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| The EMBO Journal
(2001) 20, 2768–2778, doi:10.1093/emboj/20.11.2768 |
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A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC- and DNA synthesis in vascular endothelial cells |
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| Tomoko Takahashi1, Sachiko Yamaguchi1, Kazuhiro Chida2 and Masabumi Shibuya1 |
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1 Department of Genetics, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan 2 Laboratory of Cell Regulation, Department of Animal Resource Science/Applied Biological Chemistry, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan To whom correspondence should be addressed Masabumi Shibuya, shibuya@ims.u-tokyo.ac.jp Received 27 September 2000; Revised 27 March 2001; Accepted 2 April 2001. |
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| Abstract |
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| 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- 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. |
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| Keywords: binding site, KDR, Flk-1, PLC-, tyrosine kinase receptor, vascular endothelial growth factor-A |
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