β-catenin is a protein well-known for its association with several human cancers and is known to accumulate in the nucleus with cancer progression. It is a multi-functional protein that can be found at different subcellular localisations, regulated in part by protein interactions and phosphorylation.
Here, β-catenin protein interactions in colon cancer were investigated and explore the role of phosphorylation at amino acid Y654 in regulating these interactions was explored. Recombinant human full-length β-catenin constructs were used as affinity baits to isolate protein binding partners from stable isotope labelled SW480 and HT29 colon cancer cell lines while high-resolution mass spectrometry (Orbitrap ELITE) was used for protein identification and quantitation.
For wildtype β-catenin (Y654), 379 and 123 putative protein interactors were detected in SW480 and HT29 respectively. For the phosphomimetic (Y654E) construct, 92 and 224 putative protein interactors were detected in SW480 and HT29 respectively. For the non-phosphorylatable (Y654F) construct, 129 and 48 putative proteins interactors were detected in SW480 and HT29 respectively.
A number of these candidate binding proteins were further validated by immunoprecipitation and immunofluorescence and strongest evidence was shown for mitochondrial HSP70 – providing both a novel interaction and localisation for β-catenin.