Fig. 5

Enhancers potentiate binding and ubiquitylation of S33E/S37A phosphomimetic β-catenin. a Schematic of the proposed phosphorylation cascade for WT and S37A, S33E/S37A mutant β-catenin. b Binding curve of S33E/S37A β-catenin peptide for β-TrCP measured in the TR-FRET assay in absence or presence of 40 μM NRX-103094. For reference, the binding curve for pSer33/pSer37 β-catenin peptide in the same TR-FRET assay is represented in dotted gray line. c 4 μM of fluorescently-labeled S33E/S37A β-catenin peptide (residues 17–60) was ubiquitylated in the presence of 125 nM Ube1, ATP, 1.75 μM Cdc34, 100 nM SCF^β-TrCP, and 20 μM NRX-103094. Reactions were resolved by SDS-PAGE and imaged for fluorescence. d 400 nM purified full-length β-catenin protein (as indicated) was ubiquitylated in the presence of 125 nM Ube1, ATP, 1.75 μM Cdc34, 400 nM SCF^β-TrCP, and 20 μM NRX-103094. Reactions were resolved by SDS-PAGE and analyzed by immuno-blotting with a β-catenin C-terminal antibody (* refers to degradation products). e Enhancer potencies for S33E/S37A β-catenin peptide binding to β-TrCP. Due to low intrinsic binding affinity, the pSer33/pSer37 β-catenin binding assays were carried out at 1% binding saturation. f Binding curve of S33E/S37A β-catenin peptide for β-TrCP measured in the TR-FRET assay in presence of varying NRX-252114 concentration. For reference, the binding curve for pSer33/pSer37 β-catenin peptide in the same TR-FRET assay is represented in dotted gray line. g Ubiquitylation of full-length S33E/S37A β-catenin protein in the presence of varying concentration of NRX-252114 (as described above; * refers to degradation products)