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J. Biol. Chem., Vol. 278, Issue 42, 40973-40979, October 17, 2003
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¶ **
From the
Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Dag Hammarskjölds väg 20, S-751 85 Uppsala, Sweden, the Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, Post Office Box 63, 00014 University of Helsinki, Finland, and the ||Ludwig Institute for Cancer Research, Uppsala Biomedical Center, Box 595, S-751 24 Uppsala, Sweden
Received for publication, April 30, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In adult tissues, VEGFR-3 is expressed primarily on lymphatic endothelial cells (2) and appears to exert its major functions within this system. Thus, inactivating missense point mutations in one VEGFR3 allele leads to chronic lymphedema (3). Further, overexpression of a soluble VEGFR-3 in mice leads to regression of lymph vessels and features characteristic of lymphedema, without any apparent effects on the blood vasculature (4). In several tumor models, overexpression of the VEGFR-3 ligand VEGF-C increases lymphangiogenesis and promotes spread of metastases (5–7). The same effect is achieved by overexpression of VEGF-D, another VEGFR-3 ligand (8).
VEGFR-3 is to some extent expressed also on quiescent vascular endothelial cells, primarily in fenestrated capillaries (2, 9, 10). Very low levels can be occasionally detected in the blood vascular endothelium of wound granulation tissue and in vessels stimulated with VEGFs (11, 12). Further, the endothelium of angiogenic blood vessels of several tumors express VEGFR-3 (11, 13, 14). These results suggest that VEGFR-3 could be involved in aspects of angiogenesis in adults.
The VEGFR-3 is similar in overall structure to the VEGFR-1 and VEGFR-2 (15); the extracellular, ligand-binding domain is composed of seven immunoglobulin-like folds, and the intracellular domain is characterized by an interrupted tyrosine kinase domain. In contrast to the other VEGF receptors, the VEGFR-3 extracellular domain is cleaved within the fifth immunoglobulin-homology domain; the resulting two polypeptides are held together by a disulfide bridge (16). In humans, but not in mice, a retroviral insertion between the last two exons of VEGFR-3 (17) results in two splice variants of the VEGFR-3, the shorter form of which lacks 65 amino acids in the cytoplasmic tail (18, 19).
Activation of the VEGFR-3 tyrosine kinase appears to follow the consensus scheme for receptor tyrosine kinases. Ligand binding results in receptor dimerization and sequential activation of the intrinsic kinase activity. Trans-phosphorylation between the partners in the dimer regulates kinase activity and creates docking sites for signaling molecules with characteristic domains such as Src homology (SH)-2 or phosphotyrosine binding domains. The specificity of binding is determined by the sequence adjacent to the phosphotyrosine residue. The activated VEGFR-3 associates with adaptor proteins Shc and Grb2 via tyrosine 1337 (16, 20). Moreover, VEGFR-3 activation leads to protein kinase C-dependent activation of extracellular signal-related kinases (Erk)-1 and -2, implicated in cell proliferation. Furthermore, VEGFR-3 mediates the activation of protein kinase B/Akt (21), implicated in cell survival. In accordance, VEGFR-3 transduces signals resulting in proliferation, migration, and survival of lymphatic endothelial cells (21). In this report, we have examined the potential phosphorylation of tyrosine residues in the long form of the activated VEGFR-3 and provide evidence for phosphorylation in five positions in the cytoplasmic tail. In addition, we show complex formation between VEGFR-3 and the related VEGFR-2 in primary lymphatic endothelial cells. In the heterodimeric configuration, VEGFR-2 failed to phosphorylate VEGFR-3 on two of the carboxyl-terminal sites, Tyr-1337 and Tyr-1663. This has implications for the VEGFR-3 signal transduction properties.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell Lines and Cell Culture—Porcine aorta endothelial (PAE) cells stably overexpressing VEGFR-3 or VEGFR-2 were maintained in F12/10% fetal calf serum. Human 293T cells were used for transient expression of VEGFR-3 and were maintained in Dulbecco's modified Eagle's medium/10% fetal calf serum. Primary lymphatic endothelial cells (LECs) were separated from human dermal microvascular endothelial cells as previously described (21) using antibodies against podoplanin. The cells were cultured on gelatin-coated plastic in endothelial basal medium (EBM, CC-3121; Clonetics, Walkersville, MD) supplemented with 5% fetal calf serum, 30 µg/ml endothelial growth culture supplement (ECGS, E-7060; Sigma), 10 ng/ml EGF, and 10 ng/ml VEGF-C.
