The Methionine Cycle Regulates PRMT1 Localization and Endocytosis During Wnt Signaling.

The data above were compatible with a link between one-carbon metabolism and Wnt signaling. One-carbon metabolism is required for arginine methylation, a reversible posttranslational modification that occurs almost as frequently as phosphorylation (50). Work from the E.M.D.R. laboratory and others demonstrates that arginine methylation mediates growth factor signaling through PRMT1, a type I enzyme that catalyzes ω-N^G,N^G-asymmetric dimethylation (26, 28). Derynck and coworkers showed that PRMT1-mediated arginine methylation of cytoplasmic Smad proteins is essential for BMP and TGF-β signaling (51, 52). Similarly, we found that PRMT1 was required for Wnt signaling, catalyzing arginine methylation of cytosolic substrates that become sequestered in endolysosomes (1). These data challenge the view that methylation mostly operates in the nucleus. Despite the importance of PRMT1 in cell signaling, relatively little is known about the regulation of cytoplasmic arginine methylation.

Methionine availability dictates SAM levels, which in turn directly impacts PRMT activity (53). The methyltransferase inhibitor adenosine-2′,3′-dialdehyde (Adox), strongly inhibits PRMT1 relocalization into endolysosomes by Wnt (1). To investigate whether the loss of methionine similarly decreased the sequestration of PRMT1, cells were stained with PRMT1 and GSK3 antibodies and subjected to protease protection immunofluorescence analyses. Wnt3a led to the relocalization of PRMT1 into large endolysosomal vesicles that also contained GSK3 (Fig. 2 A and B) (1). Methionine depletion dramatically decreased Wnt-induced PRMT1 sequestration and colocalization with GSK3 in vesicles (Fig. 2 C and D). Quantification of this colocalization by Pearson’s coefficient underscored the significant effect shown in Fig. 2E, indicating that methionine is essential for PRMT1 endolysosomal translocation.

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Fig. 2.

Wnt-induced endocytosis and lysosomal activity require methionine and PRMT1. (A–D) Methionine depletion inhibited PRMT1 sequestration into GSK3-containing vesicles by Wnt3a treatment in in situ protease protection analyses (compare B to D). (E) PRMT1 and GSK3 colocalization in vesicles quantified by Pearson’s correlation coefficient using ImageJ. (F–H) Methionine depletion decreased Wnt-induced endolysosomal activity, assessed by the endocytosis and digestion of BSA-DQ. (I–K) PRMT1 depletion with siRNA (siPRMT1) decreased Wnt3a-induced lysosomal activity compared with siScrambled control (siCtr) cells (compare J to K), as assessed by endocytosis of BSA-DQ. (L) Diagram of the BSA-DQ assay, which is endocytosed and fluoresces only after the protein is digested in lysosomes. (M) Lysosomal activity quantification of BSA-DQ fluorescence per cell after 30 min of methionine depletion. (N) Wnt-induced degradation of BSA-DQ in lysosomes requires the PRMT1 enzyme (ImageJ quantification per cell). (Scale bars: 10 μm.) **P < 0.01; n < 4.

Wnt-Induced Endocytosis Requires Methionine.

Canonical Wnt is emerging as a major regulator of the endocytic capacity of the cell (1, 19, 54, 55). Wnt signaling increased endocytosis and lysosomal digestion of BSA dequenched (BSA-DQ), a marker of nonreceptor-mediated endocytosis that fluoresces only after endocytosed BSA is digested in lysosomes (Fig. 2L) (1, 49). We next evaluated whether Wnt-induced BSA-DQ endocytosis and degradation after Wnt treatment was dependent on methionine levels. Wnt3a addition for 30 min increased BSA-DQ endocytosis into lysosomes in methionine-containing medium while methionine depletion significantly diminished BSA-DQ fluorescence (Fig. 2 F–H and M). To study the molecular requirements of Wnt-induced endocytosis, we next incubated cells with siRNA targeting PRMT1 and assessed endolysosomal activity during Wnt signaling. Comparison of cells treated with scrambled control siRNA (siCtr) or siPRMT1 showed greatly reduced endocytosis and lysosomal degradation of BSA (Fig. 2 I–K, quantitated in Fig. 2N), supporting the idea that PRMT1 is critical for Wnt endocytosis. Together, these data indicate that PRMT1 and methionine are required for Wnt-induced endolysosomal activity.

SAM Addition Rescues Endocytosis and Wnt Signaling.

The response to methionine starvation underscores the plasticity of metabolism that allows cells to dynamically adapt to stresses such as nutrient depletion (53). To directly test whether methionine levels regulate Wnt signaling through SAM, the methyl cofactor for PRMT1, we first examined whether SAM was sufficient to rescue the effects of methionine depletion on Wnt signaling. SAM added to the culture medium at high concentrations has been shown to enter cultured cells (36, 56). We incubated HeLa cells in medium with or without methionine and examined the effects of SAM addition on vesicle formation and protein sequestration. The lack of vesicle formation observed during methionine depletion was completely rescued in the presence of 1 mM SAM following Wnt3a treatment (Fig. 3 A, E, and I). Further, the translocation of PRMT1 and GSK3 inside protease-protected vesicles during Wnt activation was also completely rescued when cells were incubated with SAM (Fig. 3 A–L; quantitated in Fig. 3M).

