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. 2008 Apr;19(4):1594–1604. doi: 10.1091/mbc.E07-08-0832

The Postsynaptic Density 95/Disc-Large/Zona Occludens Protein Syntenin Directly Interacts with Frizzled 7 and Supports Noncanonical Wnt Signaling

Annouck Luyten *,†, Eva Mortier *,†, Claude Van Campenhout ‡, Vincent Taelman ‡, Gisèle Degeest *,†, Gunther Wuytens *,†, Kathleen Lambaerts *,†, Guido David *,†, Eric J Bellefroid ‡, Pascale Zimmermann *,†,✉
Editor: Carl-Henrik Heldin
PMCID: PMC2291413  PMID: 18256285

Abstract

Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)α and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKCα/CDC42 noncanonical Wnt signaling cascade. The syntenin–Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7–syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans.

INTRODUCTION

Wnt proteins are involved in cell proliferation, differentiation, polarity, migration, and apoptosis, controlling a variety of processes during embryonic development and adult homeostasis (Logan and Nusse, 2004). Various inborn and acquired diseases are based on aberrant Wnt signaling (Johnson and Rajamannan, 2006). Wnt signaling requires the interplay of multiple proteins (http://www.stanford.edu/∼rnusse/wntwindow.html), but the reception and transduction of Wnt signals are predominantly based on the binding of Wnt proteins to Frizzled (Fz) cell surface receptors (Yang-Snyder et al., 1996). The various Fz receptors differ in their spatial and temporal expression patterns and in their relative affinities for ligand. Receptor activation by Wnt somehow activates Dishevelled (Dsh), the most upstream component of the cytosolic signal transduction cascade. The role of Dsh is intricate because downstream of Dsh, the signaling branches into either the canonical Wnt/β-catenin or the noncanonical (β-catenin–independent) pathway (Boutros and Mlodzik, 1999). Activation of the canonical pathway drives β-catenin–dependent transcription of target genes, controls tissue-specific cell fate decisions during embryogenesis, and regulates cell proliferation in adult tissues (Logan and Nusse, 2004). The noncanonical pathway controls reorganization of the actin cytoskeleton, tissue polarity, and cell movement (Strutt, 2003). Several molecular cascades, which may overlap, seem to function in noncanonical Wnt signaling (Veeman et al., 2003; Kohn and Moon, 2005). One is similar to the planar cell polarity or PCP pathway in Drosophila, and it activates small GTPases of the Rho family and the c-Jun-NH2-terminal kinase (JNK). Another, triggered by Wnt4, Wnt5a, and Wnt11, involves activation of calcium/calmodulin-dependent kinase II and protein kinase C (PKC), and it exerts antagonistic effects on the canonical pathway (Kuhl et al., 2001; Sheldahl et al., 2003).

One major question is how Wnt signaling activates different downstream pathways. Clearly, the Wnt–Fz combination and the presence of coreceptors for Wnts, like the members of the low-density lipoprotein receptor-related proteins (Wehrli et al., 2000) or heparan sulfate proteoglycans (Reichsman et al., 1996), are important. Wnt signaling also relies on the subcellular localization of the Fz (Wu et al., 2004) and the nature of the intracellular components adapting to Fz. The Fz family members are serpentine receptors with an extracellular cysteine-rich domain important for Wnt binding, and seven membrane-spanning domains (Vinson et al., 1989; Wang et al., 2006). Fz display a C-terminal cytosolic tail with two postsynaptic density 95/disc-large/zona occludens (PDZ) binding motifs, or PDZBMs. So far, proteins containing PDZ domains are the only well established intracellular direct ligands for Fz (Tan et al., 2001; Wong et al., 2003; Yao et al., 2004; Djiane et al., 2005; Ataman et al., 2006).

Proteins containing PDZ domains (PDZ proteins) are scaffold proteins particularly abundant in multicellular organisms and probably evolved in response to the increased signaling needs of multicellularity. PDZ proteins have been implicated in the establishment and maintenance of cell polarity, in the formation and the stability of adhesion structures and in the targeting and organization of large signaling complexes at the membrane (Nourry et al., 2003; Margolis and Borg, 2005). PDZ interactions are reversible and particularly versatile.

We identified the PDZ protein syntenin as an intracellular ligand of the syndecans (Grootjans et al., 1997) and as an important regulator of cell shape (Zimmermann et al., 2001, 2005). Syndecans are abundant and ubiquitous type I transmembrane heparan sulfate proteoglycans that emerge as essential for the reception, dissemination and readout of morphogen signals during development and in pathology. The heparan sulfate chains of their extracellular domain bind numerous growth factors, including Wnts (Couchman, 2003; Bishop et al., 2007). Here, we tested for a direct interaction between syntenin and Fz, and for a role for syntenin in Fz signaling.

MATERIALS AND METHODS

Molecular Biology

Human syntenin, syntenin-2, and syndecan cytoplasmic domain constructs were described previously (Grootjans et al., 1997, 2000; Grootjans et al., 2000). Fz cytoplasmic domains were cloned in pGEX-5X-1 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Ligand overlays were performed with purified glutathione transferase (GST)-fusion proteins as described previously (Grootjans et al., 2000). Human Fz 7 cytoplasmic domain was cloned in peYFP-C1 for immunoprecipitation experiments. The human Fz 7 full cDNA clone (IMAGp958K221240Q) was purchased from RZPD Deutsches Ressourcenzentrum fur Genomforschung (Berlin, Germany), and the open reading frame was introduced into pCDNA3.1/zeo(+) (Invitrogen). Quickchange mutagenesis (Stratagene, La Jolla, CA) was used to generate mutant Fz 7 constructs. The Xsyntenin-a clone (IMAGp998K0110804Q), Xsyntenin-a' clone (IRBHp990H0541D2), and Xsyntenin-b clone (RZPDp988D1265D) were purchased from RZPD Deutsches Ressourcenzentrum fur Genomforschung, and sequence analysis confirmed the sequences present in the database. To produce the Xsyntenin-a, a', and b probes for whole-mount in situ hybridization (WISH), the full cDNA was sublconed into pGEM-T. To produce RNA for injection, Xsyntenin-a was cloned in pCS2+, flanked or not with myc or hemagglutinin (HA) tags. All constructs were verified by sequencing. The morpholino (MO) oligonucleotides (Gene Tools, Philomath, OR) used were for Xsyntenin-a, 5′-ACAGACCTGCCGACAGTCGAATAAG-3′; for Xsyntenin-a', 5′-GAGGGATATAGAGACATTTTCACAG-3′; and for Xsyntenin-b, 5′-GGATAGATAGACATGGTGTAAGACC-3′. All three MOs efficiently block in vitro transcription/translation of the respective Xsyntenins. The mismatch MO used was 5′-GACGCATAGAGAGAGATATT-3′. The MO for XFz 7 was 5′-GTGAGCAGAAATCGGCTGATACTGG-3′, as described by Sumanas et al. (2001). For reverse transcription-polymerase chain reaction (RT-PCR), total RNA extracted from embryos was reverse transcribed using RTase MuLv (20 U/μl) (Promega, Madison, WI). Histone H4 was used as an internal standard. The following PCR conditions were applied: 26 cycles for H4 and 32 cycles for Xsyntenin-a. The primers used for Xsyntenin-a, forward 5′-CCTTTGAACGCACCATCACCATG-3′ and reverse 5′-GAATTCTTAAACCTCAGGGACAGAATG-3′, generate fragments of 300 base pairs. H4 primers were as described in http://www.hhmi.ucla.edu/derobertis/index.html. Reactions were carried out using a Gene Amp TM PCR system 9600 (PerkinElmer Life and Analytical Sciences, Boston, MA).

