The Receptor Tyrosine Kinase Ror2 Associates with and Is Activated by Casein Kinase Iϵ
2004; Elsevier BV; Volume: 279; Issue: 48 Linguagem: Inglês
10.1074/jbc.m409039200
ISSN1083-351X
AutoresShuichi Kani, Isao Oishi, Hiroyuki Yamamoto, Akinori Yoda, Hiroaki Suzuki, Akira Nomachi, Kengo Iozumi, Michiru Nishita, Akira Kikuchi, Toru Takumi, Yasuhiro Minami,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoRor2, 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. 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) 1The abbreviations used are: RTK, receptor tyrosine kinase; CKIϵ, casein kinase Iϵ; GRK2, G protein-coupled receptor kinase 2; BDB, brachydactyly type B; GST, glutathione S-transferase; HA, hemagglutinin; WT, wild-type; GMCSF, granulocyte macrophage colony-stimulating factor; WCL, whole cell lysate. play important roles in developmental morphogenesis by regulating growth, differentiation, motility, adhesion, and death of many types of cells (1Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3538) Google Scholar). 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 (2Barbier A.J. Poppleton H.M. Yigzaw Y. Mullenix J.B. Wiepz G.J. Bertics P.J. Patel T.B. J. Biol. Chem. 1999; 274: 14067-14073Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 3Countaway J.L. Nairn A.C. Davis R.J. J. Biol. Chem. 1992; 267: 1129-1140Abstract Full Text PDF PubMed Google Scholar, 4Davis R.J. J. Biol. Chem. 1988; 263: 9462-9469Abstract Full Text PDF PubMed Google Scholar, 5Hunter T. Ling N. Cooper J.A. Nature. 1984; 311: 480-483Crossref PubMed Scopus (422) Google Scholar, 6Grande J. Perez M. Itarte E. FEBS Lett. 1988; 232: 130-134Crossref PubMed Scopus (23) Google Scholar, 7Roth R.A. Beaudoin J. 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Previous studies with Ror2-deficient mice have further revealed that Ror2 plays crucial roles in the development of the skeletal, genital, and cardiovascular systems (15DeChiara T.M. Kimble R.B. Poueymirou W.T. Rojas J. Masiakowski P. Valenzuela D.M. Yancopoulos G.D. Nat. Genet. 2000; 24: 271-274Crossref PubMed Scopus (198) Google Scholar, 16Oishi I. Suzuki H. Onishi N. Takada R. Kani S. Ohkawara B. Koshida I. Suzuki K. Yamada G. Schwabe G.C. Mundlos S. Shibuya H. Takada S. Minami Y. Genes Cells. 2003; 8: 645-654Crossref PubMed Scopus (607) Google Scholar, 17Takeuchi S. Takeda K. Oishi I. Nomi M. Ikeya M. Itoh K. Tamura S. Ueda T. Hatta T. Otani H. Terashima T. Takada S. Yamamura H. Akira S. Minami Y. Genes Cells. 2000; 5: 71-78Crossref PubMed Scopus (194) Google Scholar). In humans, Ror2 is responsible for two heritable skeletal disorders; recessive Robinow syndrome and dominant brachydactyly type B (BDB) (18Afzal A.R. Jeffery S. Hum. 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Genet. 2000; 25: 423-426Crossref PubMed Scopus (218) Google Scholar). Interestingly, it has recently been reported that the developmental pathology of Ror2–/– mice can explain many of the developmental malformations found in patients with Robinow syndrome (24Schwabe G.C. Trepczik B. Suring K. Brieske N. Tucker A. Sharpe P.T. Minami Y. Mundlos S. Dev. Dyn. 2004; 229: 400-410Crossref PubMed Scopus (97) Google Scholar). 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 (25Matsuda T. Suzuki H. Oishi I. Kani S. Kuroda Y. Komori T. Sasaki A. Watanabe K. Minami Y. J. Biol. Chem. 2003; 278: 29057-29064Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). 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 (16Oishi I. Suzuki H. Onishi N. Takada R. Kani S. Ohkawara B. Koshida I. Suzuki K. Yamada G. Schwabe G.C. Mundlos S. Shibuya H. Takada S. Minami Y. Genes Cells. 2003; 8: 645-654Crossref PubMed Scopus (607) Google Scholar). 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 (26Hikasa H. Shibata M. Hiratani I. Taira M. Development. 2002; 129: 5227-5239Crossref PubMed Google Scholar). 