Identification of Vascular Endothelial Growth Factor Receptor-1 Tyrosine Phosphorylation Sites and Binding of SH2 Domain-containing Molecules
1998; Elsevier BV; Volume: 273; Issue: 36 Linguagem: Inglês
10.1074/jbc.273.36.23410
ISSN1083-351X
AutoresNobuyuki Ito, Christer Wernstedt, Ulla Engström, Lena Claesson‐Welsh,
Tópico(s)Cell Adhesion Molecules Research
ResumoReceptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitroprotein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-γ binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-γ, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1. Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitroprotein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-γ binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-γ, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1. Src homology 2 phospholipase C-γ vascular endothelial growth factor vascular endothelial growth factor receptor fms-like tyrosine kinase porcine aortic endothelial cells polyacrylamide gel electrophoresis SH2-containing protein tyrosine phosphatase-2 intracellular epidermal growth factor receptor fibroblast growth factor receptor platelet-derived growth factor receptor. Receptor tyrosine kinases comprise a large family of transmembrane receptors for polypeptide growth factors (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar). Binding of the growth factor to its specific receptor triggers activation of the intrinsic receptor tyrosine kinase activity. It further provokes autophosphorylation of the receptors and tyrosine phosphorylation of various intracellular signaling molecules leading to signal transduction to downstream effector molecules (2Anderson D. Koch C.A. Grey L. Ellis C. Moran M.F. Pawson T. Science. 1990; 250: 979-982Crossref PubMed Scopus (432) Google Scholar). Phosphorylation of specific tyrosine residues in the receptors provides high affinity binding sites for a variety of Src homology 2 (SH2)1 domain-containing proteins (3Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2187) Google Scholar, 4Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T Science. 1991; 252: 668-674Crossref PubMed Scopus (1444) Google Scholar). The binding of a particular SH2 domain to tyrosine-phosphorylated proteins is dependent on the primary sequence surrounding the phosphotyrosine. Certain SH2 domain-containing proteins such as phospholipase C-γ (PLC-γ), phosphatidylinositol 3-kinase, and GTPase-activating protein possess enzymatic activities, whereas other SH2 domain molecules, i.e. adaptors like Grb2, Crk, and Nck, lack intrinsic enzymatic activities. Adaptors are believed to transduce signals by mediating protein-protein interactions with other signaling molecules such as the guanine nucleotide exchanging factor, Sos (5Birge R.B. Knudsen B.S. Besser D. Hanafusa H. Genes Cells. 1996; 1: 595-613Crossref PubMed Scopus (122) Google Scholar). Several SH2 domain-containing proteins may converge on the same signal transduction pathway; Grb2, Crk, and Nck has been shown to be involved in Ras activation through binding to the same target Sos. On the other hand, however, it has also been shown that these SH2 domain-containing molecules bind to a variety of other intracellular proteins and seem to be involved in multiple signaling pathways (5Birge R.B. Knudsen B.S. Besser D. Hanafusa H. Genes Cells. 1996; 1: 595-613Crossref PubMed Scopus (122) Google Scholar). Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that promotes endothelial cell proliferation and chemotaxis (6Ferrara N. Henzel W.J. Biochem. Biophys. Res. 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EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar), VEGF-D (also known as a fibroblast-stimulating growth factor) (20Lee J. Gray A. Yuan J. Luoh S.-M. Avraham H. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1988-1992Crossref PubMed Scopus (330) Google Scholar), and placenta growth factor (21Maglione D. Guerriero V. Viglietto G. Ferraro M.G. Aprelikova O. Alitalo K. Vecchio S.D. Lei K.-J. Chou J.Y. Persico M.G. Oncogene. 