effector of R-Ras inducing axon branching through F-actin reorganization. We observe endogenous interaction of afadin with R-Ras in cortical neurons during the stage of axonal development. Ectopic expression of afadin increases axon branch number, and the RA domains and the carboxyl-terminal F-actin binding domain are required for this action. RNA interference knockdown experiments reveal that knockdown of endogenous afadin suppressed both basal and R-Ras-mediated axon branching in cultured cortical neurons. Subcellular localization analysis shows that active R-Ras-induced translocation of afadin and its RA domains is responsible for afadin localizing to the membrane and inducing neurite development in Neuro2a cells. Overall, our findings demonstrate a novel signaling pathway downstream of R-Ras that controls axon branching.
    Full-text · Article · May 2012 · Molecular biology of the cell
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    Izumi Oinuma · Kana Kawada · Kiyoka Tsukagoshi · Manabu Negishi
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    ABSTRACT: The Rnd proteins Rnd1, Rnd2, and Rnd3/RhoE are well known as key regulators of the actin cytoskeleton in various cell types, but they comprise a distinct subgroup of the Rho family in that they are GTP bound and constitutively active. Functional differences of the Rnd proteins in RhoA inhibition signaling have been reported in various cell types. Rnd1 and Rnd3 antagonize RhoA signaling by activating p190 RhoGAP, whereas Rnd2 does not. However, all the members of the Rnd family have been reported to bind directly to p190 RhoGAP and equally induce activation of p190 RhoGAP in vitro, and there is no evidence that accounts for the functional difference of the Rnd proteins in RhoA inhibition signaling. Here we report the role of the N-terminal region in signaling. Rnd1 and Rnd3, but not Rnd2, have a KERRA (Lys-Glu-Arg-Arg-Ala) sequence of amino acids in their N-terminus, which functions as the lipid raft-targeting determinant. The sequence mediates the lipid raft targeting of p190 RhoGAP correlated with its activation. Overall, our results demonstrate a novel regulatory mechanism by which differential membrane targeting governs activities of Rnd proteins to function as RhoA antagonists.
    Full-text · Article · Feb 2012 · Molecular biology of the cell
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    ABSTRACT: Bradykinin (BK) plays a major role in producing peripheral sensitization in response to peripheral inflammation and in pain transmission in the central nerve system (CNS). Because BK activates protein kinase C (PKC) through phospholipase C (PLC)-β and myristoylated alanine-rich C kinase substrate (MARCKS) has been found to be a substrate of PKC, we explored the possibility that BK could induce MARCKS phosphorylation and regulate its function. BK stimulation induced transient MARCKS phosphorylation on Ser159 with a peak at 1  min in human neuroblastoma SH-SY5Y cells. By contrast, PKC activation by the phorbol ester phorbol 12,13-dibutyrate (PDBu) elicited MARCKS phosphorylation which lasted more than 10  min. Western blotting analyses and glutathione S-transferase (GST) pull-down analyses showed that the phosphorylation by BK was the result of activation of the PKC-dependent RhoA/Rho-associated coiled-coil kinase (ROCK) pathway. Protein phosphatase (PP) 2A inhibitors calyculin A and fostriecin inhibited the dephosphorylation of MARCKS after BK-induced phosphorylation. Moreover, immunoprecipitation analyses showed that PP2A interacts with MARCKS. These results indicated that PP2A is the dominant PP of MARCKS after BK stimulation. We established SH-SY5Y cell lines expressing wild-type MARCKS and unphosphorylatable MARCKS, and cell morphology changes after cell stimulation were studied. PDBu induced lamellipodia formation on the neuroblastoma cell line SH-SY5Y and the morphology was sustained, whereas BK induced neurite outgrowth of the cells via lamellipodia-like actin accumulation that depended on transient MARCKS phosphorylation. Thus these findings show a novel BK signal cascade-that is, BK promotes neurite outgrowth through transient MARCKS phosphorylation involving the PKC-dependent RhoA/ROCK pathway and PP2A in a neuroblastoma cell line.
    No preview · Article · Feb 2012 · Journal of Cellular Physiology

  • No preview · Article · Sep 2011 · Neuroscience Research
  • Genichi Tasaka · Izumi Oinuma · Manabu Negishi

    No preview · Article · Sep 2011 · Neuroscience Research
  • Shuhei Ueda · Manabu Negishi · Hironori Katoh

    No preview · Article · Sep 2011 · Neuroscience Research
  • Nariaki Iwasawa · Izumi Oinuma · Manabu Negishi

