Tyrosine 740 Phosphorylation of Discoidin Domain Receptor 2 by Src Stimulates Intramolecular Autophosphorylation and Shc Signaling Complex Formation
2005; Elsevier BV; Volume: 280; Issue: 47 Linguagem: Inglês
10.1074/jbc.m506921200
ISSN1083-351X
AutoresKyungmi Yang, Jeong Hak Kim, Hae Jong Kim, Insung Park, Ick Young Kim, Beom-Seok Yang,
Tópico(s)Cellular Mechanics and Interactions
ResumoDDR2 is a receptor tyrosine kinase whose activating ligands are various collagens. DDR2-mediated cellular signaling has been shown to require Src activity. However, the precise mechanism underlying the Src dependence of DDR2 signaling is unknown. Here, using baculoviral co-expression of the DDR2 cytosolic domain and Src, we show that Src targets three tyrosine residues (Tyr-736, Tyr-740, and Tyr-741) in the activation loop of DDR2 for phosphorylation. This phosphorylation by Src stimulates DDR2 cis-autophosphorylation of additional tyrosine residues. In vitro Shc binding assays demonstrate that phosphotyrosines resulting from DDR2 autophosphorylation are involved in Shc binding to the DDR2 cytosolic domain. Mutating tyrosine 740 of DDR2 to phenylalanine stimulates autophosphorylation of DDR2 to an extent similar to that resulting from Src phosphorylation of DDR2. In addition, the DDR2 Y740F mutant protein displays collagen-independent, constitutively activated signaling. These findings suggest that tyrosine 740 inhibits DDR2 autophosphorylation. Collectively, our findings are consistent with the following mechanism for Src-dependent DDR2 activation and signaling: 1) ligand binding promotes phosphorylation of Tyr-740 in the DDR2 activation loop by Src; 2) Tyr-740 phosphorylation stimulates intramolecular autophosphorylation of DDR2; 3) DDR2 autophosphorylation generates cytosolic domain phosphotyrosines that promote the formation of DDR2 cytosolic domain-Shc signaling complexes. DDR2 is a receptor tyrosine kinase whose activating ligands are various collagens. DDR2-mediated cellular signaling has been shown to require Src activity. However, the precise mechanism underlying the Src dependence of DDR2 signaling is unknown. Here, using baculoviral co-expression of the DDR2 cytosolic domain and Src, we show that Src targets three tyrosine residues (Tyr-736, Tyr-740, and Tyr-741) in the activation loop of DDR2 for phosphorylation. This phosphorylation by Src stimulates DDR2 cis-autophosphorylation of additional tyrosine residues. In vitro Shc binding assays demonstrate that phosphotyrosines resulting from DDR2 autophosphorylation are involved in Shc binding to the DDR2 cytosolic domain. Mutating tyrosine 740 of DDR2 to phenylalanine stimulates autophosphorylation of DDR2 to an extent similar to that resulting from Src phosphorylation of DDR2. In addition, the DDR2 Y740F mutant protein displays collagen-independent, constitutively activated signaling. These findings suggest that tyrosine 740 inhibits DDR2 autophosphorylation. Collectively, our findings are consistent with the following mechanism for Src-dependent DDR2 activation and signaling: 1) ligand binding promotes phosphorylation of Tyr-740 in the DDR2 activation loop by Src; 2) Tyr-740 phosphorylation stimulates intramolecular autophosphorylation of DDR2; 3) DDR2 autophosphorylation generates cytosolic domain phosphotyrosines that promote the formation of DDR2 cytosolic domain-Shc signaling complexes. The discoidin domain receptor (DDR) 2The abbreviations used are:DDRdiscoidin domain receptorDDR2 CKDdiscoidin domain receptor 2 cytosolic tyrosine kinase domainkd-DDR2kinase defective discoidin domain receptor 2GSTglutathione S-transferaseMMPmatrix metalloproteinaseIRTKinsulin receptor tyrosine kinaseHSChepatic stellate cellCKDcytosolic kinase domainCMVcytomegalovirusTBSTris-buffered salineγ-S-ATPγ-S-adenosine triphosphate 2The abbreviations used are:DDRdiscoidin domain receptorDDR2 CKDdiscoidin domain receptor 2 cytosolic tyrosine kinase domainkd-DDR2kinase defective discoidin domain receptor 2GSTglutathione S-transferaseMMPmatrix metalloproteinaseIRTKinsulin