Wnt Increases Overall Endocytosis and meArg Microautophagy.

Wnt-induced meArg accumulated within LysoTracker (which accumulates inside acidic organelles) and BSA-dequenched (DQ)-positive (which fluoresces only after BSA is degraded in endolysosomes) vesicles within 15 min of Wnt treatment (Fig. 2 A–D). The increases in colocalization between meArg and lysosomal markers were over threefold. The rapid entry of BSA into the cell after Wnt treatment could be visualized in real time through the accumulation and degradation of BSA-DQ 20 min after the addition of Wnt3a to 3T3 fibroblasts (SI Appendix, compare Movies S1 and S2). Remarkably, the degradation of BSA-DQ took place in the presence of 10% serum; since the endocytosis of BSA is nonreceptor mediated, this suggests that Wnt induces a large increase in endolysosomal trafficking. In agreement with this, total endocytosis into the liquid-phase compartment of HeLa cells was strongly increased by Wnt3a after 15 min of addition of either BSA-FITC or BSA-DQ (Fig. 2E). Quantification by flow cytometry confirmed this increase in overall endocytosis, even in the presence of cycloheximide (Fig. 2 F–H). Thus, canonical Wnt signaling emerges as a major regulator of the endocytic capacity of the cell.

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Fig. 2.

Wnt signaling increases endocytosis, and arginine-methylated proteins are sequestered by microautophagy. (A and A′) meArg colocalized with LysoTracker. Panel A′ was treated with Wnt3a for 15 min. (B) Wnt increased colocalization of meArg puncta with LysoTracker endolysosomes by Pearson’s correlation coefficient. (C and C′) Methylarginine and BSA-DQ lysosomal marker (arrowheads) colocalize with Wnt3a. Panel C′ was treated with Wnt3a for 15 min. (D) Wnt increased colocalization of meArg puncta with BSA-DQ endolysosomes by Pearson’s correlation coefficient. (E) Wnt signaling (15 min) increased endocytosis of BSA-FITC and BSA-DQ into the cell liquid phase by quantification of fluorescence per cell; n > 20 cells per sample. (F and G) Wnt signaling increased BSA-FITC and BSA-DQ uptake by flow cytometry (4-h treatment) (36). (H) Blocking protein synthesis with cycloheximide did not prevent Wnt-induced BSA-DQ endocytosis. (I–K) Wnt-induced meArg sequestration into MVBs marked by Vps4-GFP (arrowheads) was blocked by DN-Vps4. (L–N) Lys48-polyUb proteins colocalized with Vps4-GFP MVBs (arrowheads) 15 min after Wnt3a treatment and this required active Vps4. (Scale bars, 10 μm.) *P < 0.05.

To investigate the cellular mechanism by which meArg proteins are relocalized during Wnt signaling, vacuolar protein sorting-associated protein 4 (Vps4) was transfected into HeLa cells with or without Wnt treatment. Vps4, a component of the ESCRT machinery required for the final stages of intraluminal vesicle formation in MVBs (17), colocalized strongly with meArg proteins following Wnt activation (Fig. 2 I and J). A dominant-negative Vps4 (DN-Vps4-GFP) containing a point mutation within the ATPase domain inhibits microautophagy (18). In the presence of DN-Vps4-GFP, meArg did not accumulate in vesicles even in the presence of Wnt3a (Fig. 2K). Many cytosolic proteins are Lys48-polyubiquitinated and degraded in proteasomes. In the presence of Wnt, however, GSK3-phosphorylated proteins modified by Lys48-polyUb accumulate in MVBs by microautophagy (14). As in the case of meArg, Lys48-polyUb colocalized with Vps4-GFP after Wnt3a addition, but not in the presence of a DN-Vps4-GFP (Fig. 2 L–N). These results indicate that the microautophagy ESCRT machinery is required for Wnt-induced meArg and Lys48-polyUb protein sequestration in MVBs.

