Differences between the interaction of beta-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 beta-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 beta-catenin and that the phosphorylation of conserved serine residues within these repeats increases the affinity for beta-catenin. We used biophysical methods to analyse the beta-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 beta-catenin binding domain of E-cadherin. Remarkably, the mimicked phosphorylation of a serine, which is not involved in beta-catenin interaction in the E-cadherin/beta-catenin complex, led to a significant increase in the APC affinity for beta-catenin. These results indicate structural differences between the E-cadherin/beta-catenin and the APC/beta-catenin complexes and provide quantitative evidence for the importance of the APC phosphorylation for its interaction with beta-catenin.