Proteomic study of β-catenin protein interactions in colon cancer

Abstract

Empirical thesis.Running title: Proteomic study of β-catenin protein interactions in colon cancer.Bibliography: pages 210-242.1. Introduction -- 2. Methods and materials -- 3. Novel protein interactions of β-catenin in SW480 -- 4. Novel protein interactions of β-catenin in HT29 -- 5. Validation of candidate protein binding partners of β-catenin -- 6. General discussion -- 7. References -- 8. Appendices.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. The aim of this thesis was to use proteomics to investigate β-catenin protein interactions in colon cancer and explore the role of phosphorylation at amino acid Y654 in regulating these interactions. It was hypothesised that determining β-catenin protein interactions could reveal new functional roles for it.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 golgi protein COPB and mitochondrial HSP70 – providing both a novel interaction and localisation for β-catenin. These observations confirm the multi-functional roles of β-catenin in colon cancer confirming dependence on protein interactions to regulate its subcellular localisation and function.Mode of access: World wide web1 online resource (xxiii, 377 pages) colour illustration