Short Communication | Published:

ADP-ribosylation factors 1 and 6 regulate Wnt/β-catenin signaling via control of LRP6 phosphorylation

Oncogene volume 32, pages 33903396 (11 July 2013) | Download Citation

Abstract

It has been shown that inhibition of GTPase-activating protein of ADP-ribosylation factor (Arf), ArfGAP, with a small molecule (QS11) results in synergistic activation of Wnt/β-catenin signaling. However, the role of Arf in Wnt/β-catenin signaling has not yet been elucidated. Here, we show that activation of Arf is essential for Wnt/β-catenin signaling. The level of the active form of Arf (Arf-GTP) transiently increased in the presence of Wnt, and this induction event was abrogated by blocking the interaction between Wnt and Frizzled (Fzd). In addition, knockdown of Fzds, Dvls or LRP6 blocked the Wnt-mediated activation of Arf. Consistently, depletion of Arf led to inhibition of Wnt-mediated membrane PtdIns (4,5)P2 (phosphatidylinositol 4, 5-bisphosphate) synthesis and LRP6 phosphorylation. Overall, our data suggest that transient activation of Arf modulates LRP6 phosphorylation for the transduction of Wnt/β-catenin signaling.

Introduction

Signaling mediated by the Wnt family of secreted glycolipoproteins is a fundamental mechanism that directs various biological processes, including embryonic development and tissue homeostasis in adult.1 In the absence of Wnt, cytosolic β-catenin, which is a key regulator of the canonical pathway, is constantly degraded by the action of the Axin destruction complex. However, binding of Wnt to its receptor Frizzled (Fzd) and co-receptor LRP6, followed by phosphorylation of the intracellular domain of LRP6 by Casein Kinase I and GSK3β, initiates the canonical Wnt pathway.2, 3, 4 Phosphorylated LRP6 provides docking sites for Axin and the recruitment of which to the plasma membrane leads to dissociation of the β-catenin/Axin destruction complex and consequently stabilizes cytoplasmic β-catenin. Following this, stable β-catenin translocates into the nucleus where it activates Tcf/Lef1 transcription co-factors, which promote the expression of target genes.5, 6, 7

A previous study showed that the small molecule QS11, identified by chemical library screening, synergistically activates Wnt/β-catenin signaling via inhibition of ADP-ribosylation factor (Arf) GTPase-activating protein (ArfGAP).8 These data suggest that Arf-GTP has a positive role in Wnt/β-catenin signaling, although the detailed molecular mechanism remains unknown. Arf is a small GTPase belonging to the Ras superfamily. Arf cycles between GDP- and GTP-bound forms, which are inactive and active, respectively. Hydrolysis of GTP in Arf-GTP is mediated by ArfGAPs while conversion of Arf-GDP to Arf-GTP is mediated by Arf guanine nucleotide exchange factors (ArfGEFs). Six Arf isoforms have been identified in mammalian cells, and they can be divided into three classes, class I Arfs (Arfs 1–3), class II Arfs (Arfs 4 and 5) and class III (Arf 6), according to function.9 Arfs regulate vesicular trafficking and actin remodeling. Further, they act on membranes and interact specifically with a large number of effectors, including coat proteins (COPI, AP-1 and AP-3), lipid modifying enzymes (PLD, PI4K and phosphatidylinositol-4-phosphate 5-Kinase (PIP5K)) and others.10, 11, 12, 13

Initiation of Wnt/β-catenin signaling is mediated by LRP6 phosphorylation.1 Recently, it has been shown that the level of phosphatidylinositol 4, 5-bisphosphate (PtdIns (4,5)P2) increases upon activation of Wnt signaling, an event that is necessary for LRP6 phosphorylation.14 As Arf regulates the activity of lipid-modifying enzymes that control the level of PtdIns (4,5)P2,15, 16 we hypothesized that activation of Arf is necessary for Wnt/β-catenin signaling via an increase in the level of PtdIns (4,5)P2, which leads to LRP6 phosphorylation.

