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The LIM domain protein Wtip interacts with the receptor tyrosine kinase Ror2 and inhibits canonical Wnt signalling
Biochemical and Biophysical Research Communications 390 (2009) 211–216
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The LIM domain protein Wtip interacts with the receptor tyrosine kinase Ror2 and inhibits canonical Wnt signalling Nicole Verhey van Wijk a,b,1, Florian Witte a,b, Ann Carolin Feike c, Alexandra Schambony c, Walter Birchmeier d, Stefan Mundlos a,b, Sigmar Stricker a,b,* a
Max Planck-Institute for Molecular Genetics, Development and Disease Group, Berlin, Germany Institute for Medical Genetics, University Medicine Charité, Berlin, Germany Developmental Biology Unit, Biology Department, University of Erlangen–Nuremberg, Erlangen, Germany d Max Delbrück Center for Molecular Medicine, Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 17 September 2009 Available online 26 September 2009 Keywords: Wtip Ror2 Brachydactyly Wnt
a b s t r a c t Wtip is a LIM domain protein of the Ajuba/Zyxin family involved in kidney and neural crest development; Ror2 is a receptor tyrosine kinase involved in the development of skeleton, heart, lung, genitalia and kidneys. Here we describe Wtip as an intracellular interaction partner of Ror2. Full-length Ror2 recruits Wtip to the cell membrane, a mutant involved in human disease fails to do so. Both genes and proteins show overlapping expression in the mouse embryo. We show that Wtip is able to inhibit canonical Wnt signalling in mammalian cells and in Xenopus embryos linking Wtip to a crucial developmental pathway. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Ror2 is a receptor tyrosine kinase (RTK) that consists of extracellular immunoglobulin (IG)-like, cysteine-rich and kringle domains as well as an intracellular tyrosine kinase (TK) domain and, unique to the Ror family of RTKs, a C-terminal serine–proline–threonine-rich (PST) region [1]. Mutations in human ROR2 cause two distinct syndromes, brachydactyly type B (BDB) [2,3] and recessive Robinow syndrome (RRS) [4,5]. Ror2 null mice show skeletal defects including craniofacial abnormalities and a shortening of long bones, especially in the limb zeugopode, as well as defects in heart, lung and external genitalia [6,7]. In cartilage, deficiency of Ror2 or overexpression of dominant-negative isoforms severely impairs chondrocyte differentiation [8,9]. Ror2 was shown to be a receptor for Wnt5a, inducing a noncanonical cascade involving activation of cJun-N-terminal kinase (JNK) [10,11]. In addition, Wnt5a can negatively regulate canonical Wnt signalling via Ror2 [12]. Additionally, Ror2 mediates Wnt5ainduced filopodia formation via its interaction with filamin A (FLNa) [13] depending on JNK [14]. So far, only a small number of Ror2-interacting proteins have been identified. Apart from FLNa, BmpR1b, Dlxin-1, casein kinase 1e (CK1e), Glycogen-synthase ki* Corresponding author. Address: Max Planck-Institute for Molecular Genetics, Development and Disease Group, Ihnestr. 73, 14195 Berlin, Germany. Fax: +49 30 84131385. E-mail address: [email protected] (S. Stricker). 1 Present address: Division of Molecular Neurobiology, MRC National Institute for Medical Research, London NW7 1AA, United Kingdom. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.09.086
nase 3 (Gsk3), Src and the scaffolding protein 14-3-3b have been shown to interact with Ror2 [15–20]. The Wt1-interacting protein Wtip has originally been identified as an interaction partner of the Wilms tumour protein 1 (WT1) in a Y2H screen [21]. Wtip contains three LIM domains (LDs), which are generally thought to mediate protein–protein interactions [22–24] and exhibits high homology to the Ajuba/Zyxin family of LD proteins. Here, we describe the interaction of Wtip with the C-terminal part of Ror2 in yeast and in mammalian cells and provide comparative expression data of both genes and proteins. Functionally, we show that Wtip is involved in the intracellular regulation of canonical Wnt signalling. Materials and methods Yeast two-hybrid screening. The cytoplasmic part of mouse Ror2 (Ror2-CP) or distally truncated cytoplasmic mRor2 (Ror2-BDB) were fused to the Tpr dimerisation domain and in frame to the LexA domain. All constructs used for screening procedures were checked for expression by Western blotting (not shown). Bait DNA was transformed into yeast strain L40 (Clontech) and screening was performed against a mouse embryonic cDNA (E9.5–10.5) library [25,26] in VP16 yeast expression vector. Positive clones were picked, restreaked three times and assayed for LacZ activity using a filter b-galactosidase assay to reduce false positives. Antibodies, cells and transfections. Following antibodies were used: mouse anti-HA, rabbit anti-Flag (Sigma–Aldrich); mouse anti-LexA (Clontech); mouse anti-Gal4 (Santa Cruz), rabbit anti-
