Wnt-inducible Lrp6-APEX2 interacting proteins identify ESCRT machinery and Trk-fused gene as components of the Wnt signaling pathway

Abstract

The canonical Wnt pathway serves as a hub connecting diverse cellular processes, including β-catenin signaling, differentiation, growth, protein stability, macropinocytosis, and nutrient acquisition in lysosomes. We have proposed that sequestration of β-catenin destruction complex components in multivesicular bodies (MVBs) is required for sustained canonical Wnt signaling. In this study, we investigated the events that follow activation of the canonical Wnt receptor Lrp6 using an APEX2-mediated proximity labeling approach. The Wnt co-receptor Lrp6 was fused to APEX2 and used to biotinylate targets that are recruited near the receptor during Wnt signaling at different time periods. Lrp6 proximity targets were identified by mass spectrometry, and revealed that many endosomal proteins interacted with Lrp6 within 5 min of Wnt3a treatment. Interestingly, we found that Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, was strongly enriched in the proximity of Lrp6. TFG depletion with siRNA, or knock-out with CRISPR/Cas9, significantly reduced Wnt/β-catenin signaling in cell culture. In vivo, studies in the Xenopus system showed that TFG is required for endogenous Wnt-dependent embryonic patterning. The results suggest that the multivesicular endosomal machinery and the novel player TFG have important roles in Wnt signaling.

Canonical Wnt signaling induces focal adhesion and Integrin beta-1 endocytosis

“…Wnt signaling causes the uptake of large amounts of extracellular fluid by macropinosomes ( Redelman-Sidi et al., 2018 ; Tejeda-Muñoz et al., 2019 ). In addition, a gene ontology analysis of biotinylated Wnt-induced Lrp6-APEX2 target proteins indicated that LRP6-interacting proteins were involved in actin cytoskeleton remodeling ( Colozza et al., 2020 ). To examine whether Wnt induced changes in the cytoskeleton, primary human corneal stromal (HCSF) fibroblasts ( Gallego-Muñoz et al., 2018 ) were treated with Wnt3a.…”

Regulation of the Low-Density Lipoprotein Receptor-Related Protein LRP6 and Its Association With Disease: Wnt/β-Catenin Signaling and Beyond

“…FlnA interacts with LRP6, and loss of FlnA impairs internalization of LRP6 and activation of Wnt/β-catenin signaling, resulting in decreased proliferation of neural progenitor cells ( Lian et al, 2016 ; Figure 3B ). Recently, LRP6 proximity proteins induced upon short-term Wnt3a treatment were identified using an LRP6-Apex2 fusion protein ( Colozza et al, 2020 ). Among them, Trk fused gene protein (TFG) appears to have an important role in Wnt3a-mediated LRP6 internalization and activation of Wnt/β-catenin signaling ( Colozza et al, 2020 ; Figure 3B ).…”

“…Recently, LRP6 proximity proteins induced upon short-term Wnt3a treatment were identified using an LRP6-Apex2 fusion protein ( Colozza et al, 2020 ). Among them, Trk fused gene protein (TFG) appears to have an important role in Wnt3a-mediated LRP6 internalization and activation of Wnt/β-catenin signaling ( Colozza et al, 2020 ; Figure 3B ).…”

Targeting Membrane Trafficking as a Strategy for Cancer Treatment

“…Endocytosis is known to play a role in cancer by causing a loss of cell adhesion or morphological polarity, which can lead to the malignant transformation of cells [ 14 , 15 , 16 ]. For example, the small GTPases Rab proteins, which regulate vesicle transport, protein trafficking, membrane targeting and fusion, also mediate vesicle dynamics, which can work with oncogenic signaling pathways to increase tumor formation [ 17 , 18 , 19 , 20 ].…”