receptor tyrosine kinaseHSChepatic stellate cellCKDcytosolic kinase domainCMVcytomegalovirusTBSTris-buffered salineγ-S-ATPγ-S-adenosine triphosphate family, including DDR1 and DDR2, belongs to receptor tyrosine kinases (RTKs) family. Its extracellular part, containing the so-called discoidin domain, binds to various collagen proteins as their activating ligands (1Shrivastava A. Radziejewski C. Campbell E. Kovac L. McGlynn M. Ryan T.E. Davis S. Goldfarb M.P. Glass D.J. Lemke G. Yancopoulos G.D. Mol. Cell. 1997; 1: 25-34Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 2Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar, 3Vogel W. Brakebusch C. Fassler R. Alves F. Ruggiero F. Pawson T. J. Biol. Chem. 2000; 275: 5779-5784Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), and its intracellular part possesses a domain of tyrosine kinase, which shares ∼50% sequence homology with that of the Trk family of neurotrophin receptors as well as with insulin receptor (4Lai C. Lemke G. Oncogene. 1994; 9: 877-883PubMed Google Scholar, 5Laval S. Butler R. Shelling A.N. Hanby A.M. Poulsom R. Ganesan T.S. Cell Growth & Differ. 1994; 5: 1173-1183PubMed Google Scholar, 6Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J. Masiarz F. Kan Y.W. Goldfine I.D. Roth R.A. Cell. 1985; 40: 747-758Abstract Full Text PDF PubMed Scopus (957) Google Scholar). discoidin domain receptor discoidin domain receptor 2 cytosolic tyrosine kinase domain kinase defective discoidin domain receptor 2 glutathione S-transferase matrix metalloproteinase insulin receptor tyrosine kinase hepatic stellate cell cytosolic kinase domain cytomegalovirus Tris-buffered saline γ-S-adenosine triphosphate discoidin domain receptor discoidin domain receptor 2 cytosolic tyrosine kinase domain kinase defective discoidin domain receptor 2 glutathione S-transferase matrix metalloproteinase insulin receptor tyrosine kinase hepatic stellate cell cytosolic kinase domain cytomegalovirus Tris-buffered saline γ-S-adenosine triphosphate Involvement of DDR proteins in the proliferation of various cell types has been reported. Increased DDR1 expression is observed in keratinocytes of the skin and smooth muscle cells around blood vessels when the tissues are injured (7Ferri N. Carragher N.O. Raines E.W. Am. J. Pathol. 2004; 164: 1575-1585Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 8Hou G. Vogel W.F. Bendeck M.P. Circ. Res. 2002; 90: 1147-1149Crossref PubMed Scopus (124) Google Scholar). DDR1 is also expressed in monocyte-derived cells where it is believed to play a role in collagen binding and cell differentiation (9Matsuyama W. Faure M. Yoshimura T. J. Immunol. 2003; 171: 3520-3532Crossref PubMed Scopus (45) Google Scholar, 10Matsuyama W. Kamohara H. Galligan C. Faure M. Yoshimura T. FASEB J. 2003; 17: 1286-1288Crossref PubMed Scopus (43) Google Scholar). DDR2 expression is observed in mesenchymal cells and is involved in bone growth (11Islam S. Kermode T. Sultana D. Moskowitz R.W. Mukhtar H. Malemud C.J. Goldberg V.M. Haqqi T.M. Osteoarthritis Cartilage. 2001; 9: 684-693Abstract Full Text PDF PubMed Scopus (30) Google Scholar). During liver fibrosis, induction and activation of DDR2 occur in liver stellate cells, and its tyrosine kinase activity is necessary for the proliferation of stellate cells and for the increase of collagen and MMP-2 synthesis (12Olaso E. Ikeda K. Eng F.J. Xu L. Wang L.H. Lin H.C. Friedman S.L. J. Clin. Invest. 2001; 108: 1369-1378Crossref PubMed Scopus (243) Google Scholar, 13Olaso E. Labrador J.P. Wang L. Ikeda K. Eng F.J. Klein R. Lovett D.H. Lin H.C. Friedman S.L. J. Biol. Chem. 2002; 277: 3606-3613Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). In rheumatoid arthritis, DDR2 induction is also observed in activated synovial fibroblasts and is thought to stimulate the growth of these cells and MMP-1 synthesis (14Wang J. Lu H. Liu X. Deng Y. Sun T. Li F. Ji S. Nie X. Yao L. J. Autoimmun. 2002; 19: 161-168Crossref PubMed Scopus (31) Google Scholar). 