Hitoshi Yagisawa

Publications (85) View all

• Article: DGKζ is degraded through the cytoplasmic ubiquitin-proteasome system under excitotoxic conditions, which causes neuronal apoptosis because of aberrant cell cycle reentry.

  Masashi Okada, Yasukazu Hozumi, Toshiaki Tanaka, Yusuke Suzuki, Mitsuaki Yanagida, Yoshihiko Araki, Camilla Evangelisti, Hitoshi Yagisawa, Matthew K Topham, Alberto M Martelli, Kaoru Goto
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  ABSTRACT: Recent reports have described the involvement of the diacylglycerol kinase (DGK) family in various pathological conditions. In an animal model of transient ischemia, DGKζ containing a nuclear localization signal (NLS) is shown to translocate quickly from the nucleus to the cytoplasm in hippocampal neurons and to disappear gradually after reperfusion. Those neurons die a delayed neuronal death because of glutamate excitotoxicity. This study investigated the molecular mechanism and functional relation linking DGKζ and neuronal death. In primary cultured neurons, transient exposure to excitotoxic concentration of glutamate led to cytoplasmic accumulation of DGKζ followed by its down-regulation. Results showed that DGKζ down-regulation was caused by proteolytic degradation through the ubiquitin-proteasome system (UPS) rather than transcriptional inhibition. DGKζ polyubiquitination was inhibited in the presence of nuclear export inhibitor leptomycin B. Furthermore, NLS-deleted mutant DGKζΔNLS, which mainly localizes to the cytoplasm, was ubiquitinated more heavily than wild-type DGKζ. From a functional perspective, in vitro gene silencing of DGKζ via specific siRNA enhanced DNA fragmentation in cultured neurons after glutamate exposure. At the organismal level, hippocampal neurons of DGKζ-deficient mice showed vulnerability to kainate-induced seizures. In addition, DGKζ-deficient hippocampus exhibited a significant increase in Ser807/811 phosphorylated retinoblastoma protein levels together with up-regulation of the expression of type D and E cyclins, indicative of cell cycle reentry. Collectively, these results suggest that 1) glutamate excitotoxicity induces nucleocytoplasmic translocation of DGKζ followed by its degradation through the cytoplasmic UPS in hippocampal neurons and that 2) DGKζ-deficient neurons do not succumb directly to apoptosis, although they are more vulnerable to excitotoxicity because of aberrant cell cycle reentry.
  Cellular signalling 04/2012; 24(8):1573-82. · 4.09 Impact Factor
• Article: DGKζ is involved in LPS-activated phagocytosis through IQGAP1/Rac1 pathway.

  Masashi Okada, Yasukazu Hozumi, Kiyoshi Iwazaki, Kentaro Misaki, Mitsuaki Yanagida, Yoshihiko Araki, Takashi Watanabe, Hitoshi Yagisawa, Matthew K Topham, Kozo Kaibuchi, Kaoru Goto
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  ABSTRACT: Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGKζ-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGKζ or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKζ(-/-) mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGKζ is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages.
  Biochemical and Biophysical Research Communications 03/2012; 420(2):479-84. · 2.48 Impact Factor
• Article: Membrane-induced alteration of the secondary structure in the SWAP-70 pleckstrin homology domain.

  Naomi Tokuda, Katsuhisa Kawai, Young-Ho Lee, Takahisa Ikegami, Satoru Yamaguchi, Hitoshi Yagisawa, Yasuhisa Fukui, Satoru Tuzi
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  ABSTRACT: Differences in the conformation of the pleckstrin homology (PH) domain of switch-associated protein-70 (SWAP-70) in solution and at the lipid bilayer membrane surface were examined using CD, fluorescence and NMR spectroscopy. Intracellular relocalization of SWAP-70 from the cytoplasm to the plasma membrane and then to the nucleus is associated with its cellular functions. The PH domain of SWAP-70 contains a phosphoinositide-binding site and a nuclear localization signal, which localize SWAP-70 to the plasma membrane and nucleus, respectively. CD and fluorescence spectra showed that a significant conformational alteration involving formation of disordered structure occurs when the PH domain binds to D-myo-phosphatidylinositol 3,4,5-trisphosphate or D-myo-phosphatidylinositol 4,5-bisphosphate embedded in lipid bilayer vesicles. NMR spectra indicate that Ala and Trp residues located in the C-terminal α-helix of the PH domain undergo conformational alterations to form a disordered structure at the vesicle surface. These conformational alterations were not induced by association with inositol 1,3,4,5-tetrakisphosphate in solution or coexistence of phosphatidylcholine vesicles. Interaction with the plane of the lipid bilayer via association with the phosphoinositides is required for the unfolding of the C-terminal α-helix of the PH domain. The unwinding of the C-terminal α-helix could regulate the functions of SWAP-70 at the plasma membrane surface.
  Journal of biochemistry 01/2012; 151(4):391-401. · 1.95 Impact Factor
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  Available from: Giorgio Matassi

  Article: A conserved non-reproductive GnRH system in chordates.

  Takehiro G Kusakabe, Tsubasa Sakai, Masato Aoyama, Yuka Kitajima, Yuki Miyamoto, Toru Takigawa, Yutaka Daido, Kentaro Fujiwara, Yasuko Terashima, Yoko Sugiuchi, Giorgio Matassi, Hitoshi Yagisawa, Min Kyun Park, Honoo Satake, Motoyuki Tsuda
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  ABSTRACT: Gonadotropin-releasing hormone (GnRH) is a neuroendocrine peptide that plays a central role in the vertebrate hypothalamo-pituitary axis. The roles of GnRH in the control of vertebrate reproductive functions have been established, while its non-reproductive function has been suggested but less well understood. Here we show that the tunicate Ciona intestinalis has in its non-reproductive larval stage a prominent GnRH system spanning the entire length of the nervous system. Tunicate GnRH receptors are phylogenetically closest to vertebrate GnRH receptors, yet functional analysis of the receptors revealed that these simple chordates have evolved a unique GnRH system with multiple ligands and receptor heterodimerization enabling complex regulation. One of the gnrh genes is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord of vertebrates. Correspondingly, GnRH receptor genes were found to be expressed in the tail muscle and notochord of embryos, both of which are phylotypic axial structures along the nerve cord. Our findings suggest a novel non-reproductive role of GnRH in tunicates. Furthermore, we present evidence that GnRH-producing cells are present in the hindbrain and spinal cord of the medaka, Oryzias latipes, thereby suggesting the deep evolutionary origin of a non-reproductive GnRH system in chordates.
  PLoS ONE 01/2012; 7(7):e41955. · 4.09 Impact Factor
• Article: Interaction of nucleosome assembly proteins abolishes nuclear localization of DGKζ by attenuating its association with importins.

  Masashi Okada, Yasukazu Hozumi, Tohru Ichimura, Toshiaki Tanaka, Hiroshi Hasegawa, Masakazu Yamamoto, Nobuya Takahashi, Ken Iseki, Hitoshi Yagisawa, Takashi Shinkawa, Toshiaki Isobe, Kaoru Goto
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  ABSTRACT: Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGKζ, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGKζ. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGKζ binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGKζ and NAP1Ls prohibits nuclear import of DGKζ because binding of NAP1Ls to DGKζ blocks import carrier proteins, Qip1 and NPI1, to interact with DGKζ, leading to cytoplasmic tethering of DGKζ. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGKζ and provide a clue to examine functional significance of its translocation under pathological conditions.
  Experimental Cell Research 12/2011; 317(20):2853-63. · 3.58 Impact Factor