Proteomic analysis reveals APC-dependent post-translational modifications and identifies a novel regulator of β-catenin.
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 two-dimensional 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.
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Malachi A
MA
Blundon
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Danielle R
DR
Schlesinger
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Amritha
A
Parthasarathy
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Samantha L
SL
Smith
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Hannah M
HM
Kolev
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
David A
DA
Vinson
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Ezgi
E
Kunttas-Tatli
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA bmccartney@cmu.edu minden@cmu.edu.
Brooke M
BM
McCartney
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA bmccartney@cmu.edu minden@cmu.edu.
Jonathan S
0000-0003-3175-393X
JS
Minden
D000818:Animals; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004625:Embryo, Nonmammalian / Q000378:metabolism; D047108:Embryonic Development; D004843:Epistasis, Genetic; D015151:Immunoblotting; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism*
C467323:APC2 protein, Drosophila; D029721:Drosophila Proteins; D020033:Protein Isoforms; D020543:Proteome; D025521:Tumor Suppressor Proteins; D051176:beta Catenin
2D-DIGE; APC; ApepP; Post-translational modification; Wnt signaling