The Receptor Tyrosine Kinase Ror2 Associates with and Is Activated by Casein Kinase Iϵ*

Ror2, a member of the mammalian Ror family of receptor tyrosine kinases, plays important roles in developmental morphogenesis, although the mechanism underlying activation of Ror2 remains largely elusive. We show that when expressed in mammalian cells, Ror2 associates with casein kinase Iϵ (CKIϵ), a crucial regulator of Wnt signaling. This association occurs primarily via the cytoplasmic C-terminal proline-rich domain of Ror2. We also show that Ror2 is phosphorylated by CKIϵ on serine/threonine residues, in its C-terminal serine/threonine-rich 2 domain, resulting in autophosphorylation of Ror2 on tyrosine residues. Furthermore, it was found that association of Ror2 with CKIϵ is required for its serine/threonine phosphorylation by CKIϵ. Site-directed mutagenesis of tyrosine residues in Ror2 reveals that the sites of phosphorylation are contained among the five tyrosine residues in the proline-rich domain but not among the four tyrosine residues in the tyrosine kinase domain. Moreover, we show that in mammalian cells, CKIϵ-mediated phosphorylation of Ror2 on serine/threonine and tyrosine residues is followed by the tyrosine phosphorylation of G protein-coupled receptor kinase 2, a kinase with a developmental expression pattern that is remarkably similar to that of Ror2. Intriguingly, a mutant of Ror2 lacking five tyrosine residues, including the autophosphorylation sites, fails to tyrosine phosphorylate G protein-coupled receptor kinase 2. This indicates that autophosphorylation of Ror2 is required for full activation of its tyrosine kinase activity. These findings demonstrate a novel role for CKIϵ in the regulation of Ror2 tyrosine kinase.

Receptor tyrosine kinases (RTKs) 1 play important roles in developmental morphogenesis by regulating growth, differentiation, motility, adhesion, and death of many types of cells (1). It has been well documented that the interactions of RTKs with their cognate ligands trigger their dimerization or oligomerization, resulting in tyrosine autophosphorylation and tyrosine kinase activation of RTKs. This induces various intracellular signaling events. In contrast, it has been reported that tyrosine autophosphorylation and the tyrosine kinase activities of several RTKs, including the insulin and epidermal growth factor receptors, can be negatively regulated by ligand-independent transphosphorylation of these RTKs by cytoplasmic serine/ threonine kinases (2)(3)(4)(5)(6)(7)(8). However, little is known about the positive regulation of RTK tyrosine autophosphorylation and tyrosine kinase activation caused by cytoplasmic serine/threonine kinases.
The mammalian Ror family of RTKs, consisting of two structurally related proteins, Ror1 and Ror2, are orphan RTKs, characterized by several conserved domain structures, the extracellular Frizzled-like cysteine-rich domains, and the membrane-proximal Kringle domains that are assumed to mediate protein-protein interactions (9 -13). It has been reported that in nematodes and mammals, Ror family RTKs play crucial roles in various developmental processes. CAM-1, the Caenorhabditis elegans ortholog of Ror2, is implicated in cell migration, asymmetric cell division, and axon outgrowth during embryogenesis, and these processes may be either tyrosine kinase-dependent or -independent (14). Previous studies with Ror2-deficient mice have further revealed that Ror2 plays crucial roles in the development of the skeletal, genital, and cardiovascular systems (15)(16)(17). In humans, Ror2 is responsible for two heritable skeletal disorders; recessive Robinow syndrome and dominant brachydactyly type B (BDB) (18 -23). Interestingly, it has recently been reported that the developmental pathology of Ror2 Ϫ/Ϫ mice can explain many of the developmental malformations found in patients with Robinow syndrome (24).
We have recently shown that Ror2 associates with the melanoma-associated antigen family protein, Dlxin-1, which exhibits a similar developmental expression pattern with Ror2 and is known to bind to the homeodomain proteins Msx2 and Dlx5. Ror2 appears to affect transcriptional functions of Msx2 and Dlx5 by regulating intracellular distribution of Dlxin-1 in a tyrosine kinase-independent manner (25). Furthermore, our recent genetic and biochemical analyses have indicated that Ror2 interacts with Wnt5a both physically and functionally to activate the noncanonical Wnt5a/JNK pathway in a tyrosine kinase-independent manner (16). In Xenopus, Xror2, a putative Xenopus ortholog of Ror2, has also been shown to interact with Xenopus Wnts and to modulate convergent extension movements of axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt signaling in a tyrosine kinase-independent manner (26). However, nothing is known about the molecular mechanisms underlying Ror2 tyrosine kinase activation and the consequent tyrosine kinase-dependent functions of Ror2.
