Figure 5
From: Nuclear export regulation of COP1 by 14-3-3σ in response to DNA damage
Phosphorylation of COP1 at S387 is important for 14-3-3σ-mediated COP1 Degradation. (A) 14-3-3σ downregulates steady-state expression of COP1 (WT) but has no impact on COP1 (S387A). 293T cells were cotransfected with the indicated Flag-COP1 (WT), Flag- COP1 (S387A) and Flag-14-3-3σ. Cell lysates were immunoblotted with anti-Flag and anti-tubulin. (B) 14-3-3σ increases turnover rate of COP1 (WT) but has no impact on COP1 (S387A).293T cells were cotransfected with indicated plasmids. Before harvesting, cells were treated with cycloheximide (CHX) for the indicated times. Cell lysates were immunoblotted with anti-Flag and anti-tubulin. Turnover rate of COP1 is indicated as a bar graph. (C) 14-3-3σ promotes COP1 (WT) polyubiquitination but has no effect on COP1 (S387A).293T cells were cotransfected as indicated with Myc-COP1 (WT), Myc-COP1 (S387A) or Flag-14-3-3σ. Cells were treated with MG132 for 6 hrs before harvesting. Ubiquitinated COP1 was immunoprecipitated with anti-Myc and immunoblotted with anti-HA. Equal amounts of whole cell lysate were immunoblotted with anti-Myc, anti-Flag or anti-tubulin. (D) 14-3-3σ promotes cytoplasmic COP1 polyubiquitination in response to DNA damage. HCT116 14-3-3σ+/+ (lanes 1 and 2) and HCT116 14-3-3σ-/- (lanes 3 and 4) were treated with MG132 for 6 hrs before harvesting. Cytoplasmic extracts were prepared at 0 and 2 hr after cells were treated with 10 Gy IR. Cytoplasmic polyubiquitinated COP1 was detected by immunoprecipitation with anti-COP1 and immunoblotting with anti-ubiquitin in indicated cells.