Skip to main content
. 2018 Dec 4;18(1):e12872. doi: 10.1111/acel.12872

Figure 6.

Figure 6

O‐GlcNAcylation of c‐Fos at S56 and S57 regulates the transcriptional activity of c‐Fos and the expression of Bim in the presence of Aβ. SH‐SY5Y (a, b, d–g) and HEK293T (c) cell lines were treated with Aβ (at doses indicated) for 24 hr. (a, b) The interaction between c‐Fos and c‐Jun was analyzed by performing immunoprecipitation. SH‐SY5Y cells were transfected with EGFP‐c‐Fos‐WT or EGFP‐c‐Fos‐S56A‐S57A. EGFP‐c‐Fos was immunoprecipitated using an anti‐GFP antibody and probed with c‐Jun. Representative immunoblot images are shown in (a) and quantitative graph in (b) (n = 5). (c) AP‐1 luciferase assay was performed in tag free‐c‐Fos‐WT or tag free‐c‐Fos‐S56A‐S57A in AP‐1 luciferase vector co‐transfected HEK293T cells (n = 4). (d) Relative Bim mRNA expression levels were measured in tag free‐c‐Fos‐WT or tag free‐c‐Fos‐S56A‐S57A transfected SH‐SY5Y cells (n = 5). (e–g) Relative expression levels of Bim and cleaved caspase‐3 were measured using western blotting in EGFP‐c‐Fos‐WT or EGFP‐c‐Fos‐S56A‐S57A transfected SH‐SY5Y cells (n = 5). Representative immunoblot images are shown in (e). Quantitative graphs showing the relative expression of Bim and cleaved caspase‐3 ((f) and (g), respectively). Data are shown as mean ± SEM. # p < 0.05, ## p < 0.01, ### p < 0.001 among c‐Fos‐WT or c‐Fos‐S56A‐S57A transfected groups (one‐way ANOVA, Bonferroni posthoc test), *p < 0.05, **p < 0.01, ***p < 0.001 between c‐Fos‐WT and c‐Fos‐S56A‐S57A groups (two‐way ANOVA, Bonferroni posthoc test). (h) Schematic diagram of the sites and the role of c‐Fos O‐GlcNAcylation in the presence of Aβ. O‐GlcNAc sites of c‐Fos are S56 and S57. c‐Fos O‐GlcNAcylation is increased by Aβ, which is mediated by a decreased interaction between c‐Fos and OGA. O‐GlcNAcylation of c‐Fos increases its stability and the interaction with c‐Jun, consequently elevating its transcriptional activity to induce the expression of Bim, an apoptotic protein, in the presence of Aβ. Therefore, O‐GlcNAcylation of c‐Fos promotes neuronal cell death in the presence of Aβ. α‐tub: α‐tubulin; IP: Immunoprecipitation; S56A‐S57A: c‐Fos‐S56A‐S57A; Veh: Vehicle; WT: c‐Fos‐WT