DOK1 Mediates SHP-2 Binding to the αVβ3 Integrin and Thereby Regulates Insulin-like Growth Factor I Signaling in Cultured Vascular Smooth Muscle Cells
2004; Elsevier BV; Volume: 280; Issue: 5 Linguagem: Inglês
10.1074/jbc.m411035200
ISSN1083-351X
AutoresLing Yan, Laura A. Maile, Jane Badley-Clarke, David R. Clemmons,
Tópico(s)Galectins and Cancer Biology
ResumoRecruitment of the Src homology 2 domain tyrosine phosphatase (SHP-2) to the phosphorylated β3 subunit of the αVβ3 integrin is required for insulin-like growth factor I (IGF-I)-stimulated cell migration and proliferation in vascular smooth muscle cells. Because SHP-2 does not bind directly to β3, we attempted to identify a linker protein that could mediate SHP-2/β3 association. DOK1 is a member of insulin receptor substrate protein family that binds β3 and contains YXXL/I motifs that are potential binding sites for SHP-2. Our results show that IGF-I induces DOK1 binding to β3 and to SHP-2. Preincubation of cells with synthetic peptides that blocked either DOK1/β3 or DOK1/SHP-2 association inhibited SHP-2 recruitment to β3. Expression of a DOK1 mutant that does not bind to β3 also disrupts SHP-2/β3 association. As a result of SHP-2/β3 disruption, IGF-I dependent phosphorylation of Akt and p44/p42 mitogen-activated protein kinase and its ability to stimulate cell migration and proliferation were significantly impaired. These results demonstrate that DOK1 mediates SHP-2/β3 association in response to IGF-I thereby mediating the effect of integrin ligand occupancy on IGF-IR-linked signaling in smooth muscle cells. Recruitment of the Src homology 2 domain tyrosine phosphatase (SHP-2) to the phosphorylated β3 subunit of the αVβ3 integrin is required for insulin-like growth factor I (IGF-I)-stimulated cell migration and proliferation in vascular smooth muscle cells. Because SHP-2 does not bind directly to β3, we attempted to identify a linker protein that could mediate SHP-2/β3 association. DOK1 is a member of insulin receptor substrate protein family that binds β3 and contains YXXL/I motifs that are potential binding sites for SHP-2. Our results show that IGF-I induces DOK1 binding to β3 and to SHP-2. Preincubation of cells with synthetic peptides that blocked either DOK1/β3 or DOK1/SHP-2 association inhibited SHP-2 recruitment to β3. Expression of a DOK1 mutant that does not bind to β3 also disrupts SHP-2/β3 association. As a result of SHP-2/β3 disruption, IGF-I dependent phosphorylation of Akt and p44/p42 mitogen-activated protein kinase and its ability to stimulate cell migration and proliferation were significantly impaired. These results demonstrate that DOK1 mediates SHP-2/β3 association in response to IGF-I thereby mediating the effect of integrin ligand occupancy on IGF-IR-linked signaling in smooth muscle cells. Vascular smooth muscle cell (SMC) 1The abbreviations used are: SMC, smooth muscle cell; IGF-I, insulin-like growth factor I; SH, Src homology; SHPS-1, SH2 domain containing protein-tyrosine phosphatase substrate-1; SHP-2, SH2 domain tyrosine phosphatase; PTB, phosphotyrosine binding; IRS, insulin receptor substrate; GAP, GTP-activating protein; DMEM, Dulbecco's modified Eagle's medium; MAPK, mitogen-activated protein kinase; HA, hemagglutinin; WT, wild type; JNK, c-Jun NH2-terminal kinase. migration and proliferation play significant roles in atherosclerotic plaque formation (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9988) Google Scholar). Insulin-like growth factor I (IGF-I) is a potent stimulant of SMC migration and proliferation responses (2Jones J.I. Prevette T. Gockerman A. Clemmons D.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2482-2487Crossref PubMed Scopus (207) Google Scholar). We have shown previously that ligand occupancy of the αVβ3 integrin is required for SMC to respond appropriately to IGF-I (3Zheng B. Clemmons D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11217-11222Crossref PubMed Scopus (125) Google Scholar). Blocking the ligand occupancy of αVβ3 inhibits IGF-I-dependent downstream signaling including phosphorylation of IRS-1 (3Zheng B. Clemmons D.