Figure 5

Multiple lysine residues in the N-terminus are responsible for PCAF-mediated δ-catenin downregulation. (A) Schematic representation of the triple arginine mutation at Lys360, Lys371, and Lys428 (FL KR), and the deletion/arginine mutation constructs of δ-catenin 1–499, 1–499 KR, 85–499, 85–499 KR, 1–499∆N KR, and 325–499 KR. (B) Deletion mutants 85–499 KR and 325–499 KR of δ-catenin were not affected by PCAF. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibody. (C) 3-MA restored the downregulated FL KR, 85–499, and 1–499∆N KR mutants of δ-catenin except the 85–499 KR mutation. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs and incubated with 3-MA (1 mM) for 24 h, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibodies. (D) PCAF did not acetylate δ-catenin 85–499 KR mutation. HEK293T cells were transfected with full length GFP-δ-catenin or 85–499 KR mutant together with or without Flag-PCAF, and each cell lysates were subjected to immunoprecipitation with anti-acetylated-lysine, followed by immunoblotting of precipitated proteins. α-Tubulin or ß-actin was used as a loading control. Relative values of δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. *p < 0.05; **p < 0.01; NS, no significant difference compared with the control group. “−”, Mock transfection or vehicle treatment.