Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3′-kinase binding

Abstract

Progress has been made in our understanding of the mechanism by which the binding of vascular endothelial growth factor (VEGF) to cognate receptors induces a range of biological responses, but it is far from complete. Identification of receptor autophosphorylation sites will allow us to determine how activated VEGF receptors are coupled to specific downstream signalling proteins. In the present study, we have expressed human VEGF receptors in insect cells using the baculovirus expression system, identified a major autophosphorylation site on the VEGF receptor fms-like tyrosine kinase-1 (Flt-1) by HPLC-electrospray ionization (ESI)-MS, and characterized in vitro interactions between Flt-1 and phosphatidylinositol 3'-kinase (PI3-kinase). Infection of High 5 insect cells with Flt-1 recombinant virus resulted in the expression of a 170 kDa glycoprotein, which bound VEGF with a K(d) of 2 x 10(-10) M in intact insect cells. The overexpressed recombinant Flt-1 receptors exhibited tyrosine kinase activity and were constitutively phosphorylated. Analysis of Flt-1 tryptic peptides by HPLC-ESI-MS with selective phosphate ion monitoring identified a hexapeptide (YVNAFK; where single-letter amino-acid code has been used) containing a phosphotyrosine (pTyr) residue at position 1213. Using synthetic phosphopeptides, this pTyr residue was found to be directly involved in the binding of PI3-kinase in vitro even though it did not fall within a consensus pYM/VXM PI3-kinase binding motif. These results suggest that phosphorylated Flt-1 associates with PI3-kinase at pTyr(1213) to mediate the activation of this pathway in VEGF signalling.

Identification of functional VEGF receptors on human platelets

“…The relative high amount of radioactivity in the absence of added VEGF suggests that the putative KDR receptor is constitutively tyrosine phosphorylated. Ligand‐independent receptor autophosphorylation has previously been reported for both Flt‐1 and KDR [13,14]. It is also possible that this phosphorylation partly is caused by dimerization of the receptors by the KDR antiserum as previously observed for the PDGF receptors [15]; however, platelets coincubated with VEGF and thrombin showed markedly increased tyrosine phosphorylation of the putative KDR receptor (Fig.…”

“…1B). The 190 kDa and 170 kDa proteins correspond to glycosylated and non‐glycosylated form of the KDR as recently described [13]. The relative high amount of radioactivity in the absence of added VEGF suggests that the putative KDR receptor is constitutively tyrosine phosphorylated.…”

Assessing the Activity of Cediranib, a VEGFR-2/3 Tyrosine Kinase Inhibitor, against VEGFR-1 and Members of the Structurally Related PDGFR Family

“…Cediranib treatment abolished all VEGF-stimulated phosphorylation on the 4 residues described but had greatest effect against Y1048/Y1053, the signal from this peptide sequence being reduced by nearly 37-fold when compared with the untreated control, suggesting that phosphorylation at this site was the most labile. These data contrast with previous studies that have described a number of VEGFR-1 residues in the C-terminal tail as being modulated by VEGF-A treatment, in particular Y1213 (25)(26)(27) but also Y1327 and Y1333 (27), and Y1309 in response to PlGF stimulation (24). Although peptides indicating phosphorylation at Y1213 were detected in our study, these were not modulated by ligand activation.…”

Localization of vascular endothelial growth factor (VEGF) receptors in normal and adenomatous pituitaries: detection of a non-endothelial function of VEGF in pituitary tumours

“…Since VEGF-A and PlGF were reported to stimulate growth of endothelial cells through the PI3 kinase pathway (Gerber et al 1998, Yu et al 2001, the role of this signalling cascade in MtT-S cells was studied. PlGF treatment increased phosphorylation of PDK-1, Akt (both Thr308 and Ser473) and GSK-3b (Fig.…”