To gain insights into new functions of Ror2, we performed yeast two-hybrid screening using Ror2 as bait to identify a candidate molecule(s) that interacts with Ror2. From this screen, we identified casein kinase I⑀ (CKI⑀), a member of the CKI family of protein serine/threonine kinases, as a molecule that interacts with Ror2. Recently, much attention has been paid to CKI⑀ as a crucial regulator of the canonical Wnt signaling, although its exact role(s) in this regulation remains controversial (27). It has been demonstrated that CKI⑀ can phosphorylate various Wnt signaling mediators, including Dvl (Dishevelled), adenomatous polyposis coli, axin, and ␤-catenin, thereby contributing to the regulation of the canonical Wnt pathway (28 -33). Here we show that Ror2 associates with and is phosphorylated on serine/threonine residues by CKI⑀ when expressed in mammalian cells. Interestingly, serine/threonine phosphorylation of Ror2 by CKI⑀ is followed by the autophosphorylation of Ror2 tyrosine residue(s) within its cytoplasmic Pro-rich domain. Moreover, Ror2 associates with G proteincoupled receptor kinase 2 (GRK2) and tyrosine phosphorylates it following activation of Ror2 by CKI⑀. These results indicate that the tyrosine kinase activity and tyrosine autophosphorylation of Ror2 can be positively regulated by CKI⑀. We further provide evidence indicating that tyrosine autophosphorylation of Ror2 is required for activation of Ror2 tyrosine kinase.
Antibodies, Cells, and Transfection-Rabbit polyclonal anti-mouse Ror2 antibody was raised against GST mouse Ror2 (amino acids 726 -945). The mouse monoclonal antibodies M2 (Sigma) and 12CA5 (Roche Applied Science) recognize the FLAG peptide and human influenza HA protein peptide sequence. Mouse monoclonal anti-CKI⑀ antibody was purchased from Transduction Laboratories. The mouse monoclonal anti-phosphotyrosine antibodies PY20 and 4G10 were purchased from Cell Signaling and Upstate Biotechnology, Inc., respectively. Rabbit polyclonal anti-phosphoserine and anti-phosphothreonine antibodies were from Zymed Laboratories and Cell Signaling, respectively. HEK293T (293T) and NIH3T3 (3T3) cells were maintained in Dulbecco's modified Eagle's medium (Nissui) supplemented with 10% (v/v) fetal calf serum. Transient cDNA transfection was performed using the calcium phosphate method (12).
Immunoprecipitation and Immunoblotting-The cells were solubilized with lysis buffer (50 mM Tris-HCl, pH 7.4, 0.5% (v/v) Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin), and the cell lysates were prepared by centrifugation at 12,000 ϫ g for 15 min. The cell lysates were precleared for 1 h at 4°C with protein A-Sepharose (Amersham Biosciences). The precleared supernatants were then immunoprecipitated with anti-FLAG or anti-HA antibody conjugated to protein A-Sepharose beads for 2 h at 4°C. The immunoprecipitates were washed five times with 1 ml of the above lysis buffer and eluted with Laemmli sample buffer. Immunoprecipitates or whole cell lysates were separated by SDS-PAGE (9% PAGE) and transferred to polyvinylidene difluoride membrane filters (Immobilon, Millipore). The membranes were immunoblotted with the respective antibodies, and the bound antibodies were visualized with horseradish peroxidaseconjugated anti-mouse IgG antibodies using chemiluminescence reagents (Western Lightning; PerkinElmer Life Sciences) as described previously (12).
Expression and Purification of GST Fusion Proteins-The GST fusion proteins, GST-CKI⑀ WT and GST-CKI⑀ DK, expressed in Escherichia coli DH5␣ were extracted with phosphate-buffered saline containing 1% (v/v) Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin and were isolated with glutathione-Sepharose beads (Amersham Biosciences). Fusion proteins were then eluted from beads by 25 mM glutathione (reduced), followed by dialysis prior to use in kinase assays.
In Vitro Kinase Assay-For in vitro kinase assay, 293T cells were solubilized 60 h after transfection, and Ror2 WT-FLAG or Ror2 DK-FLAG proteins were immunoprecipitated as described above. Precipitates were washed five times with lysis buffer and resuspended in 50 l kinase buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , and 40 M ATP. The in vitro kinase reaction was initiated by the addition of purified GST-CKI⑀ WT or GST-CKI⑀ DK and allowed to incubate for 30 min at 30°C. The reaction was terminated by the addition of Laemmli sample buffer, and the samples were separated by SDS-PAGE (10% PAGE) and transferred to polyvinylidene difluoride membrane filters, followed by immunoblot analysis with antiphospho-serine/threonine antibodies.
In Situ Hybridization-In situ hybridization analyses were performed essentially as described previously (34). The 0.86-kb HincII/ EcoRI fragment of Ror2 or the 0.87-kb HincII/ApaI fragment of GRK2 were utilized as templates to synthesize single strand RNA probes.

Ror2
Associates with CKI⑀-To identify a Ror2-interacting protein(s), we performed a yeast two-hybrid screening using the cytoplasmic region of Ror2 as bait (25). From this screen we identified a protein serine/threonine kinase, CKI⑀ (data not shown). To determine whether Ror2 associates with CKI⑀ in mammalian cells, FLAG-tagged wild-type Ror2 (Ror2 WT) and HA-tagged CKI⑀ were coexpressed in 293T cells. As shown in Fig. 1A, HA-tagged CKI⑀ was coimmunoprecipitated with FLAG-tagged Ror2, indicating that Ror2 associates with CKI⑀ in vivo. We also found that endogenous CKI⑀ was detected in anti-Ror2 immunoprecipitates from 3T3 cells, confirming association between endogenous Ror2 and CKI⑀ (Fig. 1B).
Next, to identify a cytoplasmic domain(s) within Ror2 required for its association with CKI⑀, we generated a series of truncated mutants of Ror2 (Fig. 1C) and evaluated their abilities to associate with CKI⑀ in 293T cells. As shown in Fig. 1D, the Ror2 mutants Ror2 ⌬C and Ror2 BDB (Fig. 1C) exhibited apparently decreased levels of CKI⑀ binding compared with the Ror2 WT. To map more precisely an association domain within the C-terminal portion of Ror2, we generated additional deletion mutants of Ror2 (⌬883, ⌬S/T2, ⌬pro, ⌬S/T1, and ⌬S/T1,2) (Fig. 1C) and tested their abilities to associate with CKI⑀. As shown in Fig. 1E, among them only the Ror2 ⌬pro exhibited drastically decreased levels of CKI⑀ binding. The result indicates that the proline-rich domain of Ror2 is required critically for association with CKI⑀. On the other hand, the Ror2 RS and Ror2 Tc (Fig. 1C), both lacking the tyrosine kinase domain, failed to associate with CKI⑀, suggesting that the tyrosine kinase domain of Ror2 is also required for association (Fig. 1D). Because we have previously shown that Ror2 associates with Dlxin-1 (NRAGE) via the proline-rich domain of Ror2 (25), we also examined whether or not association of CKI⑀ and Dlxin-1 with Ror2 is competitive. It was found that association of CKI⑀ with Ror2 was unaffected by ectopic coexpression of Dlxin-1 vice versa (see first supplemental figure).
Serine/Threonine Phosphorylation of Ror2 by CKI⑀-We next examined whether or not Ror2 is phosphorylated on serine/ threonine residues by CKI⑀ in mammalian cells. To this end, FLAG-tagged Ror2 was expressed along with either HA-tagged wild-type (WT) or kinase inactive mutant (DK) of CKI⑀ in 293T cells, and serine/threonine phosphorylation of Ror2 was examined by anti-FLAG immunoprecipitation followed by anti-phosphoserine/threonine immunoblotting (see "Experimental Procedures"). As shown in Fig. 2A, Ror2 was phosphorylated on serine/threonine residues in cells coexpressing CKI⑀ WT but not CKI⑀ DK, indicating that the kinase activity of CKI⑀ is required for serine/threonine phosphorylation of Ror2. We also examined whether Dlxin-1 is phosphorylated on serine/threonine residues by CKI⑀ in the presence of Ror2. It appeared that Dlxin-1 was not phosphorylated by CKI⑀ (see second supplemental figure), suggesting that Ror2 may transduce signals via CKI⑀ and Dlxin-1 separately.
To test whether CKI⑀ could phosphorylate Ror2 directly, GST-CKI⑀ WT and GST-CKI⑀ DK proteins purified from E. coli (see "Experimental Procedures") were subjected to in vitro kinase assay using FLAG-tagged WT or kinase-inactive Ror2 mutant (DK) as substrates. As shown in Fig. 2B, GST-CKI⑀ WT, but not GST-CKI⑀ DK, could phosphorylate serine/threonine residues within both Ror2 WT and Ror2 DK in vitro, supporting the idea that CKI⑀ phosphorylates Ror2 directly.
Next, we attempted to identify serine/threonine phosphorylation sites within Ror2 by CKI⑀. Because serine/threonine phosphorylation of Ror2 was found in the Ror2 ⌬S/T1 but not Ror2 ⌬S/T2 mutants, we generated the Ror2 13S/TA mutant in which all of the serines and threonines in the S/T2 domain of Ror2 were replaced with alanines (Fig. 1C). As shown in Fig.  2C, the Ror2 13S/TA mutant could associate with CKI⑀ but failed to be phosphorylated by CKI⑀, indicating that CKI⑀ phosphorylates primarily serine/threonine residues in the S/T2 domain of Ror2.
Tyrosine Autophosphorylation of Ror2 Following Its Serine/ Threonine Phosphorylation by CKI⑀-To better understand the biological consequence of Ror2 phosphorylation by CKI⑀, we transiently coexpressed FLAG-tagged Ror2 WT with either CKI⑀ WT or CKI⑀ DK in 293T cells. Tyrosine phosphorylation of Ror2 was detected when Ror2 and CKI⑀ WT were coexpressed, but not when Ror2 was expressed alone or coexpressed with CKI⑀ DK (Fig. 3A). The results indicate that CKI⑀ kinase activity is required for tyrosine phosphorylation of Ror2. Next, FLAG-tagged Ror2 WT or Ror2 DK was expressed along with CKI⑀ WT, and Ror2 tyrosine phosphorylation was evaluated. Tyrosine phosphorylation of Ror2 WT, but not Ror2 DK, was observed under the same experimental setting, although Ror2 DK was also associated with and serine/threonine-phosphorylated by CKI⑀ to similar extents when compared with Ror2 WT (Fig. 3B). Thus, CKI⑀-mediated tyrosine phosphorylation of Ror2 requires the intrinsic tyrosine kinase activity of Ror2.
We further attempted to identify tyrosine phosphorylation sites within Ror2 induced by coexpression of CKI⑀. We have previously shown that the tyrosine kinase domains of the Ror family RTKs are most similar to those of the neurotrophin receptor Trk family of RTKs and that the four autophosphorylated tyrosine residues found in the activation loops within the tyrosine kinase domains of the Trk family RTKs are also conserved in Ror2 (Tyr 641 , Tyr 645 , Tyr 646 , and Tyr 722 ) (12). We also found that coexpression of CKI⑀ failed to induce tyrosine phosphorylation of Ror2 ⌬C (data not shown). The Ror2 ⌬C lacks the C-terminal portion of Ror2, which contains six tyrosine residues, including five (Tyr 818 , Tyr 824 , Tyr 830 , Tyr 833 , and Tyr 838 ) that are found in the Pro-rich domain. Thus, we generated the two Ror2 mutants, 4YF and 5YF (Fig. 3C and see "Experimental Procedures"), in which the tyrosines were replaced with phenylalanines. When FLAG-tagged Ror2 4YF and 5YF were expressed in 293T cells along with CKI⑀, it was found that both the Ror2 4YF and 5YF could associate with CKI⑀ and were phosphorylated on serine/threonine residues to a similar extent as Ror2 WT (Fig. 3D). Interestingly, when coexpressed with CKI⑀, Ror2 4YF but not Ror2 5YF was found to be phosphorylated on tyrosine residues (Fig. 3D). This indicates that the sites of tyrosine autophosphorylation are among the five tyrosine residues contained within the Pro-rich domain but not among the four tyrosine residues contained within the tyrosine kinase domain. We then examined whether serine/threonine phosphorylation of Ror2 by CKI⑀ is required for tyrosine autophosphorylation of Ror2. For this purpose, tyrosine phosphorylation status of the 13S/TA was monitored in the presence of CKI⑀. When Ror2 WT and 13S/TA mutant were expressed in 293T cells along with CKI⑀, tyrosine autophosphorylation of Ror2 was found in Ror2 WT but not 13S/TA (Fig. 3E). This result indicates that serine/threonine phosphorylation of Ror2 by CKI⑀ is required for subsequent tyrosine autophosphorylation of Ror2.
Tyrosine Phosphorylation of GRK2 by Ror2 Following Coexpression of CKI⑀-The Ror2-interacting proteins, CKI⑀ and WT and Ror2 DK were prepared by overexpressing the respective proteins in 293T cells followed by anti-FLAG immunoprecipitation. In vitro kinase assay was performed as described under "Experimental Procedures." Top, middle, and bottom panels indicate anti-phosphoserine/threonine, anti-FLAG, and anti-CKI⑀ immunoblot analyses of kinase reactions, respectively. C, serine/threonine phosphorylation within the serine/threoninerich 2 domain of Ror2 by CKI⑀. WCLs were prepared from 293T cells expressing FLAG-tagged WT or a series of Ror2 mutant proteins along with HA-tagged CKI⑀ protein, as shown in the panel. Serine/threonine phosphorylation of Ror2 WT and the respective Ror2 mutants were examined as described for A.
Dlxin-1 (see Fig. 5A), were not tyrosine-phosphorylated by Ror2 when coexpressed with CKI⑀ (data not shown). We therefore searched for other candidate molecule(s) that could be tyrosinephosphorylated by Ror2 under the same experimental conditions. The gene encoding the GRK2 was reported to exhibit a developmental expression pattern very similar to those of Ror2 (Fig. 4A) and Dlxin-1 (data not shown), as verified by whole mount in situ hybridization analyses on mouse embryos at embryonic day 10.5 ( Fig. 4A; data not shown), (34,35,36). In particular, Ror2 and GRK2 exhibited remarkably similar expression patterns in the pharyngeal arches and developing limb buds at embryonic day 10.5 (Fig. 4A). We therefore examined whether or not Ror2 could associate with GRK2. As shown in Fig. 4B, HA-tagged GRK2 coimmunoprecipitated with FLAG-tagged Ror2, indicating that Ror2 associates with GRK2 in vivo. In addition, it was found that the association of Ror2 with GRK2 was unaffected by coexpression of CKI⑀ (data not shown).
To examine whether or not GRK2 or Dlxin-1 could be tyrosine-phosphorylated by Ror2 following coexpression of CKI⑀, FLAG-tagged Ror2 and HA-tagged CKI⑀ were coexpressed in 293T cells along with HA-tagged GRK2 or Dlxin-1. As shown in Fig. 5A, GRK2, but not Dlxin-1, was tyrosine-phosphorylated when Ror2 and CKI⑀ were coexpressed. Under the same experimental conditions, tyrosine phosphorylation of CKI⑀ by Ror2 was not detected (data not shown). These results reveal that coexpression of Ror2 and CKI⑀ leads to the tyrosine phospho-rylation of GRK2. Next, we examined whether or not tyrosine phosphorylation of GRK2 correlates with CKI⑀-induced tyrosine autophosphorylation of Ror2. Consistent with the result shown in Fig. 5A, coexpression of Ror2 WT and CKI⑀ WT resulted in the tyrosine phosphorylation of GRK2, whereas Ror2 WT or CKI⑀ WT alone did not (Fig. 5B). Furthermore, coexpression of Ror2 WT plus CKI⑀ DK or Ror2 DK plus CKI⑀ WT failed to induce tyrosine phosphorylation of GRK2. These results indicate that tyrosine phosphorylation of Ror2, following coexpression of Ror2 and CKI⑀, leads to Ror2-mediated tyrosine phosphorylation of GRK2.
