C-terminal region of GADD34 regulates eIF2[alpha] dephosphorylation and cell proliferation in CHO-K1 cells

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 [alpha] subunit of eukaryotic initiation factor 2 (eIF2[alpha]) and glycogen synthase kinase 3[beta] (GSK3[beta]). CHO-K1-G34M cells had higher levels of eIF2[alpha] 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 eIF2[alpha] phosphorylation, while overexpression of the Q525X mutant did not produce similar reductions. Meanwhile, neither wild type nor Q525X mutation of GADD34 affected the GSK3[beta] phosphorylation status. GADD34 also did not affect the canonical Wnt signaling pathway downstream of GSK3[beta]. 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 eIF2[alpha] dephosphorylation but also cell proliferation in CHO-K1 cells.