Document Detail

Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3'-kinase binding.
MedLine Citation:
PMID:  11513746     Owner:  NLM     Status:  MEDLINE    
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.
Y Yu; J D Hulmes; M T Herley; R G Whitney; J W Crabb; J D Sato
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Biochemical journal     Volume:  358     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  2001 Sep 
Date Detail:
Created Date:  2001-08-21     Completed Date:  2001-10-04     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  England    
Other Details:
Languages:  eng     Pagination:  465-72     Citation Subset:  IM    
Surgical Research Laboratory, Children's Hospital, Boston, MA 02115, USA.
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MeSH Terms
1-Phosphatidylinositol 3-Kinase / metabolism*
Binding Sites
Cells, Cultured
Cloning, Molecular
Endothelial Growth Factors / metabolism
Endothelium, Vascular / metabolism
Lymphokines / metabolism
Mass Spectrometry
Phosphotyrosine / metabolism
Protein Binding
Proto-Oncogene Proteins / chemistry*,  genetics,  metabolism*
Receptor Protein-Tyrosine Kinases / chemistry*,  genetics,  metabolism*
Recombinant Proteins / chemistry,  metabolism
Spodoptera / genetics
Vascular Endothelial Growth Factor A
Vascular Endothelial Growth Factor Receptor-1
Vascular Endothelial Growth Factors
Grant Support
Reg. No./Substance:
0/Endothelial Growth Factors; 0/Lymphokines; 0/Proto-Oncogene Proteins; 0/Recombinant Proteins; 0/Vascular Endothelial Growth Factor A; 0/Vascular Endothelial Growth Factors; 21820-51-9/Phosphotyrosine; EC 3-Kinase; EC Protein-Tyrosine Kinases; EC Endothelial Growth Factor Receptor-1

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