VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis

Article16 June 2005free access VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis Taro Matsumoto Taro Matsumoto Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Division of Cell Regeneration and Transplantation, Advanced Medical Research Center, Nihon University School of Medicine, Ohyaguchikamimachi, Itabashi-ku, Tokyo, Japan Search for more papers by this author Svante Bohman Svante Bohman Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Johan Dixelius Johan Dixelius Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Tone Berge Tone Berge Department of Anatomy, Institute of Basal Medical Sciences, University of Oslo, Blindern, Oslo, Norway Search for more papers by this author Anna Dimberg Anna Dimberg Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Peetra Magnusson Peetra Magnusson Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Ling Wang Ling Wang Mayo Clinic Foundation, Gugg, Rochester, MN, USA Search for more papers by this author Charlotte Wikner Charlotte Wikner Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Jian Hua Qi Jian Hua Qi Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH, USA Search for more papers by this author Christer Wernstedt Christer Wernstedt Ludwig Institute for Cancer Research, Uppsala Branch, Biomedical Center, Uppsala, Sweden Search for more papers by this author Jiong Wu Jiong Wu Cell Signaling Technology, Cummings Center, Beverly, MA, USA Search for more papers by this author Skjalg Bruheim Skjalg Bruheim Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway Search for more papers by this author Hideo Mugishima Hideo Mugishima Division of Cell Regeneration and Transplantation, Advanced Medical Research Center, Nihon University School of Medicine, Ohyaguchikamimachi, Itabashi-ku, Tokyo, Japan Search for more papers by this author Debrabata Mukhopadhyay Debrabata Mukhopadhyay Mayo Clinic Foundation, Gugg, Rochester, MN, USA Search for more papers by this author Anne Spurkland Anne Spurkland Department of Anatomy, Institute of Basal Medical Sciences, University of Oslo, Blindern, Oslo, Norway Search for more papers by this author Lena Claesson-Welsh Corresponding Author Lena Claesson-Welsh Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Taro Matsumoto Taro Matsumoto Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Division of Cell Regeneration and Transplantation, Advanced Medical Research Center, Nihon University School of Medicine, Ohyaguchikamimachi, Itabashi-ku, Tokyo, Japan Search for more papers by this author Svante Bohman Svante Bohman Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Johan Dixelius Johan Dixelius Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Tone Berge Tone Berge Department of Anatomy, Institute of Basal Medical Sciences, University of Oslo, Blindern, Oslo, Norway Search for more papers by this author Anna Dimberg Anna Dimberg Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Peetra Magnusson Peetra Magnusson Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Ling Wang Ling Wang Mayo Clinic Foundation, Gugg, Rochester, MN, USA Search for more papers by this author Charlotte Wikner Charlotte Wikner Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Jian Hua Qi Jian Hua Qi Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH, USA Search for more papers by this author Christer Wernstedt Christer Wernstedt Ludwig Institute for Cancer Research, Uppsala Branch, Biomedical Center, Uppsala, Sweden Search for more papers by this author Jiong Wu Jiong Wu Cell Signaling Technology, Cummings Center, Beverly, MA, USA Search for more papers by this author Skjalg Bruheim Skjalg Bruheim Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway Search for more papers by this author Hideo Mugishima Hideo Mugishima Division of Cell Regeneration and Transplantation, Advanced Medical Research Center, Nihon University School of Medicine, Ohyaguchikamimachi, Itabashi-ku, Tokyo, Japan Search for more papers by this author Debrabata Mukhopadhyay Debrabata Mukhopadhyay Mayo Clinic Foundation, Gugg, Rochester, MN, USA Search for more papers by this author Anne Spurkland Anne Spurkland Department of Anatomy, Institute of Basal Medical Sciences, University of Oslo, Blindern, Oslo, Norway Search for more papers by this author Lena Claesson-Welsh Corresponding Author Lena Claesson-Welsh Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Author Information Taro Matsumoto1,2, Svante Bohman1, Johan Dixelius1, Tone Berge3, Anna Dimberg1, Peetra Magnusson1, Ling Wang4, Charlotte Wikner1, Jian Hua Qi5, Christer Wernstedt6, Jiong Wu7, Skjalg