Surface Plasmon Resonance Experiments

Surface plasmon resonance was measured using a Biacore 2000 instrument. N-terminal biotinylated Fz 7 synthetic peptides, corresponding to the CD of Fz 7, were immobilized on a streptavidin-sensor chip. Analytes (GST fusion proteins) were perfused at 10 μl/min in running buffer (100 mM NaCl/10 mM HEPES/0.005% Tween 20, pH 7.4). The surface was regenerated through 1-min pulses of 1 M NaCl/0.05 M NaOH. For the determination of the apparent KD, signals obtained at equilibrium (Req with different concentrations from 300 to 1500 nM GST-syntenin) were plotted as a function of protein concentration. Apparent KD values were calculated from these plots, as the concentration corresponding to Req-max/2.

Cells, Transfections, Extractions, Coimmunoprecipitation, and Fluorescence Microscopy

Cells originated from American Type Culture Collection (Manassas, VA), and they were routinely grown in DMEM/F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). For microscopic analysis, cells were plated on eight-well chamber slides (Nalge Nunc International, Rochester, NY), transfected using the FuGENE transfection reagent (Roche Diagnostics, Basel, Switzerland), and then they were fixed and stained as described previously (Zimmermann et al., 2001) by using 10 μg/ml anti Fz 7 antibody (R & D Systems, Minneapolis, MN) and Alexa 568-conjugated donkey anti-goat secondary antibodies (Invitrogen). The enrichment of enhanced green fluorescent protein (eGFP)-syntenin at the plasma membrane was scored by confocal microscopy, in three independent experiments, looking at 30 cells per transfection. Coimmunoprecipitations were performed as described previously (Djiane et al., 2005). Enhanced yellow fluorescent protein (eYFP)-Fz 7 was immunoprecipitated with goat anti-GFP antibodies (ab5449; Abcam, Cambridge, United Kingdom). Detection of eYFP-Fz 7 in Western blot was with mouse anti-GFP antibodies (G1546, 1/1000; Sigma Chemical, Pool, Dorset, United Kingdom). Endogenous syntenin was detected with purified rabbit polyclonal antibodies (Zimmermann et al., 2001). For Jun-phosphorylation assays, human embryonic kidney (HEK)293T cells were plated at a density of 150,000 cells/well in six-well dishes. Extracts were prepared 48 h after transfection in the presence of detergent and phosphatase- and protease-inhibitors: 0.1 mM sodium vanadate, 0.1 mM aprotinin, 0.1 mM leupeptin, and 0.5 mM pepstatin A in 50 mM β-glycerophosphate, 1 mM EDTA, 1 mM benzamidine, 1.5 mM EGTA, and 60 mM octylglucoside. Protein samples (10 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose filter, and incubated with 1/1000 anti-phospho c-jun (ser-63), 1/1000 anti-c-jun (Cell Signaling Technologies, Danver, MA) or 1/500 anti-actin (Sigma Chemical) antibodies.

Xenopus Embryos, In Situ Hybridization, Microinjections, and Membrane Translocation Assays

Xenopus embryos were obtained from adult frogs by hormone induced egg-laying and in vitro fertilization by using standard methods (Sive et al., 2000), and they were staged according to Nieuwkoop and Faber (1967). Synthesis of capped RNA was performed with a Message Machine kit (Ambion, Austin, TX), and injection was carried out as described previously (Bellefroid et al., 1996). For whole-mount in situ hybridization, embryos were fixed in MEMFA (0.1 mM 3-(N-morpholino)propanesulfonic acid, pH 7.4, 2 mM EGTA, 1 mM MgSO4, and 3.7% formaldehyde), and they were processed using dioxygenin-labeled antisense RNA probes (Sive et al., 2000). Membrane translocation assays were carried out as described for XDsh-myc (Umbhauer et al., 2000). Xenopus embryos were injected into the animal pole with either Xsyntenin-a-myc (200 pg) or XDsh-myc (200 pg) in the presence or absence of XFz 7 mRNA (500 pg). For the competition experiments, 100 pg of XDsh-myc, 100 pg of Xkermit-myc, 100 pg of XPKCα-GFP, 250 pg of XFz 7, 500 pg of Xsyntenin-a-HA, and 5 ng of each Xsyntenin MO or 15 ng Mismatch MO were used. Animal caps were dissected and fixed at room temperature with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 h. After washing with PBT-10% goat serum (PBS + 2 mg/ml bovine serum albumin + 0.1% Triton X-100), the caps were incubated overnight at 4°C with 1.5 μg/ml anti-myc antibodies (9E10; Santa Cruz Biotechnologies) and when relevant with 1 μg/ml anti-HA antibodies (3F10; Roche Diagnostics) or anti-GFP antibodies (G1546, 1/1000; Sigma Chemical). Alexa 488-conjugated goat anti-mouse secondary antibodies and when required Alexa 594-conjugated goat anti-rabbit secondary antibodies (Invitrogen), were incubated overnight at 4°C. Secondary antibodies did not show species cross-reactivity in our experimental settings. The caps were mounted with Vectashield (Vector Laboratories, Burlingame, CA). Images were obtained with the MRC-1024 laser scanning confocal imaging system (Bio-Rad, Hemel Hempstead, United Kingdom) or the FluoView FV1000 (Olympus, Tokyo, Japan).

RESULTS

Syntenin Interacts with the Fz Cytoplasmic Domain in a PDZ-dependent Mode

We tested for direct interaction of Fz with syntenin in ligand overlay. Therefore, we designed primers for the PCR amplification of sequences encoding the last C-terminal 25 cytosolic amino acids of all Fz (Figure 1A) of a human brain cDNA library. The amplification was successful for Fz 1, 2, 3, 5, 6, 7, and 8. We produced and purified GST fusion constructs of these peptides and overlayed these with GST-myc-syntenin. A weak signal was observed for Fz 1; no signal was observed for Fz 2, 5, and 6; and strong signals were observed for Fz 3, 7, and 8 (Figure 1B). Overlay using a syntenin construct containing solely the PDZ domains yielded similar results, whereas overlay with GST-myc or GST-myc fused to the N-terminal domain of syntenin showed no signal (data not shown). We concluded for specific interaction between the PDZ domains of syntenin and Fz 3, 7, and 8. The primary structure of the Fz C-terminal region provides no direct explanation why Fz 2, 5, and 6 do not interact with syntenin, whereas Fz 3, 7, and 8 do.

Figure 1.

Figure 1.

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Syntenin interacts with Fz in a PDZ-dependent mode. (A) Sequences of the last 25 cytosolic amino acids of all human Fz and Fz 7 mutants; overview of syntenin binding to these peptides as detected in overlay; nd, not determined. C-terminal PDZBMs are in bold. For Fz 1, 2, and 7, the membrane proximal PDZBM for Dsh is also present in the 25 last amino acids, and it is indicated in gray. (B) Overlays illustrating syntenin interaction with Fz. GST-Syndecan-2 cytoplasmic domain (WT) was used as a positive control, and GST or GST-Syndecan-2 ΔPDZBM (cytoplasmic domain deleted for the last 2 amino acids) were used as negative controls. Note the interaction of syntenin with Fz 7, 3, and 8 last 25 amino acids, and the lack of interaction with Fz 7 T/A and Fz 7 T/A V/A mutants. The quality and concentration of the fusion proteins were controlled in Coomassie as shown at the bottom. (C) Respective roles of the PDZ domains of syntenin in Fz interaction. Structure of syntenin and coordinates of the amino acids that define the different domains are shown on the right. The relevant GST fusions were overlayed with recombinant proteins containing different combinations of the PDZ domains of syntenin as indicated. Note that Fz 7 interacts preferentially with the PDZ1 domain, whereas syndecan 2, Fz 3, and 8 interact preferentially with the PDZ2 domain of syntenin. The quality and concentration of the fusion proteins were controlled in Coomassie as shown at the bottom. (D) Interaction of syntenin and syntenin PDZ domains with Fz 7 cytoplasmic domain in surface plasmon resonance. RU, response units. Note that the binding relies primarily on the PDZ1 domain of syntenin. (E) Coimmunoprecipitation of endogenous syntenin with eYFP-tagged Fz 7 cytoplasmic domain. MCF-7 cells overexpressing the Fz 7 cytoplasmic domain fused N-terminally to eYFP were extracted with detergent. The cell lysate was immunoprecipitated with anti-eYFP antibodies and protein G beads before immunoblotting (right lanes, IP Fz7) with anti-eYFP to detect the Fz 7 fusion (top) or anti-syntenin antibodies (bottom). The cell lysate was used as positive control (left), in the negative control the anti-eYFP antibodies were omitted (middle). Note that endogenous syntenin coimmunoprecipitates with the eYFP-Fz 7 fusion (right bottom lane).

