The receptor protein tyrosine kinase 7 (PTK7) was recently shown to participate in noncanonical Wnt/planar cell polarity signalling during mouse and frog embryonic development. In this study, we report that PTK7 interacts with β‐catenin in a yeast two‐hybrid assay and mammalian cells. PTK7‐deficient cells exhibit weakened β‐catenin/T‐cell factor transcriptional activity on Wnt3a stimulation. Furthermore, Xenopus PTK7 is required for the formation of Spemann's organizer and for Siamois promoter activation, events that require β‐catenin transcriptional activity. Using epistatic assays, we demonstrate that PTK7 functions upstream from glycogen synthase kinase 3. Taken together, our data reveal a new and conserved role for PTK7 in the Wnt canonical signalling pathway.
Protein tyrosine kinase 7 (PTK7)—also known as colon cancer kinase 4 (CCK4)—is a single transmembrane receptor with a kinase dead domain that was first identified in melanocytes (Lee et al, 1993) and then in fetal colon and colon carcinoma (Mossie et al, 1995; Boudeau et al, 2006). Both mouse and Xenopus PTK7‐deficient embryos show developmental defects, supporting its role in noncanonical Wnt signalling or the Wnt/planar cell polarity (PCP) pathway (Lu et al, 2004; Shnitsar & Borchers, 2008; Yen et al, 2009). Consistent with this, mouse ptk7 interacts genetically with vangl2, a core Wnt/PCP gene (Lu et al, 2004).
To gain further insights into the function of PTK7, we screened for PTK7‐binding partners by using a yeast two‐hybrid assay and identified β‐catenin as a protein that interacts with the defective tyrosine kinase domain. In the canonical Wnt pathway, ligands such as Wnt3a bind to Frizzled and low‐density lipoprotein receptor‐related proteins 5 and 6 (LRP5/6) receptors, which causes the recruitment of Dishevelled to the cell membrane and inhibits the β‐catenin degradation complex that includes the scaffold protein Axin and glycogen synthase kinase 3 (GSK3). Consequently, β‐catenin is stabilized and enters the nucleus to regulate gene expression together with T‐cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors (Angers & Moon, 2009). In Xenopus embryos, β‐catenin activates the expression of Siamois and Spemann's organizer genes (Kodjabachian & Lemaire, 2004). In this study, we show that PTK7 is required for β‐catenin‐dependent transcriptional events induced by canonical Wnt ligands, in both frog and mammalian cells. This study, together with previous research, indicates that PTK7 is an important conserved modulator of multiple Wnt pathways in normal and possibly pathological conditions, including cancer.
Results And Discussion
Physical interaction between PTK7 and β‐catenin
To gain insight into the signalling pathways associated with PTK7, we fused its cytoplasmic region (PTK7727–1070) to the Gal4 DNA‐binding domain and screened a human colon complementary DNA (cDNA) library with yeast two‐hybrid assay. Among the positive clones recovered, we focused on two that encompassed most of the peptide sequence of β‐catenin (data not shown). In yeast two‐hybrid assays, the whole intracellular region of PTK7 (PTK7727–1070) and the isolated tyrosine kinase domain (PTK7794–1070) interacted with full‐length β‐catenin (β‐catenin1–781), whereas the extracellular (PTK71–727) and juxtamembrane (PTK7727–793) regions did not (Fig 1A). Mapping experiments showed that deletion of the first 60 residues of β‐catenin (β‐catenin61–781) did not impair PTK7 binding, whereas the amino‐terminal fragment (β‐catenin1–284) had no affinity for PTK7 (Fig 1A). Next, we asked whether PTK7 and β‐catenin also interact in human cells. For this, Myc–β‐catenin was transiently expressed in COS7 cells together with Flag–PTK7 or Flag–PTK71–788, a construct containing only the extracellular and transmembrane regions of PTK7. Immunoprecipitation using a PTK7 antibody demonstrated that Flag–PTK7, but not Flag–PTK71–788, co‐immunoprecipitated