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Met (kind gift of Dr. U. Schaeper, Berlin); rabbit anti-Ror2 (kind gift of Prof. P. Knaus, Berlin), goat anti-Ror2 (R&D Systems), rabbit antiWTIP (produced by Eurogentec). Secondary antibodies: anti-goat Alexa Fluor 488, anti-rabbit Alexa Fluor 488, anti-rabbit Alexa Fluor 568 (Molecular Probes); anti-mouse and anti-rabbit IgG-peroxidase conjugate (Oncogene). HEK293 cells were cultured in aMEM (Cambrex), 10% FBS, 1 mM L-glutamine. Transient transfection of HEK293 cells was done by standard Ca/PO4 precipitation. Cos-1 cells were cultured in DMEM (4.5 g/L glucose), 5% FBS, 1 mM L-glutamine and transfected with Polyfectamine (Qiagen) according to manufacturer’s instructions. Co-immunoprecipitation. Two days after transfection, HEK293 cells were washed with cold PBS. Proteins were solubilised in lysis buffer (50 mM Hepes, pH 7.5, 50 mM NaCl, 10 mM EDTA, 10% glycerol, 1% Triton X-100) supplemented with 100 mM NaF, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 1 mM PMSF and 0.5% aprotinin. Proteins were precipitated with anti-Flag affinity agarose gel (Sigma–Aldrich), and further analysed by standard Western blotting procedure. Immunofluorescence, in-situ hybridisation and immunohistochemistry. Cos-1 cells grown on cover slips were fixed in 4% PFA for 15 min and further processed for immunofluorescence as described in Ref. [27]. In-situ hybridisation and immunohistochemistry were performed as described in Ref. [9]. A Wtip probe was generated by cloning the Wtip-Y2H (nt498–957 of Wtip CDS) fragment into pCRII-TOPO (Invitrogen) and subsequent transcription with SP6 (Roche). Probes used to detect mRor2 are described in Ref. [28]. TOPFLASH assay, injection and analysis of Xenopus laevis embryos. Cos-1 cells per well (105) were seeded in 24-well plates and transfected using ExGen 500 (Fermentas) with either 150 ng of the Tcf/Lef reporter construct TOPFLASH or FOPFLASH (negative control), pCMRV to normalise for transfection efficiency, and constructs for Dvl2, Ror2 and Wtip. Total amount of transfected DNA was kept constant by adding empty vector DNA. Cells were lysed 48 h after transfection with 1 passive lysis buffer (Promega) and luciferase activity was measured according to the Dual-Glo Luciferase Reporter Assay (Promega). Transfections were performed as duplicates, all experiments were repeated at least three times, representative experiments are shown. Xenopus axis assays were performed as described in Ref. [29].
Results Identification of Wtip as Ror2-interacting protein To identify interaction partners of Ror2 we performed a yeast two-hybrid screen with the cytoplasmic domain of Ror2 (Ror2CP) as bait. We isolated a Wtip fragment encoding amino acids 161–319 that contains the first two but not the third LIM domain of Wtip, suggesting that the third LIM domain is not crucial for binding Ror2. To confirm the interaction we analysed yeast colony growth on selection medium and LacZ expression in retransformation experiments in L40 yeast. Only yeast cells that have been transformed with Ror2-CP and Wtip-Y2H are enabled to grow on selection medium (Fig. 1A, top row) or express a LacZ reporter (Fig. 1A, bottom row). A truncated version of Ror2 lacking the distal PST domain failed to interact with Wtip, indicating that the interaction occurs via the PST domain of Ror2. Since several LIM domain proteins have been shown to interact with RTKs and tyrosine-containing motifs [30–32] we investigated the specificity of the Ror2–Wtip interaction. For that we expressed Wtip-Y2H in combination with several cytoplasmic parts of RTKs fused to a LexA domain in L40 yeast (Fig. 1B). We found that Wtip