The last 25 amino acids of Fz 3 and 8 solely contain the C-terminal PDZBM of the receptor, whereas those of Fz 7 also contain the membrane proximal or Dsh PDZBM (Figure 1A). Because all three Fz bind syntenin, we assumed syntenin would interact with the C-terminal PDZBM. To test this hypothesis, we investigated whether point mutations in the C-terminal PDZBM of Fz 7 (Figure 1A) affect the interaction with syntenin. In ligand overlay, a weak signal was still observed for the GST-Fz 7 V/A mutant, but the interaction was abolished for the GST-Fz 7 T/A and GST-Fz 7 T/A V/A mutants (Figure 1B). The Fz 7-syntenin interaction was also confirmed by surface plasmon resonance (Figure 1D), and the apparent KD for syntenin-Fz 7 interaction was determined to be 10−7 M, similar to that of syntenin-syndecan-2 interaction (data not shown). We concluded that syntenin interacts with the C-terminal PDZBM and probably not with the membrane-proximal PDZBM.

The Relative Preference for the First or the Second PDZ Domain of Syntenin Varies, Depending on the Fz

We also investigated the respective roles of the two PDZ domains of syntenin in binding Fz, and we compared this binding to the syndecan-2 interaction. This was tested in ligand overlay, by using GST-fusion constructs of single PDZ domains (GST-myc-PDZ1 and GST-myc-PDZ2) or homodimeric and heterodimeric tandem PDZ domains (GST-myc-PDZ1-PDZ1, GST-myc-PDZ2-PDZ2, GST-myc-PDZ1-PDZ2, and GST-myc-PDZ2-PDZ1). In overlay assays, PDZ2 was sufficient for syndecan-2 binding, PDZ1 was sufficient for the interaction with Fz 7, whereas Fz 3 and 8 interaction required two PDZ domains and at least one PDZ2 domain (Figure 1C). Surface plasmon resonance confirmed the interaction of Fz 7 with the PDZ1-PDZ1, PDZ1-PDZ2 and PDZ2-PDZ1 tandem, and it showed that PDZ1 is sufficient for Fz 7 binding. Contrary to overlay results, the PDZ2-tandem did not display strong interaction with Fz 7 in surface plasmon resonance (Figure 1D). Syntenin-2, a close homologue of syntenin (Mortier et al., 2005), did not bind to Fz 7 or syndecan-2 (data not shown), underscoring the specificity of the interactions. We concluded that syntenin can interact with Fz 3 and 8 via its two PDZ domains, but preferentially via the PDZ2 domain, reminiscent of the syndecan-2 mode of interaction (Grootjans et al., 2000), whereas the interaction with Fz 7 relies on the PDZ1 domain.

Syntenin Is Recruited by Fz 7 and Stimulates c-Jun Phosphorylation in Cultured Cells

To test for syntenin-Fz 7 interaction in a physiological context, we performed coimmunoprecipitation experiments. The available anti-Fz 7 antibodies were unable to detect the endogenous protein. Therefore, we performed coimmunoprecipitation experiments on cells overexpressing the cytoplasmic domain of Fz 7 tagged N-terminally with eYFP, a strategy used successfully by others (Djiane et al., 2005). Endogenous syntenin could be coimmunoprecipitated with Fz 7 in these experimental settings (Figure 1E). To further address the biology of the interaction, we transiently transfected MCF-7 cells with plasmids encoding an eGFP-syntenin fusion protein, together or without expression plasmids encoding full-length human Fz 7. The subcellular localization of these proteins was examined by fluorescence microscopy. Fz 7, as detected by antibodies, showed predominant plasma membrane localization, and its distribution was not affected by eGFP–syntenin coexpression. eGFP-syntenin expressed in isolation was distributed all over the cell (Figure 2A), with only 20% of the cells showing some concentration at the plasma membrane (Figure 2D, left column). Coexpression of wild-type Fz 7 and eGFP-syntenin in the same cells resulted in a significant enrichment of eGFP-syntenin at the plasma membrane (Figure 2, B and D, compare middle and left columns). Coexpression of eGFP-syntenin with mutant Fz 7 T/A did not result in similar translocation of eGFP-syntenin to the plasma membrane (Figure 2, C and D, compare right and middle columns), consistent with the binding data represented in Figure 1B. Similar results were obtained in HUH-7 cells (data not shown). We concluded that syntenin–Fz 7 interaction can take place at the membrane, depending on the integrity of the Fz 7 C-terminal PDZBM.

Figure 2.

Figure 2.

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Syntenin is recruited by Fz 7 and stimulates c-jun phosphorylation. Confocal micrographs of MCF-7 cells overexpressing eGFP-syntenin alone (A), overexpressing eGFP-syntenin together with Fz 7 (B), or overexpressing eGFP-syntenin together with an Fz 7 carrying a mutated C-terminal PDZBM (T/A) (C). (D) Results are expressed as the mean percentage of cells where the syntenin fluorescence was concentrated at the plasma membrane; bars represent standard deviations. Note that the recruitment of eGFP-syntenin to the plasma membrane relies on Fz 7 and on the integrity of its C-terminal PDZBM. (E) Cell lysates originating from HEK293T cells transfected as indicated on top were tested for c-jun expression and c-jun phosphorylation (ser-63). Actin was used as a loading control. Note that syntenin addition stimulates c-jun phosphorylation in a concentration dependent manner.

To test for a role for syntenin in Wnt signaling, we used standard in vitro reporter assays (Billin et al., 2000; Yao et al., 2004). Overexpression of syntenin had no significant stimulatory or inhibitory effect in TOP/FOP flash experiments, neither in MCF-7 nor in Chinese hamster ovary-K1 cells (data not shown), suggesting syntenin has no role in the canonical Wnt pathway. To further investigate a potential role for syntenin–Fz interaction in Wnt signaling, we tested for JNK activation, because it has been established that this kinase is downstream of a noncanonical Wnt signaling cascade (Boutros et al., 1998). We therefore measured the phosphorylation of the JNK target c-jun upon syntenin overexpression. In HEK293T cells, syntenin stimulates c-jun phosphorylation, in a concentration dependent manner (Figure 2E, compare lanes 4–6 with lanes 1–2). Addition of exogenous Wnt was not necessary for the stimulatory effect of syntenin, possibly because sufficient endogenous Wnt was provided or indicating syntenin functions downstream of Wnt/Fz. Similarly, Dsh overexpression stimulated c-jun phosphorylation without exogenous Wnt addition (data not shown). As expected, overexpression of Fz 7 was also stimulatory (Figure 2E, compare lane 3 with lanes 1–2). Combination of Fz 7 and syntenin overexpression was slightly more potent than either of these alone (Figure 2E, lanes 7–8). When c-jun was phosphorylated, we also observed a slight increase in total c-jun signal, consistent with the increased stability of the phosphorylated form of the protein (Yao et al., 2004). As JNK activation is not specific for Fz-mediated signaling, we tested whether syntenin-2, which does not interact with Fz 7, would also be able to stimulate c-jun phosphorylation. This was not the case (Figure 2E, lanes 10–12). Based on these results, we hypothesized that syntenin-Fz 7 interaction might play a role in noncanonical Wnt signaling. We further investigated this hypothesis in Xenopus laevis.