Methylation Is Required for GSK3 Sequestration.

To test whether methylation is required for Wnt-induced GSK3 sequestration, we used the competitive methylation inhibitor adenosine-2′,3′-dialdehyde (Adox). Preincubation of HeLa cells with 20 μM Adox prevented the relocalization of GSK3 into LAMP1-positive vesicles after treatment with Wnt3a for 15 min (Fig. 3 A–C′). In signaling assays, the β-catenin-activated luciferase reporter (BAR) activation by Wnt3a was significantly inhibited in the presence of Adox (Fig. 3D). Adox inhibited arginine methylation (Fig. 3 E–G) and prevented the accumulation of Lys48-polyUb in membrane vesicles (Fig. 3 H–J). Thus, methylation is required for the sequestration of GSK3 and cytosolic substrates during Wnt signaling.

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Fig. 3.

Arginine-methylation inhibition blocks GSK3 sequestration and Wnt signaling. (A–C′) GSK3 sequestration into endolysosomes by Wnt signaling was inhibited by the methylation inhibitor Adox (24-h preincubation, 15 min of Wnt3a). Panels indicated with prime were treated with Wnt3a. (D) Adox blocks Wnt signaling (BAR/Renilla assay) (39). (E–J) Wnt-induced meArg and Lys48-polyUb puncta colocalize (arrowheads) and are inhibited by Adox. (K) Model in which soluble cytosolic proteins are first modified by meArg, which facilitates GSK3 phosphorylation and polyubiquitination (see also Fig. 4). (Scale bars, 10 μm.) **P < 0.01.

An Arginine Methylation Site in Smad4 Is Required for Endolysosomal Localization and Wnt Regulation.

The results thus far suggested the translocation of a large number of cellular proteins modified by meArg into lysosomes under the influence of Wnt. To provide a specific example of a cytosolic Wnt-regulated protein that becomes sequestered in MVBs, we chose Smad4 (19, 20), a GSK3 substrate that our laboratory has studied in detail (21) (Fig. 4A). The choice of Smad4, also called deleted in pancreatic carcinoma 4 (DPC4), offered many advantages because it is not only an important tumor suppressor protein that is frequently inactivated in prostate, colorectal, and pancreatic carcinomas, but also a signaling protein for which sensitive luciferase reporter functional assays exist (19, 20). In addition, we had available custom-made antibodies that recognize specifically Smad4 phosphorylated by GSK3 (pSMAD4^GSK3) (21) that allow one to follow its intracellular localization.

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Fig. 4.

Smad4 provides an example of a protein that must be methylated before it can be phosphorylated by GSK3 and translocated into MVBs by Wnt signaling. (A) Diagram of how FGF/EGF, Wnt, and TGF-β/BMP signaling cross-talk at the level of Smad4. MAPK/FGF (green) primes phosphorylation by GSK3 (blue) at three sites; the meArg site discovered in this study is shown in red. (B) Wnt addition for 20 min increased Smad4 methylation in transfected HEK-293T cells. S4-Flag and GAPDH serve as loading controls. (C–E) Phospho-Smad4-Flag relocalized to vesicular structures after 15 min of Wnt3a addition, but only in the absence of the competitive methylation inhibitor Adox. (F) A potential Smad4 arginine-methylation site was mutated (R272K) to prevent arginine methylation with minimal effect on the protein. (G) Smad4-Flag-WT immunoprecipitated from transfected HEK-293T lysates was recognized by asymmetric dimethyl-Arg antibody while the Smad4-Flag-R272K mutant was not. Thus, Smad4 contains a single meArg site. (H) Smad4 phosphorylation by GSK3 requires arginine methylation. Ratios under each lane and the merge panels indicate GSK3 phosphorylated Smad4/total Smad4-Flag. (I–N) In situ protease protection assay using digitonin and proteinase K showing that wild-type Smad4-Flag was translocated inside membrane-bound organelles when Wnt was added for 15 min (J) but digested when Triton X-100 was added (K). Smad4-R272K-Flag was not translocated into membrane vesicles and was degraded by proteinase K (L and M). Panels I′–N′ show DAPI staining and differential interference contrast microscopy to visualize cellular contours in the corresponding cells shown above. (O) Smad4 wild type (WT), Smad4-R272K, and Smad4 mutated at the three GSK3 sites (phosphorylation-resistant Smad4-GM) were tested in TGF-β signaling assays. HaCaT cells permanently transfected with the CAGA12-luciferase reporter and constitutive CMV-Renilla (in which MAPK activation was primed by addition of EGF) were used. This indicates that in the context of TGF-β signaling arginine methylation is required for the integration of FGF, Wnt, and TGF-β signals. **P < 0.01; n.s., not significant statistically.

