Fig. 5: MACC1 interacts with β-catenin and induces its transcriptional activity via post-translational modification.

In a mass-spectrometry-based analysis of the MACC1 interactome in SW620 cells, several peptides interacting with MACC1 mapped to β-catenin, suggesting a direct PPI (A). Co-IP experiments on whole-cell lysates, cytoplasmic and nuclear protein of SW620 confirmed direct interaction between MACC1 and β-catenin (B). This finding was recapitulated by co-incubating recombinant MACC1 and β-catenin proteins in cell-free Co-IP assays (C). Impact of MACC1 overexpression or MACC1 knock-out is shown on TCF-reporter activity and mRNA expression of β-catenin and the β-catenin/TCF target genes Cyclin-D1, MMP7 and S100A4 in HCT116 and SW620 cells. In HCT116 cells MACC1 overexpression increases TCF reporter activity and TCF target gene expression, while β-catenin gene expression itself is unaffected. In SW620 cells MACC1 knockout decreases TCF reporter activity and TCF target gene expression, whereas β-catenin gene expression is also unaffected (D). In a DigiWest experiment, MACC1-overexpressing HCT116 cells demonstrated increased phosphorylation of p-Ser-552-β-catenin, confirmed in semiquantitative western blots, while total β-catenin protein was not altered (E). Comparative immunoprecipitation of β-catenin protein showed increased binding of TCF4 in HCT116/MACC1 cells, also shown by densitometry (F). Boxplots show means ± SEM of 3 independent experiments, test for significance with Student’s t-test.