Proteomic analysis reveals APC-dependent post translational modifications and identifies a novel regulator of β-catenin

Abstract

Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called twodimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development.

Identification of Endogenous Adenomatous Polyposis Coli Interaction Partners and β-Catenin–Independent Targets by Proteomics

“…Changes in the abundance of individual APC targets could result from alterations in PTMs and/or protein stability-as is the case for β-catenin. This is supported by previous findings in Drosophila, where loss of APC2 causes proteome-wide and β-catenin-independent changes in posttranslational modifications that also affect protein stability of some proteins (37). In addition, effects on transcription may also contribute to the differences in protein abundance observed in our study.…”

Identification of endogenous Adenomatous polyposis coli interaction partners and β-catenin-independent targets by proteomics

“…Changes in the abundance of individual APC targets could result from alterations in PTMs and/or protein stability – as is the case for β-catenin. This is supported by previous findings in Drosophila , where loss of APC2 causes proteome-wide and β-catenin-independent changes in post-translational modification that also affect protein stability of some proteins (Blundon et al, 2016). In addition, effects on transcription may also contribute to the observed differences in protein abundance.…”

Axin phosphorylation in both Wnt-off and Wnt-on states requires the tumor suppressor APC

“…These findings suggest that APC-dependent phosphorylation of Axin is important for Axin regulation during the activation of signaling following Wingless exposure and further support previous studies demonstrating that GSK3-mediated phosphorylation of Axin is important for the association between Axin and phosphorylated LRP6 [ 19 ]. The mechanism by which APC promotes the GSK3-dependent phosphorylation of Axin requires further investigation, but may involve previously proposed functions for APC in regulating protein phosphorylation, GSK3 activity, Axin multimerization, or Axin membrane association [ 18 , 51 , 63 – 65 ]; alternatively, APC may prevent Axin dephosphorylation by a phosphatase such as PP1.…”