The transcription factor Smad4 functions as a co-Smad that binds to receptor-phosphorylated Smads (R-Smads) and is an essential component of the transforming growth factor β (TGF-β) and BMP signaling pathways (22) (Fig. 4A). Smad4 is a soluble protein located in the cytoplasm or in the nucleus depending on TGF-β/BMP signaling, which causes Smad4 binding to R-Smads phosphorylated by TGF-β or BMP receptors (Fig. 4A). Until recently, Smad4 was considered a constitutive and unregulated signaling component, but work from our laboratory has shown that the linker (middle) region of Smad4 contains a site phosphorylated by mitogen-activated protein kinase (MAPK) activated by FGF or EGF receptors and three GSK3 phosphorylation sites inhibited by Wnt (21) (Fig. 4A). MAPK phosphorylation promotes maximal activity by activating a Smad transcriptional activation domain and at the same time serves as a priming site for three subsequent inhibitory phosphorylations by GSK3. The GSK3 phosphorylations down-regulate transcriptional activity and recruit the E3 polyubiquitin ligase βTRCP leading to its degradation (21). TGF-β activity signaling is maximal when the FGF/EGF pathway and the Wnt pathway (which inhibits GSK3 phosphorylation) are activated. In this way, Wnt3a increases TGF-β/BMP signaling but only in the presence of FGF/EGF (21). This regulatory mechanism at the level of Smad4 is important for germ layer specification in the embryo and probably also in the function of Smad4 as a barrier against tumor progression (21, 23).

We discovered a putative meArg site Gly-Ser-Arg (GSR) sequence within the GSK3 phosphorylation sites of Smad4 (Fig. 1A, indicated in red). Importantly, this is the same sequence previously found by Albrecht et al. (5) to be arginine-methylated within GSK3 phosphorylation sites in desmoplakin, indicating that Smad4 was an excellent candidate for studying the role of arginine methylation at a key regulatory node at the intersection of important signaling pathways such as TGF-β, FGF/EGF, and Wnt. In Western blots, Smad4 was found to be arginine methylated and this modification was increased by Wnt3a treatment (Fig. 4B). Using a custom-made antibody for phospho-Smad^GSK3, we observed that phosphorylated Smad4 was translocated into vesicular structures after 15 min of Wnt3a addition, and that this relocalization was inhibited by preventing methylation with Adox (Fig. 4 C–E). Mutation of Arg-272 into Lys in the GSR region revealed that this was the only meArg site in Smad4 (Fig. 4 F and G). Phosphorylation by GSK3 was greatly decreased or eliminated in the methylation-resistant Smad4^R272K mutant (Fig. 4H). Further, in situ proteinase K protection assays showed that while Flag-tagged Smad4^WT was translocated inside membrane-bound organelles, the methylation-resistant mutant was not sequestered (Fig. 4 I–N). In TGF-β signaling assays using the CAGA12-luciferase reporter (21), Wnt3a increased TGF-β signaling in the presence of EGF with wild-type transfected Smad4 (Fig. 4O, lanes 2 and 3) as expected, while both methylation- and GSK3 phosphorylation-resistant mutants (Smad4-GM) had maximal activity but were insensitive to Wnt stimulation (Fig. 4O, lanes 4–9). The superactivity of Smad4-GM in which the three GSK3 sites are mutated into alanines had been documented previously (21); in this mutant MAPK phosphorylation is still required for maximal activity. The fact that Smad4-R272K is just as transcriptionally active as Smad4-GM or Smad4-WT treated with Wnt supports the view that the sole arginine-methylation site in Smad4 is required for subsequent phosphorylation by GSK3.

