Figure 4
From: Nuclear export regulation of COP1 by 14-3-3σ in response to DNA damage
14-3-3σ binds phosphorylated COP1. (A) The consensus 14-3-3 binding motif is highlighted. Sequences of COP1 and other known 14-3-3 substrates are shown for comparison. Sequence alignment of COP1 containing 14-3-3 binding motifs from different species is shown in right panel. (B) Binding between 14-3-3σ and COP1 is phosphorylation-dependent. Lysates of U2OS cells stably expressing Flag-COP1 were treated with or without sodium fluoride and sodium orthovanadate. Before harvesting, cells were treated with MG132. Lysates were incubated with purified GST-14-3-3σ or GST and immunoblotted with anti-Flag. PD: pull down. (C) ATM facilitates the binding between 14-3-3σ and COP1. AT22IJE-T/pEBS7 (ATM-/-) and AT22IJE-T/YZ5 (ATM+/+) cells were treated with MG132 for 6 hrs before harvesting and were prepared at 0 (-) and 1 hr (+) after cells were treated with 10 Gy IR. Lysates were immunoprecipitated with anti-14-3-3σ and immunoblotted with anti-COP1. (D) COP1 (S387A) abolished binding to14-3-3σ in vivo. 293T cells were transfected with Myc-COP1 (WT [wild type]), Myc-COP1 (S387A) or Flag-14-3-3σ. Lysates were immunoprecipitated with anti-Myc and immunoblotted with anti-Flag.