In this report, we demonstrate that the active form of endogenous Arf1 and Arf6 transiently increased upon treatment with Wnt3a-conditioned media (Wnt3a-CM), and this event was necessary for increasing the level of PtdIns (4,5)P2 and LRP6 phosphorylation. Taken together, our results suggest that Wnt-mediated Arf activation is necessary for the formation of PtdIns (4,5)P2, which is required for LRP6 phosphorylation and transduction of Wnt/β-catenin signaling.

Results and Discussion

Arf1 and Arf6 enhances Wnt/β-catenin signaling

It has been shown that the small molecule QS11, an ArfGAP inhibitor, synergistically enhances canonical Wnt signaling.8 However, there is no direct evidence as to whether or not Arf is involved in the regulation of Wnt signaling. In light of this, we performed TCF/β-catenin reporter assay to examine the role of Arf in the regulation of Wnt signaling. When several Arf plasmids, Arf-1, 3, 4 and 6, were transiently transfected into HEK293T cells, Arf1 or Arf6 significantly enhanced the TCF reporter activity induced by treatment with Wnt3a-CM in a dose-dependent manner (Figures 1a and b). Consistently, when the level of either Arf1 or Arf6 was reduced by siRNA, Wnt-mediated reporter activity was reproducibly inhibited (Figure 1c). Knockdown of both Arf1 and Arf6 further reduced reporter activity, possibly due to functional redundancy between Arf1 and Arf6 in the regulation of canonical Wnt/β-catenin signaling (Figure 1c). Consistent with these results, knockdown of Arf1 using shRNA specific for Arf1 significantly blocked the increase in cytoplasmic β-catenin induced by Wnt3a-CM (Figure 1d). In addition, transfection of Arf1 mutant (T31N), which can not bind GTP,9 did not enhance Wnt3a-CM-mediated reporter activity, which implies that activation of Arf1 or Arf6 is necessary for enhancement (Figure 1e). Overall, these data suggest that Arf1 and Arf6 positively regulate Wnt/β-catenin signaling.

Figure 1
Figure 1

Arfs enhance Wnt/β-catenin signaling. (a) Effect of Arf family proteins on Wnt3a-induced reporter gene activity in HEK293T cells. Empty vector, HA-Arf1, HA-Arf3, HA-Arf4 or HA-Arf6 (0.5 μg) was transiently transfected into HEK293T cells with Tcf-dependent reporter plasmid. Four hours after transfection, cells were treated with control or Wnt3a-CM for 24 h, after which luciferase activity was determined. The renilla-normalized values are expressed as fold increase compared with vector-transfected control cells and correspond to the mean values (±s.d., in triplicate) of one representative of three independent experiments. (b) Arf1 and Arf6 enhance Wnt3a-mediated luciferase activity in a dose-dependent manner (0.25, 0.5, 0.75 μg). (c) Knockdown of Arfs using siRNA inhibits luciferase activity induced by Wnt3a-CM treatment (left panel). Knockdown of Arfs was confirmed by immunoblotting (right panel). (d) Knockdown of Arf1 using shRNA specific to Arf1 blocked the increase in cytoplasmic β-catenin, which was induced by Wnt3a-CM. (e) GTP-binding-deficient Arf1(T31N) mutant did not enhance Tcf reporter activity induced by Wnt3a-CM (left panel). The levels of ectopically expressed proteins (right panel).

Arfs are transiently activated by Wnt3a at early time points

As Arf1 and Arf6 positively regulate Wnt signaling, we investigated whether or not treatment of cells with Wnt3a has any effect on the nucleotide binding status of endogenous Arfs. To test whether or not Wnt3a induces activation of Arf, the level of endogenous Arf-GTP in HEK293T cells following incubation with Wnt3a-CM was measured by an Arf-GTP pull-down assay, in which Arf-GTP is recovered from cell lysates based on its selective binding to Golgi-associated γ-adaptin ear containing Arf binding protein 3 (GGA3), which is an ArfGAP protein.17 Activation of Arf1 and Arf6 was induced in about 10–30 min following incubation with Wnt3a-CM (Figures 2a and b). Importantly, the level of endogenous GTP-bound Arf1 transiently increased at early time points (10–30 min) following Wnt3a treatment and then returned to basal level (Figure 2a).

