© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 1890-1903 doi:10.1242/jcs.198580 RESEARCH ARTICLE PTK7 localization and protein stability is affected by canonical Wnt ligands Hanna Berger1, Marlen Breuer1,4, Hanna Peradziryi2, Martina Podleschny1, Ralf Jacob3,4 and Annette Borchers1,4,* ABSTRACT Wnt binding results in membrane recruitment of a destruction β Protein tyrosine kinase 7 (PTK7) is an evolutionarily conserved complex, leading to the stabilization of -catenin and expression of β transmembrane receptor with important roles in embryonic -catenin-dependent target genes (MacDonald and He, 2012; β development and disease. Originally identified as a gene MacDonald et al., 2009). In contrast, non-canonical -catenin- upregulated in colon cancer, it was later shown to regulate planar independent Wnt signaling pathways use alternative co-receptors cell polarity (PCP) and directional cell movement. PTK7 is a Wnt like the receptor tyrosine kinase-like orphan receptor 2 (Ror2) or co-receptor; however, its role in Wnt signaling remains controversial. protein tyrosine kinase 7 (PTK7) (Lu et al., 2004; Nomachi et al., Here, we find evidence that places PTK7 at the intersection of 2008; Schambony and Wedlich, 2007). The best-characterized non- canonical and non-canonical Wnt signaling pathways. In presence of canonical Wnt signaling pathway is the planar cell polarity (PCP) canonical Wnt ligands PTK7 is subject to caveolin-mediated pathway, which determines the coordinated polarity of cells in endocytosis, while it is unaffected by non-canonical Wnt ligands. the plane of an epithelial tissue, thereby regulating diverse PTK7 endocytosis is dependent on the presence of the PTK7 developmental processes (Simons and Mlodzik, 2008; Vladar co-receptor Fz7 (also known as Fzd7) and results in lysosomal et al., 2009; Wallingford, 2012). Activation of PCP signaling degradation of PTK7. As we previously observed that PTK7 activates modifies the cytoskeleton and changes cell polarity, for example, by non-canonical PCP Wnt signaling but inhibits canonical Wnt activating small GTPases or JNK-dependent transcription factors signaling, our data suggest a mutual inhibition of canonical and (Anastas and Moon, 2013) like ATF2 and their respective target PTK7 Wnt signaling. PTK7 likely suppresses canonical Wnt signaling genes (Ohkawara and Niehrs, 2011; Schambony and Wedlich, by binding canonical Wnt ligands thereby preventing their interaction 2007). Thus, receptor context is crucial to determine signaling with Wnt receptors that would otherwise support canonical Wnt outcome. signaling. Conversely, if canonical Wnt proteins interact with the A Wnt co-receptor of particular interest is the evolutionarily PTK7 receptor, they induce its internalization and degradation. conserved transmembrane receptor PTK7. Originally identified as a gene upregulated in colon carcinomas and named colon-carcinoma KEY WORDS: Wnt signaling, PTK7, Planar cell polarity, Endocytosis, kinase 4 (CCK4) (Mossie et al., 1995), PTK7 was later shown to Caveolin function in a variety of developmental and physiological processes, including the determination of PCP, and the control of cell migration INTRODUCTION and invasion as well as regeneration (Berger et al., 2017; Dunn and Wnt proteins are important for embryonic development and adult Tolwinski, 2016; Peradziryi et al., 2012). PTK7 is a single-pass tissue homeostasis, and these distinct functions require a precise transmembrane receptor with extracellular immunoglobulin fine-tuning of downstream signaling events. The demonstration that domains and an intracellular evolutionarily conserved kinase a single Wnt ligand can activate distinct signaling pathways homology domain, which lacks catalytic activity (Kroiher et al., depending on its choice of receptor (Mikels and Nusse, 2006), 2001; Miller and Steele, 2000). In vertebrates, PTK7 plays a role in suggested that receptor context determines signaling output. It is the regulation of PCP, and its loss of function results in classical now acknowledged that combinatorial co-receptor complexes PCP phenotypes, including convergent extension defects, provide a molecular code by which Wnt ligands can cause distinct disruption of stereociliary bundle orientation and inhibition of cellular responses (Niehrs, 2012; van Amerongen et al., 2008). For neural crest cell migration (Hayes et al., 2013; Lu et al., 2004; example the canonical β-catenin-dependent Wnt signaling pathway Paudyal et al., 2010; Shnitsar and Borchers, 2008; Yen et al., 2009). is activated by binding of Wnt ligands to members of the Frizzled Consistent with a role in activation of PCP signaling, PTK7 has receptor family and the low-density lipoprotein receptor-related been shown to interact with Wnt ligands as well as the Frizzled 7 proteins (LRP5 or LRP6) (Tamai et al., 2000; Wehrli et al., 2000). (Fz7; also known as Fzd7) and Ror2 receptors (Linnemannstöns et al., 2014; Martinez et al., 2015; Peradziryi et al., 2012; Podleschny et al., 2015; Shnitsar and Borchers, 2008). 