Figure 1

VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1) associate with and tyrosine phosphorylate β-catenin. A, Western blots of immunoprecipitates of HUVEC (Hu) and EOMA cell (EO) lysates with antibodies directed against VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1) reveal differentially coprecipitated β-catenin. EOMA cells display coprecipitated β-catenin, whereas negligible amounts of β-catenin were noted in the HUVEC immunoprecipitates. B, HUVEC (Hu) and EOMA (EO) cell lysates were immunoprecipitated with anti-Flk-1 antibodies, followed by Flk-1 (upper panel) Western blot analysis. The same membrane was stripped and reprobed with anti-phosphotyrosine (P-Y, second panel) and anti-β-catenin (third panel) antibodies. HUVEC and EOMA total cell lysates were immunoblotted with antivascular endothelial growth factor (anti-VEGF) antibodies (fourth panel), stripped, and reprobed with anti-actin (lower panel) antibodies. Note the robust VEGF expression, Flk-1 tyrosine phosphorylation, and association with β-catenin in EOMA cells, compared with the modest Flk-1 tyrosine phosphorylation and nondetectable β-catenin association and VEGF expression in HUVEC cells. C, HUVEC and EOMA cell proliferation rates were assessed in the absence (diamonds) and presence of VEGF (squares) and anti-VEGF–neutralizing antibody (circles). Cells (4.0 × 10⁴ per 60-mm Petri dish) were plated and cultured for 6 days in the absence or presence of 10 ng/ml VEGF or anti-VEGF. Triplicate samples were counted on days 0, 3, and 6. NS denotes a P value of ≥ 0.05. D, HUVEC were left unstimulated or stimulated with VEGF at the indicated concentrations for 15 minutes and cell lysates were immunoprecipitated with anti-Flt-1 and Flk-1 antibodies, followed by β-catenin immunoblotting. Note β-catenin association with Flt-1, but not Flk-1, in a manner that is VEGF dose dependent. E, HUVEC and EOMA cell cultures were immunoprecipitated for Flk-1 as in B, and the resulting precipitates were subjected to an in vitro kinase assay for recombinant β-catenin in the presence of [γ-³²P]ATP. Precipitated Flk-1 from HUVEC and EOMA both phosphorylated the recombinant β-catenin. However, the EOMA-derived material elicited a more robust phosphorylation compared with the HUVEC-derived precipitate, in agreement with higher PY levels noted in B (second panel).