Figure 2
Figure 2

Arfs are transiently activated by Wnt3a. (a) The level of Arf1-GTP increased upon Wnt3a-CM treatment at early time points. HEK293T cells were treated with Wnt3a-CM for the indicated times as shown in the figure. The cell lysates were incubated with bacterially expressed GST-GGA3VHS-GAT, which is an ArfGAP without GAP activity that binds to the GTP-bound forms of Arfs. Arf1-GTP and Arf1 were detected by using anti-Arf1 antibody. (b) The level of Arf6-GTP increased upon Wnt3a-CM treatment similar to that of Arf1-GTP. (c) Wnt3a induced spatiotemporal activation of Arf1 and Arf6 at early time points. HEK293T cells were transfected with Arfs FRET-based indicator plasmids and treated with control-CM or Wnt3a-CM. A series of pseudo-colored ratio images showing changes in cFRET/CFP values in HEK293T cells expressing Arfs FRET-based indicators. Treatment with Wnt3a-CM induced the activation of Arf1 and Arf6, which reached maximum levels at early points. (d, e) The net intensities of CFP and cFRET in each cell were measured, and the average emission ratio (cFRET/CFP) was calculated as in Figure 2c. The emission ratio values were normalized to those of the record-starting time.

To investigate the spatio-temporal dynamics of Arf1 and Arf6 in living cells, we developed intermolecular fluorescent resonance energy transfer (FRET)-based probes and constructs containing an internal fluorescent protein for Arf1 and Arf6. To generate the Arf6 FRET-based probe, named Arf6-seCFP-INT, we inserted seCFP into L144 and R145 of Arf6 by using the N-terminal linker GSSAGS and C-terminal linker SAGAAG, respectively, as described previously.18 Based on the crystal structure of Arf1, we prepared the Arf1 FRET-based construct, named Arf1-seCFP-INT, containing an internal seCFP with a six-amino acid linker at the C- and N-termini, as described in the Materials and methods. In addition, we made a venus-GGA3 probe consisting of venus followed by residues 148–303 from GGA3 as a marker that binds to Arfs in a GTP-dependent manner.

To test the localization of Arf1-seGFP-INT and Arf6-mCherry-INT, these constructs were expressed in HeLa cells. Arf1-seGFP-INT localized to the juxtanuclear region, whereas Arf6-mCherry-INT localized to the plasma membrane and to some extent to endosomes (Supplementary Figure 1A). In addition, when cells were treated with brefeldin A, a fungal toxin that inhibits ArfGEFs and is associated with the Golgi complex,19 Arf1-seGFP-INT dissociated from Golgi membranes, whereas the localization of Arf6-mCherry-INT was not disturbed (Supplementary Figure 1B). Next, we examined the behavior of the FRET-based probes for Arf1 and Arf6. When venus-GGA3 was co-expressed in HeLa cells with Arf1(T31N)-seCFP-INT or Arf6(T27N)-seCFP-INT, which are GDP-locked dominant negative mutants, reduced FRET signaling was observed. In contrast, when venus-GGA3 was co-expressed with Arf1(Q71L)-seCFP-INT or Arf6(Q67L)-seCFP-INT, which are GTPase-deficient activated mutants, elevated FRET signals were observed (Supplementary Figure 2). These results indicate that FRET-based probes and constructs containing specific internal fluorescent proteins are able to monitor the spatio-temporal activation dynamics of Arf1 and Arf6 within living cells.

To understand the spatio-temporal activation of Arfs in HEK293T cells treated with Wnt3a-CM, Arf1-seCFP-INT or Arf6-seCFP-INT were co-expressed with venus-GGA3 in HEK293T cells. Arf1 was gradually activated for 15 min and then deactivated thereafter (Figures 2c and d). In addition, time-lapse images of the Arf6 FRET-based probe confirmed the activation of Arf6 following treatment with Wnt3a at 10 min (Figures 2c and e). Control time courses with control-CM showed little changes in FRET signaling over the same time period (Figures 2c-e). These results are consistent with the biochemical data that demonstrated the activation of endogenous Arfs in HEK293T cells treated with Wnt3a-CM at early time points.

