A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells
Open Access
- 1 June 2001
- journal article
- research article
- Published by Springer Science and Business Media LLC in The EMBO Journal
- Vol. 20 (11), 2768-2778
- https://doi.org/10.1093/emboj/20.11.2768
Abstract
KDR/Flk‐1 tyrosine kinase, one of the two vascular endothelial growth factor (VEGF) receptors, induces mitogenesis and differentiation of vascular endothelial cells. To understand the mechanisms underlying the VEGF‐A‐induced growth signaling pathway, we constructed a series of human KDR mutants and examined their biological properties. An in vitro kinase assay and subsequent tryptic peptide mapping revealed that Y1175 and Y1214 are the two major VEGF‐A‐dependent autophosphorylation sites. Using an antibody highly specific to the phosphoY1175 region, we demonstrated that Y1175 is phosphorylated rapidly in vivo in primary endothelial cells. When the mutated KDRs were introduced into the endothelial cell lines by adenoviral vectors, only the Y1175F KDR, Tyr1175 to phenylalanine mutant, lost the ability to tyrosine phosphorylate phospholipase C‐γ and, significantly, reduced MAP kinase phosphorylation and DNA synthesis in response to VEGF‐A. Furthermore, primary endothelial cells microinjected with anti‐phosphoY1175 antibody clearly decreased DNA synthesis compared with control cells. These findings strongly suggest that autophosphorylation of Y1175 on KDR is crucial for endothelial cell proliferation, and that this region is a new target for anti‐angiogenic reagents.Keywords
This publication has 41 references indexed in Scilit:
- Sck Interacts with KDR and Flt-1 via Its SH2 DomainBiochemical and Biophysical Research Communications, 1998
- Interactions of FLT-1 and KDR with Phospholipase C γ: Identification of the Phosphotyrosine Binding SitesBiochemical and Biophysical Research Communications, 1997
- The Phosphorylated 1169-Tyrosine Containing Region of Flt-1 Kinase (VEGFR-1) Is a Major Binding Site for PLCγBiochemical and Biophysical Research Communications, 1997
- Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endotheliumNature, 1995
- Failure of blood-island formation and vasculogenesis in Flk-1-deficient miceNature, 1995
- Biological Activity and Phosphorylation Sites of the Bacterially Expressed Cytosolic Domain of the KDR VEGF-ReceptorBiochemical and Biophysical Research Communications, 1994
- SH2 domains recognize specific phosphopeptide sequencesCell, 1993
- Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factorBiochemical and Biophysical Research Communications, 1992
- What Is the Evidence That Tumors Are Angiogenesis Dependent?JNCI Journal of the National Cancer Institute, 1990
- Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cellsBiochemical and Biophysical Research Communications, 1989