Bruheim8, Hideo Mugishima2, Debrabata Mukhopadhyay4, Anne Spurkland3 and Lena Claesson-Welsh 1 1Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden 2Division of Cell Regeneration and Transplantation, Advanced Medical Research Center, Nihon University School of Medicine, Ohyaguchikamimachi, Itabashi-ku, Tokyo, Japan 3Department of Anatomy, Institute of Basal Medical Sciences, University of Oslo, Blindern, Oslo, Norway 4Mayo Clinic Foundation, Gugg, Rochester, MN, USA 5Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH, USA 6Ludwig Institute for Cancer Research, Uppsala Branch, Biomedical Center, Uppsala, Sweden 7Cell Signaling Technology, Cummings Center, Beverly, MA, USA 8Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway *Corresponding author. Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Dag Hammarskjöldsv. 20, 75185 Uppsala, Sweden. Tel.: +46 18 471 43 63; Fax: +46 18 55 89 31; E-mail: [email protected] The EMBO Journal (2005)24:2342-2353https://doi.org/10.1038/sj.emboj.7600709 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Vascular endothelial growth factor receptor-2 (VEGFR-2) activation by VEGF-A is essential in vasculogenesis and angiogenesis. We have generated a pan-phosphorylation site map of VEGFR-2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C-terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR-2-expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T-cell-specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF-A-induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd-deficient mice, indicating a critical role of Y951-TSAd signaling in pathological angiogenesis. Introduction Vascular endothelial growth factor (VEGF)-A/vascular permeability factor (VPF) is critical for vascular development and angiogenesis, that is, formation of new blood vessels from existing capillaries (Ferrara, 2002). VEGF-A is distinguished by its potent regulation by hypoxia and its occurrence in several splice variants that to different extents bind to heparan sulfate and to the extracellular matrix. Moreover, deletion of only one allele of the VEGF-A gene leads to embryonic lethality resulting from arrest in vascular development (for a review, see Matsumoto and Claesson-Welsh, 2001). The critical involvement of VEGF-A in vascular development and angiogenesis occurs through binding and activation of VEGF receptor-2 (VEGFR-2). VEGFR-2 is essential for endothelial cell function and is the earliest known marker for vascular endothelial cells (Kabrun et al, 1997). Inactivation of the VEGFR-2 gene leads to a phenotype similar to that produced by VEGF-A deletion, with early embryonic death resulting from lack of proper differentiation and/or proper localization of endothelial cells (Matsumoto and Claesson-Welsh, 2001). A growing number of diseases, such as cancer, chronic inflammation, and diabetic retinopathy, are characterized by excess angiogenesis. Progression of these diseases may depend on the attraction of blood vessels to oxygenate and nurture the growing tissue. Tumor cells often produce VEGF-A, and, in models of multistage tumor disease, onset of angiogenesis is a prerequisite for the switch from a dysplastic lesion to an expanding malignancy (the angiogenic switch; for a review, see Hanahan and Folkman, 1996). The VEGFR-2 kinase is activated according to the consensus scheme for tyrosine kinases; binding of the dimeric VEGF-A to the extracellular domains of two monomeric receptors induces dimerization and activation of the tyrosine kinase. Certain aspects of the signal-transduction properties of the VEGFR-2 kinase have been analyzed in depth. Tyrosine residues Y1054 and Y1059 in the kinase domain serve as positive regulatory sites (Dougher and Terman, 1999; Kendall et al, 1999). Phosphorylation at Y1175 in the VEGFR-2 C-terminal tail allows binding, phosphorylation, and activation of phospholipase Cγ1 (PLCγ1) (Takahashi et al, 2001), ultimately leading to Ca2+ influx and activation of protein kinase C. Phosphorylated Y1175 also binds the adapter molecule Shb, which mediates activation of phosphoinositide 3′ kinase (PI3-kinase) and assembly of focal adhesions (Holmqvist et al, 2004). Exchange of Y1175 for F in the VEGFR-2 gene results in a loss-of-function phenotype and embryonic lethality (Sakurai et al, 2005). Phosphorylation at other sites, such as Y951 in the kinase insert, has been inferred from analyses of bacterially expressed VEGFR-2 intracellular domain (Dougher-Vermazen et al, 1994); by mutational analyses, Y951 has been linked to migration of endothelial cells (Zeng et al, 2001). Using a two-hybrid screen in