Fzd, LRP6 and Dvls are necessary for Wnt-mediated Arf activation

It is well established that Wnt/β-catenin signaling is initiated by binding of Wnt to its receptor Fzd and co-receptor LRP5/6, followed by direct interaction between Fzd and Dvl, a cytoplasmic scaffolding protein.1, 5, 20, 21 To test whether or not Wnt3a-CM-mediated Arf activation requires Wnt receptors and Dvl, we carried out Arf-GTP pull-down assays following knockdown of each protein. Pan et al.14 previously showed that Fzds 2, 3, 4, 5 and 6 are expressed in HEK293T cells, and that knockdown of Fzds 2, 4 and 5 blocks Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate When the expression of Fzds 2, 4 and 5 in HEK293T cells was knocked down using shRNAs, we observed inhibition of Wnt3a-CM-mediated endogenous Arf1/6 activation (Figure 3a and Supplementary Figure 3A). To further confirm this result, we used the cysteine-rich domain of Fzd conjugated with Fc fragment of antibody (Fc-CRD) in order to block the interaction between Fzd and Wnt3a.22 When HEK293T cells were incubated with the Wnt3a-CM, lacking free Wnts following Fc-CRD treatment, activation of endogenous Arf1 was blocked, which strongly suggests that the interaction between Wnt and Fzd is necessary for the activation of Arf (Figure 3b). In addition, knockdown of LRP6 or Dvl1, 2 and 3 had inhibitory effects on Wnt3a-CM-mediated Arf1 activation (Figures 3c and d and Supplementary Figure 3B). Together, these results indicate that Wnt3a-mediated Arf1/6 activation requires Fzd, LRP6 and Dvl.

Figure 3
Figure 3

Fzd, LRP6 and Dvls are necessary for Wnt3a-mediated Arf activation. (a) Arf1 activation was inhibited by the knockdown of Fzds. HEK293T cells were transiently co-transfected with expression vectors for shFzd2, 4 and 5, and the clones stably expressing shFzds were selected using 2 μg/ml of puromycin. Subsequently, Arf activation assay was performed (left panel). Knockdown of Fzd2, 4 and 5 was shown by reverse transcription-PCR (right panel). (b) Activation of Arf1 was attenuated by the addition of Fc-CRD. HEK293T cells were transiently transfected with Fc or Fc-CRD, after which the media were incubated with Wnt3a-CM for 1 h. The mixed media were then incubated with Fc agarose beads and the supernatant was collected. HEK293T cells were treated with the collected media for the indicated times, and the levels of Arf1-GTP and Arf1 were measured (left panel). The expression of Fc or Fc-CRD was shown by western blot (right panel). (c) Arf1 activation was inhibited by the knockdown of LRP6, and the levels of proteins were measured by immunoblotting. (d) Arf1 activation was inhibited by the knockdown of Dvls, and the levels of proteins were measured by immunoblotting.

Activation of Arfs is necessary for the formation of PtdIns (4,5)P2 and LRP6 phosphorylation

In Wnt/β-catenin signaling, formation of PtdIns (4,5)P2 by PIP5K is required for LRP6 activation, which promotes the oligomerization and phosphorylation of LRP6.14, 23 However, it is unknown which factor(s) regulate PIP5K during Wnt signaling. It has been shown that PIP5Ks are regulated by diverse factors, including Arfs,24 and PIP5K can be recruited and activated in the trans-Golgi and plasma membranes by Arf1 and Arf6.15, 16 Thus, we hypothesized that the activation of Arf may be necessary for the formation of PtdIns (4,5)P2 and phosphorylation of LRP6.

