Wnt-ligand-dependent interaction of TAK1 (TGF-β-activated kinase-1) with the receptor tyrosine kinase Ror2 modulates canonical Wnt-signalling

Cellular Signalling 20 (2008) 2134–2144

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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g

Wnt-ligand-dependent interaction of TAK1 (TGF-β-activated kinase-1) with the receptor tyrosine kinase Ror2 modulates canonical Wnt-signalling Andreas Winkel a, Sigmar Stricker b,c, Przemko Tylzanowski d, Virginia Seiffart a, Stefan Mundlos b,c, Gerhard Gross a,⁎, Andrea Hoffmann a a

Helmholtz Centre for Infection Research (HZI), Inhoffenstr. 7, 38124 Braunschweig, Germany Max-Planck Institute for Molecular Genetics, Germany Institute for Medical Genetics, Charité, Berlin, Germany d Department of Rheumatology, University of Leuven, Leuven, Belgium b c

a r t i c l e

i n f o

Article history: Received 13 April 2008 Received in revised form 8 August 2008 Accepted 11 August 2008 Available online 16 August 2008 Keywords: Ror2 TAK1 Wnt PRTB Robinow Brachydactyly B

a b s t r a c t Mutations in the receptor tyrosine kinase Ror2 account for Brachydactyly type B and Robinow Syndrome. We have identified two novel factors interacting with the Ror2 intracellular domain. TAK1 (TGF-β activated kinase 1), a MAP3K, interacts with Ror2 and phosphorylates its intracellular carboxyterminal serine/ thronine/proline-rich (STP) domain. This TAK1-dependent phosphorylation of Ror2 induces phosphorylation of tyrosine-residues including a MAPK-like TGY-motif. The TAK1-dependent phosphorylation is enhanced by a second cytosolic factor, PRTB, which interacts with Ror2 and with TAK1 as well. The TAK1-dependent Tyrphosphorylation of Ror2 is not mediated by the Ror2 tyrosine kinase domain and seems predominantly triggered by cytosolic kinases. Wnt-ligand binding differentially controls the Ror2/TAK1 interaction. Wnt1binding displaces TAK1 from Ror2 while Wnt3a and Wnt5a are unable to do so thus modifying TAK1's capacity to cause phosphorylation of Ror2. Ror2 seems to act as a Wnt co-receptor enhancing Wntdependent canonical pathways while Tyr- and Ser/Thr-phosphorylation of Ror2 negatively controls the efficiency of these pathways. We propose that the level of the Wnt-ligand-regulated phosphorylation by cytosolic factors determines whether Ror2 acts as a stimulator or as an inhibitor of canonical Wnt-signalling. © 2008 Elsevier Inc. All rights reserved.

1. Introduction Ror2 belongs to a small family of receptor tyrosine kinases (RTKs) which are structurally related to the muscle-specific receptor kinase MuSK. Ror2 mutations in the ecto- and cytoplasmic domains account for Brachydactyly type B (BDB), an autosomal dominant skeletal disorder characterized by hypoplasia or aplasia of distal phalanges [1,2] and for Robinow Syndrome, an autosomal recessive multisystemic disease characterized by short stature, mesomelic limb shortening, segmental defects of the spine and a characteristic facial appearance (reviewed in [3]). The Ror2 ectodomain harbours an immunoglobulinlike domain, a Frizzled-like cysteine-rich (CRD) domain and a membrane proximal kringle domain. The CRD domain of Ror2 is able to bind members of the Wnt family [4] but relatively little is known about the signalling pathways downstream of this receptor. The Ror2 receptor binds Wnts activating the canonical pathway [5] but it was also suggested that Ror2 is involved in the non-canonical Wnt5a/JNK sig⁎ Corresponding author. Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany. Tel.: +49 531 6181 5020; fax: +49 531 6181 5012. E-mail address: [email protected] (G. Gross). 0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.08.009

nalling pathway since Ror2 interacts both physically and functionally with Wnt5a, a factor activating non-canonical Wnt-pathways [6,7]. The so-called “canonical” Wnt-β-catenin-LEF/TCF pathway involves the translocation of β-catenin from the cytoplasm to the nucleus. In the absence of extracellular Wnt proteins interacting and activating cell surface receptors such as Frizzled, β-catenin remains confined to the cytoplasm and its degradation is promoted by several molecules including GSK-3β, Axin and APC [8–10]. When Wnt proteins are present and bind to the receptors, this degradation system is inactivated and a free non-phosphorylated form of β-catenin accumulates in the cytoplasm [11]. β-catenin then translocates to the nucleus and interacts with transcription factors such as lymphoid enhancer factor-1/T-cell factor (LEF-1/TCF) proteins and activates specific target genes [12,13]. One of the non-canonical Wnt-pathways mediated by the Wnt-5a subclass triggers intracellular Ca2+ release to activate enzymes, such as protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CaMKII) but also involves the activation of a mitogen-activated protein kinase (MAPK) pathway composed of the TAK1 MAP kinase kinase kinase (MAP3K) and the Nemo like kinase (NLK) [14,15]. However, NLKactivation may occur also without stimulated Ca2+ flux. TAK1-stimulated NLK activity phosphorylates TCF to prevent the β-catenin–TCF

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complex from binding DNA resulting in interference with canonical Wnt-signalling [16]. TAK1 (TGF-β-activated kinase 1), a serine/threonine kinase, has initially been identified as a cytosolic component of MAPK (mitogenactivated protein kinase) pathways activated by ligands of the TGF-β/ BMP family of secreted factors [17] as well as by several cytokines such as IL-1 and TNF-α. Cytoplasmic factors such as TAB1, 2 and 3 regulate the activation of TAK1 [18,19]. TAK1 mediates the activation of several downstream pathways such as c-jun N-terminal kinases (JNK), p38-MAPK and NF-κB [20,21]. TAK1 may also directly interact with other signalling mediators such as the Smads (signalling mediators of the TGF-β/BMP family of secreted factors) and interfere with their biological activity [22,23]. However, molecular details of the signalling cascade(s) involving TAK1 still remain to be elucidated. Several cytoplasmic factors have been described interacting with Ror2 such as Dlxin1 [24] and casein kinase Iε (CKIε) [25]. Interestingly, CKIε interaction leads to the phosphorylation of serine/threonine residues in the cytoplasmic carboxyterminal serine/threonine/prolinerich (STP) domain which seems sufficient to activate the capacity of Ror2 to autophosphorylate tyrosine-residues [25]. In our study we show that the MAP3K TAK1 interacts with Ror2 and phosphorylates the carboxyterminal STP-region in a Wnt-ligand-dependent manner which leads to the phosphorylation of neighbouring tyrosine-residues by cytosolic factors and modulates the outcome of the TCF/LEF-1dependent transcription processes.