1Department of Biology, Molecular Embryology, Philipps-Universität Marburg, Marburg 35043, Germany. 2Institute for Clinical Research, Georg-August Furthermore, PTK7 has been reported to recruit Dishevelled UniversitätGöttingen, Göttingen 37075, Germany. 3Department of Cell Biology and (Dsh) proteins to the plasma membrane and to activate JNK and Cell Pathology, Philipps-Universität Marburg, Marburg 35037, Germany. 4DFG ATF2-dependent signaling (Martinez et al., 2015; Peradziryi et al., Research Training Group, Membrane Plasticity in Tissue Development and Remodeling, GRK 2213, Philipps-Universität Marburg, Marburg 35043, Germany. 2011; Shnitsar and Borchers, 2008). However, PTK7 function is likely not limited to the regulation of non-canonical Wnt PCP *Author for correspondence ([email protected]) signaling, because PTK7 has also been shown to interact with A.B., 0000-0002-8314-9024 components of the canonical Wnt pathway including Wnt ligands, the LRP6 receptor and β-catenin (Bin-Nun et al., 2014; Peradziryi Received 12 October 2016; Accepted 7 April 2017 et al., 2011; Puppo et al., 2011). Recently, we demonstrated that Journal of Cell Science 1890 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1890-1903 doi:10.1242/jcs.198580 PTK7 interacts with canonical Wnt ligands Wnt3a and Wnt8, but treated with Wnt3a or Wnt5a. Consistent with our previous not non-canonical Wnt5a or Wnt11. Overexpression of PTK7 findings, the amount of cell surface PTK7–GFP and ΔkPTK7– blocked activation of canonical Wnt signaling by Wnt3a and Wnt8 GFP significantly decreased after Wnt3a treatment, but remained in Xenopus double axis and luciferase assays. Conversely, PTK7 unchanged after Wnt5a stimulation (Fig. 1C,D). loss of function activated canonical Wnt activity, suggesting that In order to perform live-cell imaging of the Wnt-mediated PTK7 PTK7 inhibits canonical Wnt signaling (Peradziryi et al., 2011). internalization, total internal reflection fluorescence (TIRF) These findings are also supported by PTK7 loss-of-function studies microscopy was used. TIRF microscopy has the advantage that in zebrafish (Hayes et al., 2013), but are in conflict to others the basal plasma membrane and the cytoplasmic regions reporting activation of canonical Wnt signaling by PTK7 (Bin-Nun immediately beneath the plasma membrane can be visualized, et al., 2014; Puppo et al., 2011). Thus, the specific role of PTK7 in allowing the analysis of PTK7 vesicle formation and internalization the regulation of Wnt signaling pathways is controversial. in response to Wnt3a or Wnt5a. MCF7 cells expressing PTK7–GFP A conundrum is that PTK7 regulates PCP signaling; however, it were imaged before and after the addition of canonical Wnt3a or also serves as a receptor for canonical Wnt ligands. Our previous non-canonical Wnt5a (Movies 1,2). While few PTK7-positive data indicated that PTK7 interacts with canonical Wnt ligands, vesicles with limited mobility were observed in PTK7–GFP- leading to inhibition of canonical Wnt signaling (Peradziryi et al., expressing cells, Wnt3a treatment caused a significant increase in 2011). Currently, it remains unclear how PTK7 is affected by the number of PTK7-positive vesicles (Movie 1; Fig. 2A). These binding to canonical Wnt ligands. Here, we further analyze this by vesicles appeared larger and more mobile compared to those of studying PTK7 protein localization and stability in the presence of untreated controls (Fig. 2B,C). Furthermore, we also observed a different Wnt ligands. We find that canonical Wnt ligands lead significant increase in the internalization of PTK7-positive vesicles to caveolin-mediated endocytosis and lysosomal degradation of (Fig. 2A). An example of the formation and internalization of a PTK7, while the localization of PTK7 is not affected by the PTK7-positive vesicle in presence of Wnt3a is shown in Fig. 2D. In presence of non-canonical Wnt ligands. Thus, our data suggest that contrast, cells treated with Wnt5a resembled untreated control cells PTK7 is removed from the cell membrane in presence of canonical (Movie 2; Fig. 2A–C). In summary, these data show that in the Wnt ligands. Thereby a Wnt morphogen gradient could direct the presence of canonical Wnt ligands membrane-localized PTK7 is cellular polarization of PTK7-expressing cells. Conversely, PTK7 internalized and accumulates in vesicle-like structures, while non- can also trap canonical Wnt ligands and accordingly inhibit their canonical