To monitor Wnt3a-mediated formation of PtdIns (4,5)P2, we used an intramolecular FRET-based PtdIns (4,5)P2 indicator, Pippi-PI(4,5)P2, along with the pleckstrin homology (PH) domain of PLCδ1 (phospholipase C), which can bind locally elevated PtdIns (4,5)P2.25 To examine the binding specificity of Pippi-PI(4,5), we monitored changes in the level of FRET/CFP in NIH3T3 cells as described previously.25 The level of FRET/CFP decreased rapidly following treatment with PDGF-BB, which is known to reduce the level of PtdIns (4,5)P2 via rapid activation of PLC (Supplementary Figure 4). In addition, we confirmed the binding specificity of Pippi-PI(4,5)P2 in HEK293T cells by using the rapamycin-inducible translocation method to deplete PtdIns (4,5)P2 from the inner leaflet of the plasma membrane. Translocation of mCh-INP to the plasma membrane triggered a rapid decrease in the FRET/CFP value in rapamycin-treated cells (Supplementary Figure 5). These results show that Pippi-PI(4,5)P2 has specific binding affinity to PtdIns (4,5)P2 in HEK293T cells.

To measure the Wnt3a-mediated spatio-temporal dynamics of PtdIns (4,5)P2, HEK293T cells expressing either control shRNA or shArf1/6 were transfected with Pippi-PI(4,5)P2 construct. The transfected cells were then treated with control-CM and Wnt3a-CM, after which the level of PtdIns (4,5)P2 was measured by FRET imaging techniques. The time-course of changes in FRET is presented in Figures 4a and b. Formation of PtdIns (4,5)P2 was unaffected by treatment with control-CM. Consistent with previously published data,14 treatment with Wnt3a-CM induced formation of PtdIns (4,5)P2, which reached its highest peaks at 1030 min, whereas Arf1, 6-depleted cells did not (Figures 4a and b). These data strongly support that Wnt3a-mediated formation of PtdIns (4,5)P2 is controlled by Arfs.

Figure 4
Figure 4

Knockdown of Arfs inhibits Wnt3a-mediated formation of PtdIns (4,5)P2 and phosphorylation of LRP6. (a) Wnt3a-CM-induced formation of PtdIns (4,5)P2 was inhibited by the knockdown of Arf1 or Arf6. HEK293T cells stably expressing shGFP or shArf1, Arf6 were transfected with Pippi-PI(4,5)P2 FRET-based indicator plasmids and then treated with control-CM or Wnt3a-CM. Treatment with Wnt3a-CM induced the formation of PtdIns (4,5)P2, which reached a maximum at 1030 min, whereas Arf1, 6-depleted cells did not. (b) The net intensities of CFP and FRET in each cell were measured, and the average emission ratio (FRET/CFP) was calculated as in Figure 4a. The emission ratio values were normalized to those of the record-starting time. (c) The depletion of Arf1 or Arf6 decreased Wnt3a-mediated induction of LRP6 phosphorylation at Serine 1490. HEK293T cells transfected with siGFP, siArf1 or siArf6 were incubated for 72 h and treated with Wnt3a-CM for the indicated times, after which the lysates were immunoblotted with the indicated antibodies. (d) Schematic diagram of the involvement of Arfs in the regulation of Wnt/β-catenin signaling.

We next examined whether or not formation of PtdIns (4,5)P2 upon Arf activation is necessary for the phosphorylation of LRP6 (Figure 4c). Cells deficient in Arf1, Arf6 or both showed significantly reduced LRP6 phosphorylation at Ser1490 compared with cells transfected with siGFP (Figure 4c and Supplementary Figure 6). Taken together, these results suggest that activation of Arfs by Wnt3a-CM is necessary for the formation of PtdIns (4,5)P2 as well as LRP6 phosphorylation (Figure 4d).

Wnt-mediated LRP6 phosphorylation (or activation) is a critical initiation step for the transmission of Wnt/β-catenin signaling.3 A previous study reported that Wnt3a induces Dvl to activate PIP5K, and the resulting PtdIns (4,5)P2 formation promotes LRP6 aggregation and phosphorylation, although the underlying mechanism remains unclear.14 Here, we found that Arfs were transiently activated upon treatment with Wnt3a-CM for 1030 min (Figure 2), and formation of PtdIns (4,5)P2 was maximized at the same time points (Figure 4). These results are consistent with a report that LRP6 phosphorylation can be detected at 30 min after Wnt treatment.14 In addition, we showed that depletion of Arfs decreased Wnt3a-mediated formation of PtdIns (4,5)P2, as well as induction of LRP6 phosphorylation (Figures 4b and c). These data clearly suggest that Arf is a novel regulator of Wnt/β-catenin signaling at relatively early time points.

