Binding of Fumagillin to the Cytoplasmic Domain of FGFR‐1 Leads to Inhibition of FGF‐1‐stimulated Angiogenesis via the PI3K/AKT Signaling Pathway

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Objectives Fibroblast growth factor-1 (FGF-1), a prototypic member of the FGF family, is a potent angiogenic factor. Fumagillin, an antiangiogenic fungal metabolite, has the ability to inhibit FGF-stimulated angiogenesis in the chicken chorioallantoic membrane (CAM). In our current study we investigate the signaling mechanism by which fumagillin inhibits FGF-1-stimulated angiogenesis in CAM. Day eight chicken CAMs were transfected with a signal peptide-containing version of the FGF-1 gene construct (sp-FGF-1). Transfected CAMs were then analyzed in the presence and absence of fumagillin treatment with respect to the mRNA expression levels and protein activity of the FGF-1 receptor protein (FGFR-1), PI3K (phosphatidylinositol 3-kinase), and its immediate downstream target, AKT-1 (protein kinase B). In post treatment of sp-FGF-1- transfected CAMs by fumagillin, we found down-regulation for both PI3K and AKT-1 proteins in mRNA expression and protein activity. In contrast, we did not detect any major alterations in FGFR-1 mRNA expression level. To further investigate the site of inhibition by fumagillin along the PI3K signaling pathway, biotin-labeled fumagillin was individually incubated with recombinant FGFR-1, PI3K, and AKT-1 proteins. Only the FGF-1 receptor protein containing the cytoplasmic domain (amino acids 456-765) demonstrated binding to fumagillin. Based on these findings, we conclude that the binding of fumagillin to the cytoplasmic domain of the FGF-1 receptor containing the tyrosine residues inhibited the FGF-1-stimulated angiogenesis in vivo. Furthermore, our results suggest that fumagillin inhibits FGF-1-stimulated angiogenesis by disrupting the downstream PI3K/AKT signaling pathway.