Syntenin and Cognate Fz Are Coexpressed during Xenopus Early Development

We first examined when and where syntenin and Fz are coexpressed during X. laevis embryonic development. By in silico searches, we found three orthologues of syntenin in this organism (Figure 3). All three show higher homology to human syntenin than to human syntenin-2. These were designed as Xsyntenin-a, -a', and -b. Xsyntenin-a and -a' are 95.6% identical to one another, and they are more closely related to human syntenin than Xsyntenin-b. RT-PCR analysis, on RNA samples originating from embryos collected at different stages of development, identified a signal for Xsyntenin-a at all stages, albeit lower at stages 9–13 (Figure 4A). WISH for Xsyntenin-a, a', and b showed that maternal transcripts localized to the animal pole at stage 3 (four-cell). Signals observed for Xsyntenin-a (Figure 4B) were stronger than those obtained for Xsyntenin-a' and b (data not shown). During neurula to early tadpole stages, Xsyntenin-a transcripts were found in the neural tube, head, neural crest, and the pronephros region (Figure 4D). A similar distribution was observed for Xsyntenin-a' (Figure 4E). Xsyntenin-b expression was restricted to the cement gland and the epidermis (Figure 4F).

Figure 3.

Figure 3.

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(A) Sequence alignment of the human syntenin (Husyntenin) with the three orthologues found in X. laevis (Xsyntenin-a, -a', and -b). (B) Percentage of identity between the different domains of the various syntenins. Note the extensive conservation of the PDZ domains.

Figure 4.

Figure 4.

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Xsyntenin expressions during X. laevis early development and Xsyntenin-a corecruitment with XDsh, by XFz 7, to the plasma membrane of animal caps. (A) RNA extracts from embryos at different stages were used to analyze Xsyntenin-a expression by RT-PCR. Histone H4 amplification was used as an internal control. (B–I) Xsyntenin-a, -a', and b and XFz 3, 7, and 8 mRNA distributions (purple) at different stages (st) of development. Hd, head; nt, neural tube; pn, pronephros; ba; branchial arches; cg, cement gland. (J) Syntenin-Fz 7 interaction is conserved in Xenopus. Hu Fz7 and XFz7 cytoplasmic domain sequences are shown on the right. The sequences vary for two amino acids (underlined). Fz 7 cytoplasmic domain from Xenopus or human, mutated (T/A) or not mutated in the PDZBM, was overlayed with Xsyntenin-a (top). The quality and concentration of the fusion proteins were controlled in Coomassie (bottom). (K–U) Confocal micrographs of Xenopus animal caps at stage 9 showing the subcellular distribution of XDsh-myc (K–M and Q), Xkermit-myc (S and T), Xsyntenin-a-myc (N–P) or Xsyntenin-a-HA (R and U) as indicated at the bottom. The caps originate from embryos injected at two-cell stage with different combinations of mRNAs encoding proteins indicated on each micrograph. Note the translocation of XDsh to the plasma membrane upon XFz 7 and XFz 7 T/A expression (compare M and L with K) and the translocation of Xsyntenin-a upon XFz 7 but not XFz7 T/A expression (compare O and P with N). Note also that although Xsyntenin-a impairs Xkermit translocation (T), XDsh translocation is maintained (Q).

XFz 7 mRNA is also provided maternally, and it localizes in the animal region of early cleavage stage embryos (Medina et al., 2000) like Xsyntenin transcripts (Figure 4, compare B and C). During neurula to early tadpole stages, Xsyntenin-a and a' overlap with XFz 7 (Medina et al., 2000) in the neural tube, head, neural crest, and the pronephros region (Figure 4H). Xsyntenin-a and a' expression patterns also overlap with that of XFz 3 in developing nervous system and XFz 8 in the pronephros (Figure 4, G and I). Thus, overlapping RNA expression patterns suggested multiple opportunities for syntenin-Fz interaction during development.

XFz 7 Corecruits Xsyntenin-a and XDsh at the Membrane in X. laevis Animal Caps

After controlling that the Fz 7–syntenin interaction is conserved in Xenopus (Figure 4J), we tested for a functional relationship between XFz 7, Xsyntenin-a and XDsh in animal cap recruitment assays. These assays were previously implemented by other groups, studying Wnt signaling components that directly interact with Fz, such as XDsh and Xkermit (Umbhauer et al., 2000; Tan et al., 2001). Overexpressed XDsh distributes diffusely in animal cap cells in the absence of exogenous XFz 7 (Figure 4K), but it translocates to the plasma membrane upon simultaneous XFz 7 overexpression (Figure 4L). We found that overexpression of XFz 7 mutant for the C-terminal PDZBM induces similar XDsh translocation (Figure 4M). Overexpressed Xsyntenin-a, by itself, mainly distributes to both the cell interior and the nucleus, with a small proportion at the plasma membrane (Figure 4N). On XFz 7 overexpression, most Xsyntenin-a translocates to the plasma membrane (Figure 4O). Syntenin translocation was not observed with an XFz 7 mutant for its C-terminal PDZBM (Figure 4, compare P and O), contrary to Dsh and consistent with the in vitro mode of binding. Next, we found that the membrane translocation of XDsh is largely unaffected by Xsyntenin-a, even after injection of a fivefold excess of RNA encoding Xsyntenin-a (Figure 4, compare Q and L). Xsyntenin-a is also not displaced from the membrane upon XDsh expression (Figure 4R). In contrast, Xsyntenin-a, when used in excess, markedly influenced the XFz 7-mediated membrane recruitment of Xkermit (Figure 4, S and T), an alternative C-terminal PDZ partner of XFz 7 (Tan et al., 2001). We concluded that Fz 7 overexpression can induce the translocation of syntenin at the membrane of Xenopus animal caps without compromising the Dsh translocation.

Xsyntenin Overexpression or Down-Regulation Disrupts Convergent Extension Movements

Functional studies in Xenopus have revealed that noncanonical Wnt signaling and XFz 7 are required for convergence extension movements (CEs) during gastrulation (Djiane et al., 2000; Sumanas and Ekker, 2001). During CE, mesoderm and ectoderm cells intercalate along the medio-lateral axis, narrowing the tissues (convergence) and extending these along the anterior-posterior axis (extension). Because of the evidence for a role of syntenin in noncanonical (Figure 2G), rather than in canonical Wnt signaling, we tested for a role of syntenin in CE. For that, we first used activin-induced animal caps (Figure 5, A–D, compare left and right), a common ex vivo model for studying CE (Tada and Smith, 2000). It has previously been established that the overexpression and/or down-regulation of noncanonical Wnt signaling components such as XFz 7, XWnt 11, Strabismus, Crescent, and Syndecan-4 block CE in this assay (Djiane et al., 2000; Sumanas and Ekker, 2001; Darken et al., 2002; Shibata et al., 2005; Munoz et al., 2006). Animal hemisphere injection of a low dose (200 pg) of Xsyntenin-a mRNA did not affect cap elongation (Figure 5, E and J). However, a high dose (5 ng) of Xsyntenin-a mRNA efficiently blocked this elongation (Figure 5, F and J). To exclude that the block of elongation was due to a to a lack of mesoderm induction, we tested for the expression of the mesodermal marker cardiac actin. The expression of this marker was preserved (Figure 5G), indicating CE defects. The block of elongation was not observed when injecting a similar, high dose of GFP mRNA (Figure 5, H–J), confirming the specificity of the effect. Injection of a high dose of Xsyntenin-a mRNA also severely blocked the elongation of the body-axis in total embryos, further establishing a role for syntenin in CE (data not shown). MOs directed against Xsyntenin-a, Xsyntenin-a', or Xsyntenin-b, injected together or in isolation, did not affect the elongation of activin-treated animal caps (data not shown). Yet, when testing these MOs together on total embryo development, we observed delayed gastrulation and shortening of the trunk (Figure 5, K–Q), hallmarks of CE problems. Together, these observations indicate a role for syntenin in CE and thereby support a role for syntenin in noncanonical Wnt signaling.