As we found that knockdown of either Arf1 or 6 blocked LRP6 phosphorylation, which is necessary for the transduction of Wnt signaling, we propose that both Arf1 and 6 are required by the Wnt signaling pathway, although additional genetic evidence is needed. As Arf6 knockout mice display embryonic lethality due to impaired liver development,26 it is difficult to examine the role of Arf in Wnt signaling. As Drosophila is known to possess three Arf proteins,12 conditional ablation of Arf proteins in a specific tissue, such as the wing imaginal disc, followed by examination of Wnt target gene expression or wing development could provide more definitive answers.

The immediate molecular event of how the activation of Wnt receptor leads to stabilization of β-catenin is very controversial. It is generally postulated that activation of Wnt receptor disassembles the Axin/GSK3β/β-catenin-degradation complex, followed by sequestration of Axin to phosphorylated LRP5/6, which in turn leads to stabilization of cytoplasmic β-catenin.1 Other data suggest that proteasomal degradation of Axin or dissociation of β-TrCP, an E3 ligase for β-catenin from the Axin/β-catenin degradation complex, is an immediate molecular event for the stabilization of β-catenin.27, 28 Liu et al.29 showed that Axin/GSK3β complexes are rapidly (t1/2<3 min) disrupted upon Wnt stimulation. However, the authors only showed the dissociation of GSK3β from Axin2 at early time points (as early as 5 min) in L929 cells; data for the dissociation of GSK3β from Axin at early time points were not provided. In addition, a recent study showed that the dissociation of GSK3β from Axin upon Wnt stimulation does not occur in HEK293T cells.28 Besides Liu et al.’s report, most molecular events, including Arf1/6 activation, occur between 10–60 min following Wnt stimulation; Arf activation between 10–30 min, phosphorylation of LRP6 at 30 min,14 accumulation of cytoplasmic β-catenin between 30–60 min,28 Rac1 activation at 30–60 min,30 dissociation of β-TrCP from Axin complex at 1 h,28 and degradation of Axin between 2–4 h.27, 28 Therefore, it seems that Arf activation occurs the earliest among known molecular events following Wnt stimulation, at least in HEK293T cells. It would be interesting to identify exactly which ArfGEFs are regulated by Wnt prior to Arf activation; small molecule(s) that specifically control ArfGEFs will be extremely useful for the therapeutic control of diseases caused by the dysregulation of Wnt/β-catenin signaling. It is interesting to note that the response to Wnt stimulation was significantly slower than the kinetics for the effects of PDGF-BB (Supplementary Figure 4) and rapamycin (Supplementary Figure 5). This may suggest that a complicated complex dissociates and/or forms before the activation of downstream Wnt signaling.

Currently, it is unknown as to whether or not the reported functions of Arf are involved in the regulation of Wnt signaling. Arf has been extensively studied as an essential component in multiple intracellular trafficking pathways.31, 32 Moreover, many reports have suggested the possibility of receptor internalization and endosomal trafficking in Wnt receptor activation and signaling.33, 34, 35 Thus, it is possible that activated Arf following Wnt treatment regulates vesicle trafficking in between the plasma and Golgi membranes, which may have an effect on receptor endocytosis and recycling. It has been shown that PI3K-mediated Rac1 activation is necessary for canonical Wnt signaling.30 In addition, it was reported that PI3K can regulate the activity of Arf1,36 which in turn regulates Rac1.9 Therefore, it is reasonable to conclude that Arf may control the localization of β-catenin via regulation of Rac1, although we did not investigate this particular issue in our manuscript. As depicted in our model (Figure 4d), we propose that LRP6 phosphorylation mediated by Arf1/6 along with Arf1/Rac1-mediated nuclear localization are not mutually exclusive. As Arf may have dual roles in the regulation of Wnt signaling, careful studies should be carried out in the future.