1993; 8: 925-931PubMed Google Scholar). Although these proteins share about 30–53% homology in their primary sequences, they show distinct patterns of binding to the three known VEGF receptors; VEGF-B and -C bind to KDR/Flk-1, whereas placenta growth factor binds to Flt-1 with lower affinity than VEGF (22Park J.E. Chen H.H. Winer J. Houck K.A. Ferrara N. J. Biol. Chem. 1994; 269: 25646-25654Abstract Full Text PDF PubMed Google Scholar). In addition, VEGF-C binds with high affinity to Flt-4 (VEGFR-3) which is expressed on lymphatic endothelium (19Joukov V. Pajusola K. Kaipainen A. Chilov D. Lahtinen I. Kukk E. Saksela O. Kalkkinen N. Alitalo K. EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1160) Google Scholar). VEGF receptor expression is seen in various tissues of adult rats, and relatively high expression has been identified during embryogenesis, indicating their very important roles for embryonal development (13Peters K.G. de Vries C. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8915-8919Crossref PubMed Scopus (415) Google Scholar). Gene-targeting studies show that both of Flt-1 and KDR/Flk-1 knock-out mice die in utero by embryonic day 9.5 (23Fong G.-H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2224) Google Scholar, 24Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.-F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3371) Google Scholar). Analysis of these knock-out mice revealed an absence of yolk sac-derived blood islands and hematopoietic progenitor cells in KDR/Flk-1 null mice and disorganization of vessels in Flt-1 null mice. These data suggest that the receptors have different biological functions and indicate that Flt-1 and KDR/Flk-1 utilize different signal transduction pathways. By stimulating KDR/Flk-1-transfected PAE cells and NIH3T3 fibroblasts with VEGF, KDR/Flk-1 has been shown to autophosphorylate (8Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.- H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 25Takahashi T. Shibuya M. Oncogene. 1997; 14: 2079-2089Crossref PubMed Scopus (273) Google Scholar), and four in vitro tyrosine phosphorylation sites, Tyr-951, Tyr-996, Tyr-1054, and Tyr-1059, have been identified by bacterially expressing the cytosolic domain of KDR/Flk-1 (26Dougher-Vermazen M. Hulmes J.D. Böhlen P. Terman B.I. Biochem. Biophys. Res. Commun. 1994; 205: 728-738Crossref PubMed Scopus (108) Google Scholar). However, very little is known about signal transduction molecules involved in Flt-1 signaling. In this report, we aimed to identify the tyrosine phosphorylation sites in Flt-1 by expressing the intracellular (IC) domain of human Flt-1 in the baculosystem and to identify signal transduction molecules binding to these sites. Sf9 insect cells (PharMingen) were maintained in Grace's insect medium (Sigma) supplemented with lactalbumin hydrolysate, yeastolate, and 10% fetal calf serum at 27 °C. An endothelial cell line (MS1; kind gift of J. Arbiser, Department of Surgery, Children's Hospital, Boston) (27Arbiser J.L. Moses M.A. Fernandez C.A. Ghiso N. Cao Y. Klauber N. Frank D. Brownlee M. Flynn E. Parangi S. Byers H.R. Folkman J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 861-866Crossref PubMed Scopus (422) Google Scholar) derived from mouse pancreas and immortalized through expression of a temperature-sensitive simian virus large T oncogene was maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum under the atmosphere of 5% CO2 at 37 °C. APflMI-XbaI fragment encoding the intracellular domain (IC) of the human flt-1 cDNA (nucleotides 4961–6524) was subcloned into a baculovirus transfer vector (pVL1393; PharMingen) at a BamHI-XbaI site by use of aBamHI (5′) and PflMI (3′) double-stranded adaptor containing a starting codon downstream of the BamHI site (5′-GATCCATGCATCACCATCACCATCACGCCAGCAA-3′). This IC region contains 20 of 22 tyrosine residues in the Flt-1 IC; Tyr-794 and Tyr-815 in the juxta-membrane region are not included in this construct. The transfer vector containing h-flt-1 IC cDNA was transfected into Sf9 cells using a BaculoGoldTM transfection kit (PharMingen). The conditioned