Arginine methylation is required for canonical Wnt signaling and endolysosomal trafficking

Albrecht, Lauren V.; Ploper, Diego; Tejeda-Muñoz, Nydia; Robertis, Edward M. De

doi:10.1073/pnas.1804091115

Cited by 65 publications

(151 citation statements)

References 40 publications

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“…In summary, our searchable list of Lrp6-APEX2 biotinylated targets (Dataset S1, Tab 1 and Tab 4) offers the scientific community a valuable roadmap for the discovery of novel proteins involved in Wnt signaling and endocytosis. Importantly, it independently confirms our earlier proposals (11) that the ESCRT machinery and endocytosis (15,16) participate in Wnt signaling.…”

Section: Apex2 Hek293t Cells (Preincubated With Porcupine Inhibitor Tsupporting

confidence: 90%

“…Here, we describe the results obtained with a Human Embryonic Kidney 293T (HEK293T) cell line stably expressing a chimeric Lrp6-APEX2 receptor. Our proteomic data indicate that in presence of Wnt3a, the Lrp6-interactome was enriched in components of the ESCRT machinery that forms MVBs, providing independent support to our previous findings that endocytosis is at the core of Wnt pathway intracellular regulation (11,15,16). We also discovered that Tropomyosin-receptor kinase fused gene (Trk-fused gene, TFG), a protein involved in several pathologies including cancer (26), was a highly enriched target of Wntactivated Lrp6-APEX2 and localized to endosomal vesicles.…”

Section: Introductionsupporting

confidence: 89%

“…A guide RNA (sgRNA) targeting a sequence proximal to TFG start codon ( Fig. 4A) was subcloned into a Cas9 expression vector and transfected into HEK293T cells harboring BAR Luciferase/Renilla reporters (15). Western blot analyses confirmed complete depletion of TFG protein in a knock-out clonal cell line derived by limiting dilution (Fig.…”

Section: Apex2 Hek293t Cells (Preincubated With Porcupine Inhibitor Tmentioning

confidence: 91%

“…During the 30 years since its discovery (13,14), the Wnt pathway has been the subject of intense studies, but its molecular mechanisms have not yet been fully elucidated, in particular the role of membrane trafficking in receptor activation (11,12,(15)(16)(17). To dissect the dynamics of Wnt signaling, we decided to use a protein interaction approach to identify additional regulators of the pathway.…”

Section: Introductionmentioning

confidence: 99%

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Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

Colozza

Jami‐Alahmadi²,

Dsouza

et al. 2020

Preprint

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The canonical Wnt signaling pathway serves as a hub connecting diverse cellular physiological processes, such as 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 components of the ESCRT (Endocytic Sorting Components Required for Transport) machinery interacted with Lrp6 within 5 minutes of Wnt3a treatment. This supports the proposal of a central role of multivesicular endosomes in canonical Wnt signaling. Interestingly, proteomic analyses identified the Trk-fused gene (TFG), previously known to regulate the cell secretory pathway and to be rearranged in thyroid and lung cancers, as being strongly enriched in the proximity of Lrp6. We provide evidence that TFG specifically co-localized with MVBs after Wnt stimulation. 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. SignificanceWnt/β-catenin signaling is a conserved pathway involved in cell differentiation and in the regulation of many other processes, including cell growth and proliferation, macropinocytosis, and cell metabolism. Endocytosis is required to regulate Wnt signaling, but the precise factors at play are still elusive. Here, we describe a biotin-dependent proximity labeling approach using ascorbate peroxidase-tagged Lrp6, a Wnt co-receptor. Proteomic analysis of biotinylatedenriched targets identified numerous multivesicular endosome proteins that were recruited to the receptor shortly after addition of Wnt protein. Additionally, we identified the protein TFG as one of the strongest interactors with Lrp6. TFG co-localized with Wnt-induced multivesicular endosomes. Xenopus embryo assays revealed that TFG is required in vivo for canonical Wnt signaling. \body

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Section: Apex2 Hek293t Cells (Preincubated With Porcupine Inhibitor Tsupporting

confidence: 90%

Section: Introductionsupporting

confidence: 89%

Section: Apex2 Hek293t Cells (Preincubated With Porcupine Inhibitor Tmentioning

confidence: 91%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

Colozza

Jami‐Alahmadi²,

Dsouza

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

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“…It is known that Notum is regulated by β-catenin and change in spatial expression of Notum suggests potential changes in β-catenin activity [35]. Given the recently shown importance of methylation and one-carbon metabolism in canonical Wnt signaling by De Robertis group, we speculate that disruption of the methionine cycle may lead to failure in planarian body plan maintenance via changes in Wnt signaling [36], [37]. Although we did not find β-catenin target genes among differentially expressed genes in AdOx-adapted worms, it does not exclude possible changes in post-translational regulation.…”

Section: Discussionmentioning

confidence: 99%

S-adenosylhomocysteine hydrolase regulates anterior patterning inDugesia japonica

Reinmets

Bischof

Taketa

et al. 2020

Preprint

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BackgroundBiological methylation requires S-adenosylmethionine (SAM) and participates in a range of processes from modulation of gene expression via histone modifications to neurotransmitter synthesis. An important factor in all methylation reactions is the concentration ratio of SAM to methylation byproduct S-adenosylhomocysteine (SAH). SAH hydrolase, also known as adenosylhomocysteinase, depletes SAH and thereby facilitates metabolite recycling and maintains the methylation permissive SAM/SAH ratio. While the importance of SAH hydrolase in sustaining methylation is obvious on the cellular level, the function of this metabolic process on the organismal scale is not clear.ResultsWe used planarian Dugesia japonica to investigate the role SAH hydrolase in physiological homeostasis on the body-wide scale. Remarkably, pharmacological inhibition of the SAH hydrolase results in regression of anterior tissues and is accompanied by extensive apoptosis throughout the planarian body. Moreover, exposure to the SAHH inhibitor AdOx leads to changes in brain morphology and spatial shift in the expression of Wnt-modulator Notum. Strikingly, planarians are able to overcome these destructive patterning defects through regeneration of the anterior tissues and adaptation to the used inhibitor. Transcriptome analysis indicates that resistance to the SAHH inhibitor is at least partly mediated by changes in folate cycle and lipid metabolism.ConclusionsSAH hydrolase plays a critical role in planarian homeostasis and anterior patterning potentially through modulation of Wnt signaling. Moreover, planarian adaptation to the SAHH inhibitor via metabolic reprogramming suggests potential targets for addressing methylation-related human conditions.

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Zooming in on the WNT/CTNNB1 Destruction Complex: Functional Mechanistic Details with Implications for Therapeutic Targeting

Man

Amerongen

2021

Pharmacology of the WNT Signaling System

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Arginine methylation is required for canonical Wnt signaling and endolysosomal trafficking

Cited by 65 publications

References 40 publications

Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

S-adenosylhomocysteine hydrolase regulates anterior patterning inDugesia japonica

Zooming in on the WNT/CTNNB1 Destruction Complex: Functional Mechanistic Details with Implications for Therapeutic Targeting

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