Figure 5.

Figure 5.

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Xsyntenin overexpression and down-regulation block the elongation of activin-treated animal caps and of whole embryos. (A–I) Pictures of stage 19 animal cap explants, originating from embryos injected at two cell stage with different mRNAs as indicated on top, dissected at stage 9, and cultured in the absence or in the presence of 50 ng/ml activin (as indicated at the bottom). In situ hybridization with the mesodermal marker cardiac actin (B, D, G, and I) shows that the mesodermal differentiation is sustained in all activin-treated caps. Note the specific block of elongation induced by high dose of X-syntenin-a mRNA (F–G). (J) Results are expressed as the mean percentage of elongated caps per condition, bars represent standard deviations. (K–Q) Illustration of selected anomalies in X. laevis embryos depleted for Xsyntenin-a, a', and b. Embryos injected with Xsyntenin MOs show a delayed gastrulation (compare L with K), and they have a shorter body-axis (compare N–O with M). Injections were performed at the four-cell stage with 7, 5, or 10 ng of each Xsyntenin MO or with 22 or 30 ng mismatch MO in each blastomere. Embryos with body-axis length <60% (O) and embryos with body-axis between 60 and 80% (N) of the average length of noninjected controls were pooled for quantitative analysis (Q). The percentage of embryos showing delayed gastrulation (P) and shortened body-axis (Q) was scored in three independent experiments, with at least 80 embryos. Results are expressed as the mean percentage of embryos showing the defects at the stages indicated; bars represent standard deviations.

Xsyntenin Acts as an Activator of XFz 7 Signaling to XCDC42 and Stimulates XPKCα Membrane Recruitment

To test for the role of syntenin–Fz 7 interaction in CE, we analyzed the effect of injecting Xsyntenin RNA, together with XFz 7 MOs, on activin-induced elongation of Xenopus animal caps. Down-regulation of XFz 7 blocks CE in this assay (Sumanas and Ekker, 2001). In case syntenin supports noncanonical Wnt signaling by interacting with Fz 7, we assumed Xsyntenin-a overexpression should be able to rescue partial XFz 7 loss-of-function. Five nanograms of XFz 7 MO was the lowest dose required for an efficient block of elongation (Figure 6, A and B). This block was rescued by coinjection of a low dose of Xsyntenin-a RNA (200 pg, a dose having no effect on CE as shown in Figure 5E) but not by coinjection of 200 pg of control GFP RNA (Figure 6, C and D). This low dose of Xsyntenin-a RNA was also sufficient to rescue the effect of XFz 7 down-regulation in whole embryos (data not shown), indicating syntenin is a downstream mediator of XFz 7 signaling.

Figure 6.

Figure 6.

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Xsyntenin supports the XFz 7/ XPKCα/XCDC42 branch of the noncanonical Wnt signaling pathway. (A, C, and E) Pictures of stage 19 animal cap explants originating from embryos injected at two-cell stage with different components as indicated on top, dissected at stage 9, and cultured in the presence of 50 ng/ml activin. Note that the block of elongation observed with MOs for XFz 7 (A, right image) is rescued by the coinjection of a low dose of Xsyntenin-a RNA (C, middle picture). Note that the block of elongation induced by a high dose of Xsyntenin-a RNA is rescued by the coinjection of RNA encoding a DN form of the small GTPase XCDC42 (compare E, middle, with Figure 5F). (B, D, and F) Results are expressed as the mean percentage of elongated caps per condition; bars represent standard deviations. (G and H) Confocal micrographs of Xenopus animal caps at stage 9 showing the subcellular distribution of XPKCα-GFP as indicated at the bottom. (G) Comparison of the XPKCα-GFP distribution upon the overexpression of XFz 7 alone (left), together with Xsyntenin-a-HA (middle), or together with X-syntenin MOs (right). (H) Comparison of the XPKCα-GFP distribution upon the overexpression of Xsyntenin-a-HA (top) or the down-regulation of X-syntenins (bottom). Note that Xsyntenin enhances the plasma membrane recruitment of XPKCα-GFP.

To situate syntenin in the Fz 7 noncanonical Wnt signaling, we investigated a relationship with the CDC42 signaling cascade. Djiane et al. (2000) showed that the block of CE induced by overexpression of XFz 7 in activin-treated animal caps can be rescued by coinjecting RNA encoding a dominant-negative (DN) XCDC42. Similarly, DN XCDC42 was able to rescue the block of CE induced by a high dose of Xsyntenin-a RNA (compare Figure 5F with Figure 6E, middle), whereas it had no effect on CE by itself (Figure 6E, left). A control experiment using GFP RNA (Figure 6E, right) underscored the specificity of the DN CDC42 effect. This indicates that syntenin overexpression might act through the CDC42 pathway. To further establish a role for syntenin in the CDC42 noncanonical Wnt signaling cascade, we investigated the relationship of syntenin with PKCα. Indeed, Choi and Han (2002) showed that CDC42 is downstream of PKCα activation in noncanonical Wnt signaling. Overexpression of XFz 7 in animal caps induces membrane recruitment of XPKCα (Medina et al., 2000). We found that Xsyntenin-a-HA overexpression increases the membrane recruitment of XPKCα-GFP by XFz 7, whereas Xsyntenin down-regulation decreases this recruitment (Figure 6G). Surprisingly, Xsyntenin-a-HA overexpression alone was sufficient to induce translocation of XPKCα-GFP to the plasma membrane (Figure 6H, top). Consistently, in complementary experiments, Xsyntenin MOs decreased the discrete membrane localization of XPKCα-GFP (Figure 6H, bottom).

Together, these data suggest that syntenin works as an activator of Fz 7 noncanonical Wnt signaling upstream of PKCα and CDC42.

DISCUSSION

Here, we show that syntenin directly interacts with Fz 3, 7, and 8 (Figure 1B) and that it is coexpressed with cognate Fz during Xenopus early development (Figure 4, B–I). Syntenin seems to leave canonical Wnt signaling unaffected, because its overexpression had no effect on TOP/FOP flash reporter assays in cells, or on secondary axis formation in Xenopus (data not shown). Yet, syntenin overexpression or down-regulation both elicit a shortened trunk (Figure 5, M–O and Q). Such phenotype was also observed upon overexpression or down-regulation of components known to regulate noncanonical Wnt signaling, such as Fz 7 and strabismus (Djiane et al., 2000; Medina et al., 2000; Sumanas and Ekker, 2001; Darken et al., 2002). Moreover, syntenin overexpression increases c-jun phosphorylation in cultured cells (Figure 2E), and it blocks the activin-supported elongation of animal caps at high dose (Figure 5, F, G, and J), two additional pieces of evidence for a role in noncanonical Wnt signaling. At low dose, syntenin can rescue the Fz 7 down-regulation phenotype in activin-induced animal caps (Figure 6, C and D) and in whole embryos (data not shown), supporting a stimulating role for syntenin in noncanonical Wnt signaling. Consistently, syntenin recruits PKCα in animal caps (Figure 6H) as do Fz 7 and Wnt5a (Medina et al., 2000). Moreover, a DN CDC42 rescues the inhibitory effects of syntenin overexpression in animal caps (Figure 6, E and F), as it does for the inhibitory effects of the overexpression of noncanonical components such as Wnt11 and Fz 7 (Djiane et al., 2000). This indicates that syntenin functions downstream of Fz and upstream of the PKCα/CDC42 cascade (Choi and Han, 2002; Penzo-Mendez et al., 2003).

