FC0037
Adenomatous polyposis coli protein (APC)  -  β-catenin

Biological function
The transcriptional coactivator β-catenin mediates Wnt growth factor signaling. Phosphorylation by casein kinase 1 and glycogen synthase kinase-3β occurs in multiprotein complex (so-called ’destruction complex’), which includes axin and Adenomatus Polyposis Coli (APC) protein. Phosphorylation flags β-catenin for recognition and destruction by the ubiquitin/proteasome machinery, where each of these proteins can interact with each other. Phosphorylated APC and axin bind to the same surface of, and compete directly for β-catenin.

Structural evidence
Crystals were obtained only by substituting Ser by Asp at positions 1501, 1505, 1507. (R3-tripleD). Only 14 of the 47 residues of the APC construct were visible in the final electron density map. APC R3-tripleD binds in the repeat 5-9 groove. The typical β- catenin peptide binding motif DxθθxφxxE is observed and is structurally identical to that of E-cadherin and additional contacts are also formed. None of the sequence containing the phosphorylation-mimic Asp residues is visible in the structure, suggesting that the reported affinity enhancement is due to non-specific electrostatic effects. Of the six phosphoserines present in the construct, those at positions 1504, 1505, 1507, and 1510, part of the consensus ¹⁵⁰¹SxxSSLSSLS APC 20-mer motif, are visible, and three of the four form direct contacts with β-catenin.

Biochemical evidence
Changing APC R3 serine residues 1505 or 1507 to aspartic acid to mimic putative phosphorylation sites result in an 18-fold increase in affinity. Isothermal titration calorimetry (ITC) measurements demonstrated that homogeneously phosphorylated R3 binds to full-length β-catenin with a dissociation constant K_D of 10 nM, as compared to the 3.1 μM K_D of the untreated material. Phosphorylation results in the structuring of residues 1504-1528, which interact with β -catenin arm repeats 1-5. Residues 1500-1503 remain to be disordered.

Mechanism category
competitive binding

Posttranslational modification
The additional interactions due to phosphorylation of R3 increase the total buried surface area of the R3/β-catenin complex from 1483 Ų to 3999 Ų, consistent with the large affinity enhancement provided by phosphorylation. Consistent with these structural observations, in vitro phosphorylated APC R3 competes with axin for binding to β-catenin, whereas the non-phosphorylated APC and axin can simultaneously bind to β-catenin.

Significance
Phosphorylation of a fuzzy region gradually regulates binding to β-catenin and compete with axin interactions with the same interface.