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11217-11222Crossref PubMed Scopus (125) Google Scholar) and the trans-membrane, scaffolding protein Src homology 2 domain containing protein-tyrosine phosphatase substrate-1 (SHPS-1), as well as cell migration and proliferation (4Maile L.A. Clemmons D.R. Endocrinology. 2002; 143: 4259-4264Crossref PubMed Scopus (56) Google Scholar). One important event that occurs in response to ligand occupancy of αVβ3 is the phosphorylation of the β3 subunit, and previous studies have shown that this is required for IGF-I-dependent signaling and biologic actions (5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar). SMCs expressing a mutant form of β3 in which the two tyrosines in the cytoplasmic domain of β3 were substituted with phenylalanines did not respond to IGF-I with an increase in DNA synthesis (5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar). Therefore in SMC phosphorylation of the β3 subunit of αVβ3 integrin plays a key role in regulating IGF-I dependent cellular responses. Our prior studies have shown that ligand occupancy of αVβ3 regulates IGF-IR signaling by regulating the transfer of the protein-tyrosine phosphatase SHP-2 (4Maile L.A. Clemmons D.R. Endocrinology. 2002; 143: 4259-4264Crossref PubMed Scopus (56) Google Scholar, 5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar). In high density cultures, the β3 subunit is constitutively tyrosine phosphorylated and Src homology 2 domain tyrosine phosphatase (SHP-2) can be co-immunoprecipitated with phosphorylated β3 (5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar). This association correlates with membrane localization of SHP-2 and is required for the subsequent transfer of SHP-2 to its membrane substrate protein SHPS-1 following IGF-I stimulation (5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar). The disruption of SHP-2 and β3 association results in the elimination of SHP-2 transfer to SHPS-1 (5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar). As a result, IGF-I-dependent cell migration and DNA synthesis are both decreased (5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar, 6Maile L.A. Badley-Clarke J. Clemmons D.R. J. Cell Sci. 2001; 114: 1417-1425Crossref PubMed Google Scholar). These results suggest that the association of SHP-2 and the β3 subunit is a prerequisite for proper SHP-2 transfer and that this is required for IGF-I-stimulated biologic actions. We have previously shown that the addition of IGF-I to subconfluent cultures induces an increase in β3 phosphorylation and a corresponding increase of SHP-2 association (5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar). The incubation of SMC cultures with a Src-family kinase inhibitor PP2 inhibits β3 phosphorylation and blocks SHP-2 association with β3 (5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar), suggesting a phosphorylation-dependent association between SHP-2 and the β3 subunit. The β3 cytoplasmic domain contains one NPXY motif. This motif has been shown to interact with proteins containing phosphotyrosine binding (PTB) domains (7Songyang Z. Margolis B. Chaudhuri M. Shoelson S.E. Cantley L.C. J. Biol. Chem. 1995; 270: 14863-14866Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). SHP-2 does not contain a PTB domain, but it has two SH2 domains, which have been shown to mediate binding to phosphorylated tyrosine residues that are followed by a specific motif YXXL/I (8Case R.D. Piccione E. Wolf G. Benett A.M. Lechleider R.J. Neel B.G. Shoelson S.E. J. Biol. Chem. 1994; 269: 10467-10474Abstract Full Text PDF PubMed Google Scholar). Therefore it is likely that a linker protein containing both PTB domain and YXXL/I motif(s) modulates SHP-2 binding to β3. The adaptor proteins insulin receptor substrates 1 and 2 (IRS-1, IRS-2) contain both motifs, and IRS-1 has been shown to bind SHP-2 upon insulin receptor activation (9Myers Jr., M.G. Mendez R. Shi P. Pierce J.H. Rhoads R. White M.F. J. Biol. Chem. 1998; 273: 26908-26914Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). In addition, IRS-1 can be co-immunoprecipitated with β3 in response to insulin in rat fibroblasts that overexpress insulin receptors (10Vuori K. Ruoslahti E. Science. 