with β‐catenin (Fig 1B). We also transiently expressed green fluorescent protein (GFP) N‐terminally fused to the cytoplasmic regions of PTK7 in COS7 cells together with Myc–β‐catenin, and immunoprecipitated the protein complexes with GFP antibody. Similarly to yeast, GFP–PTK7727–1070 and GFP–PTK7794–1070 but not GFP–PTK7727–793 or GFP alone interacted with β‐catenin (Fig 1C). As GFP–PTK7727–1070 interacted more weakly than GFP–PTK7794–1070, we suspect that sequence 727–794 might destabilize the interaction. In the reverse experiment, Myc‐tagged β‐catenin constructs were coexpressed with PTK7 in COS7 cells. Myc–β‐catenin61–781, armadillo repeats (β‐catenin131–674) and the carboxy terminal (β‐catenin630–781) interacted with PTK7 (Fig 1D, upper panel). To reveal the endogenous PTK7–β‐catenin interaction, we used protein extracts from epithelial Caco2 cells. Immunoprecipitation with PTK7 antibody pulled down endogenous β‐catenin, but not β‐PAK‐interacting exchange factor (PIX), which was used as control (Fig 1E). In both Madin–Darby canine kidney (MDCK) and Caco2 polarized epithelial cells, PTK7 colocalized at the cell–cell junctions with β‐catenin and E‐cadherin (Fig 1F and data not shown, respectively). However, immunoprecipitation assays revealed that PTK7 interacts with β‐catenin but not E‐cadherin in MDCK cells (supplementary Fig S1 online), suggesting the existence of distinct pools of β‐catenin at the cell membrane. To explore the possible modulation of the PTK7–β‐catenin interaction by Wnt canonical ligands, Flag–PTK7 and Myc–β‐catenin were transiently expressed in MDCK cells together with Wnt3a–Myc or its backbone vector (pCAGGS‐Myc). Immunoprecipitation with PTK7 antibody demonstrated that Wnt3a stimulation reduces the amount of co‐immunoprecipitated β‐catenin (Fig 1G). Together, these data reveal that the tyrosine kinase domain of PTK7 is able to interact dynamically with β‐catenin, under the control of Wnt ligands.
PTK7 affects β‐catenin transcriptional activity
To test the potential implications of PTK7 for Wnt canonical signalling, we measured β‐catenin transcriptional activity by the TOP‐Flash/luciferase reporter construct, which has several TCF/LEF‐binding sites. We used the human colon cancer cell line HCT116, as it expresses PTK7 endogenously. We established two HCT116 clones in which PTK7 expression was stably knocked down by specific short hairpin RNA (shRNA), as confirmed by fluorescence‐activated cell sorting (FACS) and western blotting analysis (Fig 2A). In HCT116 control cells (shLuc), we observed a basal TCF/LEF transcriptional activity that was upregulated by twofold on Wnt3a stimulation. By contrast, decreased expression of PTK7 in the two shPTK7 clones prevented Wnt3a‐induced TCF/LEF transcriptional activity (Fig 2B).
To confirm the role of PTK7 in Wnt canonical signalling, we generated primary mouse embryonic fibroblasts (MEFs) from 15‐day‐old littermate wild‐type (+/+) or PTK7‐deficient (−/−) embryos (Lu et al, 2004). The expression of PTK7 in MEFs, according to their genotype, was confirmed by western blotting (Fig 2C). Plasmid expressing Wnt3a–Myc was nucleofected in MEFs along with the luciferase reporters. Wnt3a–Myc activated the TOP‐flash reporter more robustly in wild type than in PTK7 null MEFs (Fig 2D). We were able to rescue the Wnt3a‐induced TCF/LEF transcriptional activity by re‐expression of Flag–PTK7, but not Flag–PTK71–788 lacking the kinase domain (Fig 2D). To test whether this reduced transcriptional response involved destabilization of β‐catenin in the absence of PTK7, we measured levels of ubiquitinylated β‐catenin, a form tagged for degradation. Whereas Wnt3a lowered β‐catenin ubiquitinylation in wild‐type MEFs, it increased it in PTK7 null MEFs. Re‐expression of PTK7 reversed this effect (Fig 2E). In summary, PTK7 seems to favour β‐catenin stabilization and transcriptional activity on Wnt3a ligand stimulation.
