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Cell Stress and Chaperones

, Volume 21, Issue 1, pp 29–40 | Cite as

C-terminal region of GADD34 regulates eIF2α dephosphorylation and cell proliferation in CHO-K1 cells

  • Ryo Otsuka
  • Nagakatsu Harada
  • Shouhei Aoki
  • Kanna Shirai
  • Kazuchika Nishitsuji
  • Ayane Nozaki
  • Adzumi Hatakeyama
  • Masayuki Shono
  • Noriko Mizusawa
  • Katsuhiko Yoshimoto
  • Yutaka Nakaya
  • Hiroshi Kitahata
  • Hiroshi Sakaue
Original Paper

Abstract

GADD34 is a member of a growth arrest and DNA damage (GADD)-inducible gene family. Here, we established a novel Chinese hamster ovary (CHO)-K1-derived cell line, CHO-K1-G34M, which carries a nonsense mutation (termed the Q525X mutation) in the GADD34 gene. The Q525X mutant protein lacks the C-terminal 66 amino acids required for GADD34 to bind to and activate protein phosphatase 1 (PP1). We investigated the effects of GADD34 with or without the Q525X mutation on the phosphorylation status of PP1 target proteins, including the α subunit of eukaryotic initiation factor 2 (eIF2α) and glycogen synthase kinase 3β (GSK3β). CHO-K1-G34M cells had higher levels of eIF2α phosphorylation compared to the control CHO-K1-normal cells both in the presence and absence of endoplasmic reticulum stress. Overexpression of the wild-type GADD34 protein in CHO-K1-normal cells largely reduced eIF2α phosphorylation, while overexpression of the Q525X mutant did not produce similar reductions. Meanwhile, neither wild type nor Q525X mutation of GADD34 affected the GSK3β phosphorylation status. GADD34 also did not affect the canonical Wnt signaling pathway downstream of GSK3β. Cell proliferation rates were higher, while expression levels of the cyclin-dependent kinase inhibitor p21 were lower in CHO-K1-G34M cells compared to the CHO-K1-normal cells. The GADD34 Q525X mutant had a reduced ability to inhibit cell proliferation and enhance p21 expression of the CHO-K1-normal cells compared to the wild-type GADD34 protein. These results suggest that the GADD34 protein C-terminal plays important roles in regulating not only eIF2α dephosphorylation but also cell proliferation in CHO-K1 cells.

Keywords

CHO-K1 cell GADD34 eIF2α GSK3β Dephosphorylation Proliferation 

Notes

Acknowledgments

We thank Dr. Kenji Uchimura (Department of Biochemistry, Nagoya University Graduate School of Medicine) for kindly providing an ancestor of the CHO-K1-normal cells. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, to Nagakatsu Harada (23700906, 25350887).

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Copyright information

© Cell Stress Society International 2015

Authors and Affiliations

  • Ryo Otsuka
    • 1
  • Nagakatsu Harada
    • 2
  • Shouhei Aoki
    • 2
  • Kanna Shirai
    • 2
  • Kazuchika Nishitsuji
    • 3
  • Ayane Nozaki
    • 2
  • Adzumi Hatakeyama
    • 2
  • Masayuki Shono
    • 4
  • Noriko Mizusawa
    • 5
  • Katsuhiko Yoshimoto
    • 5
  • Yutaka Nakaya
    • 2
  • Hiroshi Kitahata
    • 1
  • Hiroshi Sakaue
    • 2
  1. 1.Department of Dental Anesthesiology, Institute of Biomedical SciencesTokushima University Graduate SchoolTokushima CityJapan
  2. 2.Department of Nutrition and Metabolism, Institute of Biomedical SciencesTokushima University Graduate SchoolTokushima CityJapan
  3. 3.Department of Molecular Pathology, Institute of Biomedical SciencesTokushima University Graduate SchoolTokushima CityJapan
  4. 4.Support Center for Advanced Medical Sciences, Institute of Biomedical SciencesTokushima University Graduate SchoolTokushima CityJapan
  5. 5.Department of Medical Pharmacology, Institute of Biomedical SciencesTokushima University Graduate SchoolTokushima CityJapan

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