did not interact with any of the kinases tested, indicating a highly specific interaction of Wtip with Ror2. Confirmation of Wtip–Ror2 interaction in eukaryotic cells First, we tested the interaction of the Wtip-Y2H-fragment with full-length Ror2 by co-immunoprecipitation (Co-IP) in HEK293 cells (Fig. 1D). Specificity of the interaction was confirmed, since WtipY2H did not interact with another tyrosine kinase, Trk-Met [26]. To determine if the full-length Wtip is also capable of associating with Ror2, the full-length Wtip-FLAG was expressed in HEK293 cells together with HA-tagged full-length and truncated Ror2 constructs [19] (see Fig. 1E). Full-length Wtip was able to specifically bind to full-length Ror2 but not to the truncated Ror2-BDB construct (Fig. 1E), confirming the data obtained in yeast. In addition, we found that Wtip under the same conditions is not able to associate with the closely related tyrosine kinase Ror1 (data not shown). In order to spot the essential domain of Ror2 responsible for interacting with Wtip, we used constructs of either the whole cytoplasmic part of Ror2 (Ror2-IC) or deleted the second serine/threonine-rich domain (Ror2-IC–DST2) or the proline- and the second ST-rich (Ror2-IC–DP/ST2) domains, respectively. As shown in Fig. 1F, full-length Wtip was able to precipitate all of these Ror2variants indicating that the interaction between Ror2 and Wtip takes place at the first ST-rich domain of Ror2. Recently, it has been described that LIM domain proteins can form homodimers [33]. Co-IP analysis of full-length Wtip-HA and full-length Wtip-FLAG in HEK293 cells revealed that Wtip is able to homodimerise. Wtip was not able to interact with a Wtip protein lacking all three LD (WtipDLD1,2,3), thus the LDs in general are indispensable for homodimerisation of Wtip (Fig. 1G). In contrast, Wtip lacking the two C-terminally located LD (WtipDLD2,3) was weakly precipitated by Wtip suggesting that LD1 is essential for homodimerisation of Wtip (Fig. 1G). To further confirm a functional interaction of Wtip and Ror2 in eukaryotic cells, we analysed the distribution of Wtip alone or in combination with full-length or truncated Ror2 in Cos-1 cells (Fig. 1H). As reported by Srichai et al. [21], Wtip was predominantly found located in cytoplasmic spots, which was also observed in other cell lines (HEK293, HeLa, not shown). Ror2 and Ror2-BDB were localised to the cell membrane. Importantly, coexpression of Ror2-HA together with Wtip-FLAG altered the subcellular localisation of Wtip (Fig. 1H, lower panel), so that it was now predominantly co-localised with Ror2 at the plasma membrane. In agreement with our Co-IP data, Ror2-BDB-HA was not able to recruit Wtip to the plasma membrane. Analysis of Wtip and Ror2 gene and protein expression in mouse embryos To gain an overall insight into the expression of Wtip and Ror2, we first performed whole-mount in-situ hybridisation on mouse embryos. At embryonic (E) stages E9.5 to E11.5, Wtip and Ror2 both are expressed in the branchial arches, the otic vesicle, the outgrowing limb buds, in somites, craniofacial mesenchyme and in the tail (Fig. 2A). To obtain information about the expression of Wtip and Ror2 during organogenesis, we analysed sagittal sections from mouse embryos stage E14.5 by section in-situ hybridisation. Wtip and Ror2 showed a broadly overlapping expression in the developing mouse embryo (Fig. 2B). To compare Wtip and Ror2 protein expression, we performed immunohistochemistry for Wtip and Ror2 on sagittal sections of E14.5 mouse embryos (Fig. 2C). Co-expression of Ror2 and Wtip proteins was verified in kidneys, in lung epithelium, in the cartilaginous condensations of the developing ribs and in intestinal loops.
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Fig. 1. Interaction of Wtip and Ror2. (A) Identification of Wtip as a binding partner of Ror2 by Y2H: confirmation of Ror2/Wtip interaction on selective medium (-THULL) and induction of LacZ. (B) Wtip interacts with Ror2, but not with 7 other RTKs. (C) Depiction of Ror2 and Wtip constructs used. (D) Wtip-Y2H interacts with full-length Ror2, but not with Met in HEK293 cells. (E) Wtip interacts with full-length Ror2 but not with a BDB truncation. (F) Wtip interacts with a form of Ror2 that carries only the first serine– threonine-rich domain. (G) Homodimerisation of Wtip via its LIM domains. (H) Ror2 but not Ror2-BDB recruits Wtip to the cell membrane. Cos-1 cells were transfected with constructs indicated, nuclear staining was performed with DAPI (blue). Top row: single transfections, bottom row: co-transfections. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)
Taken together the analysis of Wtip/Ror2 association by coimmunoprecipitation, together with the subcellular localisation of Wtip and Ror2 and their co-expression (mRNA and protein) indicates a functional interaction of these proteins in vivo.
