Fig. 5. ANKRD11 associates with hADA3 and P/CAF to enhance the acetylation and DNA-binding activity of both wild-type p53 and mutant p53^R273H. (A) Yeast two-hybrid assay showing interaction between ANKRD11^2369-2663aa and hADA3 (left panel). Yeast cells coexpressing either pGBT-ANKRD11^2369-2663aa and empty pACT2 vector, pACT-hADA3 and empty pGBT9 vector, or pACT-hADA3 and pGBT-RAC3-N showed no activation of the β-galactosidase reporter. ANKRD11 interacts with hADA3 in vitro (centre panel). GST-pull down assay shows that GST-ANKRD11^2369-2663aa but not GST alone pulled down significant amounts of ³⁵S-labeled hADA3. All proteins were resolved by SDS-PAGE and visualized by Coomassie Blue staining to confirm they were intact. ANKRD11 nuclear foci colocalize with P/CAF (right panel). ANKRD11 nuclear foci colocalize with P/CAF. COS-7 cells were transiently transfected with Flag-tagged P/CAF and full-length HA-tagged ANKRD11. Transfected cells were immunostained with anti-HA (ANKRD11; green) and anti-Flag (P/CAF; red) antibodies and DAPI (DNA; blue). (B) Restoration of expression of ANKRD11 increases p53 acetylation at Lys320. Total p53 and acetyl-lysine p53 (Lys320) protein levels were detected in MCF-7, MB-468 and MB-231 cultures stably expressing GFP-ANKRD11 or the negative control cell cultures previously described (Fig. 4A) through western blot analysis using the appropriate antibodies. MCF-7-ANK-1 was cultured in the presence or absence of mitomycin C for 6 hours and is presented as a representative MCF-7 derivative from (Fig. 4A). (C) The p53 coactivator function of ANKRD11 is dependent upon Lys320. H1299 cells were cotransfected with the p53-responsive reporter constructs encoding the CDKN1A promoter region, together with pRL-TK and constructs expressing either myc-p53, myc-p53-K320R or ANKRD11-myc as indicated. Empty pLNCX2 vector was added to equalize the total amounts of plasmid used in various treatments. Data are represented as mean ± s.e.m. of triplicates. The expression of protein levels from H1299 cells transiently transfected with either myc-p53 or myc-p53-K320R constructs was determined by western blot analysis (inset). (D) ANKRD11 enhances the DNA-binding activity of both wild-type p53 and mutant p53^R273H. Lysates from either MCF-7-ANK-1 (upper panels) or MB-468-ANK-1 (lower panel) cells stably expressing GFP-ANKRD11 or the negative control MCF-7 or MB-468 cells (described in Fig. 4A) were promoter precipitated using either beads charged with the CDKN1A promoter region or uncharged beads. Inputs or promoter precipitates were analyzed by western blot using anti-p53 (detecting wild-type and p53^R273H) and anti-acetyl-p53 (Lys320) antibodies, as indicated.