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2. Experimental procedures 2.1. Constructs Murine Ror2, Ror2 variants, TAK1 and PRTB expression constructs used in this study are shown in Fig. 1A–C. Wild-type and mutant cDNAs were integrated into the mammalian expression vector pcDNA3 (Invitrogen) or pMT7T3 [26]. Construction of the truncated Ror2 receptor Ror2ΔC and Ror2ΔTyr have been described [27]. The soluble intracellular Ror2-domain, Ror2Cterm and the point mutations in the TAK1 phosphorylation site of Ror2 (T871A, Y873A, T871A/ Y873A) were constructed by site-directed mutagenesis using the sitedirected mutagenesis kit (Stratagene) according to the manufacturer's advice. Similarly, an expression vector encoding a kinaseinactive mutant of Ror2 was constructed by replacing Lysine 507, crucial for ATP binding, with Arginine or Alanine (Ror2K507R or Ror2K507A, respectively). The extracellular epitope of the human CSF1R was fused to the murine Ror2 transmembrane- and intracellular domain. The construction of TAK1 expression vectors has been described [23]. The integrity of all constructs was checked by DNAsequencing. 2.2. Cells and transfection Human embryonic kidney 293T cells were cultured in highglucose DMEM containing 10% fetal bovine serum. Transient

Fig. 1. Ror2, TAK1 and PRTB variants used in this study. A. The Ror2 receptor domains are indicated at the left side of the panel. Ror2 variants causing Brachydactyly B and Robinow Syndrome have been described [2]. The establishment of Ror2 mutants and of the Ror2/CSF1R-chimera is described in ‘Experimental procedures’. B. The full-length murine MAP3K TAK1 was cloned by PCR from total kidney RNA [23]. Constitutive active (ca; a 23 aa deletion at the amino-terminus) or dominant negative variants (dn; a point-mutation at position 63 (K −N W)) are indicated. C. PRTB (Proline Codon-rich Transcript, Brain Expressed) variant PRTBΔN (Δaa 1–51) was isolated by a yeast two-hybrid screen for interactors with Ror2. The WT form of PRTB was completed thereafter (Experimental procedures).

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transfections of HEK293T cells were performed using FuGENE6 as described by the manufacturer (Roche Applied Science, Mannheim). The quantities of transfected DNA were kept constant by adding an appropriate amount of empty vector [28]. Murine mesenchymal progenitor cells C3H10T1/2 and C3H10T1/2-BMP2 were cultured in high-glucose DMEM containing 10% fetal bovine serum. The features of C3H10T1/2-BMP2 cells have been described [29,30]. Recombinant C3H10T1/2-BMP2 cells were stably transfected with expression vector pMT7T3 encoding Ror2 using DOSPER™ according to the manufacturer's protocol (Roche, Mannheim) together with a selection plasmid conferring G418-resistance (pAG60: BMP2-expressing C3H10T1/2 cells). Individual clones were picked, propagated, and tested for recombinant expression of Ror2 by RT-PCR using a vectorspecific and a gene-specific primer. Control cell lines (empty expression vector) were established at the same time. Selected cell clones were subcultivated in the presence of G418 (750 µg/ml) or puromycine/G418 (5 µg/ml + 750 µg/ml) and the selective pressure was maintained during the entire cultivation period.

2.6. Reporter assays HEK293T cells (2 × 105 cells per well) were seeded into 24-well plates. 24 h later, plates were transfected by Fugene6 (Roche) with TOPflash reporter [33] and expression vectors as indicated. The total amount of DNA (0.36 µg) was kept constant by supplementation with empty vector DNAs. Cells were harvested 30 h after transfection and the luciferase activity was determined with a Luciferase Assay System (Promega). We used the β-Galactosidase-vector (0.06 µg) under control of the RSV promoter for normalizing transfection efficiencies. To assess the level of non-canonical Wnt-signalling we used an expression vector encoding stabilized β-catenin [34]. 2.6.1. Yeast two-hybrid assays Yeast two-hybrid assays were performed according to the Matchmaker GAL4 Two-Hybrid System 3 (Clontech) as described [23]. For prey a cDNA bank from mesenchymal progenitors C3H10T1/2-BMP2 was used and as bait the Ror2 intracellular domain.

2.3. In vitro osteogenic differentiation

2.7. Statistical analysis

Stable C3H10T1/2 cell lines expressing recombinant BMP2 (C3H10T1/2-BMP2), BMP2 and Ror2wt (C3H10T1/2-BMP2/Ror2) or parental C3H10T1/2 cells (control) were plated at a density of 5000 cells/cm2. After reaching confluence (arbitrarily termed day 0) ascorbic acid (50 µg/ml) and β-glycerophosphate (10 mM) were added as specified [31]. Alkaline phosphatase activity in osteoblasts was visualized after fixation of cells with 3% paraformaldehyde in PBS for 30 min at 4 °C followed by wash with PBS by cellular staining with SIGMA FAST BCIP/NBT (Sigma, Deisenhofen) as described in the manufacturer's protocol.

The density of the bands was assessed with Image J 1.40 g (NIH, Bethesda, MD). F-Test was used to examine the significance of differences in expression. Differences were considered significant when P values were less than 0.05.