Wtip inhibits canonical Wnt signalling in vitro and in vivo Ror2 has been implicated in the modulation of canonical Wnt signalling [12] and the closely related LIM protein Ajuba was
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shown to repress Wnt/b-catenin signalling [34]. We therefore analysed if Wtip could play a role in the regulation of canonical Wnt signalling in cell culture and in Xenopus embryos. We first analysed if Wtip could influence Wnt/b-catenin signalling in Cos-1 cells using the TOPFLASH reporter assay. Stimulation of canonical Wnt signalling by transfection with the intracellular signalling mediator Dishevelled 2 (Dvl2) robustly activated TOPFLASH activity (Fig. 3A). Co-transfection of Wtip repressed TOPFLASH activity in a dose-dependent manner. We then used Xenopus secondary axis induction assays as in vivo model for the ability of Wtip to repress canonical Wnt signalling. Injection of XWnt8 significantly induced secondary axis formation (Fig. 3B); co-injection of Wtip repressed this activity. Altogether, these results show that Wtip is a novel intracellular inhibitor of the canonical Wnt signalling cascade. Discussion We have identified Wtip as a binding partner for the RTK Ror2. The interaction has been confirmed by Co-IP and immunocytochem-
istry. The widespread overlapping expression of both genes and proteins suggests a functional relevance for this interaction also in vivo. Wtip contains three LIM domains (LDs), which are known to mediate protein–protein interactions. Our results suggest that the interaction of Wtip and Ror2 was mediated by the first and/ or second LD of Wtip. Ror2 on the other hand bound Wtip via its first serine–threonine-rich domain of the PST terminus, a structure unique to Ror2 and its close relative Ror1 but not found in other RTKs. This suggests that the interaction of Wtip with Ror2 is highly specific. Indeed interaction of the Wtip fragment with eight other RTKs was excluded by Y2H assays. Interestingly, the domain of Ror2 responsible for Wtip binding is ablated in the human condition brachydactyly type B [3]. Thus the loss of interaction between Ror2 and Wtip could contribute to the pathogenesis of BDB. We showed that Wtip can homodimerise via its LDs, indeed homodimerisation has been shown also for other LIM domain proteins as CRP [33]. A possibility could be that Wtip provides a protein-binding platform to Ror2 by dimerisation or maybe multimerisation at the cell membrane.
Fig. 2. Comparative expression of Ror2 and Wtip mRNAs and proteins in mouse embryo development. (A) Whole-mount ISH demonstrating expression of both genes in branchial arches, limb buds and tail buds at E9.5 to E11.5. (B) Section ISH on sagittal sections of E14.5 mouse embryos. Transcripts of both genes were detected in the developing tongue (to), nasal cavity (nc), palate (pt), in condensations of the ribs (ri), lung (lu), kidney (ki), adrenal gland (ad), in the forebrain (fb), intestinal loops (in), dorsal root ganglia (drg) and in the somites (so). (C) Comparative expression of Ror2 and Wtip proteins demonstrated by immuno-labelling on E14.5 sagittal mouse embryo sections show co-localisation of both proteins in kidney, lung, rib cartilage and intestine.
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Fig. 3. Inhibition of canonical Wnt signalling by Wtip. (A) TOPFLASH assay stimulated with Dishevelled-2 (Dvl2). (B) Axis induction assay in Xenopus embryos shows secondary axis induction induced by XWnt8 is inhibited significantly by Wtip. Representative embryos are shown for each injection experiment, secondary axes are marked with arrows.
Zyxin LIM proteins are known to localise to the cell membrane especially at sites of cell–cell contacts or matrix attachment sites. In addition, Zyxin family members have been shown to shuttle between membrane and nucleus and are thought to coordinate cell attachment events with nuclear responses [21 and references therein]. We found that Ror2 was able to recruit Wtip to the cell membrane in an overexpression situation. It remains to be determined, if both proteins endogenously localise to cell–cell contact sites or focal adhesions and could contribute to their function. We show that Wtip can antagonise canonical Wnt signalling. Ror2 has also been implicated in several Wnt pathways and was shown to inhibit canonical signalling as well [11,12,35,36]. It is worthwhile noting that two key players of the canonical Wnt cascade, CK1e and Gsk3, have been shown to interact with Ror2 [15,18]. CK1e was shown to phosphorylate Ror2 on its serine–threonine-rich 2 domain resulting in Ror2 autophosphorylation [15]. Gsk3a and Gsk3b were shown to phosphorylate Ror2 after stimulation with Wnt5a [18]. Moreover it was shown that Gsk3 is necessary