1994; 266: 1576-1578Crossref PubMed Scopus (339) Google Scholar). However, although both IRS-1 and IRS-2 are expressed in primary SMC cultures, we could not detect co-immunoprecipitation of IRS-1 or IRS-2 with β3, excluding them as linker proteins for SHP-2 and β3 association. DOK1 is a member of the IRS family of proteins that contains a PH domain followed by a PTB domain in its N terminus. It has multiple tyrosine residues in its C-terminal sequence that undergo phosphorylation upon tyrosine kinase activation (11Guo D. Jia Q. Song H.Y. Warren R.S. Donner D.B. J. Biol. Chem. 1995; 270: 6729-6733Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 12Sanchez-Margalet V. Zoratti R. Sung C.K. Endocrinology. 1995; 136: 316-321Crossref PubMed Scopus (21) Google Scholar). DOK1 has been shown to function as a scaffolding protein that recruits key signaling molecules such as, the Ras-GTP-activating protein (Ras-GAP) and the adaptor protein Nck (13Ellis C. Liu X.Q. Anderson D. Abraham N. Veillette A. Pawson T. Oncogene. 1991; 6: 895-901PubMed Google Scholar, 14Murakami H. Yamamura Y. Shimono Y. Kawai K. Kurokawa K. Takahashi M. J. Biol. Chem. 2002; 277: 32781-32790Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) following its tyrosine phosphorylation. These associations have suggested that DOK1 plays a role in regulating cell functions such as migration, proliferation, and transformation (14Murakami H. Yamamura Y. Shimono Y. Kawai K. Kurokawa K. Takahashi M. J. Biol. Chem. 2002; 277: 32781-32790Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15Songyang Z. Yamanashi Y. Liu D. Baltimore D. J. Biol. Chem. 2001; 276: 2459-2465Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 16Lee S. Roy F. Galmarini C.M. Accardi R. Michelon J. Viller A. Cros E. Dumontet C. Sylla B.S. Oncogene. 2004; 23: 2287-2297Crossref PubMed Scopus (24) Google Scholar). It is not known whether SHP-2 binds DOK1; however, tyrosines 203 and 337 of DOK1 reside in YXXL motifs and therefore have the potential to bind to the SH2 domains of SHP-2. The PTB domain of DOK1 has been shown to be necessary for its regulatory role in cell transformation (15Songyang Z. Yamanashi Y. Liu D. Baltimore D. J. Biol. Chem. 2001; 276: 2459-2465Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Recently, DOK1 has been shown to bind to the NPXY motif of β3 via its PTB domain (17Calderwood D.A. Fujioka Y. de Pereda J.M. Garcia-Alvarez B. Nakamoto T. Margolis B. McGlade C.J. Liddington R.C. Ginsberg M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2272-2277Crossref PubMed Scopus (339) Google Scholar). However, the functional significance of the DOK1-β3 interaction has yet to be determined. Because of these properties DOK-1 seemed a likely candidate for mediating phosphotyrosine-dependent binding of SHP-2 to β3. In the current studies, we determined whether DOK1 mediated the association of SHP-2 with β3 and analyzed the functional consequences of disrupting this association on SHP-2 transfer to downstream signaling molecules. In addition, we further determined whether disruption of this interaction was associated with a change in IGF-IR-linked signaling and biologic actions. Human IGF-I was a gift from Genentech (South San Francisco, CA). Immobilon-P membranes were purchased from Millipore Corp. (Bedford, MA). DMEM containing 4500 mg of glucose/liter (DMEM-H) was purchased from Invitrogen. Streptomycin and penicillin were purchased from Invitrogen. A polyclonal antibody for the β3-subunit of porcine αVβ3 integrin was generated using two synthetic peptides containing the amino acid sequences encompassing positions 36–63 and 623–648. Polyclonal antibodies for SHPS-1, SHP-2, and the hemagglutinin epitope (HA) were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-phosphotyrosine (Tyr(P)) and anti-DOK1 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phospho-p44/p42MAPK, p44/p42MAPK, phospho-Akt, and Akt were from BD Transduction Laboratories (Lexington, KY). A synthetic peptide was prepared that contained the TAT sequence that confers cell permeability (18Ho A. Schwarze S.R. Mermelstein S.J. Waksman G. Dowdy S.F. Cancer Res. 2001; 61: 474-477PubMed Google Scholar) followed by 11 residues of the DOK1 sequence (underlined) YARAAARQARA201WPYTLLRRYGRD211. This DOK-1 sequence contains the known site that mediates