2.4. Immunoprecipitation and immunoblotting Human embryonic kidney (HEK) 293T cells transfected with different constructs were harvested 36 h after transfection and lysed in 1% (w/v) Nonidet P-40, 150 mM NaCI, 20 mM Tris, pH7.5, 2 mM EDTA, 50 mM NaF, 1 mM Na4P2O7, supplemented with protease inhibitors (Complete, Mini, Roche, Mannheim) and 0.1 µM okadaic acid. IP reactions were performed as described [23] in the same lysis buffer containing M2-Flag-antibody (Sigma, Deisenhofen) and protein G sepharose or mouse monoclonal c-Myc antibody beads (sc40AC, Santa Cruz, Heidelberg). Endogenous complexes were isolated with goat anti-Ror2-antibodies (R&D, Minneapolis). As a control goat anti-phosphatidylserine receptor antibodies were used [32]. Cell extracts and immunoprecipitates were analyzed by immunoblotting with the appropriate antibodies. p38 and pp38 antibodies were from Cell Signalling Technology (#9212 and #9211, respectively). In addition, antibodies used to assess MAPK-signalling were anti-JNK and anti-ERK specific for the phosphorylated and non-phosphorylaled forms (JNK and p-JNK, #9252 and #9251; ERK and p-ERK, #9102 and #9101; all from Cell Signaling Technology, Germany). 2.5. Immunocomplex kinase assays Flag-tagged Ror2 variants were co-expressed with Flag-tagged TAK1 and its activating protein, TAB1, in HEK293T cells as described. Subsequently, immunoprecipitation was performed with Flag antibody. After the final wash the agarose beads with the bound immune complexes were washed twice with kinase buffer (25 mM Tris pH 7.5/ 10 mM MgCl2/2 mM EGTA/1 mM DTT/1 mM Na3VO4/0.1 µM Okadaic acid/proteinase inhibitors), dissolved in 20 µl kinase buffer and incubated with addition of 10 µCi γ-[32P]-ATP for 20 min at 30 °C. The reaction was stopped by addition of SDS sample buffer. Samples were analyzed by SDS gel electrophoresis, and the dried gels were subjected to autoradiography.

Fig. 2. Active TAK1 interacts with Ror2 in the carboxyterminal STP-domain. HEK293T cells were transfected with expression plasmids encoding Flag- or HA-tagged proteins and cell extracts were processed as described in Experimental procedures. A. Interaction of Ror2 and TAK1 takes place in the Ror2 carboxyterminal STP-domain and is dependent on a kinase-active TAK1 variant. B. Ror2 and TAK1 form endogenous complexes in HEK293T cells. Cell extracts were subjected to immunoprecipitation (IP) using polyclonal goat anti-Ror2 antibodies (R&D, Minneapolis) or anti-phosphatidylserine receptor antibodies covalently coupled to Protein G-beads (Pierce) [32]. After blotting, Ror2 was visualized with these antibodies. TAK1 was detected with polyclonal antiTAK1antibodies (Santa Cruz; sc-7162). Two independent immunoprecipitations are shown.

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3. Results 3.1. The Ror2 receptor interacts with the signalling mediator TAK1 During a screening to identify cytosolic partners of the MAP kinase kinase kinase (MAP3K) TAK1 (TGF-β activated kinase 1) [23] we found that, based on co-immunoprecipitation studies, TAK1 was able to interact with the carboxyterminal serine/threonine/proline-rich (STP) domain of Ror2 (Figs. 1 and 2). Ror2wt or its soluble intracellular domain (Ror2Cterm) was able to co-immunoprecipitate TAK1 while the Ror2 deletion mutant lacking the STP-domain (Ror2ΔC) was only marginally able to do so (Fig. 2A). A complete deletion of the intracellular domain (Ror2ΔTyr) did not show any interaction with TAK1 at all (Fig. 2A). TAK1's kinase activity is necessary for Ror2 binding since the kinase-negative variant of TAK1 (K63W; dnTAK1) is not coimmunoprecipitated with Ror2 (Fig. 2A).

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Immunoprecipitation of endogenous Ror2 from HEK293T cells with goat anti-Ror2 antibodies indicates that this receptor interacts with TAK1 endogenously (Fig. 2B). This endogenous interaction is demonstrated in two assays which differed in the amount of endogenous Ror2 precipitation but both resulting in a notable coprecipitation of endogenous TAK1. As a control, precipitations were performed with a goat antibody unrelated to Ror2 (phosphatidylserine receptor). This control did not reveal any TAK1 co-precipitation (Fig. 2B). 3.1.1. TAK1 phosphorylates Ror2 at multiple sites While characterizing signalling pathways for Ror2 we noticed that in the presence of active, but not in the presence of kinase-inactive TAK1, Ror2wt or its soluble intracellular domain (Ror2Cterm) interacts with antibodies specific for the doubly phosphorylated form of p38 (pp38) (Fig. 3A). This might be indicative for a TAK1-dependent

Fig. 3. TAK1 phosphorylates Ror2 in its carboxyterminal STP-domain which induces Ror2 tyrosine-phosphorylation. HEK 293T cells were transfected with expression plasmids encoding Flag- or HA-tagged proteins, and cell extracts were treated as described in Experimental procedures. A. Antibodies recognizing the doubly phosphorylated form of p38 (pp38) interact with Ror2 in the presence of TAK1. The pp38 specific antibodies interact with a TGY-motif in which both threonine and tyrosine are phosphorylated. Both Ror2wt and the soluble intracellular domain of Ror2 (Ror2-Cterm) harbouring the TAK1-interacting STP-domain react with pp38 specific antibodies. B. The only intracellular TGY-motif of Ror2wt is located in the STP-domain. Mutations in this TGY-motif interfere with the TAK1-dependent recognition by pp38 antibodies. C. TAK1 phosphorylates Ror2 in a cell-free system. Immunocomplex kinase assays were performed as described in Experimental procedures. In short, Flag-tagged Ror2 variants were co-expressed with Flag-tagged TAK1 and its activating protein, TAB1, in HEK293T cells. Cells were lysed and immunoprecipitation was performed with Flag antibody. Washed immune complexes were incubated with γ-32P-ATP and analyzed by SDS gel electrophoresis and autoradiography. D. TAK1 induces tyrosine-phosphorylation of Ror2. Cellular extracts expressing Ror2 variants in the presence of TAK1 were immunoprecipitated with anti-pTyr antibodies, subjected to gel electrophoresis and then blotted with anti-Flag or pp38 antibodies (control). Anti-pTyr antibodies react with Ror2 variants in the presence of active but not kinase-negative TAK1.