for Wnt5a induced cell migration [18]. Furthermore, Haraguchi et al. [34] have recently shown that Ajuba, a LD containing protein of the Zyxin family closely related to Wtip, is phosphorylated by Gsk3b. Ajuba was shown to negatively regulate canonical Wnt signalling by enforcing the interaction of Gsk3b with b-catenin. Ajuba thereby increased the Gsk3b-mediated phosphorylation and thus destabilisation of b-catenin. At this point we cannot state on which level the inhibition of canonical Wnt signalling takes place, however it is tempting to speculate that Wtip could function along a similar mechanism. In conclusion, we have demonstrated that the receptor tyrosine kinase Ror2, via its C-terminal domain, interacts with the LIM domain protein Wtip, recruiting it to the cell surface. Furthermore, Wtip is a novel negative regulator of the canonical Wnt signalling pathway. Acknowledgments This project was funded by grants form the Deutsche Forschungsgemeinschaft to S.S. and S.M. (SFB 577) and to A.S. (965 2-
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The LIM domain protein Wtip interacts with the receptor tyrosine kinase Ror2 and inhibits canonical Wnt signalling Nicole Verhey van Wijk a,b,1, Florian Witte a,b, Ann Carolin Feike c, Alexandra Schambony c, Walter Birchmeier d, Stefan Mundlos a,b, Sigmar Stricker a,b,* a
Max Planck-Institute for Molecular Genetics, Development and Disease Group, Berlin, Germany Institute for Medical Genetics, University Medicine Charité, Berlin, Germany Developmental Biology Unit, Biology Department, University of Erlangen–Nuremberg, Erlangen, Germany d Max Delbrück Center for Molecular Medicine, Berlin, Germany b c
a r t i c l e
i n f o
Article history: Received 17 September 2009 Available online 26 September 2009 Keywords: Wtip Ror2 Brachydactyly Wnt
a b s t r a c t Wtip is a LIM domain protein of the Ajuba/Zyxin family involved in kidney and neural crest development; Ror2 is a receptor tyrosine kinase involved in the development of skeleton, heart, lung, genitalia and kidneys. Here we describe Wtip as an intracellular interaction partner of Ror2. Full-length Ror2 recruits Wtip to the cell membrane, a mutant involved in human disease fails to do so. Both genes and proteins show overlapping expression in the mouse embryo. We show that Wtip is able to inhibit canonical Wnt signalling in mammalian cells and in Xenopus embryos linking Wtip to a crucial developmental pathway. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Ror2 is a receptor tyrosine kinase (RTK) that consists of extracellular immunoglobulin (IG)-like, cysteine-rich and kringle domains as well as an intracellular tyrosine kinase (TK) domain and, unique to the Ror family of RTKs, a C-terminal serine–proline–threonine-rich (PST) region [1]. Mutations in human ROR2 cause two distinct syndromes, brachydactyly type B (BDB) [2,3] and recessive Robinow syndrome (RRS) [4,5]. Ror2 null mice show skeletal defects including craniofacial abnormalities and a shortening of long bones, especially in the limb zeugopode, as well as defects in heart, lung and external genitalia [6,7]. In cartilage, deficiency of Ror2 or overexpression of dominant-negative isoforms severely impairs chondrocyte differentiation [8,9]. Ror2 was shown to be a receptor for Wnt5a, inducing a noncanonical cascade involving activation of cJun-N-terminal kinase (JNK) [10,11]. In addition, Wnt5a can negatively regulate canonical Wnt signalling via Ror2 [12]. Additionally, Ror2 mediates Wnt5ainduced filopodia formation via its interaction with filamin A (FLNa) [13] depending on JNK [14]. So far, only a small number of Ror2-interacting proteins have been identified. Apart from FLNa, BmpR1b, Dlxin-1, casein kinase 1e (CK1e), Glycogen-synthase ki* Corresponding author. Address: Max Planck-Institute for Molecular Genetics, Development and Disease Group, Ihnestr. 73, 14195 Berlin, Germany. Fax: +49 30 84131385. E-mail address: [email protected] (S. Stricker). 1 Present address: Division of Molecular Neurobiology, MRC National Institute for Medical Research, London NW7 1AA, United Kingdom. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.09.086