medium containing recombinant baculovirus carrying the h-flt-1 IC cDNA was used for amplification to obtain high titer virus solution. For substitution of tyrosine residues to phenylalanine, site-directed mutagenesis was carried out on a fragment cDNA of h-flt-1 using the Altered Sitesin vitro Mutagenesis System (Promega Corp.). The following oligonucleotides were used for mutagenesis: 5′-CTGCAAATTTGGAAATC-3′ (Y914F); 5′-TGTCAGATTTGTAAATGC-3′ (Y1213F); 5′-TGATGACTTCCAGGGCG-3′ (Y1242F); 5′-CCCAGACTTCAACTCGGT-3′ (Y1327F); and 5′-TGGTCCTGTTCTCCACC-3′ (Y1333F). All mutations were confirmed by DNA sequencing. The mutated flt-1 receptors were subcloned into pVL1393 and transfected into Sf9 cells to yield recombinant viruses carrying mutant receptors. The rabbit anti-Flt-1 antibody, raised against a peptide corresponding to amino acids 1312–1328 in the C terminus of human Flt-1, anti-SHP-2, anti-Grb2, and anti-Nck antibodies were purchased from Santa Cruz Biotechnology Inc. The monoclonal anti-phosphotyrosine antibody (PY20), anti-Crk, and anti-p85 antibodies were from Transduction Laboratories. The rabbit anti-PLC-γ antiserum was raised against human PLC-γ and was kindly provided by Dr. Lars Rönnstrand, the Ludwig Institute for Cancer Research, Uppsala, Sweden. Peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse immunoglobulins were obtained from Amersham Pharmacia Biotech. Cell lysate was incubated with specific antibodies for 1 h at 4 °C and further incubated with protein A-Sepharose CL-4B for 30 min at 4 °C. After washing the beads, the immunocomplex was separated by SDS-PAGE, followed by transfer to Hybond-C extra membrane (Amersham Pharmacia Biotech). The filter was then blocked in 5% bovine serum albumin, 0.2% Tween 20 in phosphate-buffered saline at 4 °C overnight, and probed with specific antibodies for 1 h at room temperature. After washing, the filter was incubated with horseradish peroxidase-linked anti-rabbit or anti-mouse IgG, and reactions were visualized through enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). Insect cells (1 × 107) were infected with recombinant baculovirus carrying h-flt-1 IC for 3 days. After washing with ice-cold TBS, cells were lysed with high salt lysis buffer (20 mm Tris-HCl, pH 7.5, 500 mm NaCl, 1% Triton X-100, 10% glycerol, 2.5 mm EDTA, 100 units/ml aprotinin, 0.1 mm Na3VO4, 2.5 mm phenylmethylsulfonyl fluoride, and 1 mmdithiothreitol). After centrifugation, the supernatant was immunoprecipitated with specific antibodies against Flt-1 or PY20 on ice for 2 h. The immunocomplex was collected with protein A-Sepharose CL-4B, washed with high salt lysis buffer, and resuspended in kinase buffer (20 mm Hepes, pH 7.5, 10 mmMgCl2, 2 mm MnCl2, 0.05% Triton X-100, 1 mm dithiothreitol). In vitrophosphorylation was carried out in the presence of [γ-32P]ATP for 30 min at room temperature. The reactions were stopped by addition of 2× sample buffer (50 mm Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.1% bromphenol blue, and 2% 2-mercaptoethanol). The boiled samples were electrophoresed on SDS-containing gradient acrylamide gels (7.5–12%) and transferred to nitrocellulose. After exposure to film, bands corresponding to Flt-1 IC were excised from the filter and digested with trypsin (modified sequencing grade; Promega) or Asp-N (Boehringer Mannheim) for 12 h at 37 °C as described (28Aebersold R.H. Leavitt J. Saavedra R.A. Hood L.E. Kent S.B.H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6970-6974Crossref PubMed Scopus (630) Google Scholar). Two-dimensional phosphopeptide mapping was performed using the Hunter thin layer electrophoresis apparatus (HTLE-7000; C.B.S. Scientific Co., Inc., Del Mar, CA) according to Boyle et al. (29Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). First dimension electrophoresis was performed in pH 1.9 buffer (formic acid:glacial acetic acid:double-distilled water, 46:156:1798, v/v) for 40 min at 2000 V, and the second dimension ascending thin layer chromatography was run in isobutyric acid buffer (isobutyric acid:n-butyl alcohol:pyridine:glacial acetic acid:double-distilled water, 1250:38:96:58:558, v/v). After exposure on a Bio-Imaging Analyzer screen (Fuji), radioactive phosphopeptides on the thin layer plates were scraped off and then eluted in pH 1.9 buffer or 30% formic acid and lyophilized. The fractions were subjected to two-dimensional phosphoamino acid analysis and, in parallel, Edman degradation. For Edman degradation, phosphopeptides were coupled to Sequelon-AA membranes (Millipore) according to the manufacturer's instructions and sequenced on an Applied Biosystems Gas Phase Sequencer. The activity in released phenylthiohydantoin derivatives from each cycle was quantitated by use of the Bio-Imaging Analyzer. The eluted peptides were hydrolyzed in 6 m hydrochloric acid for 1 h at 110 °C. After lyophilization, the peptides were dissolved in pH 1.9 buffer containing phosphotyrosine, phosphoserine, and phosphothreonine as markers and separated on a cellulose plate at pH 1.9 in the first dimension and at pH 3.5 in the second dimension. After visualization of the markers by ninhydrin (BDH Laboratory Supplies) spraying, the plate was exposed to film. The following peptides with or without phosphorylation on tyrosine were synthesized: Ac-KKKDVRY1213VNAFKF (designated as Y(p)1213 with phosphotyrosine and 1213Ref without phosphotyrosine); Ac-MFDDY1242QGDSSTLLA (designated as Y(p)1242 and 1242Ref, respectively); NH2-KKKPPPDY1327NSVVLY1333STPPI (designated as Y(pp)1327/1333 with double phosphorylation, Y(p)1333 with single phosphorylation, and 1333Ref without phosphorylation). The underlined sequence KKK was added to the N terminus of the indicated peptides to increase their coupling efficiency to the support, as well as solubility, in buffer solution. The peptides encompassing Tyr-1213 and Tyr-1327/1333 were immobilized on Affi-Gel 10 (Bio-Rad) and those encompassing Tyr-1242 on Affi-Gel 15. After incubation with a mixture of [35S]methionine and [35S]cysteine at 100 μCi/ml (Promix, Amersham Pharmacia Biotech) for 3 h at 37 °C, MS1 cells were lysed in RIPA buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 1 mmphenylmethylsulfonyl fluoride, 1% aprotinin, 5 mm EDTA, 5 μg/ml leupeptin, and 0.2 mmNa3VO4). The cell lysate was precleared to reduce nonspecific binding by incubating with non-immune serum-coupled agarose gel for 30 min. The resulting cell lysate was then incubated with immobilized reference peptide or phosphorylated peptide in the presence or absence of blocking peptide for 1 h at 4 °C with end-over-end rotation. After washing the gel, binding proteins were separated by SDS-PAGE followed by fixation in destain (7% acetic acid and 10% methanol) for 30 min and incubation in Amplify (Amersham Pharmacia Biotech) for 30 min and visualized by exposing on films. Wild-type and mutants receptors, Y1213F, Y1242F, and Y1333F, were expressed in Sf9 cells. After cell lysis, the receptors were immunoprecipitated with anti-Flt-1 antibody and collected by use of immobilized protein A (Immunosorb; EC Diagnostics, Uppsala, Sweden). The beads were incubated with MS1 cell lysate for 1 h at 4 °C. The bound proteins were separated by SDS-PAGE and subjected to immunoblotting using specific antibodies. Sf9 cells infected with recombinant virus carrying the h-flt-1 intracellular (IC) domain cDNA express a 60-kDa protein corresponding to Flt-1 IC (Fig. 1 A). The protein is detected in the cell lysate but not in the conditioned medium. Immunoprecipitation of the cell lysate with anti-Flt-1 antibody followed by immunoblotting with the monoclonal anti-phosphotyrosine antibody PY20 demonstrates tyrosine phosphorylation of this protein (Fig. 1 B), indicating that the receptor tyrosine kinase is activated leading to autophosphorylation of the receptor. Immune complex kinase assays on immunoprecipitates using PY20 or anti-Flt-1 antibody also demonstrate strong autophosphorylation of the Flt-1 IC (Fig. 1 C). This is in agreement with previous reports on expression of the IC domains of EGFR and FGFR-1, respectively, in insect cells, which both were shown to be active kinases (30Hsu C.