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modification of Ror2 which is detectable by pp38-specific antibodies. Supporting this hypothesis was the fact that the Ror2 variants with a deleted STP-domain harbouring the TAK1 binding-site (Ror2ΔC, Ror2ΔTyr) do not interact with anti-pp38 antibodies (Fig. 3A). pp38-specific antibodies interact with dually phosphorylated amino acids, threonine (T) and tyrosine (Y), within the MAPK phosphorylation motif TGY. One of two TGY-motifs contained in Ror2 is located in the extracellular and the other one in the carboxyterminal STP-domain at the amino acid position 871–873 in the neighbourhood of the TAK1 binding-site. The threonine in the TGY-motif could be phosphorylated by the serine/threonine kinase TAK1, the tyrosinephosphorylation, however, would have to be performed by a tyrosine kinase, potentially by the TAK1-dependent activation of the Ror2 tyrosine kinase domain. That the TGY-motif in the carboxyterminal domain of Ror2 is involved in the TAK1-dependent modification is demonstrated by conversion of the threonine T871 to alanine and the replacement of the Y873 by alanine both interfering with recognition of Ror2 by pp38 antibodies (Fig. 3B). The fact that both T871 and Y873 in Ror2 have to be phosphorylated to be recognized by the pp38 antibodies leads to the conclusion that the TGY-motif is modified by two separate kinasing events. That TAK1 interaction with Ror2 is able to induce phosphorylation is also demonstrated in cell-free immunocomplex kinase assays where a TAK1-dependent 32P-phosphorylation of Ror2 or its intracellular domain is observed (Ror2-Cterm) (Fig. 3C). This is not observed in a kinase-inactive TAK1 variant (dnTAK1). We suggest an initial TAK1-dependent phosphorylation of the threonine in the TGY-motif based on experiments involving immunoprecipitation of tyrosine-phosphorylated proteins with anti-pTyr antibodies in the presence or absence of active TAK1 (Fig. 3D). Immunoprecipitation of the total tyrosine-phosphorylated proteins demonstrated that Ror2wt or the soluble carboxyterminal domain of Ror2 (Ror2-Cterm) were precipitated in the presence of active but not in the presence of kinase-inactive TAK1 (Fig. 3D). As observed before, an interaction of Ror2 with anti-pp38 antibodies could only be de-

monstrated in the presence of active but not in the presence of kinaseinactive TAK1 (Fig. 3D). Finally, in addition to the TAK1-dependent phosphorylation of the TGY-motif, TAK1 directs the phosphorylation of more residues in the STP-domain of Ror2. This finding is based on the observation that the TAK1-dependent tyrosine-phosphorylation of Ror2 persists in variants with a mutated TGY-motif (T871A/Y873A; Fig. 3D) and in immunocomplex kinase assays. Thus, TAK1 predominantly interacts with Ror2 in the STP-domain and modifies it at several positions including a TGY-motif at position 871, the latter being recognized by pp38 antibodies. These TAK1 dependent modifications influence the signalling capacity of Ror2 (see below). 3.2. The Ror2 receptor interacts with PRTB In addition to the characterization of Ror2–TAK1 interaction we performed a yeast two-hybrid screen to identify factors interacting with the intracellular domain of Ror2. The intracellular Ror2 signalling domain served as bait and a cDNA library established from murine mesenchymal progenitors C3H10T1/2-BMP2 as prey [26]. As an interactor we found PRTB (Proline Codon-rich Transcript, Brain Expressed) lacking its aminoterminal region (PRTBΔN; see Fig. 1). PRTB has been described as a 17.3 kDa, ubiquitously expressed proline-rich factor, expressed at higher levels in brain, heart and bone (Fig. 1) [35,36]. Interestingly, this coincides with the Ror2 expression profile. Both, full-length PRTB and the partial PRTBΔN construct bind Ror2 as confirmed by co-immunoprecipitations after overexpression in HEK293T cells (Fig. 4A). Ror2 deletion analysis showed that the binding region for PRTB is located in the carboxyterminal STP-region (Fig. 4B). Since PRTB and TAK1 interact with Ror2 in the identical domain we also investigated a potential PRTB/TAK1 interaction. We found that PRTB not only binds to Ror2, but also interacts with TAK1. Again, TAK1's kinase activity is necessary for PRTB-binding since the wild-type form of TAK1 (TAK1wt) or the constitutively active TAK1 (TAK1ca) co-

Fig. 4. PRTB interacts with Ror2 and TAK1. HEK293T cells were transfected with expression plasmids encoding Flag- or Myc-tagged proteins as indicated. After transfection the cells were harvested, lysed and subjected to immunoprecipitation (IP) with anti-Flag antibodies. A. Ror2wt interacts with the aminoterminal deletion of PRTB (PRTBΔN) and with fulllength PRTB as well. PRTBΔN was first isolated in a yeast two-hybrid screen for Ror2 interactors. B. PRTB interacts with Ror2 in the carboxyterminal STP-domain. C. In addition to Ror2, PRTB also interacts with TAK1. Only active TAK1 variants (TAK1wt and TAK1ca) interact with PRTB, a kinase-negative TAK1 variant (TAK1dn) does not.