nase 3 (Gsk3), Src and the scaffolding protein 14-3-3b have been shown to interact with Ror2 [15–20]. The Wt1-interacting protein Wtip has originally been identified as an interaction partner of the Wilms tumour protein 1 (WT1) in a Y2H screen [21]. Wtip contains three LIM domains (LDs), which are generally thought to mediate protein–protein interactions [22–24] and exhibits high homology to the Ajuba/Zyxin family of LD proteins. Here, we describe the interaction of Wtip with the C-terminal part of Ror2 in yeast and in mammalian cells and provide comparative expression data of both genes and proteins. Functionally, we show that Wtip is involved in the intracellular regulation of canonical Wnt signalling. Materials and methods Yeast two-hybrid screening. The cytoplasmic part of mouse Ror2 (Ror2-CP) or distally truncated cytoplasmic mRor2 (Ror2-BDB) were fused to the Tpr dimerisation domain and in frame to the LexA domain. All constructs used for screening procedures were checked for expression by Western blotting (not shown). Bait DNA was transformed into yeast strain L40 (Clontech) and screening was performed against a mouse embryonic cDNA (E9.5–10.5) library [25,26] in VP16 yeast expression vector. Positive clones were picked, restreaked three times and assayed for LacZ activity using a filter b-galactosidase assay to reduce false positives. Antibodies, cells and transfections. Following antibodies were used: mouse anti-HA, rabbit anti-Flag (Sigma–Aldrich); mouse anti-LexA (Clontech); mouse anti-Gal4 (Santa Cruz), rabbit anti-
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Met (kind gift of Dr. U. Schaeper, Berlin); rabbit anti-Ror2 (kind gift of Prof. P. Knaus, Berlin), goat anti-Ror2 (R&D Systems), rabbit antiWTIP (produced by Eurogentec). Secondary antibodies: anti-goat Alexa Fluor 488, anti-rabbit Alexa Fluor 488, anti-rabbit Alexa Fluor 568 (Molecular Probes); anti-mouse and anti-rabbit IgG-peroxidase conjugate (Oncogene). HEK293 cells were cultured in aMEM (Cambrex), 10% FBS, 1 mM L-glutamine. Transient transfection of HEK293 cells was done by standard Ca/PO4 precipitation. Cos-1 cells were cultured in DMEM (4.5 g/L glucose), 5% FBS, 1 mM L-glutamine and transfected with Polyfectamine (Qiagen) according to manufacturer’s instructions. Co-immunoprecipitation. Two days after transfection, HEK293 cells were washed with cold PBS. Proteins were solubilised in lysis buffer (50 mM Hepes, pH 7.5, 50 mM NaCl, 10 mM EDTA, 10% glycerol, 1% Triton X-100) supplemented with 100 mM NaF, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 1 mM PMSF and 0.5% aprotinin. Proteins were precipitated with anti-Flag affinity agarose gel (Sigma–Aldrich), and further analysed by standard Western blotting procedure. Immunofluorescence, in-situ hybridisation and immunohistochemistry. Cos-1 cells grown on cover slips were fixed in 4% PFA for 15 min and further processed for immunofluorescence as described in Ref. [27]. In-situ hybridisation and immunohistochemistry were performed as described in Ref. [9]. A Wtip probe was generated by cloning the Wtip-Y2H (nt498–957 of Wtip CDS) fragment into pCRII-TOPO (Invitrogen) and subsequent transcription with SP6 (Roche). Probes used to detect mRor2 are described in Ref. [28]. TOPFLASH assay, injection and analysis of Xenopus laevis embryos. Cos-1 cells per well (105) were seeded in 24-well plates and transfected using ExGen 500 (Fermentas) with either 150 ng of the Tcf/Lef reporter construct TOPFLASH or FOPFLASH (negative control), pCMRV to normalise for transfection efficiency, and constructs for Dvl2, Ror2 and Wtip. Total amount of transfected DNA was kept constant by adding empty vector DNA. Cells were lysed 48 h after transfection with 1 passive lysis buffer (Promega) and luciferase activity was measured according to the Dual-Glo Luciferase Reporter Assay (Promega). Transfections were performed as duplicates, all experiments were repeated at least three times, representative experiments are shown. Xenopus axis assays were performed as described in Ref. [29].
Results Identification of Wtip as Ror2-interacting protein To identify interaction partners of Ror2 we performed a yeast two-hybrid screen with the cytoplasmic domain of Ror2 (Ror2CP) as bait. We isolated a Wtip fragment encoding amino acids 161–319 that contains the first two but not the third LIM domain of Wtip, suggesting that the third LIM domain is not crucial for binding Ror2. To confirm the interaction we analysed yeast colony growth on selection medium and LacZ expression in retransformation experiments in L40 yeast. Only yeast cells that have been transformed with Ror2-CP and Wtip-Y2H are enabled to grow on selection medium (Fig. 1A, top row) or express a LacZ reporter (Fig. 1A, bottom row). A truncated version of Ror2 lacking the distal PST domain failed to interact with Wtip, indicating that the interaction occurs via the PST domain of Ror2. Since several LIM domain proteins have been shown to interact with RTKs and tyrosine-containing motifs [30–32] we investigated the specificity of the Ror2–Wtip interaction. For that we expressed Wtip-Y2H in combination with several cytoplasmic parts of RTKs fused to a LexA domain in L40 yeast (Fig. 1B). We found that Wtip
