Wnt signalling induces accumulation of phosphorylated β-catenin in two distinct cytosolic complexes.
Wnt/β-catenin signalling controls development and adult tissue homeostasis and causes cancer when inappropriately activated. In unstimulated cells, an Axin1-centred multi-protein complex phosphorylates the transcriptional co-activator β-catenin, marking it for degradation. Wnt signalling antagonizes β-catenin proteolysis, leading to its accumulation and target gene expression. How Wnt stimulation alters the size distribution, composition and activity of endogenous Axin1 complexes remains poorly understood. Here, we employed two-dimensional blue native/SDS-PAGE to analyse endogenous Axin1 and β-catenin complexes during Wnt signalling. We show that the size range of Axin1 complexes is conserved between species and remains largely unaffected by Wnt stimulation. We detect a striking Wnt-dependent, cytosolic accumulation of both non-phosphorylated and phosphorylated β-catenin within a 450 kDa Axin1-based complex and in a distinct, Axin1-free complex of 200 kDa. These results argue that during Wnt stimulation, phosphorylated β-catenin is released from the Axin1 complex but fails to undergo immediate degradation. Importantly, in APC-mutant cancer cells, the distribution of Axin1 and β-catenin complexes strongly resembles that of Wnt-stimulated cells. Our findings argue that Wnt signals and APC mutations interfere with the turnover of phosphorylated β-catenin. Furthermore, our results suggest that the accumulation of small-sized β-catenin complexes may serve as an indicator of Wnt pathway activity in primary cancer cells.
Department of Cell Biology, Center for Molecular Medicine, Heidelberglaan 100, Utrecht 3584CX, The Netherlands.
Jan P
JP
Gerlach
Department of Surgery, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3584CX, The Netherlands.
Benjamin L
BL
Emmink
MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Hisashi
H
Nojima
Department of Surgery, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3584CX, The Netherlands.
Onno
O
Kranenburg
Department of Cell Biology, Center for Molecular Medicine, Heidelberglaan 100, Utrecht 3584CX, The Netherlands m.m.maurice@umcutrecht.nl.
Madelon M
MM
Maurice
D060466:Axin Protein / Q000378:metabolism*; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism*
C555451:AXIN1 protein, human; D060466:Axin Protein; C495270:CTNNB1 protein, human; D051176:beta Catenin
Axin1; Wnt signalling; blue native/SDS-PAGE; colon cancer; protein complexes; β-catenin