-Y.J. Mohammadi M. Nathan M. Honegger A. Ullrich A. Schlessinger J. Hurwitz D.R. Cell Growth Differ. 1990; 1: 191-200PubMed Google Scholar, 31Mohammadi M. Dikic I. Sorokin A. Burgess W.H. Jaye M. Schlessinger J. Mol. Cell. Biol. 1996; 16: 977-989Crossref PubMed Scopus (345) Google Scholar). Moreover, the pattern of autophosphorylation of the insect cell-derived EGFR and FGFR-1 IC was shown to correspond exactly to in vivo phosphorylated intact receptors. To investigate tyrosine phosphorylation sites in Flt-1, the 60-kDa protein corresponding to Flt-1 IC was labeled with 32P through an immune complex kinase assay as shown in Fig. 1 C. After SDS-polyacrylamide gel electrophoresis and transfer to a membrane, the 60-kDa band was excised, digested with trypsin, and subjected to two-dimensional phosphopeptide mapping. Fig. 2 shows a two-dimensional phosphopeptide mapping of the Flt-1 IC which was immunoprecipitated with the anti-Flt-1 antibody prior to the immune complex kinase assay. The immunoprecipitated material was separated by thin layer electrophoresis at pH 1.9 and chromatography. Several spots appeared to theright of the application spot (Δ) in the two-dimensional maps, after infection of the Sf9 cells with the Flt-1 IC virus (Fig. 2), indicating that these Flt-1-derived spots have neutral or positive charges at pH 1.9. Two spots (a and b) showed strong signals, whereas other spots such as c were fainter. This pattern was quite similar to that obtained after immunoprecipitation with PY20 (data not shown). Each spot was scraped from the plate to elute the tryptic peptides, followed by phosphoamino acid analysis. Fig. 3 (insets) shows that the peptides from spots a and bcontain phosphotyrosine but not phosphoserine nor phosphothreonine. Spot c contains both phosphotyrosine and phosphothreonine, whereas other fainter spots contain phosphoserine or phosphothreonine but not phosphotyrosine. Tryptic peptides eluted from spots a, b, and c were subjected to Edman degradation.Figure 3Edman degradation and phosphoamino acid analysis of trypsin-digested Flt-1 IC. Radioactive peptide fragments were eluted from each spot on the two-dimensional map (Fig. 2). A part of each sample was hydrolyzed in 6 mhydrochloric acid for 1 h at 110 °C and separated on a cellulose plate at pH 1.9 in the first dimension and at pH 3.5 in the second dimension (insets). S, T, and Y indicate phosphoserine, phosphothreonine, and phosphotyrosine, respectively. For Edman degradation, the remaining radioactive peptide samples were coupled to Sequelon-AA membranes and sequenced on an Applied Biosystems Gas Phase Sequencer. The activity in the released phenylthiohydantoin derivatives from each cycle was quantitated by use of a Bio-Imaging Analyzer. A–C show the results of Edman degradation and phosphoamino acid analysis(insets) of material from spots a, b,and c, respectively, on the two-dimensional map in Fig. 2. The amino acid sequences of Flt-1-derived tryptic peptides that could be aligned with the radiochemical sequencing are shown beloweach panel.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As shown in Fig. 3, radioactive peaks appeared for spota at cycle 16, for spot b at cycle 1, and for spot c at cycles 1 and 16. Complete trypsin digestion of the human Flt-1 IC would be expected to give rise to around 50 different peptides. Among these, only two peptides, encompassing Tyr-1242 and Tyr-1333, have a tyrosine residue at position 16. However, the peptide encompassing Tyr-1333 should be negatively charged at pH 1.9 and, therefore, would not migrate to the position of spot a on the two-dimensional mapping. On the other hand, the peptide encompassing Tyr-1242 has +1 charge at pH 1.9, which would be compatible with the position of spot a. In order to confirm this notion, the mutant Y1242F Flt-1 receptor IC domain was expressed in insect cells and subjected to immune complex kinase assays followed by two-dimensional phosphopeptide mapping in the same way as for the wild-type Flt-1 (Figs. 4 and 5). The fact that the spot ain the wild-type receptor analysis was missing from the two-dimensional map of the mutant