did not interact with any of the kinases tested, indicating a highly specific interaction of Wtip with Ror2. Confirmation of Wtip–Ror2 interaction in eukaryotic cells First, we tested the interaction of the Wtip-Y2H-fragment with full-length Ror2 by co-immunoprecipitation (Co-IP) in HEK293 cells (Fig. 1D). Specificity of the interaction was confirmed, since WtipY2H did not interact with another tyrosine kinase, Trk-Met [26]. To determine if the full-length Wtip is also capable of associating with Ror2, the full-length Wtip-FLAG was expressed in HEK293 cells together with HA-tagged full-length and truncated Ror2 constructs [19] (see Fig. 1E). Full-length Wtip was able to specifically bind to full-length Ror2 but not to the truncated Ror2-BDB construct (Fig. 1E), confirming the data obtained in yeast. In addition, we found that Wtip under the same conditions is not able to associate with the closely related tyrosine kinase Ror1 (data not shown). In order to spot the essential domain of Ror2 responsible for interacting with Wtip, we used constructs of either the whole cytoplasmic part of Ror2 (Ror2-IC) or deleted the second serine/threonine-rich domain (Ror2-IC–DST2) or the proline- and the second ST-rich (Ror2-IC–DP/ST2) domains, respectively. As shown in Fig. 1F, full-length Wtip was able to precipitate all of these Ror2variants indicating that the interaction between Ror2 and Wtip takes place at the first ST-rich domain of Ror2. Recently, it has been described that LIM domain proteins can form homodimers [33]. Co-IP analysis of full-length Wtip-HA and full-length Wtip-FLAG in HEK293 cells revealed that Wtip is able to homodimerise. Wtip was not able to interact with a Wtip protein lacking all three LD (WtipDLD1,2,3), thus the LDs in general are indispensable for homodimerisation of Wtip (Fig. 1G). In contrast, Wtip lacking the two C-terminally located LD (WtipDLD2,3) was weakly precipitated by Wtip suggesting that LD1 is essential for homodimerisation of Wtip (Fig. 1G). To further confirm a functional interaction of Wtip and Ror2 in eukaryotic cells, we analysed the distribution of Wtip alone or in combination with full-length or truncated Ror2 in Cos-1 cells (Fig. 1H). As reported by Srichai et al. [21], Wtip was predominantly found located in cytoplasmic spots, which was also observed in other cell lines (HEK293, HeLa, not shown). Ror2 and Ror2-BDB were localised to the cell membrane. Importantly, coexpression of Ror2-HA together with Wtip-FLAG altered the subcellular localisation of Wtip (Fig. 1H, lower panel), so that it was now predominantly co-localised with Ror2 at the plasma membrane. In agreement with our Co-IP data, Ror2-BDB-HA was not able to recruit Wtip to the plasma membrane. Analysis of Wtip and Ror2 gene and protein expression in mouse embryos To gain an overall insight into the expression of Wtip and Ror2, we first performed whole-mount in-situ hybridisation on mouse embryos. At embryonic (E) stages E9.5 to E11.5, Wtip and Ror2 both are expressed in the branchial arches, the otic vesicle, the outgrowing limb buds, in somites, craniofacial mesenchyme and in the tail (Fig. 2A). To obtain information about the expression of Wtip and Ror2 during organogenesis, we analysed sagittal sections from mouse embryos stage E14.5 by section in-situ hybridisation. Wtip and Ror2 showed a broadly overlapping expression in the developing mouse embryo (Fig. 2B). To compare Wtip and Ror2 protein expression, we performed immunohistochemistry for Wtip and Ror2 on sagittal sections of E14.5 mouse embryos (Fig. 2C). Co-expression of Ror2 and Wtip proteins was verified in kidneys, in lung epithelium, in the cartilaginous condensations of the developing ribs and in intestinal loops.
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Fig. 1. Interaction of Wtip and Ror2. (A) Identification of Wtip as a binding partner of Ror2 by Y2H: confirmation of Ror2/Wtip interaction on selective medium (-THULL) and induction of LacZ. (B) Wtip interacts with Ror2, but not with 7 other RTKs. (C) Depiction of Ror2 and Wtip constructs used. (D) Wtip-Y2H interacts with full-length Ror2, but not with Met in HEK293 cells. (E) Wtip interacts with full-length Ror2 but not with a BDB truncation. (F) Wtip interacts with a form of Ror2 that carries only the first serine– threonine-rich domain. (G) Homodimerisation of Wtip via its LIM domains. (H) Ror2 but not Ror2-BDB recruits Wtip to the cell membrane. Cos-1 cells were transfected with constructs indicated, nuclear staining was performed with DAPI (blue). Top row: single transfections, bottom row: co-transfections. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)
Taken together the analysis of Wtip/Ror2 association by coimmunoprecipitation, together with the subcellular localisation of Wtip and Ror2 and their co-expression (mRNA and protein) indicates a functional interaction of these proteins in vivo.
