Wnt signaling has multiple functions beyond the transcriptional effects of β-catenin stabilization. We review recent investigations that uncover new cell physiological effects through the regulation of Wnt receptor endocytosis, Wnt-induced stabilization of proteins (Wnt-STOP), macropinocytosis, increase in lysosomal activity, and metabolic changes. Many of these growth-promoting effects of canonical Wnt occur within minutes and are independent of new protein synthesis. A key element is the sequestration of glycogen synthase kinase 3 (GSK3) inside multivesicular bodies and lysosomes. Twenty percent of human proteins contain consecutive GSK3 phosphorylation motifs, which in the absence of Wnt can form phosphodegrons for polyubiquitination and proteasomal degradation. Wnt signaling by either the pharmacological inhibition of GSK3 or the loss of tumor-suppressor proteins, such as adenomatous polyposis coli (APC) and Axin1, increases lysosomal acidification, anabolic metabolites, and macropinocytosis, which is normally repressed by the GSK3-Axin1-APC destruction complex. The combination of these cell physiological effects drives cell growth.

Significance: There is a critical need for new drugs targeting Wnt-driven diseases. For example, 80% of colorectal cancers are initiated by Wnt-activating APC mutations which then require multiple additional mutations to progress into invasive cancer. Here we present cell biological studies showing that Wnt pathway activation, or mutation of the tumor suppressors APC or Axin, greatly increased macropinocytosis. In the presence of Wnt, membrane ruffles at the plasma membrane engulfed large volumes of extracellular fluids which were channeled for degradation in lysosomes. The experiments suggest that inhibition of multivesicular body formation, methylation, or the Na⁺/H⁺exchanger, may help prevent Wnt-driven cancer progression.

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Here, we report that Wnt-induced macropinocytosis is regulated through glycogen synthase kinase 3 (GSK3) and the β-catenin destruction complex. We find that mutation of Axin1, a tumor suppressor and component of the destruction complex, results in the activation of macropinocytosis. Surprisingly, inhibition of GSK3 by lithium chloride (LiCl), CHIR99021, or dominant-negative GSK3 triggers macropinocytosis. GSK3 inhibition causes a rapid increase in acidic endolysosomes that is independent of new protein synthesis. GSK3 inhibition or Axin1 mutation increases lysosomal activity, which can be followed with tracers of active cathepsin D, β-glucosidase, and ovalbumin degradation. Microinjection of LiCl into the blastula cavity of Xenopus embryos causes a striking increase in dextran macropinocytosis. The effects of GSK3 inhibition on protein degradation in endolysosomes are blocked by the macropinocytosis inhibitors EIPA or IPA-3, suggesting that increases in membrane trafficking drive lysosomal activity.

Significance: Endocytosis plays a fundamental role in Wnt signaling, leading to the sequestration of cytosolic GSK3 kinase inside multivesicular endosomes. Here we show an unexpected role for the enzyme protein arginine methyltransferase 1 (PRMT1) during Wnt signaling. In the case of the soluble tumor suppressor transcription factor Smad4, modification of a particular arginine was required before this protein could be phosphorylated by GSK3. Wnt3a addition caused rapid endocytosis that strikingly expanded the cellular liquid-phase compartment within 5–20 min. Wnt-induced vesicles contained arginine-methylated proteins, GSK3 and PRMT1 itself, within membrane-bound organelles. The translocation of these proteins into multivesicular endosomes via microautophagy was essential for canonical Wnt signaling.