binding to β3 (17Calderwood D.A. Fujioka Y. de Pereda J.M. Garcia-Alvarez B. Nakamoto T. Margolis B. McGlade C.J. Liddington R.C. Ginsberg M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2272-2277Crossref PubMed Scopus (339) Google Scholar). A second peptide that contained the TAT sequence followed by a SH2 domain recognition sequence within DOK-1 was also prepared, YARAAARQARA334KPLYWDLYE342. These two peptides are referred to hereafter as DOK1-β3 and DOK1-SHP-2 blocking peptides, respectively. The peptides were synthesized by the Protein Chemistry Core Facility at the University of North Carolina at Chapel Hill. Purity and sequence confirmation were determined by mass spectrometry. Cell Culture—pSMCs were prepared from porcine aortas as described previously (19Parker A. Gockerman A. Busby W.H. Clemmons D.R. Endocrinology. 1995; 136: 2470-2476Crossref PubMed Google Scholar). The cells were maintained in DMEM-H with 10% fetal bovine serum (Hyclone, Logan, UT) streptomycin (100 ng/ml), and penicillin (100 units/ml). The smooth muscle cells that were used in these experiments were used between passages 4–16. Generation of pLenti Expression Vectors—The full-length human DOK1 cDNA was generated by reverse transcription-PCR from mRNA that had been derived from human fibroblasts (GM10 Coriell Inst., Camden, NJ). The full-length DOK1 sequence was PCR-amplified and cloned into pcDNA-3.1 vector to generate pcDNA-DOK1 wild type (WT). The forward and reverse primers that were used to generate the PCR product were 5′-CACCATGTACCCATACGATGTTCCAGATTACGCTGACGGAGCAGTGATGGAAGGGCCGCT-3′ and 5′-TCAGGTAGATCCCTCTGACTTGACCCCA-3′. The wild type DOK1 construct contains a hemagglutinin sequence at the 5′-end of the coding sequence (underlined). Arg207 and Arg208 were mutated to alanines to generate what is referred to hereafter as the DOK1-AA mutant. The mutant construct was generated by first synthesizing two DNA fragments that contained the mutations using pcDNA-DOK1WT as a template. The primers used to generate the first fragment were the forward primer from above plus 5′-CCTTGTCCCGGCCATAGGCAGCCAACAGAGTGTAGGGCC-5′. The primers used to generate the second fragment were 5′-GGCCCTACACTCTGTTGGCTGCCTATGGCCGGGACAAGG-3′ plus the reverse primer from above. The two fragments were designed to overlap across the region of the mutation (bold letters). They were annealed and subsequently extended by Taq polymerase (Clontech) to generate a full-length DOK1 sequence containing the alanine substitutions. The final PCR products containing a Kozac sequence (CACC) followed by a sequence encoding the HA epitope at the 5′-end of the DOK1 coding sequence were cloned into the pLenti6/V5-D-TOPO expression vector (Invitrogen). The complete sequence was verified by DNA sequencing. Generation of Virus Stocks—293FT cells (Invitrogen) were prepared for generation of virus stocks of each individual pLenti construct. Cells were plated at 5 × 106/75 cm2 flask (Corning Inc., Corning, NY) the day before transfection in the growth medium (DMEM-H with 10% FBS with streptomycin at 100 ng/ml and penicillin at 100 units/ml). On the day of transfection, the culture medium was replaced with 5 ml of Opti-MEM I (Invitrogen) without antibiotics or serum. DNA-Lipofectamine™ 2000 complexes for each transfection sample were prepared and added along with total 8 ml of Opti-MEM I medium according to the manufacturer's protocol (Invitrogen). The next day the medium containing the DNA-Lipofectamine™ 2000 complexes was removed and replaced with 12 ml of growth medium. The virus-containing supernatants were harvested at 48-h post-transfection, filtered through a 0.2-μm filter, and stored as 1-ml aliquots at –80 °C. Establishment of SMCs Expressing pLenti Constructs—pSMCs (passage 4–5) were seeded at 3 × 105/well in 6-well plates (Falcon, catalog number 353046) the day before transduction. The viral stocks were thawed, and the viral complexes precipitated as follows. For each 1 ml of virus stock, 1 μl of an 80 mg/ml solution of chondroitin sulfate (Sigma, C4384) was added, then mixed gently, and incubated at 37 °C for 10 min. 1 μl of 80 mg/ml Polybrene (Sigma, H9286) was subsequently added and incubated at 37 °C for 10 min. The mixture