Generation of Mutated VEGFR-3—VEGFR-3 tyrosine mutants were generated by the GeneEditor in vitro site-directed mutagenesis kit (Promega) using oligonucleotides in which one nucleotide change in the tyrosine-encoding sequence was introduced, resulting in Tyr > Phe amino acid change in the protein sequence.
VEGFR-3 kinase dead (R3-KD; R1041P and K879G) mutants were generated as above using oligonucleotides that introduced desired nucleotide changes in the VEGFR-3 kinase domain-encoding sequence. The R1041P represents a mutation found in lymphedema (25), whereas in the K879G mutant the ATP-binding Lys has been changed into Gly. Both mutant proteins were found to be kinase-inactive when expressed in 293T cells (25) (data not shown).
Transient Transfections—Vectors (pcDNA3.1/Zeo; Invitrogen) encoding wild type and mutant VEGFR-3 were transfected into human 293T cells using the calcium phosphate method. In brief, 6 x 106 cells in 10-cm Petri dishes were treated with 25 µM chloroquine for 1.5 h. Vector cDNA (4–10 µg) in 250 mM CaCl2 was mixed with 2x concentrated Hank's balanced salt solution, incubated for 20 min, and then added to the cells. 5–6 h later, cells were treated for 2 min with culture medium containing 10% glycerol. The cells were harvested 48 h post-transfection.
Immunocomplex Kinase Assay and SDS-PAGE—The cells were starved overnight in serum-free medium supplemented with 0.1% bovine serum albumin and treated for 8 min with or without VEGF or VEGF-C using 50 ng/ml washed in Tris-buffered saline/100 µM Na3VO4 on ice. The cells were lysed in ice-cold Nonidet P-40 lysis buffer (1% Nonidet P40, 20 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 2.5 mM EDTA, 10 Kallekrein inhibitory units aprotinin/ml, 1 mM phenylmethylsulfonyl fluoride, and 100 µm Na3VO4). An aliquot of the cell lysate was saved for control blotting. Lysates were clarified by centrifugation and incubated for 2 h on ice with in-house anti-VEGFR-3 antibodies. The mouse and rabbit antibodies were precipitated with protein G-Sepharose (17–0618-01; Amersham Biosciences) and protein A-Sepharose (Immunosorb A; Medicago AB, Uppsala, Sweden), respectively. The precipitate was washed three times in lysis buffer and twice in kinase buffer (20 mM Hepes, 10 mM MgCl2, 2 mM MnCl2, 0.05% Triton X-100). The precipitate was incubated for 10 min in kinase buffer containing 20 µCi of [
-32P]ATP (Amersham Biosciences) at 37 °C and heated in sample buffer (8% SDS, 0.4 M Tris-HCl, pH 8.0, 1 M sucrose, 10 mM EDTA, 0.02% bromphenol blue, 4% 
-mercaptoethanol). The samples were separated by SDS-PAGE, using 7% polyacrylamide gel, transferred to a nitrocellulose membrane, and detected by a BioImager (BioImager, BAS-1800II; Fujifilm, Tokyo, Japan) screen that was subsequently scanned using the BioImager.
Immunoblotting—Samples were prepared essentially as described above but without the kinase reaction, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane. The membranes were incubated with indicated primary antibodies and subsequently with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive sites were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences).
Phosphopeptide Mapping—The detailed procedure has been described previously (26, 27). Briefly, the phosphorylated bands on the membrane from the in vitro complex assay were localized using the BioImager scan, cut out and treated for 30 min at 37 °C in 0.5% polyvinylpyrrolidone in 100 mM acetic acid, washed four times in H2O, and digested by 1 µg of trypsin (V542A; Promega, Madison, WI) in 200 µl of 50 mM NH4CO3 at 37 °C overnight. The supernatant was lyophilized in a centrifugal vacuum concentrator, dissolved in 50 µl of performic acid (formic acid, 30% H2O2, 9:1), incubated for 1 h at room temperature, diluted with 500 µl of H2O, and frozen to –135 °C. The frozen samples were lyophilized again and dissolved in 50 mM NH4CO3 supplemented with 1 µg of trypsin and incubated overnight at 37 °C. 140 µl of pH 1.9 buffer (2.2% formic acid and 7.8% acetic acid in H20) was added, and any particles were precipitated by centrifugation. 180 µl of the supernatant was lyophilized and dissolved in 7 µl of pH 1.9 buffer. The samples were centrifuged, and 6 µl of each sample was gently dried onto a cellulose-covered thin-layer chromatography glass plate (1.05716; Merck, Darmstadt, Germany) as a spot of 
5 mm in diameter, localized 5 cm from the left side and 3 cm from the bottom of the plate. The peptides were separated by electrophoresis (x-axis) using pH 1.9 buffer, and the plate was dried. Separation in the second dimension (y-axis) was performed by ascending chromatography using isobutyric acid buffer (62.5% isobutyric acid, 1.9% n-butanol, 4.8% pyridine, 2.9% acetic acid in H2O) overnight. The plate was dried, and the peptides with incorporated 32P were detected by the BioImager. Using the resulting phosphopeptide map for localization, cellulose-containing peptide spots of interest were scraped off the chromatography plate. The peptides were extracted by pH 1.9 buffer, lyophilized, and used for Edman degradation or phosphoamino acid analysis.
Edman Degradation and Radio Amino Acid Sequencing—For Edman degradation, phosphopeptides were coupled to sequelon-amino acid membranes (Millipore, Sundbyberg, Sweden) and sequenced using a gas phase sequencer (Applied Biosystems, Foster City, CA). The fractions were spotted onto thin-layer chromatography plates, detected by BioImager, and analyzed by the BioImager software.
Phosphoamino Acid Analysis—The samples extracted from the thinlayer plates were hydrolyzed in 6 M HCl at 110 °C and lyophilized, dissolved in H2O, and lyophilized again. The samples were dissolved in 7 µl of pH 1.9 buffer supplemented with non-radioactive phosphorylated serine, threonine, and tyrosine as markers and spotted onto a thin-layer chromatography plate. Electrophoresis using first the pH 1.9 buffer (x-axis) and subsequently the pH 3.5 buffer (acetic acid 5% and pyridine 0.5% in H2O; y-axis) provided two-dimensional separation of the hydrolyzed amino acid residues. The dried plates were sprayed with ninhydrin for detection of the marker phosphoamino acid residues. The plate was dried, and the incorporated 32P was visualized using the BioImager. By overlaying the resulting image with the ninhydrin pattern of the marker amino acids, the identity of the amino acid residues with incorporated 32P was determined.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Position of Phosphorylated Residues—Peptides giving reproducible spots in the phosphopeptide maps were extracted, hydrolyzed in hydrochloric acid, and separated by two-dimensional electrophoresis on thin-layer plates. Unlabeled phosphoamino acid residues served as references (indicated by circles in the insets in Fig. 2C). Peptides displaying tyrosine phosphorylation (circled in Fig. 2B) were subjected to Edman degradation. Chemical identification of amino acid residues was not feasible because of the minute amounts of protein in the assay. Instead, the material in each cycle was spotted individually on thin-layer chromatography plates, and the 32P content in each fraction was quantified after exposure and detection using a BioImager. The positions of the radioactive peaks were the basis for tentative identification of a tryptic peptide containing a tyrosine residue in the corresponding position. Peptides from some distinct spots displayed identical radio sequences (spots denoted c in Fig. 2). This may be because of serine phosphorylation of the corresponding peptide, which contains five serine residues. Serine phosphorylation was indeed detected in the left-most spot (data not shown). The predicted change in the phosphopeptide map position because of such a modification is in compliance with the observed positions. An alternative explanation for multiple spots is partial oxidation of cysteine residues with consequences for the charge and thereby the migration position of the peptide.