A. Winkel et al. / Cellular Signalling 20 (2008) 2134–2144 Fig. 5. PRTB enhances TAK1-dependent phosphorylation of Ror2. HEK293T cells were transfected with expression plasmids encoding Flag-, HA-, or Myc-tagged proteins as indicated. After transfection the cells were harvested, lysed and subjected to immunoprecipitation (IP) with anti-Flag antibodies. A. PRTB and TAK1 do not synergize for Ror2 binding. B. Coexpression of PRTB and TAK1 synergize to enhance TAK1-dependent phosphorylation of the carboxyterminal TGYmotif in Ror2. C. The formation of dimeric Ror2/TAK1 or trimeric Ror2/TAK1/PRTB complexes only marginally modifies the capacity of these factors to stimulate MAPK-signalling pathways. ERK, JNK and p38 signalling was evaluated in western blots with antibodies specific for activated (phosphorylated) and non-activated MAPK-signalling mediators.

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Fig. 6. Wnt1- but not Wnt3a or Wnt5a ligands interfere with TAK1/Ror2 interaction. HEK 293T cells were transfected with expression plasmids encoding Flag- or HA-tagged proteins, and cell extracts were treated as described in Experimental procedures. Wnt1 and Wnt3a direct the canonical signalling pathway while Wnt5a predominantly initiates the noncanonical pathway. A. Ror2 recognizes Wnts directing the canonical and the non-canonical pathway. A chimeric Ror2/CSF1R consisting of the CSF1R-ectodomain and the intracellular domain of Ror2 serves as a control. B. Wnt1 but not Wnt3a or Wnt-5a efficiently displaces TAK1 from Ror2 in a dose-dependent fashion indicative for a transitory Wnt1-sensitive TAK1/Ror2 complex. C. Coprecipitated TAK1-band intensities were densitometrically assessed with Image J (Experimental procedures) and are presented as means ± SD from three independent experiments. Statistical difference between the level of dose-dependent Ror2/TAK1 co-precipitation in the presence of Wnt1 is indicated by ⁎ (F-Test, P b 0.05).

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immunoprecipitate with PRTB while a kinase-negative variant of TAK1 (K63W; dnTAK1) is unable to do so (Fig. 4C). TAK1 and PRTB do not synergize or compete for binding to Ror2 since the presence or absence of PRTB does not significantly change the level of TAK1-binding to Ror2 (Fig. 5A). The formation of these dimeric Ror2/TAK1 or trimeric Ror2/ TAK1/PRTB complexes did only marginally change the capacity of these factors to stimulate MAPK pathways (ERKs, JNKs, p38; Fig. 5C). Nevertheless, PRTB seems to enhance the capacity of TAK1 to phosphorylate Ror2 including the ensuing phosphorylation step at the tyrosine Y873 (~9-fold; Fig. 5B). In conclusion, the TAK1-dependent phosphorylation events are significantly enhanced by PRTB. 3.3. Wnt1 but not Wnt3a and Wnt5a interfere with TAK1 binding to Ror2 The Ror2 receptor binds members of the Wnt-family of growth factors which leads to the activation and modification of canonical and non-canonical Wnt-signalling pathways [5,6]. We therefore asked whether or not Wnt-ligand binding influences the TAK1 interaction with Ror2. Secreted factors of the Wnt-family bind the frizzled-like domain of Ror2 [4]. Co-immunoprecipitations demonstrate that Wnt1, Wnt3a and Wnt5a bind the extracellular domain of Ror2 (Fig. 6) while the exchange of the extracellular Ror2 domain with the extracellular CSF-1 receptor domain does not allow Wnt-ligand binding (Fig. 6A). Surprisingly, the TAK1/Ror2 interaction is sensitive to ligand-binding. Wnt1, but not Wnt3a or Wnt5a, is able to efficiently displace TAK1 from Ror2 in a dose-dependent fashion (Fig. 6B) which indicates that TAK1/Ror2 complexes may be modified not only by intracellular factors such as PRTB (see above) but also by extracellular events. Wnt3a and Wnt5a displace TAK1 to a much lesser extent than Wnt1 (Fig. 6B). The F-Test was used to demonstrate that Wnt1 is able to significantly displace TAK1 from Ror2. Under the tested conditions (three independent experiments) Wnt1 exhibits a significant TAK1-displacement at the highest expression rate in contrast to Wnt-3a or Wnt-5a (P b 0.05, Fig. 6C). Then we were investigating whether tyrosine-phosphorylation in the presence of TAK1 is caused by autophosphorylation mediated by the Ror2 tyrosine kinase domain. This has been observed for the casein kinase Iε which also interacts with the Ror2 carboxyterminal STP-domain [25]. In our study, however, TAK1-dependent tyrosinephosphorylation in Ror2 is not relieved in the tyrosine kinase-negative Ror2K507R mutant (Fig. 7). This result has been obtained by the immunoprecipitation of Ror2wt or its tyrosine-negative mutants with pp38 antibodies and assessing the amount of precipitated Ror2 with anti-Flag antibodies. There is no obvious difference in the TAK1dependent phosphorylation capacity in Ror2wt and the Ror2 tyrosine kinase-negative mutants. Moreover, the Wnt1-dependent sensitivity of the TAK1 interaction/phosphorylation is also observed in the Ror2 tyrosine kinase-negative mutant. Similar results were obtained when this experiment was performed with another Ror2 tyrosine kinase mutant (Ror2K507A) or when anti-pTyr-antibodies were used for immunoprecipitation (data not shown). These experiments indicate that the TAK1-dependent tyrosine-phosphorylation event is mediated predominantly by (a) cytoplasmic tyrosine kinase(s) and not by the Ror2 tyrosine kinase itself. 3.4. Ror2 regulates Wnt-signalling To assess the ability of Ror2 to affect Wnt-signalling we used the TOPflash luciferase reporter assay that measures activation of the canonical Wnt-pathway. In the absence of Wnt-ligands, Ror2 or its variants have no measurable influence on the TOPflash reporter system (Fig. 8A). The presence of Ror2 reproducibly stimulates Wnt1dependent reporter gene activation from the TOPflash reporter system in HEK293T cells (Fig. 8A): Addition of Wnt1 leads to a stimulation of the TOPflash reporter system which is further potentiated by the presence of Ror2wt. Strikingly, the deletion of the carboxyterminal

Fig. 7. TAK1-dependent modification at tyrosine-residues is not mediated by autophosphorylation of the Ror2-tyrosine kinase domain. HEK293T cells were transfected with expression plasmids encoding Flag- and HA-tagged factors as indicated. TAK1-dependent modification at tyrosine-residues does not involve the Ror2-tyrosine kinase domain activity. Ror2, phosphorylated by TAK1 interaction, was immunoprecipitated with pp38 antibodies. The level of phosphorylation at the TGY-motif is not reduced in mutations affecting autoactivation of the Ror2-tyrosine kinase domain (Ror2K507R).