yeast, Wu et al (2000) identified an adapter molecule designated VEGF receptor-associated protein (VRAP) as a binding partner to Y951. VRAP is equivalent to the T cell-specific adapter molecule (TSAd)/Rlk and Itk-binding protein (RIBP)/Lck adapter (LAD), which we have designated TSAd in this study. TSAd gene inactivation results in impaired T-cell-receptor activation and immune-response deficiency (Rajagopal et al, 1999). The overall objective of this study was to create a pan-phosphorylation map of VEGFR-2 and to determine the roles of the phosphorylation sites in endothelial cell function in vivo. For this purpose, we have determined phosphorylation sites in VEGFR-2 under conditions that allow resolution of all possible sites except Y1175, which is a previously identified phosphorylation site in VEGFR-2. Thereby, a pan-phosphorylation site map of VEGFR-2 can be constructed, with five major phosphorylation sites: Y951 in the kinase insert; Y1054 and Y1059 in the C-terminal part of the kinase domain; Y1175 and Y1214 in the C-terminal tail. We show that Y951 is used only in certain endothelial cells during development and that phosphorylated Y951 binds and mediates tyrosine phosphorylation of TSAd, which is expressed in endothelial cells in tumor vasculature. Moreover, we show that Y951-mediated coupling of VEGFR-2 and TSAd is critical for VEGF-A-mediated actin reorganization in endothelial cells and for tumor vascularization and growth. Results Phosphorylation of tyrosine residues in VEGFR-2 The intracellular domain of VEGFR-2 contains 19 tyrosine residues, 11 of which are located in the noncatalytic part of the receptor, potentially serving as phosphorylation sites (Figure 1A). To create a pan-phosphorylation site map of VEGFR-2, we labeled immunoprecipitated VEGFR-2 using γ-32P-ATP in a kinase reaction; we have shown that this procedure faithfully mimics phosphorylation in intact cells (Ito et al, 1998; Dixelius et al, 2003). The 32P-labeled VEGFR-2 sample was digested with trypsin, and the digest was separated in two dimensions by electrophoresis and liquid chromatography. This approach allows resolution of all potential phosphotyrosine-containing VEGFR-2 tryptic peptides except the one encompassing Y1175 (Figure 1A) that is negatively charged under the employed conditions. The resulting phosphopeptide map is shown in Figure 1B. A number of phosphopeptides (indicated as spots a–h in the schematic representation in Figure 1B) were reproducibly identified. Phosphoamino-acid analyses showed that spots a–f contained phosphotyrosine, whereas spots g and h contained phosphoserine and phosphothreonine (Figure 1C). Figure 1.Generation of a pan-phosphorylation site map of VEGFR-2. (A) Schematic drawing of the VEGFR-2 intracellular domain. The 19 tyrosine residues in the intracellular domain are indicated by their amino-acid sequence numbers. Tryptic digestion of VEGFR-2 potentially generates 14 tryptic peptides from the intracellular domain. The electrophoretic migration of these peptides depends on their charge at pH 1.9. (B) Tryptic phosphopeptide map of 32P-labeled VEGF-A-stimulated VEGFR-2 at pH 1.9. Tryptic peptides of immunoprecipitated and 32P-labeled VEGFR-2 were separated by electrophoresis in the first dimension and by liquid chromatography in the second dimension. Tryptic phosphopeptide map of Wt VEGFR-2 (left) and schematic representation of the map with spots indicated by letters a–h (right). (C) Phosphoamino-acid analyses of all major phosphorylated spots showing phosphorylation on tyrosine in spots a–f and on serine and threonine in spots g and h. (D) Phosphopeptide maps of VEGFR-2 mutated at indicated tyrosine residues (Y951F, Y1054F, and Y1214F) to the right shows loss of spots a, b, and c, respectively (indicated by arrows). Phosphopeptide map of mutant Y996F is identical to that of Wt VEGFR-2. The identities of spots a, b, and c were confirmed by radiochemical sequencing of the peptides, shown to the left for each spot, which yielded peaks of radioactivity in the fraction corresponding to the position of the tyrosine residue in each peptide. (E) Phosphopeptide maps of Wt VEGFR-2, a triple-mutated Y1305/1309/1319F VEGFR-2, and a C-terminally truncated VEGFR-2 (Ct truncated). Note the absence of spots d and e in the triple mutant map and the additional loss of spot c (Y1214) in the truncated mutant map. (F) Schematic drawing of VEGFR-2 with potential phosphorylation sites of the receptor. P indicates major phosphorylation sites. 