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 Iϵ (CKIϵ), 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 CKIϵ as a crucial regulator of the canonical Wnt signaling, although its exact role(s) in this regulation remains controversial (27Ding Y. Dale T. Trends Biochem. Sci. 2002; 27: 327-329Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). It has been demonstrated that CKIϵ can phosphorylate various Wnt signaling mediators, including Dvl (Dishevelled), adenomatous polyposis coli, axin, and β-catenin, thereby contributing to the regulation of the canonical Wnt pathway (28Amit S. Hatzubai A. Birman Y. Andersen J.S. Ben-Shushan E. Mann M. Ben-Neriah Y. Alkalay I. Genes Dev. 2002; 16: 1066-1076Crossref PubMed Scopus (592) Google Scholar, 29Gao Z.H. Seeling J.M. Hill V. Yochum A. Virshup D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1182-1187Crossref PubMed Scopus (192) Google Scholar, 30Kishida M. Hino S. Michiue T. Yamamoto H. Kishida S. Fukui A. Asashima M. Kikuchi A. J. Biol. Chem. 2001; 276: 33147-33155Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 31Lee E. Salic A. Kirschner M.W. J. Cell Biol. 2001; 154: 983-993Crossref PubMed Scopus (127) Google Scholar, 32Peters J.M. McKay R.M. McKay J.P. Graff J.M. Nature. 1999; 401: 345-350Crossref PubMed Scopus (384) Google Scholar, 33Schwarz-Romond T. Asbrand C. Bakkers J. Kuhl M. Schaeffer H.J. Huelsken J. Behrens J. Hammerschmidt M. Birchmeier W. Genes Dev. 2002; 16: 2073-2084Crossref PubMed Scopus (162) Google Scholar). Here we show that Ror2 associates with and is phosphorylated on serine/threonine residues by CKIϵ when expressed in mammalian cells. Interestingly, serine/threonine phosphorylation of Ror2 by CKIϵ is followed by the autophosphorylation of Ror2 tyrosine residue(s) within its cytoplasmic Pro-rich domain. Moreover, Ror2 associates with G protein-coupled receptor kinase 2 (GRK2) and tyrosine phosphorylates it following activation of Ror2 by CKIϵ. These results indicate that the tyrosine kinase activity and tyrosine autophosphorylation of Ror2 can be positively regulated by CKIϵ. We further provide evidence indicating that tyrosine autophosphorylation of Ror2 is required for activation of Ror2 tyrosine kinase. Plasmid Constructions—Wild-type and mutant cDNAs were constructed in the mammalian expression vector pcDNA3 (Invitrogen). Expression vectors encoding the FLAG-tagged Ror proteins were constructed as described previously (25Matsuda T. Suzuki H. Oishi I. Kani S. Kuroda Y. Komori T. Sasaki A. Watanabe K. Minami Y. J. Biol. Chem. 2003; 278: 29057-29064Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The Ror2 mutant constructs (ΔC, BDB, RS, Tc, Δ883, Δpro, ΔS/T1, ΔS/T2, and ΔS/T1,2) were generated by deleting amino acids 788–944, 749–944, 502–944, 434–944, 883–944, 783–859, 744–782, 860–882, 744–782 and 860–882, respectively, in the C-terminal region of Ror2. An expression vector encoding a kinasedead mutant of Ror2 was constructed by replacing lysine 507, crucial for ATP binding, with arginine. Ror2 mutants bearing substitutions of serine and threonine with alanines, Ror2 13S/TA (S860A, S861A, S864A, S866A, S868A, S870A, S879A, and S882A; T869A, T871A, T875A, T876A, and T881A) were constructed by site-directed mutagenesis. Ror2 mutants bearing substitutions of tyrosines with phenylalanines, Ror2 4YF (Y641F, Y645F, Y646F, and Y722F) and Ror2 5YF (Y818F, Y824F, Y830F, Y833F, and Y838F), were also constructed by site-directed mutagenesis. The cDNA fragment corresponding to CKIϵ was obtained by PCR and inserted into pcDNA3. The expression vector pcDNA-HA-CKIϵ DK, encoding a kinase-dead mutant of CKIϵ, was constructed by replacing lysine 38, crucial for ATP binding, with arginine. The plasmids encoding the GST fusion proteins, GST-CKIϵ WT and GST-CKIϵ DK, were constructed using the pGEX plasmids (Amersham Biosciences). Bovine GRK2 cDNA (kindly provided by Dr. Haga, Gakushuin University and Dr. Lefkowitz, Duke University) was subcloned into pcDNA3 together with the influenza hemagglutinin (HA) protein epitope tag at its C terminus (pcDNA-GRK2-HA). 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-CKIϵ 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 (12Oishi I. Takeuchi S. Hashimoto R. Nagabukuro A. Ueda T. Liu Z.-J. Hatta T. Akira S. Matsuda Y. Yamamura H. Otani H. Minami Y. Genes Cells. 1999; 4: 41-56Crossref PubMed Scopus (115) Google Scholar). 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 Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin), and the cell lysates were prepared by centrifugation at 12,000 × 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 (12Oishi I. Takeuchi S. Hashimoto R. Nagabukuro A. Ueda T. Liu Z.