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Fig. 3.

SAM addition to the culture medium rescues inhibition of Wnt signaling. (A–L) Methionine depletion decreased Wnt-induced vesicle formation and inhibited the Wnt-induced sequestration of PRMT1 and GSK3 after 30 min of Wnt3a treatment, and this effect was rescued by the addition of 1 mM SAM to the culture medium. (M) Quantification of the rescue of PRMT1 and GSK3 protease protection in vesicles by Pearson’s correlation coefficient. (N) Addition of 1 mM SAM increased Wnt-induced BAR luciferase activity when methionine was depleted. (O) Schematic depiction of the effect of addition of nicotinamide (vitamin B3) to the culture medium. Nicotinamide becomes readily methylated (generating 1-methylnicotinamide) and serves as a sink to store methyl groups and decrease SAM levels (59, 60). (P) Addition of 8 mM nicotinamide to the culture medium inhibited BAR/β-catenin luciferase activity. (Scale bars: 10 μm.) **P < 0.01; n < 3.

We next examined the effect of SAM addition on canonical Wnt signaling using the BAR/β-catenin luciferase reporter assay. Cells were incubated in medium with or without methionine, stimulated with Wnt3a, and supplemented with SAM. Wnt/β-catenin signaling in the absence of methionine was markedly increased by SAM addition to the culture medium (Fig. 3N). This indicated that the effects of methionine depletion on the Wnt pathway were mediated by a deficiency in SAM, the universal methyl donor. This also indicates that the inhibition of Wnt signaling by lowering methionine was not caused indirectly by a generalized defect in protein synthesis, since it could be replaced by a downstream metabolite. Intriguingly, SAM led to a twofold increase in Wnt signaling compared with cells without SAM even in the absence of methionine (Fig. 3N, lane 4). Perhaps the rapid endocytosis induced by Wnt may facilitate SAM incorporation into the cell (57).

Nicotinamide as a Methyl Sink.

Beyond the one-carbon metabolic pathway, additional metabolites can be exploited to alter global cellular methylation by directly affecting SAM concentrations (58). Nicotinamide (NAM, also known as vitamin B3) is the precursor to NAD⁺ and has been reported to act as a “methyl sink” to decrease SAM levels, draining methyl units from the cytoplasm to produce 1-methyl-NA (1MNA) (Fig. 3O) (59, 60). To examine whether decreasing SAM levels in this way would also affect Wnt signaling, we performed Wnt luciferase assays in permanently transfected BAR reporter HEK293T cells following treatment with nicotinamide. Compared with control cells, nicotinamide significantly decreased Wnt signaling activity (Fig. 3P). Taken together, the data with SAM rescue and the nicotinamide sink strongly support a model whereby SAM levels are intrinsically linked to the Wnt signaling pathway by promoting arginine methylation, sequestration of cytosolic PRMT1 and GSK3 in MVBs (1), and increasing endocytosis and lysosomal degradation of extracellular proteins.

Methionine Depletion and Nicotinamide Experiments.

Briefly, cells were grown in DMEM or RPMI 1640 supplemented with 10% FCS, glutamine, and 100 units/mL penicillin. For immunofluorescence methionine depletion experiments, HeLa cells were plated on coverslips at a density of 60% and incubated in either normal RPMI or methionine-free RPMI for the indicated times, with or without 100 ng/mL Wnt3a for 30 min. For luciferase assays, HEK293T cells stably transfected with BAR/Renilla DNA (49) were plated onto 12 wells and methionine depletion was performed over a 12-h time course, with or without Wnt3a (100 ng/mL) stimulation. Methionine depletion was performed with methionine-free RPMI 1640 supplemented with 10% of either dialyzed or nondialyzed FCS; both gave similar results. Nicotinamide has been previously reported to reduce SAM levels (59). Luciferase assays were performed in cells treated with nicotinamide (8 mM) with or without Wnt3a for 12 h.

Methotrexate Experiments.

Folate cycle inhibition with methotrexate (20 μM) was performed in HeLa cells for 1 h in folate-free RPMI medium 1640, before adding Wnt3a and assessing by immunofluorescence analyses. Cells displayed normal morphology. BAR-Renilla HEK293T cells were used to measure Wnt/β-catenin activity in the presence of 20 μM methotrexate after preincubation for 2 h in folate-free RPMI medium 1640 followed by overnight treatment with Wnt3a for 12 h. Cellular toxicity was assessed at methotrexate concentrations ranging from 5 μM to 60 μM.

Rescue Experiments.