Next, we examined whether tyrosine autophosphorylation of Ror2 is required for Ror2-mediated tyrosine phosphorylation of GRK2. As shown in Fig. 5C, tyrosine phosphorylation of GRK2 was observed when Ror2 WT or Ror2 4YF, but not Ror2 5YF, was coexpressed with CKI⑀. This indicates that autophosphorylation of one or more of the five tyrosine residues within the Pro-rich domain of Ror2 is required for tyrosine phosphorylation of GRK2 by Ror2. Finally, we also examined whether serine/threonine phosphorylation of Ror2 by CKI⑀ is indeed required for Ror2-mediated tyrosine phosphorylation of GRK2. As expected, tyrosine phosphorylation of GRK2 was detected in cells expressing Ror2 WT, but not 13S/TA, along with CKI⑀ (Fig. 5D). The result indicates that serine/threonine phosphorylation of Ror2 by CKI⑀ is also required for tyrosine phosphorylation of GRK2 by activated Ror2. FLAG-tagged WT or DK Ror2 was expressed transiently in 293T cells with or without CKI⑀ protein, as shown in the panel. Tyrosine and serine/threonine phosphorylation of Ror2 were monitored as described in A. C, schematic representations of the Ror2 mutants (Ror2 4YF and Ror2 5YF) containing different substitutions of tyrosines with phenylalanines. Tyrosines 641, 645, 646, and 722 were replaced with phenylalanines in the 4YF mutant and tyrosines 818, 824, 830, 833, and 838 were replaced with phenylalanines in the 5YF mutant. The numbers indicated represent the positions of the respective amino acid residues. D, five tyrosine residues within the proline-rich domain of Ror2 are required for tyrosine phosphorylation of Ror2 following expression of CKI⑀. FLAG-tagged WT or the tyrosine-substituted Ror2 mutants (4YF and 5YF) were expressed transiently in 293T cells with or without CKI⑀ protein, as shown in the panel. Tyrosine and serine/threonine phosphorylation of Ror2 were monitored as described in A. E, tyrosine phosphorylation of following expression of CKI⑀ requires Ror2 serine/threonine phosphorylation in the serine/threonine rich 2 domain of Ror2. FLAG-tagged WT or Ror2 13S/TA was expressed transiently in 293T cells along with CKI⑀ protein, as shown in the panel. Tyrosine and serine/threonine phosphorylation of Ror2 were monitored as described for A.

DISCUSSION
The receptor tyrosine kinase Ror2 plays important roles in developmental morphogenesis, including the development of skeletal, genital, and cardiorespiratory systems (15,17). Genetic analyses in nematodes have revealed that CAM-1, the C. elegans ortholog of Ror2, possesses both tyrosine kinase-dependent and -independent functions in development (9,13,14). However, little is known about the tyrosine kinase-dependent functions of Ror2, in particular, the mechanisms underlying tyrosine kinase activation of Ror2. In fact, Wnt5a stimulation of Ror2, antibody-mediated cross-linking of Ror2, and granulocyte macrophage colony-stimulating factor (GMCSF)-induced dimerization of a chimeric GMCSF/Ror2 receptor (GMCSF receptor extracellular region/Ror2 transmembrane and intracellular regions) fail to induce Ror2 tyrosine autophosphorylation or tyrosine kinase activity (data not shown). Here we show that tyrosine autophosphorylation and tyrosine kinase activation of Ror2 are induced by the cytoplasmic protein serine/threonine kinase CKI⑀-mediated phosphorylation of Ror2 (Fig. 3).