These experiments identify Smad4 as a GSK3 and arginine methylase soluble substrate that undergoes methylation-dependent translocation into protease-protected organelles after the addition of Wnt. Methylation-resistant Smad4 was maximally active but Wnt insensitive, suggesting indirectly that MAPK phosphorylation takes place, even though this construct is not phosphorylated by GSK3. The results are consistent with a biochemical pathway in which meArg modification of proteins facilitates GSK3 phosphorylation which, in turn, facilitates protein degradation in lysosomes (see diagram in Fig. 3K).

Cell Lines and Tissue Culture.

CAGA12-HaCaT, HeLa, HEK-293T, L cells (ATCC, CRL-2648), as well as L-Wnt3a cells (ATCC, CRL-2647) (37), or NIH 3T3 permanently transfected with a LAMP1-RFP lentivector (38), were cultured in DMEM supplemented with 10% FBS (GIBCO), 1% penicillin/streptomycin, and 1% glutamine. The cells were maintained at 37 °C and 5% CO₂.

Plasmids.

Plasmids used in this study were wild-type Vps4-GFP and dominant-negative DN-Vps4-GFP (18); constitutively active LRP6 (CA-LRP6) (7); BAR-Luc and Renilla reporters, a generous gift from Randall Moon, University of Washington, Seattle (39); wild-type Smad4-Flag and GM Smad4-Flag (21), and R272K Smad4-Flag generated in this study by PCR-based site-directed mutagenesis (QuikChange II Site-Directed Mutagenesis, Stratagene). The primers employed to generate the arginine methylation-resistant Smad4-R272K were: 5′-ctaccacctggactggaagtaagactgcaccatac-3′ and 5′-gtatggtgcagtcttacttccagtccaggtggtag-3′. Mutations were confirmed by sequencing. Human PRMT1-pCMV6 was purchased from OriGene (RC224239) and subcloned into pCS2+ using ClaI and StuI restriction enzymes; Xenopus PRMT1 cDNA (26) was a generous gift from Yun-Bo Shi, NIH. A pCS2+ xPRMT1 was generated by PCR amplification followed by an intermediate step of cloning xPRMT1 in the pGEM-4Z vector and excision with BamHI and ClaI restriction enzymes. The human PRMT1 siPRMT1 oligos (Santa Cruz Biotechnology, sc-41069) targeted three regions with the following sequences: 1: 5′-gcaactccatgtttcataa-3′; 2: 5′-gttccagtatctctgatta-3′; and 3: 5′-cgacatgtcttgcatcaaa-3′, all of which were not conserved in the Xenopus sequence which was used to rescue Wnt signaling inhibition by PRMT1 siRNA. The negative control was Scrambled siRNA (Thermo Fisher, D-001810).

Wnt Treatments and Endocytosis.

