Wnt Signaling Requires Sequestration of Glycogen Synthase Kinase 3 inside Multivesicular Endosomes

TLDR

Canonical Wnt signaling depends on inhibiting GSK3, yet the mechanism remains unknown. The study investigates whether Wnt signaling sequesters GSK3 into multivesicular bodies to inhibit its cytosolic activity. Wnt signaling sequesters GSK3 into multivesicular bodies, lowering its cytosolic activity, protecting it from protease, and extending the half‑life of β‑catenin and other proteins, and the requirement of MVB formation proteins HRS/Vps27 and Vps4 shows that MVBs are essential for Wnt signal transduction.

Abstract

Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here, we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt colocalized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmunoelectron microscopy showed that these corresponded to MVBs. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to β-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal-transduction pathway.