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Molecular mechanism of growth and differentiation of neutrophilic granulocyte mediated by G-CSF

Research Project

Project/Area Number 12680696
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Cell biology
Research InstitutionOsaka University

Principal Investigator

FUKUNAGA Rikiro  Osaka University, Graduate School of Medicine, Associate Professor, 医学系研究科, 助教授 (40189965)

Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
Keywordshematopoiesis / cell growth / differentiation / signal transduction / transcription factor / protein kinase / chromatin remodeling factor / granulocyte colony-stimulating factor / SWI2 / SNF2型ATPase / 好中球 / MAPキナーゼ / 好中性顆粒球
Research Abstract

Our goal is to understand the molecular mechanism of growth and differentiation of neutrophilic granulocytes, which are mediated by granulocyte colony-stimulating factor (G-CSF). For this purpose, we have investigated the myeloid-specific zinc-finger protein MZF-2 that plays a role in granulocyte differentiation. Mutational analysis of mouse MZF-2 demonstrated that MZF-2 protein carries a transcription-inhibitory region at the N-terminus, a transactivation domain (TA domain) in the middle of the molecule, and a DNA-binding zinc-finger domains in the C-terminal region. We also found that the TA domain was phosphorylated by ERK MAP kinase. To determine sites phosphorylated by ERK, we constructed GST-MZF-2 fusion proteins with various alanine substitution of putative ERK phosphorylation sites. An in vitro phosphorylation analysis using the mutant substrates revealed that ERK phosphorylated three serine residues (Ser257, Ser275 and Ser295) in the TA domain. A mutant MZF-2 protein containin … More g alanine substitutions of the these serine residues showed higher transactivation activity than the wild-type protein when expressed in LGM-1 myeloid cells, suggesting that ERK MAP kinase negatively regulates the transcriptional activity of MZF-2 in vivo. To identify nuclear factors involved in the MZF-2-dependent gene activation, we screened mouse CDNA library for MZF-2 binding protein by using the ras recruitment two-hybrid system, and isolated a CDNA encoding a huge (3035 amino acid residues) protein, which showed a significant homology to. Drosophila Domino protein. The cloned protein, designated as mammalian Domino (mDomino), has a SWI/SNF-type ATP /helicase structure with a domain containing two polyglutamine tracts in the C-terminal region. Mutational analyzes using deletion mutants of mDomino revealed that MZF-2 interacted with the C-terminal, glutamine-rich domain. Moreover, the MZF-2-dependent reporter activation was enhanced when the mDomino C-terminal domain was co-expressed. These results suggest that chromatin remodeling caused by mDomino/MZF-2 complex may regulate myeloid-specific gene activation in granulocyte development. Less

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (9 results)

All Other

All Publications (9 results)

  • [Publications] Hefner, Y. et al.: "Serine 727 phosphorylation and activation of cytosolic----------"Journal of Biological Chemistry. 275. 37542-37551 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Fukumoto, T. et al.: "The Fused protein kinase regulates Hedgehog-stimulated------------"Journal of Biological Chemistry. 276. 38441-38448 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Seger, R. (編): "MAP Kinase Protocols : Methods in Molecular Biology Series"Humana Press, Totowa, USA(印刷中)(未定).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Hefner, Y., Borsch-Haubold, A. G., Murakami, M., Wilde, J. I., Pasquet, S., Schieltz, D., Ghomashchi, F., Yates III, J. R., Armstrong, C. G., Paterson, A., Cohen, P., Fukunaga, R., Hunter, T., Kudo, I., Watson, S. p. and Gelb, M. H.: "Serine 727 Phosphorylation and Activation of Cytosolic Phospholipase A2 by MNK1-related Protein Kinases"J. Biol. Chem.. 275. 37542-37551 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Fukumoto. T., Watanabe-Fukunaga, R., Fujisawa, K., Nagata, S. and FuruKunaga, R.: "The Fused protein kinase regulates Hedgehog-stimulated transcriptional activation in Drosophila Schneider 2 cells"J. Biol. Chem.. 276. 38441-38448 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Fukunaga, R. and Hunter, T.: ""Identifying MAP kinase substrates by expression screening with solid-phase phosphorylation". in : MAP Kinase Protocols : Methods in Molecular Biology, (Seger, R., Ed.)"Humana Press, Totowa, USA. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Fukumoto, T.et al.: "The Fused protein kinase regulates Hedgehog-stimulated transcriptional activation in Drosophila Schneider 2 cells"Journal of Biological Chemistry. 276. 38441-38448 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Seger, R.(編): "MAP Kinase Protocols : Methods in Molecular Biology Series"Humana Press, Totowa, USA(印刷中).

    • Related Report
      2001 Annual Research Report
  • [Publications] Hefner,Y. et al.: "Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases."Journal of Biological Chemistry. 275. 37542-37551 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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