Our structure-function analyses of Ror2 indicate that Ror2 associates with CKI⑀ primarily via its C-terminal proline-rich domain (Fig. 1, D and E) and that Ror2 is phosphorylated by CKI⑀ in its C-terminal S/T2 domain (Fig. 2C). Because all of the deletional mutants of Ror2 lacking the proline-rich domain, Ror2 ⌬C, BDB, RS, Tc (data not shown), and ⌬pro (Fig. 2C), exhibited both drastically decreased levels or complete loss of CKI⑀ binding and of serine/threonine phosphorylation by CKI⑀ ( Fig. 2C; data not shown), it is likely that association of Ror2 with CKI⑀ is required for its phosphorylation by CKI⑀. Furthermore, we present evidence indicating that phosphorylation of Ror2 by CKI⑀ is prerequisite to induce autotyrosine phosphorylation and activation of Ror2 (Figs. 3 and 5). In fact, the Ror2 13S/TA mutant, which is assumed to lack serine/threonine phosphorylation sites by CKI⑀, can associate with CKI⑀ but fail to be autotyrosine-phosphorylated or activated by CKI⑀ (Figs.  2C, 3E, and 5D). It is of importance to determine the pivotal serine/threonine residues within the S/T2 domain of Ror2 that are phosphorylated by CKI⑀. Ror2 mutants lacking the Cterminal portion (e.g. Ror2 ⌬C and BDB) are also neither autotyrosine-phosphorylated nor activated by CKI⑀ (data not shown). Interestingly, mutant Ror2 proteins from BDB patients lack this C-terminal portion of Ror2, suggesting that the pathogenesis of BDB may be attributable to a defect in tyrosine autophosphorylation and activation of Ror2 tyrosine kinase activity.
To clarify the mechanism of tyrosine kinase activation of Ror2 following phosphorylation by CKI⑀, we attempted to identify the sites of tyrosine autophosphorylation within the cytoplasmic region of Ror2. It had been reported that autophosphorylation of the tyrosine residues in the activation loop of the tyrosine kinase domains of the Trk family RTKs is required for activating the Trk family RTKs (38), the closest relatives of Ror family RTKs. However, we mapped the Ror2 tyrosine autophosphorylation sites to the proline-rich domain, not the activation loop (Fig. 3D). Further study will be required to identify more precisely the autophosphorylated tyrosine residue(s). In this study, we also identified the cytoplasmic protein serine/threonine kinase GRK2, as a substrate for activated Ror2 (Fig. 5). Interestingly, the Ror2 5YF, but not 4YF, failed to tyrosine phosphorylates GRK2 (Fig. 5C), indicating that autophosphorylation of tyrosine residue(s) within the proline-rich domain of Ror2 is required for (full) activation of Ror2 tyrosine kinase. We have previously shown that Ror1, in addition to Ror2, plays an important role in developmental morphogenesis and that most functions of Ror1 during mouse development can be compensated for by Ror2 (37). We found that Ror1 could also associate with CKI⑀ in vivo and is phosphorylated on serine/threonine residues by CKI⑀ (data not shown). However, at present tyrosine autophosphorylation and tyrosine kinase activation of Ror1 have not been detected, probably because of a very low level of Ror1 expression compared with Ror2 in our transfection experiments. The results also suggest that the mechanism underlying activation of Ror2 tyrosine kinase may be distinct from those of other Trk family RTKs.
Our results demonstrate that the tyrosine kinase activity of Ror2 is regulated by CKI⑀. It has been reported that several RTKs can be transphosphorylated by cytoplasmic protein kinases. For example, the insulin receptor is phosphorylated by cAMP-dependent protein kinase (7), protein kinase C (8), and casein kinase 2 (6), and the epidermal growth factor receptor is phosphorylated by cAMP-dependent protein kinase (2), protein kinase C (4,5), and calmodulin-dependent protein kinase II (3). In these cases, serine/threonine phosphorylation of these RTKs results in the drastic down-regulation of their auto-tyrosine phosphorylation and tyrosine kinase activities. Compared with these RTKs, Ror2 RTK is rather unique in that serine/threonine phosphorylation of Ror2 by CKI⑀ results in the stimulation of its tyrosine autophosphorylation and tyrosine kinase activity. Importantly, it has been reported that CKI⑀ regulates the canonical Wnt pathway by interacting both physically and functionally with various Wnt signal mediators (28 -30, 32, 33). Taken together with our findings, we envisage that there may be significant cross-talk between the Ror2 and canonical Wnt signal pathways. Although we have previously shown that Ror2 is also involved in the noncanonical Wnt pathway (16), it is currently unclear whether or not CKI⑀ is likewise involved in noncanonical Wnt signaling. The rat Frizzled-2 (rFz2) is a putative receptor for Wnt5a and has been shown to activate several protein serine/threonine kinases, including protein kinase C, CaMKII, TAK1, and NLK, which mediate the noncanonical Wnt signaling pathway following engagement of upstream receptors (39 -42). It would therefore be of interest to examine whether or not these protein kinases may also be able to phosphorylate and regulate Ror2 tyrosine kinase.
