title: C-terminal region of GADD34 regulates eIF2α dephosphorylation and cell proliferation in CHO-K1 cells.
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.
authors: Department of Dental Anesthesiology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8504, Japan.
authors: Ryo
authors: R
authors: Otsuka
authors: Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8503, Japan. nharada@tokushima-u.ac.jp.
authors: Nagakatsu
authors: N
authors: Harada
authors: Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8503, Japan.
authors: Shouhei
authors: S
authors: Aoki
authors: Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8503, Japan.
authors: Kanna
authors: K
authors: Shirai
authors: Department of Molecular Pathology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8503, Japan.
authors: Kazuchika
authors: K
authors: Nishitsuji
authors: Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8503, Japan.
authors: Ayane
authors: A
authors: Nozaki
authors: Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8503, Japan.
authors: Adzumi
authors: A
authors: Hatakeyama
authors: Support Center for Advanced Medical Sciences, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8503, Japan.
authors: Masayuki
authors: M
authors: Shono
authors: Department of Medical Pharmacology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8504, Japan.
authors: Noriko
authors: N
authors: Mizusawa
authors: Department of Medical Pharmacology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8504, Japan.
authors: Katsuhiko
authors: K
authors: Yoshimoto
authors: Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8503, Japan.
authors: Yutaka
authors: Y
authors: Nakaya
authors: Department of Dental Anesthesiology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8504, Japan.
authors: Hiroshi
authors: H
authors: Kitahata
authors: Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho, Tokushima City, 770-8503, Japan.
authors: Hiroshi
authors: H
authors: Sakaue
mesh_terms: D000818:Animals; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation / Q000235:genetics*; D018389:Codon, Nonsense / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis*; D059865:Endoplasmic Reticulum Stress / Q000502:physiology; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000235:genetics* / Q000378:metabolism*; D060449:Wnt Signaling Pathway / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D018389:Codon, Nonsense; D050759:Cyclin-Dependent Kinase Inhibitor p21; D015852:Eukaryotic Initiation Factor-2; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D054645:Protein Phosphatase 1
keywords: CHO-K1 cell; Dephosphorylation; GADD34; GSK3β; Proliferation; eIF2α