STP-domain (Ror2ΔC) or the entire kinase domain (Ror2ΔTyr) leads to a considerable upregulation of the Wnt1-dependent signalling activity (Fig. 8A), especially, if one considers that the deletion mutants are significantly less efficiently expressed than the Ror2 full-length constructs. This indicates that the intracellular Ror2 domain, the Ror2 Tyr-kinase and the STP-domain as well might confer a negative impact upon canonical Wnt-signalling. Although Mutations in Ror2 interfering with TAK1-dependent phosphorylation in the carboxyterminal TGY-motif enhance canonical signalling based on the activity observed with the TOPflash reporter system (Fig. 8A). This indicates that any phosphorylation of the intracellular domain of Ror2 may exert a significant negative influence on canonical Wnt-signalling even though the kinase TAK1 only shows a reduced affinity for Ror2 in the presence of Wnt1. Wnt3a exerts opposite effects in comparison with Wnt1 on the TOPflash reporter system in HEK293T: Ror2wt stimulates Wnt1—but interferes with Wnt3a-dependent promoter activation, (Fig. 8A, B). A similar observation has been described before for Wnt1 in comparison with Wnt3 [5]. However, the deletion of the carboxyterminal STPdomain or the Tyr-kinase domain alleviates this Wnt3a-dependent inhibitory effect. This is indicative for the capacity of the Ror2 intracellular domain to modulate Wnt-canonical pathways in a liganddependent fashion. The differential effect of Wnt1 and Wnt3a may be explained by the fact that both the extracellular and the intracellular domain of Ror2 as well are able to modify canonical Wnt-signalling. The Ror2 extracellular domain alone seems to be sufficient to exert a stimulatory effect (Fig. 8A). The level of the Ror2 Tyr- and Ser/Thr-phosphorylation, however, may modify this rate of stimulation in a liganddependent fashion. In case of Wnt1-ligand binding the level of the Ror2 Tyr- and Ser/Thr-phosphorylation is low due to the fact that in the presence of Wnt1, in contrast to Wnt3a, Ror2 is less susceptible to the interaction with intracellular factors like TAK1 (Fig. 7A). Wnt3a is significantly less capable to displace TAK1 from the STP-domain than Wnt1 (Fig. 6B) which leads to a higher Wnt3a-dependent level of Ror2 Tyr- and Ser/Thr-phosphorylation (Fig. 7A). This may be an explanation for the negative impact of Wnt3a- in comparison with Wnt1-

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Fig. 8. Intracellular deletion mutants of Ror2 stimulate Wnt1-dependent canonical signalling. HEK 293T cells were transiently cotransfected with the TOPflash promoter-luciferase reporter with expression vectors encoding Wnt1, Ror2 variants, TAK1 and PRTB. Relative luciferase activity was assessed as described in Experimental procedures. The data are presented as means ± of at least three independent experiments with n = 12. The expression levels for Ror2 variants and PRTB were assessed by western analysis and are indicated. A. Wnt1-dependent activity in the presence of Ror2 variants and the intracellular signalling mediator TAK1. The expression rate of Ror2 deletion variants is significantly lower in comparison with ROR2wt. Nevertheless, apparently lower expression levels of Ror2 mutants lead to higher rates of Wnt1-dependent reporter gene activation. The Ror2wt-controls in the absence of Wnt1 were done in a separate experiment. ++ indicates twice the amount of Ror2-expression vector added. B. Wnt3a-dependent activity in the presence of Ror2 variants or the intracellular signalling mediator TAK1. Ror2wt interferes with Wnt3a-dependent reporter activation. Deletion of the carboxyterminal STP-domain or the Tyr-kinase domain alleviates this inhibitory effect indicative for the capacity of the Ror2 intracellular domain to modulate Wnt-canonical pathways. TAK1 expression was monitored with another anti-HA antibody in comparison with panel A. This antibody also reacts with an artificial band of TAK1- size (Santa Cruz, lot # 2206).

Fig. 9. TAK1 modifies Ror2's capacity to serve as a Wnt co-receptor. A. The extracellular domain of the Ror2 receptor exerts a Wnt-dependent stimulation of the canonical Wntsignalling pathway. It is unclear whether or not Ror2 enhances the capacity of Wnts to interact with frizzled receptors (FZ) but it seems that the extracellular Ror2 domain is sufficient to exert a stimulatory activity (bold +). B. Intracellular factors like TAK1 or the TAK1/PRTB complex leads to a rearrangement of the Ror2 intracellular domain enabling Tyrphosphorylation of Ror2 by cytosolic kinases. Tyr- and Ser/Thr-phosphorylation reduces Ror2's capacity to serve as a co-receptor for the Wnt-signalling pathway. Wnt1, but not Wnt3a or Wnt5a, is able to efficiently displace TAK1 from Ror2. Wnt1 binds to Ror2 and strongly interferes with interaction of intracellular factors like TAK1/PRTB. Therefore, the presence of Wnt1 causes a low level of Ror2 Tyr- and Ser/Thr-phosphorylation only resulting in a reduced amplification of the canonical pathway (fine +) if compared to the scenario described in A. In contrast, Wnt3a allows a prolonged interaction of Ror2 with intracellular factors leading to a substantial phosphorylation of Ror2. The higher level of Ror2 Tyr- and Ser/Thr-phosphorylation may efficiently reduce Wnt3a-dependent canonical signalling (bold −).

