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Vol. 19, Issue 4, 1594-1604, April 2008
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*Department of Human Genetics, K.U. Leuven, B-3000 Leuven, Belgium; Department for Molecular and Developmental Genetics, VIB, B-3000 Leuven, Belgium; and Institut de Biologie et de Medecine Moléculaires, Université Libre de Bruxelles, B-6041 Gosselies, Belgium
Submitted August 28, 2007;
Revised January 3, 2008;
Accepted January 30, 2008
Monitoring Editor: Carl-Henrik Heldin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 PKC/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 |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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 XPKC-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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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; 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).
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) 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.
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Membrane Recruitment). 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.
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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 PKC. Indeed, Choi and Han (2002) showed that CDC42 is downstream of PKC activation in noncanonical Wnt signaling. Overexpression of XFz 7 in animal caps induces membrane recruitment of XPKC (Medina et al., 2000). We found that Xsyntenin-a-HA overexpression increases the membrane recruitment of XPKC-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 XPKC-GFP to the plasma membrane (Figure 6H, top). Consistently, in complementary experiments, Xsyntenin MOs decreased the discrete membrane localization of XPKC-GFP (Figure 6H, bottom).
Together, these data suggest that syntenin works as an activator of Fz 7 noncanonical Wnt signaling upstream of PKC and CDC42.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; 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 PKC 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 PKC/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 PKC (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 PKC 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 PKC (Oh et al., 1998; Lim et al., 2003). Syntenin might serve as a scaffold for Fz 7-syndecan-4-PKC 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.
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) 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/PKC 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).
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Pascale Zimmermann (pascale.zimmermann{at}med.kuleuven.be)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PDZBM (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).