By what direct mechanisms could syntenin operate to stimulate Fz 7-dependent noncanonical Wnt signaling? Mutational analysis shows that the syntenin–Fz 7 interaction requires the C-terminal PDZBM of Fz 7 (Figure 1B) and that this motif must be intact to recruit syntenin to the membrane in cells (Figure 2, A–C) and in Xenopus animal caps (Figure 4, N–P). This leaves the membrane proximal PDZBM available for Dsh interaction. Consistently, syntenin does not affect the membrane recruitment of Dsh by Fz 7 (Figure 4, Q–R). Syntenin overexpression or down-regulation shows no significant effect on Dsh membrane localization in animal caps (data not shown) in contrast to what it does to PKCα (Figure 6H). This means that the stimulation of noncanonical Wnt signaling by syntenin might be Dsh-independent. This is reminiscent of what has been reported by Winklbauer et al. (2001), where Fz 7 signals through trimeric G proteins and PKCα independently from Dsh to control cell-sorting behavior in the mesoderm. Domain mapping shows that the syntenin–Fz 7 interaction requires the PDZ1 domain of syntenin (Figure 1, C and D). Because syndecans interact preferentially with the PDZ2 domain of syntenin (Grootjans et al., 2000), one could envision that syntenin serves as a scaffold bridging syndecan-signaling complexes to Fz (Figure 7). Syntenin can be recruited to the plasma membrane by syndecans (Zimmermann et al., 2002) and by Fz 7 (Figure 2B). In addition, syndecan, Fz 7 and syntenin largely colocalize in clusters at the membrane when coexpressed in cultured cells, suggesting they might form one functional unit (data not shown). Remarkably, the syntenin expression pattern in Xenopus early embryos overlaps with that reported for syndecan-4 (Munoz et al., 2006). Syndecan-4 can bind and activate PKCα (Oh et al., 1998; Lim et al., 2003). Syntenin might serve as a scaffold for Fz 7-syndecan-4-PKCα complexes, and this might explain how it stimulates noncanonical Wnt signaling (Figure 7A). Interestingly, Munoz et al. (2006) recently showed that syndecan-4 is required for noncanonical Wnt signaling in Xenopus and that syndecan-4 interacts, functionally and physically, with Fz 7 and Dsh (Munoz et al., 2006). Although we do not have evidence for the importance of Dsh in Fz 7-syntenin signaling (see above), we cannot exclude it. Regardless, our in vitro findings show syntenin can provide a direct link between Fz 7 and syndecan. Additionally, syntenin effects might take place via syndecan-2, a syndecan that activates CDC42 (Granes et al., 1999) (Figure 7B). We could not identify a clear increase in CDC42-GTP content upon syntenin overexpression in MCF-7 cells (data not shown). Yet, syntenin might be important for the appropriate targeting of CDC42 to Fz signaling complexes rather than for increasing CDC42-GTP steady-state levels.

Figure 7.

Figure 7.

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Model for the role of syntenin scaffolding in noncanonical Wnt signaling. Syntenin interacts with Fz 7 via its PDZ1 domain and with Syndecan-4 (A) or Syndecan-2 (B) via its PDZ2 domain. Syndecan and/or Fz 7 can recruit syntenin to the plasma membrane. (A) Syndecan-4 interacts directly with PKCα. Syndecan-4 triggers the activation of PKCα. PKCα functions upstream of CDC42 in noncanonical Wnt signaling. (B) Syndecan-2 might also activate CDC42. See Discussion for details and references.

Syntenin seems to be indispensable in vertebrates, because its down-regulation interferes with Xenopus (Figure 5, K–Q) and zebrafish (Lambaerts, K., Mortier, E., Degeest, G., Luyten, A., and Zimmerman, P., unpublished data) early development. Yet, it is not essential for all aspects of noncanonical Wnt/Fz signaling, because Drosophila melanogaster and Caenorhabditis elegans lack a syntenin orthologue in their genome. Interestingly, it was shown that syntenin is overexpressed in several human cancer cell lines (Koo et al., 2002) and that it is a positive regulator of metastasis by promoting cell invasion and migration, in particular in melanoma cells (Koo et al., 2002; Boukerche et al., 2005). For this process, the PDZ domains of syntenin seem essential (Koo et al., 2002; Meerschaert et al., 2007). Although the canonical Wnt pathway plays a role in carcinogenesis (Polakis, 2000), more recently the noncanonical Wnt pathway has been implicated in metastasis (Weeraratna et al., 2002; Katoh, 2005; Dissanayake et al., 2007). Aberrant regulation of Wnt5a/PKCα activation has also been implicated in melanoma metastasis (Weeraratna et al., 2002; Dissanayake et al., 2007). Syntenin PDZ domains might thus represent an attractive drug target for cancer therapy as it has been proposed for Dsh (Shan et al., 2005; Fujii et al., 2007).

ACKNOWLEDGMENTS

We thank Massimo Nichane for advice and help in the completion of this work. We thank Dr. Stephane Plaisance (University of Leuven, Belgium) for providing the pcDNA3-c-jun expression vector, Dr. H. Steinbesser (Max-Planck Institute for Developmental Biology, Tübingen, Germany) for the pBS(SK) XFz 7 WISH probe, Dr. D. Shi (Université P. et M. Curie, Paris, France) for the XFz3 WISH probe, Dr. M. Asashima (University of Tokyo, Japan) for the XFz8 WISH probe, Dr. M. Umbhauer (Université P. et M. Curie) for the pCS2+-XDsh-myc and pCS2+-XFz 7 expression constructs, Dr. P.S. Klein (University of Pennsylvania, Philadelphia, PA) for the pCS2+-XKermit-myc expression construct, and Dr. Jacob Souopgui (Université Libre de Bruxelles, Belgium) for the pCS2+-XCDC42 DN construct. A.L. was the recipient of a fellowship from the Vlaams Instituut voor de bevordering van het Wetenschappelijk–Technologisch onderzoek in de Industrie (I.W.T.), Belgium. This work was supported by the Fund for Scientific Research-Flanders (FWO), the Interuniversity Attraction Poles of the Prime Ministers Services, the Belgian Federation against Cancer, FB insurance Fortis, the Flanders Institute for Biotechnology (VIB), the Concerted Actions Program, the Research Fund, and the cell imaging core of the K.U. Leuven.

Footnotes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-08-0832) on February 6, 2008.