'Y-F identical to wt' indicates that a mutated VEGFR-2 with a Y-to-F replacement at the particular residue showed the same phosphopeptide map pattern as Wt VEGFR-2 and therefore does not constitute a phosphorylation site. Download figure Download PowerPoint To determine the positions of 32P-tyrosine residues, peptides eluted from phosphotyrosine-containing spots were subjected to radiochemical sequencing. As seen in Figure 1D, sequencing of peptides from spots a, b, and c resulted in elution of radioactive residues in position 2 (spot a) and position 3 (spots b and c). This outcome allowed the tentative assignment of spot a as the phosphoY951 (pY951)-containing peptide. Similarly, spots b and c most likely corresponded to pY1054- and/or pY1214-containing peptides. To confirm spot a as pY951 and to distinguish between pY1054 and pY1214, Y-to-F mutations in VEGFR-2 were created and phosphopeptide maps were produced as described above. We thereby confirmed that spot a corresponded to pY951 and showed that spots b and c corresponded to pY1054 and pY1214, respectively (Figure 1D). The tryptic peptide encompassing pY1214 also contains Y1223, which if phosphorylated should give rise to a peak in cycle 12. This peak was missing; moreover, the Y1214F mutant completely lacked spot c. Phosphorylation at Y1223 should have been noticed as a persistent, albeit less intense, spot c slightly shifted upwards and to the left. As a control, we generated a phosphopeptide map of a VEGFR-2 Y996F mutant, in which all phosphopeptide spots seen in the wild-type (Wt) map were retained (bottom panel in Figure 1D). The identity of spots d–f was sought with similar strategies. However, no radioactive peaks were obtained when subjecting spots d and e to 20 consecutive Edman degradation cycles. Re-cleavage of the d and e tryptic peptides with cyanogen bromide or endoproteinase Asp-N also did not result in interpretable sequences (data not shown). Nevertheless, we noted that a truncated VEGFR-2 lacking five tyrosine residues of the six in the C-terminal tail (Ct truncated) did not give rise to spots d and e in the phosphopeptide map (Figure 1E). In addition, spot c was missing, which is compatible with the assignment of this spot as pY1214. We therefore examined the contribution of Y1305, Y1309, and Y1319 to spots d and e. A triple mutant carrying F instead of Y at all three positions also lacked spots d and e (Figure 1E). In contrast, individual mutations at these sites still allowed phosphorylation at d and e, but showed changes in the mobility of weakly labeled 32P-peptides, which were not amenable to radioactive Edman degradation (data not shown). Our interpretation of these data is that the C-terminal Y1305, Y1309, and Y1319 are phosphorylated only at a low stoichiometry after VEGF-A stimulation. By mutation of all three sites, the folding of the C-terminal tail may be disturbed, which in turn affects the accessibility to the VEGFR-2 kinase of other sites most likely in the kinase domain. Sequencing of spot f showed multiple radioactive peaks in repeated experiments, most likely because of incomplete cleavage at certain potential cleavage sites. However, the migration position of spot f fits with that of Y1059, which, previously, has been identified as a phosphorylation site (Dougher and Terman, 1999; Kendall et al, 1999). Mutation of Y1059 to F renders the receptor kinase inactive (Zeng et al, 2001), and we were therefore unable to definitively demonstrate phosphorylation of Y1059 and its assignment as spot f. Thus, in conclusion, individual mutations of Y951, Y1054 and Y1214 showed that these are major phosphorylation sites in VEGFR-2. The three C-terminal sites Y1305, Y1309, and Y1319 are phosphorylated only to a minor extent, yielding phosphopeptide spots visible after prolonged exposure. Furthermore, we did not see any changes in the VEGFR-2 phosphopeptide map when we analyzed individual Y-to-F mutations at Y801 and Y822 in the juxtamembrane domain and Y938 and Y996 in the kinase insert (see Figure 1F for a summary of the mapping data). Phosphorylation of VEGFR-2 at Y951, Y1175, and Y1214 in embryoid body (EB) vessels To demonstrate phosphorylation of VEGFR-2 in intact endothelial cells, we employed phospho-specific antibodies raised against the individual sites to stain vessel structures in EBs (see online Supplementary Figure 1 for characterization of the specificity of the phosphosite-specific antibodies). Murine embryonic stem cells (R1) were aggregated in hanging drops to create EBs. We and others have previously documented vascular development in EBs, with spatial and temporal expression patterns of endothelial cell markers very closely mimicking in vivo patterns (Magnusson et al, 2004). Thus, 8 days after