Fig. 4: PPP1R15A promotes translation initiation by regulating the phosphorylation level of EIF2S1 and 4EBP1. | Cell Death & Disease

Fig. 4: PPP1R15A promotes translation initiation by regulating the phosphorylation level of EIF2S1 and 4EBP1.

From: Protein phosphatase 1 regulatory subunit 15 A promotes translation initiation and induces G2M phase arrest during cuproptosis in cancers

Fig. 4

A The flowchart for screening out EIF2S1 and 4EBP1. B PPP1R15A and EIF2S1 were co-localized by immunofluorescence. Scale bars represent 10 μm. C Western blot showed the phosphorylation level of EIF2S1 at 24 h pulse-treated with indicated concentrations of elesclomol. D, E PPP1R15A knockdown and GBZ offset the decrease of 4EBP1 phosphorylation level caused by elesclomol. Western blot showed the phosphorylation level of 4EBP1 at 48 h in cells transfected with Sh-PPP1R15A, pulse-treated with 50 nM elesclomol or treated with 20 μM GBZ for 48 hours. F Diagram of EIF2S1 and 4EBP1 phosphorylation site mutation. G S51D decreased rate of protein synthesis in cells. S51D, mutation of EIF2S1 S51 to the phosphomimetic amino acid aspartic acid. 293 T cells were transfected with Si-EIF2S1#2. H, I Rate of translation in cells with indicated treatment. For GBZ treatment, the cells were treated with 20 μM GBZ for 48 hours. S51A, mutation of EIF2S1 S51 to the non-phosphorylatable amino acid alanine; T70A, mutation of 4EBP1 T70 to the non-phosphorylatable amino acid alanine; T70D, mutation of 4EBP1 T70 to the phosphomimetic amino acid aspartic acid. 293 T cells were transfected with indicated siRNA. J Cell viability transfected with mutation of EIF2S1 and 4EBP1 were assessed 24 hours after pulse treatment of 50 nM elesclomol for 2 hours. K Mechanism map of increased translation rate induced by elesclomol. For elesclomol treatment, media were supplemented with 1 μM CuCl2. Data are presented as the means ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

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