PTK7 is required for organizer gene expression
In the Xenopus embryo, the Wnt/β‐catenin pathway triggers the formation of Spemann's organizer, an embryonic structure required for axis formation (Kodjabachian & Lemaire, 2004). We used published morpholinos (MOs) targeting PTK7 to address its function in organizer formation. As expected, PTK7 morphant embryos showed neural‐tube closure defects at tailbud stage (Lu et al, 2004). However, they also often displayed incomplete blastopore closure and a reduced nervous system, as shown by sox2 staining, particularly in the brain region (Fig 3A), suggesting reduced organizer activity. To address this possibility, we analysed organizer gene expression before gastrulation movements, thus excluding indirect consequences of aberrant morphogenesis. Both whole‐mount in situ hybridization (WISH; 81%, n=82) and reverse transcriptase–quantitative PCR (RT–qPCR) analyses showed that the organizer genes chordin (chd), goosecoid (gsc), hex, noggin (nog), siamois (sia) and xnr3 were downregulated in the absence of PTK7 (Fig 3B,C). The extent of this repression was comparable to, although weaker than, that caused by β‐catenin knockdown (Heasman et al, 2000; Fig 3B,C). This repression was partly reverted (38%, n=87) in PTK7 morphant embryos that received a separate injection of messenger RNAs (mRNAs) encoding PTK7 or PTK7–Venus, both of which lacked the MO‐binding sites (Fig 3B). By contrast, the isolated cytosolic or extracellular domains of PTK7 were not able to rescue PTK7 morphants (data not shown). Of the organizer genes studied here, siamois is known to be directly controlled by Wnt canonical signalling, as its promoter contains binding elements for the TCF3–β‐catenin complex (Fan et al, 1998). We found that both β‐catenin and PTK7 MOs resulted in a fivefold reduction in the level of expression of the co‐injected siamois‐luciferase reporter plasmid (p‐Sia‐Luc, Fig 3D). To confirm the specificity of PTK7 knockdown, we injected embryos with a dominant‐negative form of PTK7 that lacks the intracellular domain (Lu et al, 2004). We found that this mutant could also suppress organizer and neural gene expression (Fig 3E). Taken together, these results suggest that, similarly to that in mammalian cells, β‐catenin transcriptional activity is compromised in embryonic cells depleted of PTK7 activity.
PTK7 functions upstream from GSK3
To assess directly the requirement of PTK7 in activation of the canonical Wnt pathway, Xenopus embryos were injected with Wnt8 mRNA in a ventral position to induce the formation of an ectopic organizer (Fig 4A). When Wnt8 was injected into embryos pre‐loaded with PTK7 or β‐catenin MOs, ectopic organizer gene expression and siamois reporter activation were prevented (Fig 4A–C). As PTK7 protein is normally found at the plasma membrane, it might act upstream from the cytosolic components of the canonical Wnt pathway. Thus, we activated Wnt signalling at the level of Axin and GSK3—two proteins that control β‐catenin availability in the cell. For this, we injected dominant‐negative forms of Axin (dnAxin; Zeng et al, 1997) or GSK3 (dnGSK3; He et al, 1995) and treated embryos with lithium chloride, a potent inhibitor of GSK3 (Klein & Melton, 1996). The WISH and RT–qPCR analyses and the luciferase assays with the siamois reporter revealed that PTK7 was largely dispensable for organizer gene activation caused by all three reagents. By contrast and as expected, β‐catenin knockdown suppressed gene activation induced by such treatments (Fig 4G–N). It was proposed recently that PTK7 in association with the Frizzled 7 receptor initiates PCP signalling through recruitment of Dishevelled at the plasma membrane (Shnitsar & Borchers, 2008). We therefore tested the requirement for PTK7 in organizer gene expression induced by Dishevelled (Sokol et al, 1995). As shown in Fig 4D–F, PTK7 knockdown suppressed the response induced by Dishevelled mRNA injection, suggesting that these proteins might also be partners in canonical Wnt signalling. Interestingly, we found that overexpression of Dishevelled in MDCK cells, competes for the binding of PTK7 to β‐catenin (supplementary Fig S2 online). Together with the observed reduction in PTK7–β‐catenin interaction induced by Wnt3A (Fig 1G), we suggest that PTK7 might act as a platform to deliver stabilized β‐catenin upon receptor activation. Finally, we confirmed that a stabilized form of β‐catenin that cannot be phosphorylated by GSK3 (ΔN‐βcat; Yost et al, 1996) could activate the siamois promoter in the absence of PTK7 (Fig 4O). Taken together, these data indicate that PTK7 functions upstream from the β‐catenin destruction complex in the Wnt canonical signalling pathway.