Y1242F indicates that the peptide encompassing Tyr-1242 matches with spot a, and the Tyr-1242 is one of the tyrosine phosphorylation sites in Flt-1 (Fig. 5).Figure 5Two-dimensional phosphopeptide mapping of mutant receptors. IC domains of the mutant receptors as well as the wild-type receptor (wt) were expressed in Sf9 cells and immunoprecipitated with the anti-Flt-1 antibody. The precipitates were subjected to kinase reactions in the presence of [γ-32P]ATP, separated by SDS-PAGE, and transferred onto nitrocellulose membrane. The bands corresponding to IC domains of the wild-type and mutated receptors were excised and digested with trypsin, followed by two-dimensional mapping performed as described in the legend to Fig. 2. The two-dimensional maps show the wild-type (A) and mutant receptors (B, Y1242F;C, Y1213F; and D, Y914F). Arrows in B–D indicate spots missing from the mutant receptor two-dimensional maps.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Similarly, the two tryptic peptides encompassing Tyr-914 and Tyr-1213 are candidates for the spot b, since only these two peptides have a tyrosine residue at position 1 (Fig. 3 B). However, both peptides have +1 charge at pH 1.9 precluding the tentative assignment of one of them as spot b. The two-dimensional mapping of Y914F and Y1213F mutant receptors show that the spotb in the wild-type receptor is completely abolished in mutant Y1213F two-dimensional map, whereas it is still present in the two-dimensional mapping of Y914F (Fig. 5, C and D). These results indicate that Tyr-1213 is a second tyrosine phosphorylation site in Flt-1. It is noteworthy that spota in the wild-type receptor is missing in the mutant Y914F. Since Tyr-914 is located in the first kinase domain, it is possible that the mutation of this tyrosine may affect the kinase activity of Flt-1. Accordingly, the overall phosphorylation level of the Y914F mutant IC domain in the immune complex kinase assay was considerably lower than those of the wild-type and other mutant receptors (Fig. 4). We infer from these data that tyrosine phosphorylation of the Flt-1 IC was due to autophosphorylation and not phosphorylation by other kinases present in the Sf9 cells. The only tryptic peptide matching spot c is that encompassing Tyr-1242, in which Thr-1227 as well as Tyr-1242 are phosphorylated (Fig. 3 C). In agreement, spot c as well as spot a were missing from the two-dimensional map of the mutant Y1242F receptor (Fig. 5 B). Thus, Tyr-1213 and Tyr-1242 are the major tyrosine phosphorylation sites in Flt-1. To confirm these results in mammalian cells, porcine aortic endothelial (PAE) cells expressing Flt-1 were stimulated with VEGF (50 ng/ml), subjected to an immune complex kinase assay, and digested with trypsin, followed by two-dimensional mapping (Fig. 6). The two-dimensional mapping revealed spots at positions very similar to those obtained from the Flt-1 IC domain expressed in the baculosystem (see Fig. 2). Edman degradation confirmed that the spots a and b in Fig. 6 exactly correspond to the peptides encompassing Tyr-1242 and Tyr-1213, respectively (data not shown), indicating that Tyr-1213 and Tyr-1242 are tyrosine phosphorylation sites in Flt-1 expressed in mammalian cells. Most peptide fragments derived from the Flt-1 IC by trypsin digestion will be neutral or positively charged at pH 1.9. However, a peptide encompassing tyrosines 1327 and 1333 (IACCSPPPDY1327NSVVLY1333STPPI) will be negatively charged at pH 1.9 and would therefore not migrate to the right of the application spot (Δ). To investigate if the peptide YY1327/1333 is tyrosine-phosphorylated, the Flt-1 IC was digested with endopeptidase Asp-N instead of trypsin and subjected to two-dimensional phosphopeptide mapping (Fig. 7). In the wild-type receptor, a few weakly phosphorylated spots (Fig. 7 A) as well as strong signals are seen. Phosphoamino acid analysis revealed that spot d contains phosphotyrosine but not phosphoserine and phosphothreonine (Fig. 7 B, inset). Edman degradation of peptide material eluted from
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