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This phosphorylation causes a considerable change in the three-dimensional structure of the activation loop and stimulates RTK activity by providing ATP and peptide substrate unrestricted access to the active site pocket (17Hubbard S.R. Till J.H. Annu. Rev. Biochem. 2000; 69: 373-398Crossref PubMed Scopus (859) Google Scholar, 19Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (768) Google Scholar). Further autophosphorylation of tyrosine residues in the juxtamembrane and C-terminal region occurs by the stimulated kinase activity of these receptors (17Hubbard S.R. Till J.H. Annu. Rev. Biochem. 2000; 69: 373-398Crossref PubMed Scopus (859) Google Scholar, 21White M.F. kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 22Cann A.D. Kohanski R.A. Biochemistry. 1997; 36: 7681-7689Crossref PubMed Scopus (22) Google Scholar). Treatment of cells with the ligand collagen triggers tyrosine phosphorylation of the cytosolic domain of DDR1 and DDR2 (1Shrivastava A. Radziejewski C. Campbell E. Kovac L. McGlynn M. Ryan T.E. Davis S. Goldfarb M.P. Glass D.J. Lemke G. Yancopoulos G.D. Mol. Cell. 1997; 1: 25-34Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 2Vogel W. Gish G.D. Alves F. Pawson T. Mol. Cell. 1997; 1: 13-23Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar). Potential phosphatase activity notwithstanding, the appearance of tyrosine phosphorylation in DDR proteins upon ligand treatment requires hours. In contrast, tyrosine phosphorylation of most other RTKs occurs on the order of minutes after ligand binding. The reason for this apparent kinetic difference is unclear. In addition it has been suggested that DDR2-mediated signaling requires Src tyrosine kinase activity (23Ikeda K. Wang L.H. Torres R. Zhao H. Olaso E. Eng F.J. Labrador P. Klein R. Lovett D. Yancopoulos G.D. Friedman S.L. Lin H.C. J. Biol. Chem. 2002; 277: 19206-19212Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). In this study, we explore the precise mechanism for activation of DDR2 cytosolic signaling using in vitro analysis. Plasmid Construction—All baculoviral expression vectors were constructed using pBacPAK8 vector (Clontech). For convenient glutathione S-transferase (GST) tagging of expressed proteins, the pBacPAK8 vector was modified to contain the GST gene by subcloning a PCR-amplified cDNA fragment of GST, from pGEX4T1 plasmid having a BglII restriction site at the 5′ position and BamHI or XhoI sites at the 3′ position, into BamHI or BamHI-XhoI cutting sites in pBacPAK8 plasmid (pBacPAK 8-GST-BamHI or pBacPak 8-GST-XhoI). For the baculoviral expression vector of GST-tagged DDR2 cytosolic kinase domain (GST-DDR2 CKD), a PCR-amplified cDNA fragment covering human DDR2 from amino acids 441–815 was subcloned into the XhoI-NotI site of pBacPAK8-GST-XhoI vector. Baculoviral expression vectors of GST-tagged full-length human CDK4 (GST-CDK4), DDR1 cytosolic kinase domain (amino acids from 454–914, GST-DDR1 CKD), and full-length mouse Akt1 (GST-Akt1) were made by subcloning each PCR-amplified cDNA into a BamHI-NotI site of pBacPAK 8-GST-BamHI vector. Full-length human c-Src gene and kinase-inactive c-Src gene were PCR-amplified from the vector of pUSE human c-Src wild-type or pUSE human c-Src-negative (Upstate Biotechnology) and were subcloned into the XhoI-EcoRI site of pBacPAK 8, respectively, to obtain their baculoviral expression vectors. Mammalian expression vector bearing human full-length DDR2 under the control of the CMV promoter (CMV-DDR2) was constructed by ligating two DNA fragments of NcoI/BsrDI-digested N-terminal half and BsrDI/EcoRI-digested C-terminal half of human DDR2 cDNA amplified by PCR along with NotI/EcoRI-digested CMV-sport 6 vector DNA. The human MMP-1 promoter region from -152 to +3 was PCR-amplified from human genomic DNA prepared from HepG2 cells and subcloned into the KpnI-HindIII site of pGL2 basic vector (Promega) to generate MMP-1 promoter-driven luciferase reporter vector. The sequence of all PCR-amplified DNAs was verified by extensive