Analyses of VEGFR-3 Tyrosine to Phenylalanine Mutants— The above results indicated that all tyrosine residues in the carboxyl-terminal tail, with the possible exception of Tyr-1333, were phosphorylated. To further exploit these findings, we created six receptor variants, point-mutated in the carboxyl-terminal tail, each with a single amino acid exchange from tyrosine to phenylalanine. These receptors were denoted Y1230F-R3, Y1231F-R3, Y1265F-R3, Y1333F-R3, Y1337F-R3, and Y1363F-R3. Phosphopeptide mapping of the mutant receptors transiently expressed in 293 cells confirmed the preliminary identification of the phosphopeptides (Fig. 3). In the phosphopeptide map of Y1265F-R3 one spot was lost, whereas the map of Y1337F-R3 showed loss of three spots (Fig. 3). The corresponding spots in the Y1333F-R3 map were shifted along the y axis, indicating increased hydrophobicity, in agreement with the expected change as a result of the exchange of tyrosine for phenylalanine. The Tyr-1337 residue was still phosphorylated in this peptide. The peptides containing Tyr-1230/Tyr-1231 and Tyr-1363 were not fully separated, but analysis of this region in the phosphopeptide map (boxed in Fig. 3A) by scanning densitometry confirmed that loss of tyrosine phosphorylation as a consequence of the different mutations resulted in the expected phosphopeptide pattern (Fig. 3H).
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VEGFR-2 and VEGFR-3 Heterodimerization in Primary Cells—We wished to verify that the phosphorylation pattern of VEGFR-3 overexpressed in PAE or 293 cells mimicked that of the endogenously expressed VEGFR-3 in LECs. Fig. 4 shows that the phosphopeptide map of LEC-derived VEGFR-3 was essentially indistinguishable from that of the overexpressed recombinant receptor, confirming the relevance of our approach. Minor differences in migration positions of a collection of spots in the right margin were anticipated, because there had been some variations in this region of the TLC plate between repeated maps of VEGFR-3 overexpressed in the 293 cells.
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The immunocomplex kinase assay of VEGFR-3 immunoprecipitated from VEGF-C-stimulated LECs demonstrated that a phosphorylated protein of 220 kDa was co-immunoprecipitated by the VEGFR-3-specific antibodies. We hypothesized that this component could correspond to VEGFR-2. This was confirmed by phosphopeptide mapping (data not shown). To investigate under which conditions the association between VEGFR-2 and VEGFR-3 occurred, LECs were treated with VEGF, specific for VEGFR-2, or VEGF-C, a ligand for both receptors. Immunoprecipitation with antibodies specific for the respective receptors, followed by immunocomplex kinase assay, demonstrated that the association was evident only after stimulation with VEGF-C (Fig. 5A). The specificity of the antibodies was confirmed by immunoprecipitation and blotting of cell lysates derived from PAE cells overexpressing either VEGFR-2 or VEGFR-3 (Fig. 5B). The different levels of kinase activity and the different properties of the antisera used precluded a determination of the relative stoichiometry of heterodimers versus homodimers in the VEGF-C-treated LECs.
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Distinct Pattern of Phosphorylation of VEGFR-3 in the Heterodimeric Configuration—To examine whether VEGFR-3 was phosphorylated similarly in the homodimeric and the heterodimeric configuration, a kinase-dead mutant VEGFR-3 (R1041P; here denoted R3-KD) was expressed alone or in combination with VEGFR-2 or VEGFR-3 in 293 cells. The V5-tagged R3-KD was specifically recognized by anti-V5 antibodies, ensuring that the analysis was focused on R3-KD dimerized with kinase-active VEGFR-2 or VEGFR-3. In this setup, phosphorylation of R3-KD was dependent on the coexpressed kinase-active receptors. As shown in Fig. 6, wild type VEGFR-3-mediated phosphorylation of R3-KD resulted in a phosphopeptide map very similar to the previous VEGFR-3 maps (see Fig. 3). In contrast, the map of R3-KD phosphorylated by VEGFR-2 lacked spots corresponding to peptides containing tyrosine residues Tyr-1337 and -1363. Repeating the analysis using another kinase-inactive VEGFR-3 (K879G) gave identical results (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Our preliminary results suggest that the tyrosine residues 1063 and 1068 in the second kinase domain become phosphorylated upon receptor activation (data not shown). The positions of these sites correspond to those previously implicated in positive regulation of tyrosine kinase activity, e.g. Tyr-1054 and -1059 in VEGFR-2 (28). We therefore suggest that Tyr-1063 and -1068 in VEGFR-3 serve a positive regulatory role in the activation of the VEGFR-3 kinase. Of the remaining tyrosine residues in the intracellular domain of VEGFR-3, five are located in the first and second parts of the kinase domain, and three are located in the juxtamembrane domain (Tyr-812, -830 and -833). Tyrosine residues at positions conserved relative to Tyr-812 are found both in VEGFR-1 (Tyr-794) and VEGFR-2 (Tyr-801) (29). All three VEGFR-3 juxtamembrane tyrosine residues are contained within the same tryptic peptide. Edman degradation of this peptide has not allowed an unambiguous conclusion on the phosphorylation of these tyrosine residues.