REFERENCES

  1. Ataman B., Ashley J., Gorczyca D., Gorczyca M., Mathew D., Wichmann C., Sigrist S. J., Budnik V. Nuclear trafficking of Drosophila Frizzled-2 during synapse development requires the PDZ protein dGRIP. Proc. Natl. Acad. Sci. USA. 2006;103:7841–7846. doi: 10.1073/pnas.0600387103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bellefroid E. J., Bourguignon C., Hollemann T., Ma Q., Anderson D. J., Kintner C., Pieler T. X-MyT1, a Xenopus C2HC-type zinc finger protein with a regulatory function in neuronal differentiation. Cell. 1996;87:1191–1202. doi: 10.1016/s0092-8674(00)81815-2. [DOI] [PubMed] [Google Scholar]
  3. Billin A. N., Thirlwell H., Ayer D. E. Beta-catenin-histone deacetylase interactions regulate the transition of LEF1 from a transcriptional repressor to an activator. Mol. Cell. Biol. 2000;20:6882–6890. doi: 10.1128/mcb.20.18.6882-6890.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bishop J. R., Schuksz M., Esko J. D. Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature. 2007;446:1030–1037. doi: 10.1038/nature05817. [DOI] [PubMed] [Google Scholar]
  5. Boukerche H., Su Z. Z., Emdad L., Baril P., Balme B., Thomas L., Randolph A., Valerie K., Sarkar D., Fisher P. B. mda-9/Syntenin: a positive regulator of melanoma metastasis. Cancer Res. 2005;65:10901–10911. doi: 10.1158/0008-5472.CAN-05-1614. [DOI] [PubMed] [Google Scholar]
  6. Boutros M., Mlodzik M. Dishevelled: at the crossroads of divergent intracellular signaling pathways. Mech. Dev. 1999;83:27–37. doi: 10.1016/s0925-4773(99)00046-5. [DOI] [PubMed] [Google Scholar]
  7. Boutros M., Paricio N., Strutt D. I., Mlodzik M. Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell. 1998;94:109–118. doi: 10.1016/s0092-8674(00)81226-x. [DOI] [PubMed] [Google Scholar]
  8. Choi S. C., Han J. K. Xenopus Cdc42 regulates convergent extension movements during gastrulation through Wnt/Ca2+ signaling pathway. Dev. Biol. 2002;244:342–357. doi: 10.1006/dbio.2002.0602. [DOI] [PubMed] [Google Scholar]
  9. Couchman J. R. Syndecans: proteoglycan regulators of cell-surface microdomains? Nat. Rev. Mol. Cell Biol. 2003;4:926–937. doi: 10.1038/nrm1257. [DOI] [PubMed] [Google Scholar]
  10. Darken R. S., Scola A. M., Rakeman A. S., Das G., Mlodzik M., Wilson P. A. The planar polarity gene strabismus regulates convergent extension movements in Xenopus. EMBO J. 2002;21:976–985. doi: 10.1093/emboj/21.5.976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dissanayake S. K., et al. The Wnt5A/protein kinase C pathway mediates motility in melanoma cells via the inhibition of metastasis suppressors and initiation of an epithelial to mesenchymal transition. J. Biol. Chem. 2007;282:17259–17271. doi: 10.1074/jbc.M700075200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Djiane A., Riou J., Umbhauer M., Boucaut J., Shi D. Role of frizzled 7 in the regulation of convergent extension movements during gastrulation in Xenopus laevis. Development. 2000;127:3091–3100. doi: 10.1242/dev.127.14.3091. [DOI] [PubMed] [Google Scholar]
  13. Djiane A., Yogev S., Mlodzik M. The apical determinants aPKC and dPatj regulate Frizzled-dependent planar cell polarity in the Drosophila eye. Cell. 2005;121:621–631. doi: 10.1016/j.cell.2005.03.014. [DOI] [PubMed] [Google Scholar]
  14. Fujii N., et al. An antagonist of dishevelled protein-protein interaction suppresses beta-catenin-dependent tumor cell growth. Cancer Res. 2007;67:573–579. doi: 10.1158/0008-5472.CAN-06-2726. [DOI] [PubMed] [Google Scholar]
  15. Granes F., Garcia R., Casaroli-Marano R. P., Castel S., Rocamora N., Reina M., Urena J. M., Vilaro S. Syndecan-2 induces filopodia by active cdc42Hs. Exp. Cell Res. 1999;248:439–456. doi: 10.1006/excr.1999.4437. [DOI] [PubMed] [Google Scholar]
  16. Grootjans J. J., Reekmans G., Ceulemans H., David G. Syntenin-syndecan binding requires syndecan-synteny and the co-operation of both PDZ domains of syntenin. J. Biol. Chem. 2000;275:19933–19941. doi: 10.1074/jbc.M002459200. [DOI] [PubMed] [Google Scholar]
  17. Grootjans J. J., Zimmermann P., Reekmans G., Smets A., Degeest G., Durr J., David G. Syntenin, a PDZ protein that binds syndecan cytoplasmic domains. Proc. Natl. Acad. Sci. USA. 1997;94:13683–13688. doi: 10.1073/pnas.94.25.13683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johnson M. L., Rajamannan N. Diseases of Wnt signaling. Rev. Endocr. Metab. Disord. 2006;7:41–49. doi: 10.1007/s11154-006-9003-3. [correction published in Rev. Endocr. Metab. Disord (2007) 8, 183] [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Katoh M. WNT/PCP signaling pathway and human cancer (review) Oncol. Rep. 2005;14:1583–1588. [PubMed] [Google Scholar]
  20. Kohn A. D., Moon R. T. Wnt and calcium signaling: beta-catenin-independent pathways. Cell Calcium. 2005;38:439–446. doi: 10.1016/j.ceca.2005.06.022. [DOI] [PubMed] [Google Scholar]
  21. Koo T. H., Lee J. J., Kim E. M., Kim K. W., Kim H. D., Lee J. H. Syntenin is overexpressed and promotes cell migration in metastatic human breast and gastric cancer cell lines. Oncogene. 2002;21:4080–4088. doi: 10.1038/sj.onc.1205514. [DOI] [PubMed] [Google Scholar]
  22. Kuhl M., Geis K., Sheldahl L. C., Pukrop T., Moon R. T., Wedlich D. Antagonistic regulation of convergent extension movements in Xenopus by Wnt/beta-catenin and Wnt/Ca2+ signaling. Mech. Dev. 2001;106:61–76. doi: 10.1016/s0925-4773(01)00416-6. [DOI] [PubMed] [Google Scholar]
  23. Lim S. T., Longley R. L., Couchman J. R., Woods A. Direct binding of syndecan-4 cytoplasmic domain to the catalytic domain of protein kinase C alpha (PKC alpha) increases focal adhesion localization of PKC alpha. J. Biol. Chem. 2003;278:13795–13802. doi: 10.1074/jbc.M208300200. [DOI] [PubMed] [Google Scholar]
  24. Logan C. Y., Nusse R. The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 2004;20:781–810. doi: 10.1146/annurev.cellbio.20.010403.113126. [DOI] [PubMed] [Google Scholar]
  25. Margolis B., Borg J. P. Apicobasal polarity complexes. J. Cell Sci. 2005;118:5157–5159. doi: 10.1242/jcs.02597. [DOI] [PubMed] [Google Scholar]
  26. Medina A., Reintsch W., Steinbeisser H. Xenopus frizzled 7 can act in canonical and non-canonical Wnt signaling pathways: implications on early patterning and morphogenesis. Mech. Dev. 2000;92:227–237. doi: 10.1016/s0925-4773(00)00240-9. [DOI] [PubMed] [Google Scholar]
  27. Meerschaert K., Bruyneel E., De Wever O., Vanloo B., Boucherie C., Bracke M., Vandekerckhove J., Gettemans J. The tandem PDZ domains of syntenin promote cell invasion. Exp. Cell Res. 2007;313:1790–1804. doi: 10.1016/j.yexcr.2007.03.014. [DOI] [PubMed] [Google Scholar]
