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

Abstract

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.

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.

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.

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.

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.

Figure 5

Attentuation of TSAd expression by specific siRNA in HUVE cells leads to a block in VEGF-induced stress fiber formation and motility, but not mitogenesis. (A) VEGF-A-induced actin stress fiber formation in HUVE cells transfected with 50 nM TSAd siRNA and control siRNA visualized by TRITC-phalloidin. Bar: 25 μm. (B) Quantification of VEGF-A-induced stress fiber formation in cells transfected with different concentrations of TSAd and control siRNA. (C) VEGF-A-induced wound closure in HUVE cell monolayer in cells transfected with 50 nM TSAd or control siRNA. Bar: 250 μm. (D) Quantification of the VEGF-A-induced migration of cells into the wounded area by HUVE cells transfected with TSAd or control siRNA at indicated concentrations. (E) VEGF-A-induced migration in a modified Boyden chamber of HUVE cells transfected with TSAd and control siRNA. (F) BrdU incorporation in HUVE cells transfected with TSAd or control siRNA. Similar results were obtained in two independent experiments for each assay. Bars in panels B and D–F show mean±s.d. of triplicate wells. ^*P<0.05, and ^**P<0.01 (Mann–Whitney U-test). NS=not significant.

Figure 6

Expression of TSAd in endothelial cells in EBs and in tumor vessels. (A) Coexpression of VEGFR-2 and TSAd/Lad in embryonic vessels is shown by immunostaining of whole-mount EBs treated with VEGF-A for 8 days. Bar: 100 μm. (B) Expression of TSAd in human kidney tumor vessels (top panel), in vessels in adjacent fibrous kidney tissue (middle panel), and in normal kidney vessels (bottom panel) as visualized by costaining with FITC-labeled UEA-1. Bar: 10 μm.

Figure 7

Decreased tumor growth rate and vascularization in TSAd^−/− mice. (A) Tumor growth over time in C57BL/6 Wt (n=5), TSAd^+/− (n=4), and TSAd^−/− (n=10) mice after injection of T241 fibrosarcoma cells (day 0). Error bars indicate the s.d. (B) Upper panel, visualization of vessels in C57BL/6 Wt, TSAd^+/−, and TSAd^−/− mice by staining for CD31. Bar: 100 μm. Lower panel, quantification of CD31-positive vessel area in sections from tumors from C57BL/6 Wt, TSAd^+/−, and TSAd^−/− mice. Error bars indicate the s.d.