The kinase insert domain plays an important role in signal transduction by the PDGF receptors. This sequence is of varying length in different receptor tyrosine kinases, and it is usually not conserved between otherwise related receptors, such as the PDGF 
- and -receptors (30), which has prompted the suggestion that the kinase insert is important in receptor type-specific signaling. It is therefore interesting that neither the VEGFR-1 nor the VEGFR-3 kinase insert contains any tyrosine residues. In contrast, the VEGFR-2 kinase insert contains three tyrosine residues, of which at least one is a phosphorylation site (28).
Our analysis of VEGFR-3 tyrosine phosphorylation in primary lymphatic endothelial cells allowed the following conclusions. 1) The phosphorylation pattern of VEGFR-3 was faithfully reproduced between the receptor overexpressing cells and primary LECs. 2) In these primary cells, VEGF-C, but not VEGF, treatment induced formation of VEGFR-2 and VEGFR-3 heterodimers. 3) VEGFR-3 phosphorylation-site usage was altered in the heterodimeric configuration. Thus, the two most carboxyl-terminal tyrosine residues in VEGFR-3 are substrates only for the VEGFR-3. VEGFR-3 in adult blood vessels and at least some lymphatic endothelia occurs in areas with VEGFR-2 expression (2, 31), indicating that VEGFR-2/ VEGFR-3 heterodimers may form in vivo. Interestingly, recent data suggest that VEGFR-3 modulates sensitivity to VEGFR-2 signaling to promote vascular integrity in blood vascular endothelial cells co-expressing the two receptors (32, 33). It is possible that such cross-talk between the receptors is dependent on heterodimerization.
Growth factors of the VEGF and PDGF families are dimeric proteins in which each monomer contributes one receptor binding site. The different PDGF variants induce homo- or heterodimers of the PDGF receptors in a manner dictated by the receptor-specificity of the monomers. Similarly, treatment with VEGF, which is a common ligand for VEGFR-1 and VEGFR-2, induces receptor heterodimerization and functional signaling units (34). PDGF - and -receptor heterodimers have been shown to have signal transduction properties distinct from the respective - and - homodimeric forms, because of receptor tyrosine phosphorylation specific for the heterodimeric receptors (35). The differences in the phosphorylation-site pattern between homo- and heterodimeric VEGFR-3 suggest that the signal transduction properties and biological function are distinct for the heterodimerized VEGFR-3. In particular, Shc and Grb2, which are known to bind to Tyr-1337 (16), are most likely not substrates for heterodimeric VEGFR-3. VEGF-C is produced as a 60-kDa protein with affinity for VEGFR-3 but poor affinity for VEGFR-2 (22). A stepwise proteolysis of VEGF-C results in a 20-kDa fragment with high affinity for both receptors. Thus, VEGF-C proteolysis provides a mechanism whereby VEGFR-2/VEGFR-3 heterodimerization and, in turn, VEGFR-3 signaling, could be modulated.
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FOOTNOTES |
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¶ Present address: Dept. of Molecular Neurobiology, Max-Planck Institute of Neurobiology, Am Klopferspitz 18A, D-82152 Martinsried, Germany.
** To whom correspondence should be addressed. Fax: 46-18-55-89-31; E-mail: Lena.Welsh{at}genpat.uu.se.
1 The abbreviations used are: VEGFR, vascular endothelial growth factor receptor; PAE, porcine aorta endothelial; LEC, lymphatic endothelial cell; R3-KD, VEGFR-3 kinase dead; PDGF, platelet-derived growth factor.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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