sequencing. Site-directed Mutagenesis—Site-directed mutagenesis in GST-DDR2 CKD was performed by PCR. Briefly, to generate a baculoviral expression vector of GST-tagged kinase-defective DDR2 kinase domain (GST-DDR2 CKD-K608A), lysine 608 to alanine 608 mutation was introduced into a GST-DDR2 cytosolic domain by replacing NcoI-BamHI fragment within DDR2 cytosolic domain gene with a PCR-amplified fragment to contain the mutation of K608A from overlapping PCR technique using four primers of gccgtcaccatggacctg (forward primer containing an NcoI site), gcccggccctggatccgg (reverse primer containing a BamHI site), gtggctgtggcaatgctccga (forward primer containing a K608A mutation), and tcggagcattgccacagccac (reverse primer containing a K608A mutation). To introduce site-directed mutations to the three tyrosine residues (Tyr-736/Tyr-740 and Tyr-741) in the activation segment region of wild-type DDR2 tyrosine kinase domain or kinase-defective DDR2 tyrosine kinase domain, we PCR-amplified fourteen NcoI/BamHI fragments, seven from DDR2 CKD and seven from kinase-defective DDR2 CKD-K608A cDNA, using seven sets of primer pairs consisting of 5′-primer (actcagtgcctgccgtcacc) containing NcoI site and each of seven different 3′-primers containing each mutation as well as a BamHI site such as Y740F primer (cccggccctggatccggtaatagtcaccactgaacaggttc), Y736F primer (cccggccctggatccggtaaaagtcacc), Y471F primer (cccggccctggatccggaaatagtc), Y736/740F primer (cccggccctggatccggtaaaagtcaccactgaacaggttg), Y736F/Y741F primer (cccggccctggatccggaaatagtcaccactgaacaggtc), Y740F/Y741F primer (cccggccctggatccggaaaaagtcacc), and Y736F/Y740F/Y741F primer (cccggccctggatccggaaaaagtcaccactgaacaggttcc), respectively. Each of fourteen PCR fragments was subcloned into the NcoI/BamHI site in pBacPAK8-GST-DDR2 CKD. The K608A and Y740F mutations were introduced into the full-length DDR2 mammalian expression vector (CMV-DDR2) to generate CMV-DDR2-K608A and CMV-DDR2-Y740F expression vector by replacing the wild-type sequence with the cDNA fragment containing each corresponding mutation from the mutated GST-DDR2 CKD baculoviral expression vector. Cell Lines and Culture—Spodoptera frugiperda SF9 insect cells (Clontech) were maintained in TNM-FH insect medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum, 50 μg/ml gentamycin (Sigma), and 2 mm glutamine (Invitrogen) at 27 °C. HSC T6 and NIH 3T3 cells were cultured using Dulbecco's modified Eagle's medium, and SK-N-SH cells were maintained using RPMI in the presence of 10% fetal bovine serum, 150 μg/ml penicillin-streptomycin (Invitrogen), under 5% CO2 at 37 °C. HSC T6 and SK-N-SH cells were kindly provided by Dr. S. L. Friedman (Mount Sinai School of Medicine) and Dr. Y.-H. Seo (Medical College of Seoul National University, Korea) respectively. Baculoviral Expression and Purification of Proteins—Each generated baculoviral expression vector plasmid DNA was transfected into sf9 cells along with viral genomic DNA from a baculovirus generation kit purchased from Clontech, according to the manufacturer's manual. The viral stock was amplified to a titer of ∼108 plaque-forming units/ml. sf9 cells were infected with multiplicity of infection 10 and left for 48 h before harvest. GST-tagged DDR2 kinase domain proteins were purified using a glutathione-agarose bead affinity column and subsequent Superdex 200 prep grade gel filtration (Amersham Biosciences) by fast-protein liquid chromatography system. Infected sf9 cells from 400-ml cultures were suspended in 20 ml of lysis buffer consisting of 20 mm Tris-HCl (pH 8.0), 0.15 m NaCl, 1 mm dithiothreitol, 10 mm NaF, 0.1 mm EDTA, 0.1 m sodium vanadate, 0.02% IGE-PAL (Sigma), a proteinase inhibitor mixture tablet (Roche Applied Science) and lysed by sonication for 1 min. The lysate was centrifuged at 12,000 rpm for 30 min using a Sorval SS34 rotor (Beckmann), and the supernatant was applied to 1-ml bed volume of a glutathione-agarose bead column (Amersham Biosciences) pre-equilibrated with 20 mm Tris-HCl (pH 8.0), 0.15 m NaCl buffer and washed with the