Future studies should address the mechanism of action of PTK7 in the Wnt–β‐catenin pathway. Overexpression of full‐length PTK7 or its intracellular domain is not sufficient to activate the siamois reporter and organizer gene expression, suggesting that PTK7 acts as a permissive factor in this pathway (data not shown). Pseudo‐tyrosine kinase receptors, such as PTK7, might participate with enzymatically active complexes by interacting with functional kinases at the plasma membrane (Boudeau et al, 2006). Previous studies have shown that the enzymatically active Ror2 tyrosine kinase receptor can bind to distinct Wnt ligands and stimulate canonical and noncanonical responses (Angers & Moon, 2009). Thus, Ror2 is a potential partner for PTK7. Overall, identification of additional PTK7 partners might shed more light on the mechanism of action of this important modulator of Wnt pathways.
Plasmids and short hairpin RNA, antibodies, cell culture, transfection, luciferase assay, FACS analysis, yeast two‐hybrid screens, immunofluorescence and protein procedures. See supplementary information online and references.
Xenopus procedures. Eggs commercially obtained from NASCO (California, USA) females were fertilized in vitro, dejellied, cultured and injected as described (Marchal et al, 2009). PTK7 MOs were purchased from GeneTools LLC (Oregon, USA), resuspended in sterile water to a concentration of 10 mg/ml and further diluted before injection. Among the three published MOs targeting Xenopus PTK7 (Lu et al, 2004), we found that oligonucleotide A was toxic, so we used the combination of oligonucleotides B and C. These two MOs target the 5′ UTR of PTK7 messengers, a region that is not present in the mRNA used for the rescue assay. The MO targeting Xenopus β‐catenin was reported by Heasman et al (2000). Synthetic capped mRNAs were produced with Ambion (Applied Biosystems, France) mMessage mMachine kit. Wnt8, ΔN‐βcat, PTK7, PTK7‐Venus and PTK7‐ECD mRNAs were synthesized with T3 polymerase after plasmid linearization with SfiI. References for Dsh, dnAxin, dnGSK3 and ΔN‐βcat expression constructs are included in the results section. Embryos were processed for RT–qPCR and WISH with digoxygenin‐labelled probes (Roche) as described previously (Marchal et al, 2009).
Quantitative RT–PCR. See supplementary information online and references.
Supplementary information is available at EMBO reports online (http://www.emboreports.org).
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplementary Figure 1 and 2
We thank J. Muster, R. Moon, H. Sasaki, S. Sokol, P. Lemaire and H. Yasuo for plasmids. We also thank A. Zurubrica for helpful discussion. This study was supported by Centre National de la Recherche Scientifique, Inserm, Institut Paoli‐Calmettes, Association pour la Recherche contre le Cancer, Agence Nationale de la Recherche (V.T. & L.K.), Ligue Nationale Contre le Cancer (Label 2010 J.‐P.B.), Fondation de France (T.P.), European Consortium for Anticancer Antibody Development and Infrastrutures en Biologie Sante et Agronomie (Marseille Proteomic Platform).
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