C-terminal region of GADD34 regulates eIF2α dephosphorylation and cell proliferation in CHO-K1 cells.
C-terminal region of GADD34 regulates eIF2α dephosphorylation and cell proliferation in CHO-K1 cells.
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GADD34 的 C 末端区域调节 CHO-K1 细胞中的 eIF2α 去磷酸化和细胞增殖。
DOI：
10.1007/s12192-015-0633-9
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发表时间：
2016
影响因子：
3.8
通讯作者：
Sakaue H
中科院分区：
文献类型：
--
作者：
Otsuka R;Harada N;Aoki S;Shirai K;Nishitsuji K;Nozaki A;Hatakeyama A;Shono M;Mizusawa N;Yoshimoto K;Nakaya Y;Kitahata H;Sakaue H
GADD34是生长停滞和DNA损伤（GADD）诱导基因家族的成员。在这里，我们建立了一种新的中国仓鼠卵巢(CHO-K1)- k1衍生细胞系CHO-K1- g34m，该细胞系携带GADD34基因的无意义突变（称为Q525X突变）。Q525X突变蛋白缺乏GADD34结合和激活蛋白磷酸酶1 （PP1）所需的c端66个氨基酸。我们研究了携带或不携带Q525X突变的GADD34对PP1靶蛋白磷酸化状态的影响，包括真核起始因子2 （eIF2α）和糖原合成酶激酶3β (GSK3β)的α亚基。无论是否存在内质网应激，CHOK1-G34M细胞的eIF2α磷酸化水平都高于对照cho - k1 -正常细胞。在chok1正常细胞中，野生型GADD34蛋白的过表达大大降低了eIF2α的磷酸化，而过表达Q525X突变体则没有产生类似的降低。同时，GADD34的野生型和Q525X突变都不影响GSK3β的磷酸化状态。GADD34也不影响GSK3β下游的典型Wnt信号通路。CHO-K1-G34M细胞的细胞增殖率较高，而细胞周期蛋白依赖性激酶抑制剂p21的表达水平低于cho - k1正常细胞。与野生型GADD34蛋白相比，GADD34 Q525X突变体抑制cho - k1正常细胞增殖和增强p21表达的能力降低。这些结果表明GADD34蛋白c端不仅在CHO-K1细胞中调控eIF2α去磷酸化，而且在细胞增殖中起重要作用。
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)-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). CHOK1-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 wild type GADD34 protein in CHOK1-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.