PMID- 11513746
OWN - NLM
STAT- MEDLINE
DCOM- 20011004
LR  - 20190501
IS  - 0264-6021 (Print)
IS  - 1470-8728 (Electronic)
IS  - 0264-6021 (Linking)
VI  - 358
IP  - Pt 2
DP  - 2001 Sep 1
TI  - Direct identification of a major autophosphorylation site on vascular endothelial 
      growth factor receptor Flt-1 that mediates phosphatidylinositol 3'-kinase 
      binding.
PG  - 465-72
AB  - 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.
FAU - Yu, Y
AU  - Yu Y
AD  - Surgical Research Laboratory, Children's Hospital, Boston, MA 02115, USA.
FAU - Hulmes, J D
AU  - Hulmes JD
FAU - Herley, M T
AU  - Herley MT
FAU - Whitney, R G
AU  - Whitney RG
FAU - Crabb, J W
AU  - Crabb JW
FAU - Sato, J D
AU  - Sato JD
LA  - eng
GR  - EY06603/EY/NEI NIH HHS/United States
GR  - P40-RR15452/RR/NCRR NIH HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - England
TA  - Biochem J
JT  - The Biochemical journal
JID - 2984726R
RN  - 0 (Endothelial Growth Factors)
RN  - 0 (Lymphokines)
RN  - 0 (Proto-Oncogene Proteins)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Vascular Endothelial Growth Factor A)
RN  - 0 (Vascular Endothelial Growth Factors)
RN  - 21820-51-9 (Phosphotyrosine)
RN  - EC 2.7.1.- (Phosphatidylinositol 3-Kinases)
RN  - EC 2.7.10.1 (Receptor Protein-Tyrosine Kinases)
RN  - EC 2.7.10.1 (Vascular Endothelial Growth Factor Receptor-1)
SB  - IM
MH  - Animals
MH  - Binding Sites
MH  - Cells, Cultured
MH  - Cloning, Molecular
MH  - Endothelial Growth Factors/metabolism
MH  - Endothelium, Vascular/metabolism
MH  - Glycosylation
MH  - Humans
MH  - Lymphokines/metabolism
MH  - Mass Spectrometry
MH  - Phosphatidylinositol 3-Kinases/*metabolism
MH  - Phosphorylation
MH  - Phosphotyrosine/metabolism
MH  - Protein Binding
MH  - Proto-Oncogene Proteins/*chemistry/genetics/*metabolism
MH  - Receptor Protein-Tyrosine Kinases/*chemistry/genetics/*metabolism
MH  - Recombinant Proteins/chemistry/metabolism
MH  - Spodoptera/genetics
MH  - Transfection
MH  - Vascular Endothelial Growth Factor A
MH  - Vascular Endothelial Growth Factor Receptor-1
MH  - Vascular Endothelial Growth Factors
PMC - PMC1222080
EDAT- 2001/08/22 10:00
MHDA- 2001/10/05 10:01
PMCR- 2002/03/01
CRDT- 2001/08/22 10:00
PHST- 2001/08/22 10:00 [pubmed]
PHST- 2001/10/05 10:01 [medline]
PHST- 2001/08/22 10:00 [entrez]
PHST- 2002/03/01 00:00 [pmc-release]
AID - 10.1042/0264-6021:3580465 [doi]
PST - ppublish
SO  - Biochem J. 2001 Sep 1;358(Pt 2):465-72. doi: 10.1042/0264-6021:3580465.