Wtip inhibits canonical Wnt signalling in vitro and in vivo Ror2 has been implicated in the modulation of canonical Wnt signalling [12] and the closely related LIM protein Ajuba was
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shown to repress Wnt/b-catenin signalling [34]. We therefore analysed if Wtip could play a role in the regulation of canonical Wnt signalling in cell culture and in Xenopus embryos. We first analysed if Wtip could influence Wnt/b-catenin signalling in Cos-1 cells using the TOPFLASH reporter assay. Stimulation of canonical Wnt signalling by transfection with the intracellular signalling mediator Dishevelled 2 (Dvl2) robustly activated TOPFLASH activity (Fig. 3A). Co-transfection of Wtip repressed TOPFLASH activity in a dose-dependent manner. We then used Xenopus secondary axis induction assays as in vivo model for the ability of Wtip to repress canonical Wnt signalling. Injection of XWnt8 significantly induced secondary axis formation (Fig. 3B); co-injection of Wtip repressed this activity. Altogether, these results show that Wtip is a novel intracellular inhibitor of the canonical Wnt signalling cascade. Discussion We have identified Wtip as a binding partner for the RTK Ror2. The interaction has been confirmed by Co-IP and immunocytochem-
istry. The widespread overlapping expression of both genes and proteins suggests a functional relevance for this interaction also in vivo. Wtip contains three LIM domains (LDs), which are known to mediate protein–protein interactions. Our results suggest that the interaction of Wtip and Ror2 was mediated by the first and/ or second LD of Wtip. Ror2 on the other hand bound Wtip via its first serine–threonine-rich domain of the PST terminus, a structure unique to Ror2 and its close relative Ror1 but not found in other RTKs. This suggests that the interaction of Wtip with Ror2 is highly specific. Indeed interaction of the Wtip fragment with eight other RTKs was excluded by Y2H assays. Interestingly, the domain of Ror2 responsible for Wtip binding is ablated in the human condition brachydactyly type B [3]. Thus the loss of interaction between Ror2 and Wtip could contribute to the pathogenesis of BDB. We showed that Wtip can homodimerise via its LDs, indeed homodimerisation has been shown also for other LIM domain proteins as CRP [33]. A possibility could be that Wtip provides a protein-binding platform to Ror2 by dimerisation or maybe multimerisation at the cell membrane.
Fig. 2. Comparative expression of Ror2 and Wtip mRNAs and proteins in mouse embryo development. (A) Whole-mount ISH demonstrating expression of both genes in branchial arches, limb buds and tail buds at E9.5 to E11.5. (B) Section ISH on sagittal sections of E14.5 mouse embryos. Transcripts of both genes were detected in the developing tongue (to), nasal cavity (nc), palate (pt), in condensations of the ribs (ri), lung (lu), kidney (ki), adrenal gland (ad), in the forebrain (fb), intestinal loops (in), dorsal root ganglia (drg) and in the somites (so). (C) Comparative expression of Ror2 and Wtip proteins demonstrated by immuno-labelling on E14.5 sagittal mouse embryo sections show co-localisation of both proteins in kidney, lung, rib cartilage and intestine.
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Fig. 3. Inhibition of canonical Wnt signalling by Wtip. (A) TOPFLASH assay stimulated with Dishevelled-2 (Dvl2). (B) Axis induction assay in Xenopus embryos shows secondary axis induction induced by XWnt8 is inhibited significantly by Wtip. Representative embryos are shown for each injection experiment, secondary axes are marked with arrows.
Zyxin LIM proteins are known to localise to the cell membrane especially at sites of cell–cell contacts or matrix attachment sites. In addition, Zyxin family members have been shown to shuttle between membrane and nucleus and are thought to coordinate cell attachment events with nuclear responses [21 and references therein]. We found that Ror2 was able to recruit Wtip to the cell membrane in an overexpression situation. It remains to be determined, if both proteins endogenously localise to cell–cell contact sites or focal adhesions and could contribute to their function. We show that Wtip can antagonise canonical Wnt signalling. Ror2 has also been implicated in several Wnt pathways and was shown to inhibit canonical signalling as well [11,12,35,36]. It is worthwhile noting that two key players of the canonical Wnt cascade, CK1e and Gsk3, have been shown to interact with Ror2 [15,18]. CK1e was shown to phosphorylate Ror2 on its serine–threonine-rich 2 domain resulting in Ror2 autophosphorylation [15]. Gsk3a and Gsk3b were shown to phosphorylate Ror2 after stimulation with Wnt5a [18]. Moreover it was shown that Gsk3 is necessary for Wnt5a induced cell migration [18]. Furthermore, Haraguchi et al. [34] have recently shown that Ajuba, a LD containing protein of the Zyxin family closely related to Wtip, is phosphorylated by Gsk3b. Ajuba was shown to negatively regulate canonical Wnt signalling by enforcing the interaction of Gsk3b with b-catenin. Ajuba thereby increased the Gsk3b-mediated phosphorylation and thus destabilisation of b-catenin. At this point we cannot state on which level the inhibition of canonical Wnt signalling takes place, however it is tempting to speculate that Wtip could function along a similar mechanism. In conclusion, we have demonstrated that the receptor tyrosine kinase Ror2, via its C-terminal domain, interacts with the LIM domain protein Wtip, recruiting it to the cell surface. Furthermore, Wtip is a novel negative regulator of the canonical Wnt signalling pathway. Acknowledgments This project was funded by grants form the Deutsche Forschungsgemeinschaft to S.S. and S.M. (SFB 577) and to A.S. (965 2-
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