GRK2 is known as a key modulator in the internalization of seven transmembrane-spanning G protein-coupled receptors. Following agonist stimulation, GRK2 phosphorylates the most C-terminal cytoplasmic region of G protein-coupled receptors, resulting in the recruitment of ␤-arrestin and eventual agonistinduced internalization of the G protein-coupled receptors (43). It has previously been shown that the kinase activity and/or stability of GRK2 can also be modulated by tyrosine phosphorylation by the Src protein tyrosine kinase (44 -47). Therefore, it is important to determine whether tyrosine phosphorylation of GRK2 by Ror2 may affect GRK2 kinase activity and consequent endocytosis of G protein-coupled receptors. We have shown that Ror2 forms a complex with rFz2 or human Frizzled 5 (hFz5), putative seven transmembrane-spanning type receptors for Wnt5a (16). Furthermore, it has recently been reported that mouse Frizzled 4 (mFz4), another putative seven transmembrane-spanning type receptor for Wnt5a, can be internalized following stimulation with Wnt5a and phorbol myristoyl FIG. 5. Tyrosine phosphorylation of GRK2 by Ror2 following coexpression of CKI⑀. A, tyrosine phosphorylation of GRK2 following coexpression of Ror2 and CKI⑀. FLAG-tagged Ror2 and HA-tagged CKI⑀ proteins were coexpressed transiently in 293T cells with or without HA-tagged GRK2 or Dlxin-1, as shown in the panel. Anti-HA or anti-FLAG immunoprecipitates (IP) from the respective WCLs were analyzed by anti-phosphotyrosine (top and bottom panels), anti-HA (second panel), anti-CKI⑀ (third panel), and anti-FLAG (fourth panel) immunoblotting. B, tyrosine phosphorylation of GRK2 induced by coexpression of Ror2 and CKI⑀ requires Ror2 and CKI⑀ kinase activities. HA-tagged GRK2 protein was expressed transiently in 293T cells singly or in combination with FLAG-tagged Ror2 proteins (WT or DK) and/or HA-tagged CKI⑀ proteins (WT or DK), as shown in the panel. WCLs and anti-HA immunoprecipitates were analyzed by immunoblotting as described for A. C, tyrosine residues within the C-terminal proline-rich domain of Ror2 are required for tyrosine phosphorylation of GRK2 by Ror2 in the presence of CKI⑀. FLAG-tagged WT or the tyrosine-substituted Ror2 mutants (4YF and 5YF) were expressed transiently in 293T cells along with HA-tagged GRK2 and CKI⑀ proteins, as shown in the panel. Anti-HA or anti-FLAG immunoprecipitates from the respective WCLs were analyzed by immunoblotting as described for A. D, serine/threonine phosphorylation within the serine/threonine rich 2 domain of Ror2 are required for tyrosine phosphorylation of GRK2 by Ror2 in the presence of CKI⑀. FLAG-tagged Ror2 WT or Ror2 13S/TA were expressed transiently in 293T cells along with both HA-tagged GRK2 and CKI⑀ proteins, as shown in the panel. Anti-HA or anti-FLAG immunoprecipitates from the respective WCLs were analyzed by immunoblotting as described for A. acetate, a potent activator of protein kinase C, and that phosphorylated Dvl 2 recruits ␤-arrestin 2 to mediate internalization of mFz4 (48). The Ror2-associated CKI⑀ may be located in proximity to the putative seven transmembrane-spanning type receptors for Wnt5a (mFz4, rFz2, and hFz5) (16). In addition, CKI⑀ has been shown to phosphorylate the Dvl proteins (30,32,49). Therefore, it is conceivable that the Dvl proteins may be phosphorylated by Ror2-associated CKI⑀, recruit ␤-arrestin to these Wnt5a receptors, and thereby mediate internalization of these receptors. Alternatively, activated Ror2 tyrosine kinase may phosphorylate and regulate the function of GRK2, which in turn phosphorylates these receptors, resulting in their internalization. Further study will be required to clarify the biological significance of Ror2 tyrosine kinase activation.