Recombinant murine Wnt3a protein (315–20) from PeproTech was used for Wnt signaling studies (80 ng/mL). Similar results were obtained with Wnt conditioned medium obtained from L cells stably expressing mouse Wnt3a (ATCC, CRL-2647) or control L cells were used to collect Wnt medium as described (37). A major difference between this study and our previous studies is that for immunoblotting, protease protection, and immunofluorescence studies, Wnt3a was added to cells for short time periods before fixation, ranging from 5 to 20 min at 37 °C. For luciferase assays, Wnt3a was added for 16 h. To measure endocytosis following Wnt activation, we assessed LysoTracker and BSA-DQ or BSA-FITC. Cells were plated onto 12-well dishes (60% confluency) and incubated with 5 μg/mL of BSA-DQ or BSA-FITC in the presence or absence of Wnt3a. For flow cytometry, following 4 h of Wnt activation, cells were washed twice with PBS, fixed in suspension with 4% (wt/vol) paraformaldehyde (PFA) for 15 min at room temperature, and washed with PBS at room temperature. Endocytosis was measured as the intensity of fluorescence from BSA-DQ and BSA-FITC by flow cytometry. Quantification of fluorescence by flow cytometry was performed in an LSRII flow cytometer (Becton Dickinson) as described previously (36). Ten thousand events per sample were collected by FACSDivav6.0 and analyzed by CellQuest software.

For immunofluorescence analyses, cells plated on 12-well dishes were incubated with LysoTracker (1:1,000), BSA-DQ, or BSA-FITC for 15 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).

Protease Protection Assays.

Protease protection assays were performed to assess whether proteins were contained within membrane-bound organelles (7, 8, 16). For in situ protease protection assays, which to our knowledge are unique, cells were incubated with Wnt3a for 5- to 20-min short time points, 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. As a control to show that proteinase K was active, Triton X-100 (0.1%), which dissolves all membranes, was added together with the protease. The reaction was stopped by fixing with PFA, followed by immunofluorescence analyses as described below. To examine protease-protected proteins by immunoblotting analyses, 2 million cells were plated on 10-cm dishes and transfected with 5 μg of CA-LRP6 to activate Wnt signaling. Then, 24 h after transfection, cells still attached to the culture dish were incubated with a 6.5 μg/mL digitonin solution containing 100 mM potassium phosphate pH 6.7, 5 mM MgCl₂, 250 mM sucrose (15 min on ice), washed with PBS, and incubated with proteinase K (1 μg/mL, 10 min), with or without Triton X-100 (0.1%). Finally, the reaction was stopped by adding cell lysis buffer 2× Laemmli sample buffer and boiling samples at 95 °C, before immunoblot analyses. Actin and GAPDH served as cytosolic protein controls.

Immunofluorescence Assays.

Immunofluorescence analyses were performed as described previously (7, 14). 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 (Sigma, P6148) 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 lasers used to capture the images were 488 nm, 568 nm, and 405 nm (DAPI) using Alexa 568- and Alexa 488-conjugated secondary antibodies. To detect asymmetric dimethylarginine in endolysosomes, two antibodies were used. All results shown here were with mouse monoclonal Abcam 21C7; however, rabbit polyclonal Sigma-Aldrich ASYM24 resulted in similar results with respect to Wnt-induced endolysosomes, but had more nuclear staining (presumably because it also reacts with monomethylarginine modifications).

We thank Prof. Yun-Bo Shi (NIH) for the Xenopus PRMT1 constructs; Roberto Zoncu (University of California, Berkeley) for Lamp1-RFP-lentivirus; Caroline Hill (Francis Crick Institute) for CAGA-HaCaT; Randy Moon (University of Washington) for reporter genes; Richard L. Watson (Cedars-Sinai Medical Center), Duane G. Albrecht (University of Texas at Austin), undergraduate students Jane Kwong and Maggie Bui, and other members of the E.M.D.R. laboratory for comments on the manuscript. L.V.A. is supported by an NIH postdoctoral fellowship (NIH F32 GM123622) and N.T.-M., by a University of California Institute for Mexico and the United States (UC MEXUS) and El Consejo Nacional de Ciencia y Tecnologia (CONACYT) postdoctoral fellowship (FE-17-65). This work was supported by the Norman Sprague Endowment and the Howard Hughes Medical Institute, of which E.M.D.R. is an investigator.