Materials and methods

Plasmids, shRNAs and reagents

pCS2-Arf1-EGFP, pCS2-HA3-Arf1, pCS2-Arf6-EGFP, pCS2-HA3-Arf6 and pCS2-Arf1(T31N)-EGFP were constructed by using mouse embryonic cDNA via a PCR-based method. ARF1-seCFP-INT, ARF1-seGFP-INT, ARF6-seCFP-INT and ARF6-mCherry-INT were constructed as described previously.18 For mammalian expression, the cDNAs of ARF1-seCFP-INT, ARF1-seGFP-INT, ARF6-seCFP-INT, ARF6-mCherry-INT and venus-GGA3 were subcloned into pcDNA3 (Invitrogen, Carlsbad, CA, USA), respectively. ARF1(T31N)-seCFP-INT, ARF1(Q71L)-seCFP-INT, ARF6(T27N)-seCFP-INT and ARF6(Q67L)-seCFP-INT were generated by modified QuikChange mutagenesis.37 For simultaneous imaging of the rapamycin-induced depletion of PtdIns (4,5)P2 and FRET-based PtdIns (4,5)P2 indicator, the CFP part of CF-INP was replaced with mCherry to generate mCh-INP. pGEX-GGA3 (1–316) and pSuper vector, which was used for the expression of shRNAs, were kindly provided by Drs Parent (Université de Sherbrooke, Quebec, Canada) and Agami (The Netherlands Cancer Institute, Amsterdam, Netherlands), respectively.17, 38 Previously published nucleotide sequences for the shRNAs were used in our experiments; GFP,39 Arf1, Arf6,31 Frizzled2, 4, 5, LRP6 and Dvl1, 2, 3.14 A plasmid for FRET-based PtdIns (4,5)P2 indicator (Pippi-PI(4,5)P2) was provided by Professor Michiyuki Matsuda of Kyoto University, Kyoto, Japan.25 For the depletion of PtdIns (4,5)P2, PM-localized FK506-binding protein (FKBP12)-rapamycin-binding (FRB) construct and a cytosolic INP54p enzyme conjugated with FKBP12 (CF-INP) construct were provided by Professor Won Do Heo of the Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea.40

Antibodies

Anti-β-catenin monoclonal antibody (Transduction Laboratories, Rockville, MD, USA), anti-HA monoclonal antibody, anti-α-tubulin monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-β-actin monoclonal antibody (Sigma, St Louis, MO, USA), anti-LRP6 polyclonal antibody (Abcam, Cambridge, MA, USA), anti-phospho LRP6 (Ser 1490) polyclonal antibody (Cell Signaling, Danvers, MA, USA), anti-Arf1 monoclonal antibody (Epitomics, Burlingame, CA, USA) and anti-Arf6 monoclonal antibody (Santa Cruz Biotechnology Inc.) were used to detect their corresponding proteins. Horse Radish Peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies (Santa Cruz Biotechnology Inc.) were used, and the proteins were detected by using an enhanced chemiluminescence reagent (ELPIS, Daejeon, Korea).

Cell culture and transient transfection

HEK293T and L929 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% antibiotics (Gibco BRL). For the transient transfection experiments, each plasmid DNA was transfected into HEK293T cells by the calcium phosphate precipitation method. The amount of DNA in each transfection was kept constant by the addition of an appropriate amount of empty expression vector.

Preparation of conditioned media(CM)

To obtain the Wnt3a-CM, Wnt3a-producing L929 cells (which were kindly provided by Dr Nusse, School of Medicine, Stanford, CA, USA) were cultured in a 175 mm flask containing DMEM supplemented with 10% fetal bovine serum. When these cells were 100% sub-confluent (about 48–72 h), the medium was harvested, centrifuged at 2000, rpm for 5 min, and then filtered through a cellulose acetate syringe filter (0.22 μM, SCA). Conditioned medium derived from L929 cells was used as a control for Wnt3a-CM. To obtain the CM lacking Wnt3a from Wnt3a-CM, CM containing Fc or Fc-CRD (obtained by the transient transfection of Fc or Fc-CRD plasmid into HEK293T cells) were incubated with Wnt3a-CM for 1 h, followed by pelleting with anti-IgG agarose beads.22 The supernatant was collected and used.

