
<ArticleTitle>Direct identification of a major autophosphorylation site on <prot><prot>vascular endothelial growth factor</prot> receptor</prot> <prot>Flt-1</prot> that mediates <prot>phosphatidylinositol 3'-kinase</prot> binding.</ArticleTitle>
<AbstractText>Progress has been made in our understanding of the mechanism by which the binding of <prot>vascular endothelial growth factor</prot> (<prot>VEGF</prot>) 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 <prot><prot>VEGF</prot> receptor</prot>s are coupled to specific downstream signalling proteins. In the present study, we have expressed human <prot><prot>VEGF</prot> receptor</prot>s in insect cells using the baculovirus expression system, identified a major autophosphorylation site on the <prot><prot>VEGF</prot> receptor</prot> <prot>fms-like tyrosine kinase-1</prot> (<prot>Flt-1</prot>) by HPLC-electrospray ionization (ESI)-MS, and characterized in vitro interactions between <prot>Flt-1</prot> and <prot>phosphatidylinositol 3'-kinase</prot> (<prot>PI3-kinase</prot>). Infection of High 5 insect cells with <prot>Flt-1</prot> recombinant virus resulted in the expression of a 170 kDa glycoprotein, which bound <prot>VEGF</prot> with a K(d) of 2 x 10(-10) M in intact insect cells. The overexpressed recombinant <prot><prot>Flt-1</prot> receptor</prot>s exhibited tyrosine kinase activity and were constitutively phosphorylated. Analysis of <prot>Flt-1</prot> 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 <prot>PI3-kinase</prot> in vitro even though it did not fall within a consensus pYM/VXM <prot>PI3-kinase</prot> binding motif. These results suggest that phosphorylated <prot>Flt-1</prot> associates with <prot>PI3-kinase</prot> at pTyr(1213) to mediate the activation of this pathway in <prot>VEGF</prot> signalling.</AbstractText>
