Beta-catenin is a protein associated with several human cancers. The progression of cancer is correlated with nuclear accumulation of beta-catenin, a process which is regulated by phosphorylation. Protein phosphorylation also alters function through changing protein-protein interactions. In the context of colon cancer we are interested in furthering our understanding of these two aspects of beta-catenin regulation. 

We generated recombinant human full-length beta-catenin constructs to achieve phospho-mimic and phospho-dead Y654 in the Arm 10-12 domain. These constructs were used along with wildtype beta-catenin to enrich for protein binding-partners in SILAC labelled SW480 and HT29 colon cancer cell lines in triplicate.

Eluates were recovered, separated by SDS-PAGE and analysed by mass spectrometry (Orbitrap ELITE). Proteome Discoverer 1.3 was used for data analysis and candidates selected based on presence in all replicates at least 2-fold more than the control. Detection of at least 2 peptides and replicates having a p-value <0.05 (one-sided t-test) was also applied.

For SW480, candidates include 225 for the wildtype, 49 for the phospho-mimic and 62 for the phospho-dead. For HT29, candidates include 66 for the wildtype, 146 for the phospho-mimic and 22 for the phospho-dead. Several well-known beta-catenin interactors were detected including catenin alpha-1, catenin delta-1, adenomatous polyposis coli protein (APC), four and a half LIM domains protein 2, ruvB-like 1, and paxillin.

Novel candidate interactors were grouped into processes and functions including nuclear import and export, regulation of cell cycle and mitochondrial proteins. We selected 3 candidate proteins (OPA1, Cdk1, Hsp70) as potential novel interactors and attempted to validate by immunofluorescence microscopy and an in situ proximity ligation assay (DuoLink). This presentation will report our progress towards confirming these proteins as novel beta-catenin effectors.