column equilibration buffer. The bound proteins were eluted with 10 ml of washing buffer containing 20 mm reduced glutathione and subsequently concentrated to 1 ml using Vivaspin concentrator (Vivascience, Germany). The concentrated sample was applied to a HiLoad 16/60 Superdex 200 prep grade column pre-equilibrated with a buffer of 20 mm Tris-HCl (pH 8.0) containing 0.15 m NaCl, 1 mm dithiothreitol, 10 mm NaF, and 0.1 mm EDTA and separated with the flow rate of 0.5 ml/min using a fast-protein liquid chromatography system. The eluted GST-DDR2 kinase domain protein fractions were combined and concentrated using Vivaspin concentrator. For a brief purification of GST-tagged proteins bound to glutathione beads, infected sf9 cells were lysed in 500 μl of the lysis buffer described above by sonication and centrifuged at 12,000 rpm using microcentrifuge for 10 min. 50 μl of 50% slurry of glutathione bead was added to the supernatant, and the mixture was slowly rotated for 10 min at 4 °C. Finally the GST-tagged protein-bound bead was obtained by washing with 1× TBS for three times. Autophosphorylation and Tyrosine Kinase Activity of DDR2—To measure the autophosphorylation activity of DDR2, the reaction was performed using 200 ng of DDR2 kinase domain protein in 20 μl of reaction mixture containing 20 mm Tris-HCl (pH 8.0), 5 mm MgCl2, 0.5 mm dithiothreitol, 0.01 mm ATP, and 0.2 μCi of [32P]ATP. After 15-min incubation at 30 °C, the reaction was stopped by adding a half volume of 3× Laemmli buffer with subsequent boiling for 2 min. The stopped mixture was run in 10% SDS-PAGE gel, the portion of gel containing unreacted free ATP was removed, and the remaining gel was stained using Coomassie Brilliant Blue (Sigma) and dried. The 32P radioactivity in the stained DDR2 kinase protein band was visualized by autoradiography and quantitated using a BAS 32P-image analyzer. For measuring the autophosphorylation rate (the phosphate transfer rate from ATP to DDR2 cytosolic domain), the radioactivities in the total reaction mixture and DDR2 band stained by Coomassie Brilliant Blue in the SDS-PAGE gel were measured, respectively, by scintillation counting, and the transferred mole number of phosphate was calculated from the ratio of the two radioactivity values as described previously (24Kim D.-M. Yang K. Yang B.-S. Exp. Mol. Med. 2003; 35: 421-430Crossref PubMed Scopus (13) Google Scholar). Measurement of DDR2 tyrosine kinase activity toward heterologous peptide substrates was performed using 100 ng of the purified kinase in 20 μl of reaction mixture containing 20 mm Tris-HCl (pH 8.0), 5 mm MgCl2, 0.5 mm dithiothreitol, 0.01 mm ATP, 4 μg of peptide substrate such as histone H2B (Sigma) or poly(D4Y)n (Promega) and 0.2 μCi of [32P]ATP. After 15-min incubation at 30 °C, the reaction was stopped by adding a half volume of 30% phosphoric acid. When H2B was used as a peptide substrate, the reaction mixture was spotted onto p81 paper (Millipore). For poly(D4Y)n as a substrate, reactions were spotted on avidin-coated membrane (Promega). The spotted filter was washed with 0.1 m Tris-HCl (pH 8.0) five times for 10 min each, and the radioactivity of each spot was visualized and quantitated using a BAS image analyzer (Fuji). Antibodies and Immunoblotting—Phosphotyrosine-specific antibody and antibody against human DDR2 were purchased from Cell Signaling Biotechnology and abcam (UK), respectively. Human c-Src specific antibody and monoclonal antibody against Shc were obtained from Upstate Biotechnology. Samples were boiled for 2 min in 1× Laemmli sample buffer and loaded in 10% SDS-PAGE. Proteins were transferred to Immobilon-P membrane (Millipore) and blocked by 5% skim milk in TBS for 1 h. The membrane was immunoblotted with antibodies for 2 h and subsequently washed with 1× TBS five times. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, and protein bands were detected by chemiluminescence (Amersham Biosciences). Shc Binding Assay—As a source of Shc protein, total lysate of HSC T6 cells was used. For the preparation of the lysate, 2 × 107 HSC T6 cells were harvested and lysed by sonication in 5 ml of lysis buffer consisting of 20 mm Tris-HCl (pH 8.0), 0.1 m NaCl, 5 mm dithiothreitol, 10 mm NaF, 0.1 m orthovanadate, 0.02% IGE-PAL (Sigma) and a proteinase inhibitor mixture pellet (Roche Applied Science). The supernatant was obtained by centrifugation at 15,000 rpm for 10 min at 4 °C and used for Shc binding assays as follows. Various GST-DDR2 CKD proteins bound to glutathione beads were obtained as described above. For the preparation of autophosphorylated GST-DDR2 CKD bound to beads, kinase reaction mixtures containing Tris-HCl (pH 7.5), 0.1 m NaCl, 5 mm MgCl2, 1 mm dithiothreitol, and 100 μm of ATP were added to the protein-bound glutathione beads, incubated at 30 °C for 15 min, and washed three times with cold 1× TBS. Mock autophosphorylation was carried using γ-S-ATP instead of ATP. For Shc binding, HSC T6 cell lysate was added to the protein-bound beads, and the mixture was slowly rotated for 1 h at 4 °C. Next, the beads were washed with cold 1× TBS for three times, and the bound proteins on the bead were eluted by boiling in 1× Laemmli buffer for 2 min. The eluted proteins were resolved by 10% SDS-PAGE. Western blotting using Shc-specific antibody was carried out to detect Shc protein. Transfections, MMP-1 Promoter Assay, and Enzyme-linked Immunosorbent Assay of MMP-2—1 × 105 NIH 3T3 cells were plated into 24-well dishes and left overnight. Cells were transfected with plasmid DNAs of 0.2 μg of MMP-1 promoter-Luciferase vector along with 1 μg of CMV-DDR2 (wild-type) or CMV-DDR2-Y740F or CMV-DDR2-K608A, respectively, per well using CytoPure transfection reagent (Q Biogene) according to the manufacturer's protocol. After 24 h, cells were treated with 20 μg/ml type I collagen (BD Biosciences). 12 h later, cells were harvested and luciferase activity was measured (Promega). To construct stable cell lines expressing wild-type DDR2 or DDR2-Y740F, 10 μg of each expression plasmid was transfected into 106 SK-N-SH cells in a 6-well plate along with 1 μg of hygromycin resistance gene plasmid using CytoPure transfection reagent. After 48 h, transfected cells were transferred to 10-cm diameter culture dish and subject to selection with a gradual increase of hygromycin B (Invitrogen) from 200 to 600 μg/ml until visible colonies appeared. Isolated stable cell colonies were tested for the expression of transfected DDR2 by Western blotting using the antibody specific to DDR2. For measuring MMP-2 expression, 1 × 105 cells of each clone as well as parental SK-N-SH cells were plated into both of pre-collagen coated and non-coated 24-well dishes in serum-free RPMI medium and further incubated for 36 h. The culture medium from each well was harvested and tested for the level of MMP-2 using an enzyme-linked immunosorbent assay kit purchased from R&D Systems according to the manufacturer's protocol. Src Phosphorylates the DDR2 Tyrosine Kinase Domain—When we expressed the human DDR2 tyrosine kinase domain (amino acids 441–815) as a GST fusion in sf9 cells by baculoviral infection, the expressed GST-DDR2 CKD did not undergo detectable tyrosine phosphorylation in sf9 cells as shown by Western blotting using phosphotyrosine-specific antibody. However, when GST-DDR2 CKD was co-expressed with human c-Src tyrosine kinase in sf9 cells, significant induction of GST-DDR2 CKD tyrosine phosphorylation was detected (Fig. 1A). Control GST-tagged proteins, including GST-CDK4, did not display detectable tyrosine phosphorylation in the same experiment (Fig. 1B), indicating that tyrosine phosphorylation occurred within the DDR2 tyrosine kinase domain. In contrast, co-expression of GST-DDR2 CKD with a kinase-negative Src did not induce tyrosine phosphorylation of GST-DDR2 CKD (Fig. 1A). These data indicate that Src can specifically phosphorylate the kinase domain of DDR2. Note that, in all of these experiments, similar amounts of purified GST-DDR2 CKD were used as shown by Coomassie staining, and the expression of