leukemia inhibitory factor (LIF) withdrawal, which allowed differentiation of endothelial cells, vessel-like structures expressing CD31 and VEGFR-2 were formed in response to treatment with VEGF-A (Figure 2A). Costaining with antibodies against the VEGFR-2 protein and the phosphosite-specific antibodies against pY1175 or pY1214 showed that, wherever VEGFR-2 was expressed, it was phosphorylated at these two positions (Figure 2B). In contrast, some but not all of the VEGFR-2-expressing vessels were positive for phosphorylation at Y951 (Figure 2B, bottom panel). Costaining of EBs with antibodies against VEGFR-2 protein, pY951, and α-smooth muscle cell actin (ASMA) showed that endothelial cells lacking VEGFR-2 phosphorylated at Y951 more often were covered by pericyte-like, ASMA-positive cells, whereas endothelial cells containing VEGFR-2 phosphorylated at Y951 lacked associated pericytes (Figure 2C). Figure 2.Selective tyrosine phosphorylation at Y951 in VEGFR-2 during vascular development. (A) Visualization of the vascular tree in EBs cultured for 8 days in the absence (Basal) and presence of VEGF-A and immunostained to detect vessels using antibodies against CD31 or VEGFR-2. (B) VEGF-A-treated EBs coimmunostained with antibodies against VEGFR-2 (red) and pY1175 (green; upper panel) or VEGFR-2 (red) and pY1214 (green; middle panel). To the right in each panel a merged picture is shown, indicating that VEGFR-2 is phosphorylated at Y1175 and Y1214 in all cells expressing the receptor. EBs immunostained with antibodies against VEGFR-2 (red) and pY951 (green; lower panel) show detection of pY951 only in a subset of vessels (arrow; see merged picture to the right). (C) Costaining of EBs with VEGFR-2 (red), pY951 (green), and ASMA (blue) show that VEGFR-2-positive vessel structures surrounded by ASMA-positive pericytes lack phosphorylation at Y951. Bars: 100 μm. Download figure Download PowerPoint pY951 regulates actin reorganization downstream of VEGFR-2 To determine which VEGF-A-induced signals are transduced via pY951 in VEGFR-2, we generated stable porcine aortic endothelial (PAE) cell lines expressing Wt VEGFR-2, mutated Y951F VEGFR-2, or, as a control, Y996F VEGFR-2. These lines were examined by immunoblotting and phosphatidyl inositol phosphate assay for the capacity to mediate the VEGF-A signal to known VEGFR-2 downstream targets such as extracellular regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK), PLCγ1, and PI3-kinase/Akt pathways. However, no difference was detected in activation of these pathways between cells expressing the Wt VEGFR-2 or the mutants Y951F and Y996F (data not shown). VEGF-A is known to stimulate DNA synthesis and cell migration, involving actin stress fiber reorganization. We sought a role for Y951 in VEGF-A signal transduction regulating these responses by introduction of biotinylated, phosphorylated Y951 peptide, or the unphosphorylated counterpart as a control into human umbilical vein endothelial (HUVE) cells. The cells were stimulated with VEGF-A and stained with TRITC-phalloidin to visualize the actin cytoskeleton. VEGF-A treatment induced formation of stress fibers in untreated cells and in cells transfected with the unphosphorylated peptide (Figure 3A). In contrast, VEGF-A-induced actin stress fiber formation was blocked to basal level in cells containing the phosphorylated Y951 peptide (Figure 3A and B). Staining of cells by FITC-coupled streptavidin showed that most cells in the culture were successfully transfected; moreover, staining with phosphotyrosine antibodies revealed that phosphorylation remained for about 120 min after transfection (see Supplementary Figure 2). We therefore argued that VEGF signals dependent on pY951 would be efficiently blocked for up to 2 h, allowing analysis of long-term responses to VEGF, such as DNA synthesis. Thus, whereas VEGF-A-induced actin reorganization was blocked by the pY951 peptide to a level seen in unstimulated cells (Figure 3B), incorporation of BrdU was not significantly affected by introduction of the peptide (Figure 3C). To show that the Y951-dependent effect on actin stress fiber formation represents a motility response, we employed primary HUVE cells transduced with retrovirus encoding a fusion protein composed of the epidermal growth factor receptor (EGFR) extracellular domain fused to the transmembrane and intracellular part of VEGFR-2 of either Wt or mutant Y951F forms, designated EGDR and Y951F EGDR, respectively (see online Supplementary Figure 3 for characterization of EGDR and Y951F EGDR HUVE cells). We have previously shown that the Y951F EGDR-expressing HUVE