was centrifuged at 10,000 rpm for 5 min to pellet the virus, and the supernatant was removed. For transduction, the pellet was resuspended in 1 ml of growth medium and 1 μl of Polybrene (40 mg/ml) was added, and then the mixture was incubated with the cells for 24 h. The virus-containing medium was removed and changed to 2 ml of growth medium for another 24 h, then replaced with selection medium (growth medium containing 4 μg/ml blasticidin, Invitrogen). The cultures were grown until they reached confluent density. The expression of the HA-tagged DOK1 proteins was detected by immunoprecipitation and immunoblotting with an anti-HA antibody (1: 1000) followed by an horseradish peroxidase-conjugated anti-rabbit secondary antibody. Immunoprecipitation and Immunoblotting—Cells were seeded at 5 × 105 cells/10-cm plate (BD Biosciences) and grown for 7 days to reach confluency. Subconfluent cultures were used 3 days after plating. The cultures were incubated in serum-free DMEM-H for 12–16 h prior to the addition of IGF-I (100 ng/ml). For the experiments in which the cell-permeable peptides were added, 10 μg/ml of each peptide was added directly to the serum-free media for 1 h prior to adding IGF-I. The cell monolayers were lysed in a modified radioimmune precipitation assay buffer (1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm EGTA, 150 mm NaCl, and 50 mm Tris-HCl (pH 7.5)) in the presence of protease inhibitors (10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml pepstatin) and phosphatase inhibitors (25 mm sodium fluoride and 2 mm sodium orthovanadate). The cell lysates were centrifuged at 14,000 × g for 10 min at 4 °C. The supernatant was exposed to a 1:330 dilution of anti-DOK1, anti-β3, or anti-SHPS-1 antibody overnight at 4 °C. The immunoprecipitates were immobilized using protein A-Sepharose beads for 2 h at 4°C and washed three times with the same buffer. The precipitated proteins were eluted in 40 μl of 2× Laemmli sample buffer, boiled for 5 min, and separated with a 7.5 or 8% SDS-PAGE. The proteins were then transferred to Immobilon-P membranes that were blocked for 1 h in 1% bovine serum albumin in Tris-saline buffer with 0.2% Tween 20. The blots were incubated overnight at 4 °C with the indicated antibodies (1:500 for Tyr(P) and SHP-2 or 1:1000 for antibodies against β3-subunit or DOK1). To detect the phosphorylation of Akt and p44/p42MAPK, 30 μl of cell lysate was removed prior to immunoprecipitation and mixed with 25 μlof2× Laemmli sample buffer then separated by SDS-PAGE using an 8% gel. Anti-phospho-p44/p42MAPK (1:1000) and anti-phospho-Akt (1:1000) were used to detect activated MAPK and Akt. Total p44/p42MAPK and Akt protein were detected using a monoclonal anti-Erk antibody (1:1000) or anti-Akt (1:1000). The proteins were detected using enhanced chemiluminescence (Pierce Chemical Co.), and their abundance was analyzed using the GeneGnome CCD image system (Syngene, Ltd., Cambridge, UK). The images obtained were also scanned using an Agfa Scanner. Densitometric analyses of the images were undertaken using NIH Image, version 1.61. All experiments were conducted at least three times. Cell Migration Assay—pSMCs were seeded in 6-well dishes and grown to confluency. A razor blade was used to scrape an area of cells, leaving a denuded area and a sharp visible wound line. The wounded monolayers were then incubated with 0.2% fetal bovine serum-DMEM-H in the presence or absence of the DOK1-β3, DOK1-SHP2 peptide (10 μg/ml) with or without 100 ng/ml IGF-I for 48 h at 37 °C. The cells were then fixed and stained (Diff Quick, Dade Behring, Newark, DE), and the number of cells migrating into the wound area was counted. Eight of the previously selected 1-mm areas at the edge of the wound were counted for each data point. Each experiment was repeated three times and the results are the means ± S.E. of eight determinations in each of the three separate experiments. Cell Proliferation Assay—Assessment of SMC proliferation was performed as described previously (20Nam T.J. Busby Jr., W.H. Rees C. Clemmons D.R. Endocrinology. 