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 170kDa glycoprotein, which bound VEGF with a Kd of 2×10−10M 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 pTyr1213 to mediate the activation of this pathway in VEGF signalling.
Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3′-kinase binding
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Ying YU, Jeffrey D. HULMES, Mark T. HERLEY, Ronald G. WHITNEY, John W. CRABB, J. Denry SATO; Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3′-kinase binding. Biochem J 1 September 2001; 358 (2): 465–472. doi: https://doi.org/10.1042/bj3580465
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