The nutrient-sensing metabolite S-adenosylmethionine (SAM) controls one-carbon metabolism by donating methyl groups to biochemical building blocks, DNA, RNA, and protein. Our recent work uncovered a requirement for cytoplasmic arginine methylation during Wnt signaling through the activity of protein arginine methyltransferase 1 (PRMT1), which transfers one-carbon groups from SAM to many protein substrates. Here, we report that treatments that decrease levels of the universal methyl donor SAM were potent inhibitors of Wnt signaling and of Wnt-induced digestion of extracellular proteins in endolysosomes. Thus, arginine methylation provides the canonical Wnt pathway with metabolic sensing properties through SAM. The rapid accumulation of Wnt-induced endolysosomes within 30 minutes was inhibited by the depletion of methionine, an essential amino acid that serves as the direct substrate for SAM production. We also found that methionine is required for GSK3 sequestration into multivesicular bodies through microautophagy, an essential step in Wnt signaling activity. Methionine starvation greatly reduced Wnt-induced endolysosomal degradation of extracellular serum proteins. Similar results were observed by addition of nicotinamide (vitamin B3), which serves as a methyl group sink. Methotrexate, a pillar in the treatment of cancer since 1948, decreases SAM levels. We show here that methotrexate blocked Wnt-induced endocytic lysosomal activity and reduced canonical Wnt signaling. Importantly, the addition of SAM during methionine depletion or methotrexate treatment was sufficient to rescue endolysosomal function and Wnt signaling. Inhibiting the Wnt signaling pathway by decreasing one-carbon metabolism provides a platform for designing interventions in Wnt-driven disease.

Arginine methylation has emerged as a widespread and reversible protein modification with the potential to regulate a multitude of cellular processes, but its function is poorly understood. Endolysosomes play an important role in Wnt signaling, in which glycogen synthase kinase 3 (GSK3) becomes sequestered inside multivesicular bodies (MVBs) by the process known as microautophagy, causing the stabilization of many proteins. Up to 20% of cellular proteins contain three or more consecutive putative GSK3 sites, and of these 33% also contain methylarginine (meArg) modifications. Intriguingly, a cytoskeletal protein was previously known to have meArg modifications that enhanced subsequent phosphorylations by GSK3. Here, we report the unexpected finding that protein arginine methyltransferase 1 (PRMT1) is required for canonical Wnt signaling. Treatment of cultured cells for 5-30 min with Wnt3a induced a large increase in total endocytic vesicles which were also positive for asymmetric dimethylarginine modifications. Protease protection studies, both biochemical and in situ in cultured cells, showed that many meArg-modified cytosolic proteins became rapidly translocated into MVBs together with GSK3 and Lys48-polyubiquitinated proteins by ESCRT-driven microautophagy. In the case of the transcription factor Smad4, we showed that a unique arginine methylation site was required for GSK3 phosphorylation and Wnt regulation. The enzyme PRMT1 was found to be essential for Wnt-stimulated arginine methylation, GSK3 sequestration, and canonical Wnt signaling. The results reveal a cell biological role for PRMT1 arginine methylation at the crossroads of growth factor signaling, protein phosphorylation, membrane trafficking, cytosolic proteolysis, and Wnt-regulated microautophagy.

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Wnt treatment markedly increased the endocytosis and degradation in lysosomes of BSA. In this study, we report that in addition to receptor-mediated endocytosis, Wnt also triggers the intake of large amounts of extracellular fluid by macropinocytosis, a nonreceptor-mediated actin-driven process. Macropinocytosis induction is rapid and independent of protein synthesis. In the presence of Wnt, large amounts of nutrient-rich packages such as proteins and glycoproteins were channeled into lysosomes after fusing with smaller receptor-mediated vesicles containing glycogen synthase kinase 3 (GSK3) and protein arginine ethyltransferase 1 (PRMT1), an enzyme required for canonical Wnt signaling. Addition of Wnt3a, as well as overexpression of Disheveled (Dvl), Frizzled (Fz8), or dominant-negative Axin induced endocytosis. Depletion of the tumor suppressors adenomatous polyposis coli (APC) or Axin dramatically increased macropinocytosis, defined by incorporation of the high molecular weight marker tetramethylrhodamine (TMR)-dextran and its blockage by the Na⁺/H⁺ exchanger ethylisopropyl amiloride (EIPA). Macropinocytosis was blocked by dominant-negative vacuolar protein sorting 4 (Vps4), indicating that the Wnt pathway is dependent on multivesicular body formation, a process called microautophagy. SW480 colorectal cancer cells displayed constitutive macropinocytosis and increased extracellular protein degradation in lysosomes, which were suppressed by restoring full-length APC. Accumulation of the transcriptional activator β-catenin in the nucleus of SW480 cells was inhibited by methyltransferase inhibition, EIPA, or the diuretic amiloride. The results indicate that Wnt signaling switches metabolism toward nutrient acquisition by engulfment of extracellular fluids and suggest possible treatments for Wnt-driven cancer progression.

Vitamin B micronutrients are essential regulators of one carbon metabolism that ensures human health. Vitamin B9, or folate, lies at the heart of the folate cycle and converges with the methionine cycle to complete the one carbon pathway. Additionally, vitamin B6 contributes by orchestrating the flux of one carbon cycling. Dysregulation of vitamin B contributes to altered biochemical signaling that manifests in a spectrum of human diseases. This review presents an analysis of the past, present, and future work, highlighting the interplay between folate and vitamin B6 in one carbon metabolism. Emerging insights include advances in metabolomic-based mass spectrometry and the use of live-cell metabolic labeling. Cancer is used as a focal point to dissect vitamin crosstalk and highlight new insights into the roles of folate and vitamin B6 in metabolic control. This collection of vitamin-based research detailing the trends of one carbon metabolism in human disease exemplifies how the future of personalized medicine could unfold using this new base of knowledge and ultimately provide next-generation therapeutics.

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.

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.