2000; 141: 1100-1106Crossref PubMed Scopus (94) Google Scholar). Cells were incubated in the presence or absence of the DOK1-β3, DOK1-SHP2 peptide (10 μg/ml) with or without IGF-I (50 ng/ml) for 48 h, and the cell number in each well was counted. Each treatment was analyzed in triplicate, and the results represent mean values of six independent experiments. Statistical Analysis—Student's t test was used to compare the differences between control and treatment groups or control cells and cells expressing mutant proteins. p ≤ 0.05 was considered statistically significant. DOK1 Is Associated with Phosphorylated β3 Subunit in pSMCs—Consistent with our previous finding (5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar), tyrosine phosphorylation of the β3 subunit was detected at high level in the basal state in confluent cultures, and IGF-I stimulation decreased the level of phosphorylated β3 (89.8 ± 5% reduction, mean ± S.E., n = 3, p < 0.01). In contrast, in subconfluent cultures, there was a low level of β3 phosphorylation basally, and IGF-I stimulated an increase in β3 phosphorylation (4.22 ± 0.13-fold increase compared with basal level, mean ± S.E., n = 3, p < 0.01) (Fig. 1). When DOK1 and β3 association was evaluated, the amount of β3 that associated with DOK1 correlated with levels of β3 phosphorylation. IGF-I decreased the amount of DOK1 associated with β3to14 ± 5% of the basal level in high density cultures and stimulated a 3.00 ± 1.06-fold increase in subconfluent cultures (mean ± S.E., n = 3, p < 0.01 in both cases). These results suggested the association between DOK1 and the β3 subunit was phosphorylation-dependent, and they are consistent with previous studies showing a direct association between DOK1 and the β3 subunit (17Calderwood D.A. Fujioka Y. de Pereda J.M. Garcia-Alvarez B. Nakamoto T. Margolis B. McGlade C.J. Liddington R.C. Ginsberg M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2272-2277Crossref PubMed Scopus (339) Google Scholar). In contrast, we could not detect an association between β3 and IRS-1 or IRS-2 or between β3 and Grb-2-associated binder 2 (Gab2). Inhibition of DOK1-β3 Association Blocks SHP-2 Association with the β3 Subunit—To test the hypothesis that DOK1 may mediate SHP-2 association with the β3 subunit, we incubated subconfluent SMCs with a synthetic peptide that contained the region of sequence that had been shown to mediate DOK1-β3 association prior to IGF-I stimulation (17Calderwood D.A. Fujioka Y. de Pereda J.M. Garcia-Alvarez B. Nakamoto T. Margolis B. McGlade C.J. Liddington R.C. Ginsberg M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2272-2277Crossref PubMed Scopus (339) Google Scholar). It would be predicted that because this peptide is added in excess, it would bind phosphorylated β3 and prevent the binding of DOK1 to β3. In control cultures, IGF-I induced DOK1 binding to β3 after 5 min (Fig. 2A, first panel) and at the same time point, there was a corresponding increase of SHP-2 association with β3 (Fig. 2A, third panel). Exposure to the peptide abolished the IGF-I-induced increase in DOK1 binding to β3, and it markedly inhibited SHP-2 association with β3. However, there was no significant impairment of IGF-I-induced β3 phosphorylation. Quantitative analysis of the tyrosine phosphorylation of β3 showed that IGF-I induced a 4.36 ± 0.84-fold increase in control cultures and a 4.08 ± 1.59-fold increase in β3 phosphorylation in the presence of the blocking peptide (mean ± S.E., n = 3, p = 0.86). These results suggested that DOK1 might be mediating SHP-2 association with β3. To confirm this hypothesis, we generated SMCs expressing a DOK1 mutant that had had arginines 207 and 208 substituted with alanines (DOK1-AA). The expression of a mutant containing these substitutions blocked phosphotyrosine-mediated binding of DOK1 and altered the function of DOK1 in NIH-3T3 cells (15Songyang Z. Yamanashi Y. Liu D. Baltimore D. J. Biol. Chem. 2001; 276: 2459-2465Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Fig. 2B shows that DOK1WT and DOK1-AA were expressed in SMCs at similar levels. In cells expressing wild type DOK1, IGF-I increased the binding of DOK1 to β3, and this was associated with a corresponding increase in the association of SHP-2 with β3. However, in cells expressing the DOK1-AA mutant, the ability of IGF-I to stimulate an increase