Methionine depletion and methotrexate treatments were rescued by adding 1 mM SAM. For immunofluorescence assays, plated cells were incubated with methotrexate or in methionine-free medium for 2 h. The addition of 1 mM SAM to plated cells was performed for 30 min before Wnt3a stimulation for 30 min. For luciferase assays, cells were preincubated with methotrexate or methionine-free medium for 2 h before the addition of 1 mM SAM, and subsequently treated with Wnt3a protein (12 h). For rescue experiments with methionine, cells were incubated in methionine-depleted RPMI medium for 12 h and subsequently in standard RPMI 1640 for 12 h.

Cell Lines and Tissue Culture.

HeLa and HEK293T were cultured in DMEM or RPMI 1640 supplemented with 10% FCS (GIBCO), 1% penicillin, and 1% glutamine. Cells were maintained at 37 °C and 5% CO₂.

Plasmids.

Plasmids used in this study were BAR-luciferase and Renilla (under the control of the elf1a promoter) reporters, a generous gift from Randall Moon, University of Washington, Seattle. Human PRMT1 siPRMT1 oligos (1) (sc-41069; Santa Cruz Biotechnology) targeted three regions with the following sequences: (i) 5′-gcaactccatgtttcataa-3′; (ii) 5′-gttccagtatctctgatta-3′; and (iii) 5′-cgacatgtcttgcatcaaa-3′. The negative control was Scrambled siRNA (D-001810, Thermo Fisher Scientific).

Wnt Treatments.

Recombinant murine Wnt3a protein (PeproTech, 315-20) was used for Wnt signaling studies (100 ng/mL). For protease protection studies, cells processed after 30 min of Wnt3a treatment at 37 °C. For luciferase assays, Wnt3a was added for 12 h.

Protease Protection and Endocytosis Assays.

In situ protease protection assays were performed to determine whether proteins were contained within membrane-bounded organelles as described previously (1). Briefly, cells were incubated with Wnt3a for 30 min, placed on ice, washed twice with PBS, incubated with 6.5 μg/mL digitonin in PBS for 15 min on ice, washed twice with ice-cold PBS to remove digitonin, and subsequently incubated with proteinase K (1 μg/mL) for 10 min (1). PFA fixation stopped the reaction and immunofluorescence preparation was performed as described below. To measure endocytosis and lysosomal activity following Wnt activation, we used BSA-DQ. Cells were plated onto 12-well dishes (60% confluency) and incubated with 5 μg/mL of BSA-DQ in the presence or absence of Wnt3a. Endocytosis was measured as the intensity of fluorescence from lysosome-digested BSA-DQ. For immunofluorescence analyses, cells plated on 12-well dishes were incubated with BSA-DQ for 30 min in the presence or absence of Wnt3a. Cells were subsequently washed with PBS and subjected to standard immunofluorescence processing which included fixation with 4% PFA for 15 min at room temperature in the dark, washing with PBS, and mounting with ProLong Gold antifade reagent with DAPI (Life Technologies).

Immunofluorescence Assays.

Immunofluorescence analyses were performed as described previously (1). In short, cells were grown in 10-cm dishes and replated onto 12-well dishes containing glass coverslips. Cells were allowed to attach to coverslips for 24 h before Wnt treatment. Cells were washed twice with PBS to remove medium, fixed in 4% (wt/vol) PFA (P6148, Sigma) in PBS for 20 min, and permeabilized with 0.2% (vol/vol) Triton X-100 in PBS for 10 min on ice. Next, coverslips were washed with PBS, blocked for 1 h in BSA blocking buffer composed of 5% (vol/vol) goat serum plus 0.5% (wt/vol) BSA at room temperature, and incubated with primary antibodies in blocking buffer overnight at 4 °C (1:100–1:1,000, diluted in blocking buffer). Finally, cells were washed three times with PBS, incubated with secondary antibodies produced in donkey for 45 min (1:1,000, diluted in blocking buffer), and mounted onto glass slides in ProLong Gold antifade reagent with DAPI (Life Technologies) to stain cell nuclei. Images were acquired on a Zeiss Imager Z.1 microscope with Apotome using a 63× oil immersion objective. Zeiss and ImageJ (NIH) imaging software were used for image analyses. The excitation filters used to capture the images were 488 nm, 568 nm, and 405 nm (DAPI) using Alexa 568- and Alexa 488-conjugated secondary antibodies.

We thank Randy Moon (University of Washington) for reporter genes and Richard L. Watson (Cedar Sinai), Duane G. Albrecht (University of Texas, Austin), Douglas Geissert, Samantha Rundell, Alyssa Dsouza, Yi Ding, Gabriele Colozza, and Nydia Tejeda-Munoz [University of California, Los Angeles (UCLA)] for comments on the manuscript. L.V.A. is supported by an NIH postdoctoral fellowship (NIH F32 GM123622) and M.H.B. is funded by a UCLA Clinical and Translational Institute undergraduate fellowship (CTSI UL1TR000124). This work was supported by the Norman Sprague Endowment in Molecular Oncology and the Howard Hughes Medical Institute, of which E.M.D.R. is an investigator.