-J. Hatta T. Akira S. Matsuda Y. Yamamura H. Otani H. Minami Y. Genes Cells. 1999; 4: 41-56Crossref PubMed Scopus (115) Google Scholar). Expression and Purification of GST Fusion Proteins—The GST fusion proteins, GST-CKIϵ WT and GST-CKIϵ DK, expressed in Escherichia coli DH5α were extracted with phosphate-buffered saline containing 1% (v/v) Triton X-100, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/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 μl kinase buffer containing 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, and 40 μm ATP. The in vitro kinase reaction was initiated by the addition of purified GST-CKIϵ WT or GST-CKIϵ 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 (34Matsuda T. Nomi M. Ikeya M. Kani S. Oishi I. Terashima T. Takada S. Minami Y. Mech. Dev. 2001; 105: 153-156Crossref PubMed Scopus (119) Google Scholar). 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 CKIϵ—To identify a Ror2-interacting protein(s), we performed a yeast two-hybrid screening using the cytoplasmic region of Ror2 as bait (25Matsuda T. Suzuki H. Oishi I. Kani S. Kuroda Y. Komori T. Sasaki A. Watanabe K. Minami Y. J. Biol. Chem. 2003; 278: 29057-29064Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). From this screen we identified a protein serine/threonine kinase, CKIϵ (data not shown). To determine whether Ror2 associates with CKIϵ in mammalian cells, FLAG-tagged wild-type Ror2 (Ror2 WT) and HA-tagged CKIϵ were coexpressed in 293T cells. As shown in Fig. 1A, HA-tagged CKIϵ was coimmunoprecipitated with FLAG-tagged Ror2, indicating that Ror2 associates with CKIϵ in vivo. We also found that endogenous CKIϵ was detected in anti-Ror2 immunoprecipitates from 3T3 cells, confirming association between endogenous Ror2 and CKIϵ (Fig. 1B). Next, to identify a cytoplasmic domain(s) within Ror2 required for its association with CKIϵ, we generated a series of truncated mutants of Ror2 (Fig. 1C) and evaluated their abilities to associate with CKIϵ in 293T cells. As shown in Fig. 1D, the Ror2 mutants Ror2 ΔC and Ror2 BDB (Fig. 1C) exhibited apparently decreased levels of CKIϵ 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 (Δ883, ΔS/T2, Δpro, ΔS/T1, and ΔS/T1,2) (Fig. 1C) and tested their abilities to associate with CKIϵ. As shown in Fig. 1E, among them only the Ror2 Δpro exhibited drastically decreased levels of CKIϵ binding. The result indicates that the proline-rich domain of Ror2 is required critically for association with CKIϵ. On the other hand, the Ror2 RS and Ror2 Tc (Fig. 1C), both lacking the tyrosine kinase domain, failed to associate with CKIϵ, 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 (25Matsuda T. Suzuki H. Oishi I. Kani S. Kuroda Y. Komori T. Sasaki A. Watanabe K. Minami Y. J. Biol. Chem. 2003; 278: 29057-29064Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), we also examined whether or not association of CKIϵ and Dlxin-1 with Ror2 is competitive. It was found that association of CKIϵ with Ror2 was unaffected by ectopic coexpression of Dlxin-1 vice versa (see first supplemental figure). Serine/Threonine Phosphorylation of Ror2 by CKIϵ—We next examined whether or not Ror2 is phosphorylated on serine/threonine residues by CKIϵ 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 CKIϵ 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 CKIϵ WT but not CKIϵ DK, indicating that the kinase activity of CKIϵ is required for serine/threonine phosphorylation of Ror2. We also examined whether Dlxin-1 is phosphorylated on serine/threonine residues by CKIϵ in the presence of Ror2. It appeared that Dlxin-1 was not phosphorylated by CKIϵ (see second supplemental figure), suggesting that Ror2 may transduce signals via CKIϵ and Dlxin-1 separately. To test whether CKIϵ could phosphorylate Ror2 directly, GST-CKIϵ WT and GST-CKIϵ 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-CKIϵ WT, but not GST-CKIϵ DK, could phosphorylate serine/threonine residues within both Ror2 WT and Ror2 DK in vitro, supporting the idea that CKIϵ phosphorylates Ror2 directly. We also examined the phosphorylation status of the Ror2 ΔS/T1, ΔS/T2, ΔS/T1,2, and Δpro in mammalian cells coexpressing CKIϵ. The Ror2 ΔS/T2, ΔS/T1,2, and Δpro exhibited a complete loss