in DOK1 binding to β3 was abolished, and the association between β3 and SHP-2 was decreased (Fig. 2C). These results provided in vivo evidence that DOK1 binds to β3 via its PTB domain and this binding mediates the recruitment of SHP-2 to the β3 subunit. Disruption of DOK1 and SHP-2 Association Inhibits SHP-2 Binding to β3—Because we showed that DOK1 is required for SHP-2 binding to β3 and DOK1 contains the YXXL motifs that are potential binding sites for SHP-2, we hypothesized that DOK1 would bind to SHP-2 through this domain and that this interaction was required for SHP-2 binding to β3. Therefore we determined whether 1) DOK1 bound SHP-2 through its YXXL motifs and 2) whether disrupting the binding altered the interaction between β3 and SHP-2. Fig. 3A illustrates the regions of DOK1 that contain the PH domain at the N terminus followed by the PTB domain. Multiple tyrosine residues, including Tyr203 and Tyr337, that are located within YXXL motifs are also shown. In the basal state, there is low level of SHP-2 association with DOK1, and the level is significantly enhanced after IGF-I stimulation for 5 min (2.73 ± 0.28-fold increase compared with basal state, p < 0.05, Fig. 3, B and C). Pretreatment of cultures with the synthetic peptide that contains a DOK-1 SH2 recognition sequence (i.e. Y337WDL) abolished the association between SHP-2 and DOK1 (Fig. 3D, first panel). In contrast to the disrupting peptide used in Fig. 2A this peptide did not block DOK1 and β3 subunit association (Fig. 3D, second panel). β3 and SHP-2 association was also inhibited following peptide exposure (Fig. 3D, fourth panel). These results further support the conclusion that SHP-2 binding to β3 is mediated by DOK1. Inhibiting SHP-2-β3 Association Impairs SHP-2 Recruitment to SHPS-1—Our previous studies have shown that inhibiting of SHP-2 and β3 association by inhibiting β3 phosphorylation leads to impaired SHP-2 transfer to SHPS-1 upon IGF-I stimulation (5Ling Y. Maile L.A. Clemmons D.R. Mol. Endocrinol. 2003; 17: 1824-1833Crossref PubMed Scopus (55) Google Scholar). Therefore we analyzed SHP-2 recruitment to SHPS-1 in the presence of either the DOK1-β3 or the DOK1-SHP2 blocking peptide. In control cultures, IGF-I induced SHP-2 association with SHPS-1 after 5 min. However, this association was abolished following exposure to either the DOK1-β3 or the DOK1-SHP-2 blocking peptide (Fig. 4, A and B). Compared with SMCs expressing DOK1-WT, in which IGF-I induces a significant increase in SHP-2 binding to SHPS-1, expression of the DOK1-AA mutant also inhibited the increase in the amount of SHP-2 that is transferred to SHPS-1 after IGF-I stimulation (Fig. 4, C and D). These results suggested that the degree of DOK1-AA expression is sufficient to exert a dominant negative effect on SHP-2 transfer to SHPS-1 following IGF-I stimulation. IGF-I-mediated Phosphorylation of Akt and p44/p42MAPK Is Decreased in the Presence of DOK1-β3 and DOK1-SHP2 Blocking Peptide or in Cells Expressing the DOK1-AA Mutant— Impaired recruitment of SHP-2 to SHPS-1 or expression of a SHP-2 mutant with attenuated phosphatase activity have been linked to deficient MAPK and phosphatidylinositol 3-kinase activation in response to growth factor stimulation including insulin and IGF-I (21Ivins Zito C. Kontaridis M.I. Fornaro M. Feng G.S. Bennett A.M. J. Cell. Physiol. 2004; 199: 227-236Crossref PubMed Scopus (84) Google Scholar, 22Takada T. Matozaki T. Takeda H. Fukunaga K. Noguchi T. Fujioka Y. Okazaki I. Tsuda M. Yamao T. Ochi F. Kasuga M. J. Biol. Chem. 1998; 273: 9234-9242Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 23Maile L.A. Badley-Clarke J. Clemmons D.R. Mol. Biol. Cell. 2003; 14: 3519-3528Crossref PubMed Google Scholar). We therefore analyzed IGF-I-induced phosphorylation of Akt and p44/p42MAPK in control cultures and in SMCs that had been exposed to either the DOK1-β3 blocking peptide or the DOK1-SHP2 blocking peptide prior to IGF-I addition. Fig. 5 shows that IGF-I induced a significant increase of phosphorylation of Akt and p44/p42MAPK after 5 and 10 min in control cultures. In the presence of the DOK1-β3 blocking peptide, however, the IGF-I-induced respo
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