  28. Mortier E., Wuytens G., Leenaerts I., Hannes F., Heung M. Y., Degeest G., David G., Zimmermann P. Nuclear speckles and nucleoli targeting by PIP2-PDZ domain interactions. EMBO J. 2005;24:2556–2565. doi: 10.1038/sj.emboj.7600722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Munoz R., Moreno M., Oliva C., Orbenes C., Larrain J. Syndecan-4 regulates non-canonical Wnt signalling and is essential for convergent and extension movements in Xenopus embryos. Nat. Cell Biol. 2006;8:492–500. doi: 10.1038/ncb1399. [DOI] [PubMed] [Google Scholar]
  30. Nieuwkoop P. D., Faber J. The Netherlands: North Holland Publishing Company; 1967. A Laboratory Manual, Amsterdam. [Google Scholar]
  31. Nourry C., Grant S. G., Borg J. P. PDZ domain proteins: plug and play! Sci. STKE. 2003;2003:RE7. doi: 10.1126/stke.2003.179.re7. [DOI] [PubMed] [Google Scholar]
  32. Oh E. S., Woods A., Lim S. T., Theibert A. W., Couchman J. R. Syndecan-4 proteoglycan cytoplasmic domain and phosphatidylinositol 4,5-bisphosphate coordinately regulate protein kinase C activity. J. Biol. Chem. 1998;273:10624–10629. doi: 10.1074/jbc.273.17.10624. [DOI] [PubMed] [Google Scholar]
  33. Penzo-Mendez A., Umbhauer M., Djiane A., Boucaut J. C., Riou J. F. Activation of Gbetagamma signaling downstream of Wnt-11/Xfz7 regulates Cdc42 activity during Xenopus gastrulation. Dev. Biol. 2003;257:302–314. doi: 10.1016/s0012-1606(03)00067-8. [DOI] [PubMed] [Google Scholar]
  34. Polakis P. Wnt signaling and cancer. Genes Dev. 2000;14:1837–1851. [PubMed] [Google Scholar]
  35. Reichsman F., Smith L., Cumberledge S. Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction. J. Cell Biol. 1996;135:819–827. doi: 10.1083/jcb.135.3.819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shan J., Shi D. L., Wang J., Zheng J. Identification of a specific inhibitor of the dishevelled PDZ domain. Biochemistry. 2005;44:15495–15503. doi: 10.1021/bi0512602. [DOI] [PubMed] [Google Scholar]
  37. Sheldahl L. C., Slusarski D. C., Pandur P., Miller J. R., Kuhl M., Moon R. T. Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos. J. Cell Biol. 2003;161:769–777. doi: 10.1083/jcb.200211094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shibata M., Itoh M., Hikasa H., Taira S., Taira M. Role of crescent in convergent extension movements by modulating Wnt signaling in early Xenopus embryogenesis. Mech. Dev. 2005;122:1322–1339. doi: 10.1016/j.mod.2005.06.002. [DOI] [PubMed] [Google Scholar]
  39. Sive H. L., Grainer R. M, Harland R. M. A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor, Laboratory Press; 2000. [Google Scholar]
  40. Strutt D. Frizzled signalling and cell polarisation in Drosophila and vertebrates. Development. 2003;130:4501–4513. doi: 10.1242/dev.00695. [DOI] [PubMed] [Google Scholar]
  41. Sumanas S., Ekker S. C. Xenopus frizzled-7 morphant displays defects in dorsoventral patterning and convergent extension movements during gastrulation. Genesis. 2001;30:119–122. doi: 10.1002/gene.1044. [DOI] [PubMed] [Google Scholar]
  42. Tada M., Smith J. C. Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. Development. 2000;127:2227–2238. doi: 10.1242/dev.127.10.2227. [DOI] [PubMed] [Google Scholar]
  43. Tan C., Deardorff M. A., Saint-Jeannet J. P., Yang J., Arzoumanian A., Klein P. S. Kermit, a frizzled interacting protein, regulates frizzled 3 signaling in neural crest development. Development. 2001;128:3665–3674. doi: 10.1242/dev.128.19.3665. [DOI] [PubMed] [Google Scholar]
  44. Umbhauer M., Djiane A., Goisset C., Penzo-Mendez A., Riou J. F., Boucaut J. C., Shi D. L. The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/beta-catenin signalling. EMBO J. 2000;19:4944–4954. doi: 10.1093/emboj/19.18.4944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Veeman M. T., Axelrod J. D., Moon R. T. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev. Cell. 2003;5:367–377. doi: 10.1016/s1534-5807(03)00266-1. [DOI] [PubMed] [Google Scholar]
  46. Vinson C. R., Conover S., Adler P. N. A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains. Nature. 1989;338:263–264. doi: 10.1038/338263a0. [DOI] [PubMed] [Google Scholar]
  47. Wang H. Y., Liu T., Malbon C. C. Structure-function analysis of Frizzleds. Cell Signal. 2006;18:934–941. doi: 10.1016/j.cellsig.2005.12.008. [DOI] [PubMed] [Google Scholar]
  48. Weeraratna A. T., Jiang Y., Hostetter G., Rosenblatt K., Duray P., Bittner M., Trent J. M. Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell. 2002;1:279–288. doi: 10.1016/s1535-6108(02)00045-4. [DOI] [PubMed] [Google Scholar]
  49. Wehrli M., Dougan S. T., Caldwell K., O'Keefe L., Schwartz S., Vaizel-Ohayon D., Schejter E., Tomlinson A., DiNardo S. arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature. 2000;407:527–530. doi: 10.1038/35035110. [DOI] [PubMed] [Google Scholar]
  50. Winklbauer R., Medina A., Swain R. K., Steinbeisser H. Frizzled-7 signalling controls tissue separation during Xenopus gastrulation. Nature. 2001;413:856–860. doi: 10.1038/35101621. [DOI] [PubMed] [Google Scholar]
  51. Wong H. C., Bourdelas A., Krauss A., Lee H. J., Shao Y., Wu D., Mlodzik M., Shi D. L., Zheng J. Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled. Mol. Cell. 2003;12:1251–1260. doi: 10.1016/s1097-2765(03)00427-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wu J., Klein T. J., Mlodzik M. Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity. PLoS Biol. 2004;2:E158. doi: 10.1371/journal.pbio.0020158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yang-Snyder J., Miller J. R., Brown J. D., Lai C. J., Moon R. T. A frizzled homolog functions in a vertebrate Wnt signaling pathway. Curr. Biol. 1996;6:1302–1306. doi: 10.1016/s0960-9822(02)70716-1. [DOI] [PubMed] [Google Scholar]
  54. Yao R., Natsume Y., Noda T. MAGI-3 is involved in the regulation of the JNK signaling pathway as a scaffold protein for frizzled and Ltap. Oncogene. 2004;23:6023–6030. doi: 10.1038/sj.onc.1207817. [DOI] [PubMed] [Google Scholar]
  55. Zimmermann P., Meerschaert K., Reekmans G., Leenaerts I., Small J. V., Vandekerckhove J., David G., Gettemans J. PIP(2)-PDZ domain binding controls the association of syntenin with the plasma membrane. Mol. Cell. 2002;9:1215–1225. doi: 10.1016/s1097-2765(02)00549-x. [DOI] [PubMed] [Google Scholar]
  56. Zimmermann P., Tomatis D., Rosas M., Grootjans J., Leenaerts I., Degeest G., Reekmans G., Coomans C., David G. Characterization of syntenin, a syndecan-binding PDZ protein, as a component of cell adhesion sites and microfilaments. Mol. Biol. Cell. 2001;12:339–350. doi: 10.1091/mbc.12.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zimmermann P., Zhang Z., Degeest G., Mortier E., Leenaerts I., Coomans C., Schulz J., N′Kuli F., Courtoy P. J., David G. Syndecan recycling [corrected] is controlled by syntenin-PIP2 interaction and Arf6. Dev. Cell. 2005;9:377–388. doi: 10.1016/j.devcel.2005.07.011. [DOI] [PubMed] [Google Scholar]

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