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dependent activation on the canonical pathway in the presence of Ror2 which will be discussed below (Figs. 8 and 9). The ability of Ror2 to regulate canonical Wnt-signalling should influence bone formation and maintenance because canonical Wnt signaling cascades are known to modulate bone mass, osteoblast survival and differentiation (recently reviewed in [37]). It has been demonstrated that Ror2 is expressed in the osteoblast developmental sequence being undetectable in pluripotent stem cells significantly increasing in osteoblasts and disappearing again in osteocytes [5]. Here we confirm that the forced expression of Ror2 in mesenchymal progenitors C3H10T1/2 stably expressing BMP2 positively modulate osteoblast-like cell formation by enhanced alkaline phosphatase expression, a marker gene for osteoblast-like cells (Suppl. Fig. 2). PRTB seems to exert a general positive effect on canonical pathways independent of the absence or presence of its binding-site in the STPdomain of Ror2 (Suppl. Fig. 1A). A direct effect of the TAK1–Ror2 or TAK1–PRTB–Ror2 interaction is difficult to evaluate with TOPflash assays since TAK1 alone directly negatively regulates the TCF/LEF system [6] and, therefore efficiently suppresses the TOPflash-mediated canonical Wnt activity independent of Ror2 (Fig. 7A, B; Suppl. Fig. 1A right panel and B lane 7). In contrast, an inactive TAK1-variant (dnTAK1) does not exert a major influence in the TOPflash assay-system (Suppl. Fig. 3). 4. Discussion In this study we confirm that Wnt1, Wnt3a and Wnt5a bind the RTK Ror2 (Fig. 6A). This has been observed before [5] but it has been questioned for Wnt3a [6]. So far, it has been shown that Ror2 stimulates the non-canonical Wnt-signalling pathway in mice [6] and the planar cell polarity pathway in Xenopus [4,7]. Here we document that Ror2 stimulates the Wnt1—but not the Wnt3a-dependent canonical pathway (Fig. 8A, B). Similar observations have been obtained recently for the Ror2dependent stimulation of Wnt1 in comparison with Wnt3 pathways [5]. Ror2 had opposing effects on canonical Wnt signaling: It potentiated Wnt1 activity but inhibited Wnt3 function as assessed by changes in Wnt-responsive reporter gene activity. The authors postulated that the Tyr-kinase activity of Ror2 is required for its ability to stimulate the Wnt1-dependent pathway and they suggest an as yet unknown Wnt3- and Ror2-dependent pathway which interferes with canonical signalling cascades. The results presented here may imply that this second signaling cascade may involve disruption of the TAK1/ ROR2 interaction (see below, Fig. 9). There are a number of inconsistent reports on the functional role of the Ror2 intracellular domain harbouring the Tyr-kinase domain. On one side, mice lacking the entire Ror2 receptor [38] or those lacking only the Tyr-kinase domain appear to have comparable phenotypes [39]. Also, point mutations in the Tyr-kinase domain indicate an important role for the Ror2 Tyr-kinase moiety [40]. On the other hand, in Xenopus [4] and Caenorhabditis [41,42] the role of the Ror2 Tyrkinase domain seems dispensable. We here find that the Brachydactyly B-causing Ror2 deletion mutations lacking the carboxyterminal STP-domain (Ror2ΔC) or the entire intracellular domain of Ror2 (Ror2ΔTyr) exert a higher stimulation of the canonical pathway than the entire receptor (Fig. 8). This indicates that the Ror2 extracellular domain is sufficient for a stimulation of the canonical Wnt-pathway and that Ror2 acts as a Wnt co-receptor which, possibly, enables a more efficient Wnt-ligand presentation to the frizzled receptors (Fig. 9A). Full-length Ror2 shows a reduced capacity for a stimulation of Wnt-canonical pathways in comparison with the deleted entire intracellular or the carboxyterminal STP-domain (Fig. 8). Therefore, it seems justified to argue that the intracellular Ror2 domain possesses an inherent capacity to negatively regulate the stimulatory potential exerted by the extracellular domain. Thereby, the tyrosine-phosphorylation of the carboxyterminal STP-domain and the Ser/Thr-phosphorylation as well may act as controlling elements for the interaction

