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.
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-gamma 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.
Takahashi
T
T
Department of Genetics, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
Yamaguchi
S
S
Chida
K
K
Shibuya
M
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051966:Phospholipase C gamma; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017978:Receptors, Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A
D016228:Endothelial Growth Factors; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; C075278:platelet-derived growth factor A; D016222:Fibroblast Growth Factor 2; D004247:DNA; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma