FIG. 2.

β-Catenin is acetylated at lysine residue K345 by p300. (A) Arm domain 6 of β-catenin is acetylated in vitro by p300. (Left panel) Schematic representation of GST-arm repeat constructs used in the in vitro acetylation assay. GST-full-length armadillo construct (GST-arm 1-12) or serial deletion constructs were incubated with GST-p300 recombinant protein and [¹⁴C]acetyl-coenzyme A. Reaction products were separated on SDS-10% PAGE. The gel was Coomassie blue-stained (bottom right panel) and then dried and exposed to X-ray film (upper right panel). (B) K345 is an acetyl acceptor in vitro. The amino acid sequence of the arm 6 domain contains two lysines (upper panel). GST-arm 1-12 (wt), GST-arm 1-12 K345R, or GST-arm 1-12 K354R was used for in vitro acetylation assays with GST-p300 recombinant protein. Reaction products were analyzed by SDS-PAGE and autoradiography. (C) In vitro acetylation assays with PCAF recombinant protein revealed acetylated PCAF and histone H3 but no acetylated form of β-catenin. (D) Lysine 345 is acetylated in vivo. HeLa cells were transfected with T41A-β-catenin or T41A-β-catenin K345R. Proteins were immunoprecipitated with β-catenin antibodies and resolved on SDS-PAGE. Blots were hybridized either with anti-acetyl-lysine antibodies or with anti-β-catenin antibodies. WB, Western blotting.