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with other factors influencing canonical Wnt-signalling cascades. Factors which phosphorylate this STP-domain at Ser/Thr residues like TAK1 (this study) or CKIε [25] induces a significant Tyr-phosphorylation. It has been postulated that CKIε leads to autoactivation of the Ror2 Tyr-kinase domain [25]. Here we show that the TAK1-dependent Ser/Thr-phosphorylation in a similar region as observed for CKIε does not similarly lead to an autoactivation of the Ror2 Tyr-kinase domain. In either case the Tyr- and Ser/Thr-phosphorylation of the intracellular moiety of Ror2 may result in the differential effect of Ror2 on the Wnt1- or Wnt3a-mediated canonical pathways. This model is depicted in Fig. 9. We postulate that the extracellular domain of Ror2 may act as a Wnt co-receptor which enables a more efficient Wnt-ligand presentation to the frizzled receptors. This results in the stimulation of the canonical Wnt-signalling pathway (bold +; Fig. 9A). We have shown in this study that Wnt1- and Wnt3a-ligands exert significant differential effects on the interaction of the Ror2-STP-domain with the cytosolic factor TAK1. Wnt1 displaces TAK1 efficiently from Ror2 while Wnt3a is not able to do so (Fig. 6B) which also results in a differential level of Ror2 Tyr- and Ser/Thr-phosphorylation by Wnt1 in comparison with Wnt3a (Fig. 7A). Wnt1-ligand binding allows a low level of Tyr- and Ser/Thr-phosphorylation only. This low level would only moderately interfere with the stimulation of the canonical Wntpathways mediated by the Ror2 extracellular epitope and would yet result in a net amplification of the canonical pathway (fine +; Fig. 9B). An efficient level of Tyr- and Ser/Thr-phosphorylation (Wnt3a), however, could overcome the stimulatory potential of the Ror2 extracellular domain and thus interfere with the canonical pathway (bold −; Fig. 9B). We have observed that another factor, PRTB, which interacts with Ror2 in its STP-domain is able to substantially increase the extent of phosphorylation mediated by TAK1 (Fig. 5B). However, we consider it unlikely that PRTB itself is able to act as a kinase based on own experiments (data not shown) and also on the fact that PRTB does not possess either a Ser/Thr- or a Tyr-kinase domain. Whether or not the reported ROR2/CKIε association [25] is similarly sensitive to Wnt-ligands like the Ror2/TAK1 interaction described here remains to be demonstrated. In conclusion, we propose the model that the level of Tyr- and Ser/Thr-phosphorylation determines whether the Ror2 receptor may act as a stimulator or as an inhibitor of the canonical Wnt-signalling pathway. On the basis of this model, one may hypothesize that Brachydactyly B is likely caused by uncontrolled canonical Wnt-signalling and that canonical Wntsignalling in the organism is fine tuned by Ror2 which is controlled in an individual Wnt-ligand-dependent fashion. The situation for Wnt5a is more complex. The fact that Wnt5a causes induction of the non-canonical Wnt-pathways by primarily inhibiting the level of LEF/TCF-mediated transcription and not by affecting βcatenin protein levels has been described before [14–16]. Recently it also has been shown that Wnt5a and Ror2 activate non-canonical PI3 kinase and cdc42 pathways [7]. In our study Wnt5a binds to Ror2 and allows the prolonged interaction of intracellular factors (TAK1) and the concomitant phosphorylation of the intracellular domain of Ror2 (Fig. 7A). In our study, Wnt5a interferes with β-catenin-induced reporter gene expression (Suppl. Fig. 1B). The overexpressed Ror2 intracellular deletion mutants do not significantly influence this process probably due to a direct negative impact of TAK1 on the β-catenin-dependent activation of TCF/LEF transcription factors [15]. Surprisingly, in a very recent study it was been shown that purified Wnt5a is also able to initiate canonical pathways by interacting with the receptor Frizzled 4 [43]. In such a scenario where Wnt5a acts as stimulator of the canonical pathway, Ror2 would operate similarly as discussed before for Wnt3a: Tyr- and Ser/Thr-phosphorylation of Ror2 might substantially interfere with the Wnt5a-dependent stimulation of the canonical pathway. PRTB seems to exert a general positive effect on canonical pathways independent of the absence or presence of its binding-site in the STP-domain of Ror2 (Suppl. Fig. 1A). Unfortunately, a direct effect of

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the TAK1–Ror2 or TAK1–PRTB–Ror2 interaction is difficult to evaluate with TOPflash assays since TAK1 alone directly negatively regulates the TCF/LEF system [6] and, therefore efficiently suppresses the TOPflash-mediated canonical Wnt activity independent of Ror2 (Fig. 7A, B; Suppl. Fig. 1A right panel and B lane 7). Overall, a picture emerges in which Ror2 has an important role as a signal modulator integrating the available ligands, receptors and intracellular components in a given cellular context, thus determining the cellular readout of Wnt-signalling. Further research will have to focus on the intracellular components that interact with and mediate the phosphorylation-dependent activities of Ror2. In conclusion, we demonstrate that the MAP3K TAK1 interacts with Ror2 and phosphorylates the carboxyterminal domain of the tyrosine kinase receptor Ror2 in a Wnt-ligand-dependent manner. This leads to the phosphorylation of neighbouring tyrosine-residues by cytosolic factors and modulates the outcome of the TCF/LEF-1dependent transcription processes. Acknowledgements We thank Dr. Jens Böse, GBF, Braunschweig, for the generous gift of goat anti-phosphatidylserine receptor antibodies. We are grateful for the cDNA encoding the human CSF1R vector which we obtained from Prof. Axel Ullrich, Max-Planck-Institute, Martinsried. This study was funded in part by the SFB 599 and SFB 578 by the German Research Foundation (DFG) and the EU-FR6 integrated project GENOSTEM (G. G.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellsig.2008.08.009. References [1] M. Oldridge, A.M. Fortuna, M. Maringa, P. Propping, S. Mansour, C. Pollitt, T.M. DeChiara, R.B. Kimble, D.M. Valenzuela, G.D. Yancopoulos, A.O. Wilkie, Nat. Genet. 24 (2000) 275. [2] G.C. Schwabe, S. Tinschert, C. Buschow, P. Meinecke, G. Wolff, G. Gillessen-Kaesbach, M. Oldridge, A.O. Wilkie, R. Komec, S. Mundlos, Am. J. Hum. Genet. 67 (2000) 822. [3] M.A. Patton, A.R. Afzal, J. Med. Genet. 39 (2002) 305. [4] H. Hikasa, M. Shibata, I. Hiratani, M. Taira, Development 129 (2002) 5227. [5] J. Billiard, D.S. Way, L.M. Seestaller-Wehr, R.A. Moran, A. Mangine, P.V. Bodine, Mol. Endocrinol. 19 (2005) 90. [6] I. Oishi, H. Suzuki, N. Onishi, R. Takada, S. Kani, B. Ohkawara, I. Koshida, K. Suzuki, G. Yamada, G.C. Schwabe, S. Mundlos, H. Shibuya, S. Takada, Y. Minami, Genes Cells 8 (2003) 645. [7] A. Schambony, D. Wedlich, Dev. Cell 12 (2007) 779. [8] P. Polakis, Genes Dev. 14 (2000) 1837. [9] J.R. Miller, A.M. Hocking, J.D. Brown, R.T. Moon, Oncogene 18 (1999) 7860. [10] J.H. van Es, R.H. Giles, H.C. Clevers, Exp. Cell Res. 264 (2001) 126. [11] M. Peifer, P. Polakis, Science 287 (2000) 1606.

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