cells undergo proliferation in response to EGF to the same extent as Wt EGDR cells (Zeng et al, 2001). A wound assay was performed which showed that EGF-treated HUVE cells expressing Wt EGDR migrated to fill the wounded cell monolayer over a 24-h time period (Figure 3D), whereas the mutant Y951F EGDR-expressing cells failed to respond to EGF in this assay (Figure 3E). Figure 3.Role of VEGFR-2 Y951 in VEGF-A-induced motility and mitogenicity responses. (A) HUVE cells were transfected or not with phosphorylated or unphosphorylated peptide covering Y951 in VEGFR-2. Visualization of actin filaments by TRITC-phalloidin showed stress fiber formation in response to VEGF-A (15 min at 37°C) and block of the response in cells containing the pY951 peptide. Bar: 10 μm. (B) Quantification of the fraction of stress fiber-forming cells. Similar results were obtained in two independent experiments. Note that there was a significant decline of VEGF-A-mediated stress fiber formation in pY951 peptide-treated cells. (C) VEGF-A-induced BrdU incorporation was similar in HUVE cells transfected with pY951 or Y951 peptides. (D) EGF-induced wound closure in HUVE cell monolayer after transduction with retrovirus encoding the EGF receptor/VEGFR-2 fusion protein (EGDR), Wt or Y951F forms. Bar: 250 μm. (E) Quantification of the number of cells moving into the wounded area in (D). Similar results were obtained twice in independent experiments. In panels B, C and E, * indicates P<0.05, and ** indicates P<0.01 (Mann–Whitney U-test). NS=not significant. Bars show mean±s.d. of triplicate wells. Download figure Download PowerPoint TSAd signals downstream of pY951 An adapter molecule denoted TSAd/RIBP/Lad/VRAP has been implicated as a binding partner of VEGFR-2/Y951 in a yeast two-hybrid screen (Wu et al, 2000). TSAd was initially discovered in T lymphocytes and is highly expressed in peripheral blood mononuclear cells (PBMC) (Spurkland et al, 1998). The use of TSAd-specific nucleotide oligomers allowed PCR amplification of fragments from PBMC as well as from HUVE cells (see Supplementary Figure 4). Analysis of expression levels of TSAd protein in resting and activated PBMC in comparison to control and VEGF-A-treated HUVE cells showed that the TSAd protein was expressed at levels in HUVE cells similar to those in activated PBMC and that TSAd expression in HUVE cells was unaffected by VEGF-A treatment (see Supplementary Figure 4). Using PAE cells expressing Wt VEGFR-2 or mutated Y951F VEGFR-2, we noted that TSAd and Wt VEGFR-2 engaged in a transient complex in response to VEGF-A (Figure 4A), which was lost in the Y951F VEGFR-2-expressing cells, thus validating previous data (Wu et al, 2000). We also used HUVE cells expressing EGDR and Y951F EGDR to show pY951-dependent complex formation between TSAd and VEGFR-2 (see Supplementary Figure 3). VEGF-A treatment induced tyrosine phosphorylation of TSAd in primary HUVE cells (Figure 4B). Transient VEGF-A-induced complex formation between VEGFR-2 and TSAd was detected both after immunoprecipitation (IP) of TSAd followed by blotting for VEGFR-2, and after IP of VEGFR-2 followed by blotting for TSAd. VEGF-A stimulation also mediated an enhanced complex formation between Src and TSAd. Figure 4.Complex formation and tyrosine phosphorylation of TSAd. (A) TSAd/VEGFR-2 complex formation in PAE cells expressing Wt VEGFR-2 or the Y951F mutant receptor (Y951F). Cells were stimulated with VEGF-A or not for 5 or 10 min and processed for immunoprecipitation (IP) of TSAd and immunoblotting (IB) for VEGFR-2. Whole-cell lysate aliquots were blotted for VEGFR-2 to show equal loading of protein. (B) HUVE cells treated with VEGF-A or not for 5 or 10 min were analyzed for complex formation between TSAd and VEGFR-2 or Src by IP and IB, as indicated. The relative migration rate of protein standards is shown to the left. The relative changes in band intensity are given below each blot. Note the VEGF-A-induced tyrosine phosphorylation of TSAd and complex formation between VEGFR-2 and TSAd, as well as TSAd and Src. Download figure Download PowerPoint We next examined whether the loss of VEGF-A-induced actin stress fiber formation and motility in the absence of Y951 phosphorylation (Figure 3) resulted from loss of coupling of TSAd to Y951 in VEGFR-2. Expression of TSAd in HUVE cells was suppressed by introduction of TSAd-specific siRNA. RT–PCR of TSAd transcript levels showed a complete loss of transcripts after introduction of 50 nM TSAd siRNA (see Supplementary Figure 5). HUVE cells that received TSAd siRNA failed to form stress fibers in response to VEGF-A, whereas cells receiving