of serine/threonine phosphorylation of Ror2, whereas the Ror2 ΔS/T1 showed somewhat weak, yet apparent serine/threonine phosphorylation of Ror2, compared with the Ror2 WT (Fig. 2C). Next, we attempted to identify serine/threonine phosphorylation sites within Ror2 by CKIϵ. Because serine/threonine phosphorylation of Ror2 was found in the Ror2 ΔS/T1 but not Ror2 ΔS/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 CKIϵ but failed to be phosphorylated by CKIϵ, indicating that CKIϵ phosphorylates primarily serine/threonine residues in the S/T2 domain of Ror2. Tyrosine Autophosphorylation of Ror2 Following Its Serine/Threonine Phosphorylation by CKIϵ—To better understand the biological consequence of Ror2 phosphorylation by CKIϵ, we transiently coexpressed FLAG-tagged Ror2 WT with either CKIϵ WT or CKIϵ DK in 293T cells. Tyrosine phosphorylation of Ror2 was detected when Ror2 and CKIϵ WT were coexpressed, but not when Ror2 was expressed alone or coexpressed with CKIϵ DK (Fig. 3A). The results indicate that CKIϵ kinase activity is required for tyrosine phosphorylation of Ror2. Next, FLAG-tagged Ror2 WT or Ror2 DK was expressed along with CKIϵ 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 CKIϵ to similar extents when compared with Ror2 WT (Fig. 3B). Thus, CKIϵ-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 CKIϵ. 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 (Tyr641, Tyr645, Tyr646, and Tyr722) (12Oishi I. Takeuchi S. Hashimoto R. Nagabukuro A. Ueda T. Liu Z.-J. Hatta T. Akira S. Matsuda Y. Yamamura H. Otani H. Minami Y. Genes Cells. 1999; 4: 41-56Crossref PubMed Scopus (115) Google Scholar). We also found that coexpression of CKIϵ failed to induce tyrosine phosphorylation of Ror2 ΔC (data not shown). The Ror2 ΔC lacks the C-terminal portion of Ror2, which contains six tyrosine residues, including five (Tyr818, Tyr824, Tyr830, Tyr833, and Tyr838) 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 CKIϵ, it was found that both the Ror2 4YF and 5YF could associate with CKIϵ and were phosphorylated on serine/threonine residues to a similar extent as Ror2 WT (Fig. 3D). Interestingly, when coexpressed with CKIϵ, 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 CKIϵ is required for tyrosine autophosphorylation of Ror2. For this purpose, tyrosine phosphorylation status of the 13S/TA was monitored in the presence of CKIϵ. When Ror2 WT and 13S/TA mutant were expressed in 293T cells along with CKIϵ, 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 CKIϵ is required for subsequent tyrosine autophosphorylation of Ror2. Tyrosine Phosphorylation of GRK2 by Ror2 Following Coexpression of CKIϵ—The Ror2-interacting proteins, CKIϵ and Dlxin-1 (see Fig. 5A), were not tyrosine-phosphorylated by Ror2 when coexpressed with CKIϵ (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), (34Matsuda T. Nomi M. Ikeya M. Kani S. Oishi I. Terashima T. Takada S. Minami Y. Mech. Dev. 2001; 105: 153-156Crossref PubMed Scopus (119) Google Scholar, 35Sefton M. Blanco M.J. Penela P. Mayor F. Nieto M.A. Mech. Dev. 2000; 98: 127-131Crossref PubMed Scopus (11) Google Scholar, 36Al-Shawi R. Ashton S.V. Underwood C. Simons J.P. Dev. Genes Evol. 2001; 211: 161-171Crossref PubMed Scopus (102) Google Scholar). 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 CKIϵ (data not shown).Fig. 4Ror2 associates with GRK2 in vivo.A, developmental expression patterns of Ror2 and GRK2. In situ hybridization of whole mouse embryos (embryonic day 10.5) was performed as described under "Experimental Procedures." Ror2 (left panel) and GRK2 (right panel) were expressed in a similar manner in the pharyngeal arches and developing limbs. B, association of Ror2 with GRK2 in 293T cells. FLAG-tagged Ror2 protein (WT) was expressed transiently in 293T cells with or without HA-tagged GRK2 protein, as shown in the panel. WCLs or anti-FLAG immunoprecipitates (IP) from the respective WCLs were analyzed by anti-FLAG (top panel) or anti-HA immunoblotting (middle and bottom panels).View Large Image Figure ViewerDownload (PPT) To examine whether or not GRK2 or Dlxin-1 could be tyrosine-phosphorylated by Ror2 following coexpression of CKIϵ, FLAG-tagged Ror2 and HA-tagged CKIϵ were coexpressed
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