TOPFLASH Luciferase assay

HEK293T cells were transiently transfected with the following plasmids: 0.5 μg of reporter plasmid (pSuperTOPFLASH, kindly provided by Dr Moon, University of Washington), 50 ng of pRL-TK, and 250, 500 and 750 ng of pCS2-HA3-Arf1 plasmid or pCS2-HA3-Arf6. Four hours after transfection, cells were treated with control CM and Wnt3a-CM for 24 h. Luciferase activities were measured using a Dual-Luciferase reporter assay kit (Promega, Madison, WI, USA).

Arf activation assay

To detect Arf1-GTP, we used a protocol published by Santy and Casanova.9 Cells were lysed with 50 mM Tris, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol with 0.1 mM PMSF and 1 μg/ml of leupeptin. Lysates were centrifuged at 12 000 rpm for 5 min, after which the supernatant was collected. Equal amounts of lysates were incubated with purified beads bound to GST-GGA3. After 30 min, precipitates were washed three times with wash buffer (50 mM Tris, pH7.5, 100 mM NaCl, 2 mM MgCl2, 1% NP-40, 10% glycerol with 0.1 mM PMSF, and 1 μg/ml of leupeptin). Bound proteins were eluted in SDS-PAGE sample buffer. This sample was assayed for the active form of endogenous Arf1 or Arf6 by Western blotting. Total level of Arf1 or Arf6 was used as a control.

Live cell and FRET imaging

HEK293T cells were cultured on a collagen-coated 35 mm glass-base dish (Asahi techno glass, Tokyo, Japan) and grown overnight at 37 °C in DMEM supplemented with 10% fetal bovine serum. For time-lapse and FRET analysis were carried out as described previously.25 HEK293T cells transfected with plasmids indicated in figures were imaged using a Nikon Ti-E inverted microscope (Nikon, Tokyo, Japan) equipped with a Perfect Focus System (PFS), Cascade 512B (EMCCD) camera (Roper Scientific, Trenton, NJ, USA), and excitation and emission filter wheels. All systems were controlled by MetaMorph software (Universal Imaging, Downingtown, PA, USA). Filter sets and ND filters were purchased from Semrock (Rochester, NY, USA). Images were acquired in 2 × 2 binning mode with a 200 ms exposure time. For imaging with a pair of intermolecular FRET probes, fluorescent images were acquired sequentially using CFP, FRET and YFP filter channels. After background subtraction, pseudo-color images of FRET/CFP were created using eight colors from red to blue to represent the FRET/CFP ratio in intensity modulated display mode with MetaMorph software (Universal Imaging, Downingtown, PA, USA). Corrected FRET (cFRET) was calculated by using the following equation: cFRET=(FRET−0.72) × (CFP−0.06) × YFP.

Disclaimer

Our work is original research, has not been previously published and has not been submitted for publication elsewhere while under consideration.

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Acknowledgements

This work was supported by grants from the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2006-2004046 and 2012R1A2A2A01012472 to E-HJ; 2010-0029206 to I-SK; 2011-0003980 to S-YK). WK, TK, and MK were supported by the Brain Korea 21 program.

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Author notes

    • W Kim
    • , S Y Kim
    •  & T Kim

    These authors contributed equally to this work

Affiliations

  1. Department of Life Science, The University of Seoul, Seoul, Korea

    • W Kim
    • , T Kim
    • , M Kim
    •  & E Jho
  2. Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu, Korea

    • S Y Kim
    • , D-J Bae
    • , H-I Choi
    •  & I-S Kim
  3. Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea

    • I-S Kim

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The authors declare no conflict of interest.

Corresponding authors

Correspondence to I-S Kim or E Jho.

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https://doi.org/10.1038/onc.2012.373

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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