    No preview · Article · Sep 2011 · Neuroscience Research
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    ABSTRACT: Pombe Cdc15 homology proteins, characterized by Fer/CIP4 homology Bin-Amphiphysin-Rvs/extended Fer/CIP4 homology (F-BAR/EFC) domains with membrane invaginating property, play critical roles in a variety of membrane reorganization processes. Among them, Rapostlin/formin-binding protein 17 (FBP17) has attracted increasing attention as a critical coordinator of endocytosis. Here we found that Rapostlin was expressed in the developing rat brain, including the hippocampus, in late developmental stages when accelerated dendritic spine formation and maturation occur. In primary cultured rat hippocampal neurons, knockdown of Rapostlin by shRNA or overexpression of Rapostlin-QQ, an F-BAR domain mutant of Rapostlin that has no ability to induce membrane invagination, led to a significant decrease in spine density. Expression of shRNA-resistant wild-type Rapostlin effectively restored spine density in Rapostlin knockdown neurons, whereas expression of Rapostlin deletion mutants lacking the protein kinase C-related kinase homology region 1 (HR1) or Src homology 3 (SH3) domain did not. In addition, knockdown of Rapostlin or overexpression of Rapostlin-QQ reduced the uptake of transferrin in hippocampal neurons. Knockdown of Rnd2, which binds to the HR1 domain of Rapostlin, also reduced spine density and the transferrin uptake. These results suggest that Rapostlin and Rnd2 cooperatively regulate spine density. Indeed, Rnd2 enhanced the Rapostlin-induced tubular membrane invagination. We conclude that the F-BAR protein Rapostlin, whose activity is regulated by Rnd2, plays a key role in spine formation through the regulation of membrane dynamics.
    Preview · Article · Jul 2011 · Journal of Biological Chemistry
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    ABSTRACT: Disruption of cell-extracellular matrix interaction causes epithelial cells to undergo apoptosis called anoikis, and resistance to anoikis has been suggested to be a critical step for cancer cells to metastasize. EphA2 is frequently overexpressed in a variety of human cancers, and recent studies have found that overexpression of EphA2 contributes to malignant cellular behavior, including resistance to anoikis, in several different types of cancer cells. Here we show that Ephexin4, a guanine nucleotide exchange factor for the small GTPase RhoG that interacts with EphA2, plays an important role in the regulation of anoikis. Knockdown of Ephexin4 promoted anoikis in HeLa cells, and experiments using a knockdown-rescue approach showed that activation of RhoG, phosphatidylinositol 3-kinase (PI3K), and Akt was required for the Ephexin4-mediated suppression of anoikis. Indeed, Ephexin4 knockdown caused a decrease in RhoG activity and Akt phosphorylation in HeLa cells cultured in suspension. In addition, Ephexin4 was involved in the EphA2-mediated suppression of anoikis. Taken together, these results suggest that Ephexin4 mediates resistance to anoikis through activation of RhoG and PI3K downstream of EphA2.
    No preview · Article · Jul 2011 · Experimental Cell Research
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    Izumi Oinuma · Yuri Ito · Hironori Katoh · Manabu Negishi
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    ABSTRACT: Plexins are receptors for axonal guidance molecules semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin-B1, suppresses PI3K signaling through the R-Ras GTPase-activating protein (GAP) activity, inducing growth cone collapse. Phosphatidylinositol 3-phosphate level is critically regulated by PI3K and PTEN (phosphatase and tensin homologue deleted chromosome ten). Here we examined the involvement of PTEN in the Plexin-B1-induced repulsive response. Phosphorylation of PTEN at Ser-380 is known to suppress its phosphatase activity. Sema4D induced the dephosphorylation of PTEN at Ser-380 and stimulated PTEN phosphatase activity in hippocampal neurons. Knockdown of endogenous PTEN suppressed the Sema4D-induced growth cone collapse. Phosphorylation mimic PTEN mutant suppressed the Sema4D-induced growth cone collapse, whereas phosphorylation-resistant PTEN mutant by itself induced growth cone collapse. Plexin-B1-induced PTEN dephosphorylation through R-Ras GAP activity and R-Ras GAP activity was by itself sufficient for PTEN dephosphorylation and activation. We also suggested that the Sema4D-induced PTEN dephosphorylation and growth cone collapse were mediated by the inhibition of casein kinase 2 alpha activity. Thus, we propose that Sema4D/Plexin-B1 promotes the dephosphorylation and activation of PTEN through the R-Ras GAP activity, inducing growth cone collapse.
    Full-text · Article · Sep 2010 · Journal of Biological Chemistry

Publication Stats

12k Citations
974.85 Total Impact Points


  • 1980-2015
    • Kyoto University
      • • Graduate School of Biostudies
      • • Division of Pharmaceutical Sciences
      • • Department of Pharmacology
      • • Division of Chemistry
      Kioto, Kyōto, Japan
  • 2010
    • Nagoya University
      • Department of Chemistry
      Nagoya, Aichi, Japan
  • 2004
    • National Cancer Center, Japan
      • Endoscopy Division
      Edo, Tōkyō, Japan
  • 2002
    • The University of Calgary
      • Faculty of Medicine
      Calgary, Alberta, Canada
  • 2001
    • Fourth Military Medical University
      Xi’an, Liaoning, China
  • 1994
    • Duke University Medical Center
      • Department of Medicine
      Durham, North Carolina, United States
  • 1990-1993
    • Gunma University
      • School of Medicine
      Maebashi, Gunma, Japan
  • 1989-1991
    • Osaka Bioscience Institute
      Ōsaka, Ōsaka, Japan