Background

The canonical Wnt signaling pathway controls numerous developmental processes. Over-activation of this pathway often leads to the development of cancer.

In experiments aimed at identifying new Wnt regulators, we isolated different members of the 14-3-3 family of proteins as interactors of the positive Wnt signaling regulator - Dishevelled-2 (Dvl-2). The 14-3-3 proteins are a family of conserved proteins that modulate diverse biological processes, including signal transduction pathways.

Method

In order to understand the connection between 14-3-3 and the Wnt pathway we used different assays: western blot analysis, immunoprecipitation, luciferase reporter assay, Immunofluorescence, live cell imaging, exosome purification and wound healing assay.

Results

We have found that 14-3-3ζ positively regulates the Wnt pathway and interacts with Dvl-2, Axin, Glycogen synthase kinase-3β (GSK3β) and adenomatous polyposis coli (APC), all known regulators of the Wnt pathway.

Moreover, we provide evidence showing that the kinase activity of GSK-3β is crucial for the binding between GSK-3β and 14-3-3ζ as well as 14-3-3ζ and Dvl-2. Interestingly, using fixed cells and live cell imaging we show that in a colorectal cancer cell line expressing high levels of Wnt signaling, 14-3-3ζ and β-catenin co-localize in exosomes that are formed only when both β-catenin and 14-3-3ζ are co-expressed. Isolation of these secreted bodies revealed that they express β-catenin as well as 14-3-3ζ. In a wound healing assay we find that co-expression of 14-3-3ζ and β-catenin increases cell migration.

Conclusion

Taken together, our results suggest that 14-3-3ζ binds Dvl-2 in a GSK-3β kinase dependent manner which in turn leads to β-catenin accumulation and formation and secretion of exosomes. We thus speculate the 14-3-3 family of protein may have novel functions in activation of the Wnt cascade and can alter the tumor microenvironment in a manner that may promote disease progression.