Differences Between the Interaction of β-Catenin with Non-phosphorylated and Single-mimicked Phosphorylated 20-amino acid Residue Repeats of the APC Protein

The tumour suppressor protein adenomatous polyposis coli (APC) regulates the level and the intracellular localisation of the proto-oncoprotein β-catenin. There are indications that a region comprising seven homologous 20-amino acid residue repeats within the APC protein is responsible for the interaction with β-catenin and that the phosphorylation of conserved serine residues within these repeats increases the affinity for β-catenin. We used biophysical methods to analyse the β-catenin binding of single repeats or repeat combinations as non-phosphorylated or phosphorylated recombinant proteins. The non-phosphorylated repeats showed similar affinities, no matter whether they were tested as single recombinant repeats or in combination with neighbouring repeats. This result makes a cooperative influence between the repetitive motifs unlikely. The phosphorylation of the APC protein was mimicked by specific serine/aspartate mutations, which align to serine residues in the cytoplasmic β-catenin binding domain of E-cadherin. Remarkably, the mimicked phosphorylation of a serine, which is not involved in β-catenin interaction in the E-cadherin/β-catenin complex, led to a significant increase in the APC affinity for β-catenin. These results indicate structural differences between the E-cadherin/β-catenin and the APC/β-catenin complexes and provide quantitative evidence for the importance of the APC phosphorylation for its interaction with β-catenin.