co-transfected Src proteins in sf9 cells was confirmed by Western blotting (data not shown). Tyrosine phosphorylation by Src shows some specificity toward the DDR2 kinase domain among other tested kinases, including the human DDR1b kinase domain (amino acids 454–913), human CDK4, and murine Akt1 (Fig. 1B). When GST-tagged forms of each of these kinases were co-expressed with Src in sf9 cells, Src-dependent tyrosine phosphorylation of GST-CDK4 and GST-Akt1 was undetectable, and such phosphorylation of GST-DDR1b CKD was much weaker than Src-dependent tyrosine phosphorylation of GST-DDR2 CKD (Fig. 1B). Similar amounts of each GST-tagged kinase were used in these experiments, and the identity of each purified protein was confirmed by Western blotting with specific antibodies (data not shown). To determine whether the GST-DDR2 CKD tyrosine phosphorylation we detected upon Src co-expression was due to Src activity, GST-DDR2 CKD autophosphorylation, or both, a parallel experiment was performed with a GST-tagged kinase-defective mutant of the DDR2 kinase domain (kd-GST-DDR2 CKD). This kinase-defective mutant was generated by converting lysine 608, a conserved catalytic amino acid in the active site, to alanine. This point mutation completely abolished the tyrosine kinase activity of the purified protein (data not shown). After co-expression with Src in sf9 cells, the kd-GST-DDR2 CKD protein displayed considerably lesser tyrosine phosphorylation than GST-DDR2 CKD, but tyrosine phosphorylation of kd-GST-DDR2 CKD was not abolished (Fig. 1C). This finding suggested that initial tyrosine phosphorylation of the DDR2 tyrosine kinase domain by Src occurs, and this in turn stimulates autophosphorylation of the DDR2 tyrosine kinase domain. The purified c-Src could phosphorylate the purified kd-GST-DDR2 CKD protein in an in vitro phosphorylation reaction as well (data not shown). This is consistent with the idea that Src directly phosphorylates the DDR2 tyrosine kinase domain in our sf9 co-expression experiments. Tyrosine Phosphorylation by Src Stimulates the Autophosphorylation Activity of DDR2 and Its Tyrosine Kinase Activity toward Exogenous Substrates—To explore the functional significance of the observed DDR2 CKD phosphorylation by Src, we tested whether this modification stimulates the autophosphorylation activity of DDR2. To obtain tyrosine-phosphorylated GST-DDR2 CKD by Src (GST-DDR2 CKD-pY), sf9 cells were co-infected with baculoviruses encoding Src and GST-DDR2 CKD. In preliminary experiments, when the ratio of Src to GST-DDR2 CKD baculoviral titers was 1:3 or greater, the level of tyrosine phosphorylation in a specific amount of co-expressed GST-DDR2 CKD remained constant (data not shown). Therefore, we reasoned that saturation of GST-DDR2 CKD tyrosine phosphorylation by Src could be achieved by co-infecting the viruses in a ratio of 1:1 with a multiplicity of infection of 10. Expressed GST-DDR2 CKD was purified in to excess of 90% using glutathione-agarose affinity chromatography and a subsequent Superdex 200 fast-protein liquid chromatography. Src protein was undetectable in the purified GST-DDR2 CKD-pY, as judged by Western blotting (data not shown). Unphosphorylated GST-DDR2 CKD was purified from sf9 cells in the same manner after 2 days of baculovirus infection with only GST-DDR2 CKD. Following the purifications outlined above, the autophosphorylation activities of Src phosphorylated DDR2 (GST-DDR2 CKD-pY) and non-Src phosphorylated DDR2 (GST-DDR2 CKD) were assayed in kinase reactions containing [γ-32P]ATP. Reactions were carried out at 4 °C for 4 h or at 30 °C for 30 min. Non-Src-phosphorylated GST-DDR2 CKD displayed negligible autophosphorylation at 4 °C and weak autophosphorylation at 30 °C. In contrast, the autophosphorylation activity of Src-phosphorylated GST-DDR2 CKD-pY was increased ∼3-fold at 4 °C and 7-fold at 30 °C relative to that of GST-DDR2 CKD (Fig. 2A). These data suggest that phosphorylation by Src reduces a thermodynam
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