Essential role of Flk-1 (VEGF receptor 2) tyrosine residue 1173 in vasculogenesis in mice

Abstract

Flk-1 (human counterpart, KDR) tyrosine kinase, which is one of the two VEGF receptors, is crucial for vascular development. Recently, we showed that, among tyrosine residues of KDR, tyrosine residues 1175 (Y1175, corresponding to Y1173 in murine Flk-1) and Y1214 (Y1212 in Flk-1) are autophosphorylated in response to VEGF, and that Y1175 is important for VEGF-dependent phospholipase C␥͞PKC͞mitogen-activated protein kinase activation leading to DNA synthesis in cultured endothelial cells. However, the importance of these tyrosine residues in Flk-1͞KDR in vivo is not yet known. To examine the role of these Flk-1 tyrosine residues in vivo, we generated knock-in mice substituting Y1173 and Y1212 of the Flk-1 gene with phenylalanine, respectively. As a result, Flk-1 1173F homozygous mice died between embryonic days 8.5 and 9.5 without any organized blood vessels or yolk sac blood islands, and hematopoietic progenitors were severely reduced, similar to the case of Flk-1 null mice. In contrast, Flk-1 1212F homozygous mice were viable and fertile. These results suggest that the signaling via Y1173 of Flk-1 is essential for endothelial and hematopoietic development during embryogenesis.Flk-1͞KDR ͉ single tyrosine-phenylalanine mutation ͉ tyrosine kinase receptor V EGF is essential for many angiogenic processes both in normal and pathological conditions (1, 2). VEGF binds two tyrosine kinase receptors, Flt-1 (also known as VEGF receptor 1) (3, 4) and Flk-1 (also known as VEGF receptor 2, KDR as human counterpart) (5-7) with high affinity. Flt-1-and Flk-1-deficient mice both die in utero between embryonic days (E) 8.5 and E9.5 but have different phenotypes. Flt-1-deficient embryos showed an overgrowth of endothelial cells and disorganization of blood vessels (8). Moreover, Flt-1 tyrosine kinase-deficient mice showed normal vascular development (9), suggesting that the Flt-1 extracellular domain acts as a negative regulator of VEGF, and that Flt-1 tyrosine kinase is not necessary for vasculogenesis during development. On the other hand, Flk-1-deficient mice lack both mature endothelial and hematopoietic cells, indicating that Flk-1 is crucial for vascular development (10).Ligand binding to Flk-1͞KDR induces autophosphorylation of Flk-1͞KDR intracellular tyrosine residues and activates several signaling pathways, leading to cell proliferation, survival, migration, and permeability (11). Recently, we have shown that tyrosine residues 1175 (Y1175) and Y1214, but not Y801, on KDR are highly autophosphorylated in response to VEGF, and that Y1175 is crucial for VEGF-dependent cell proliferation via the phospholipase C␥ (PLC␥)͞PKC͞mitogen-activated protein kinase (MAPK) pathway in cultured endothelial cells (12). However, the importance of these tyrosine residues in Flk-1͞KDR in vivo remains to be elucidated.To develop a pharmaceutical drug(s) that efficiently suppresses angiogenesis in diseases such as cancer, it is important to identify the tyrosine residue(s) that is involved in the critical signaling pathway via Flk-1͞KDR for ...

Vascular endothelial growth factor in eye disease

“…PLCγ activates protein kinase C via the production of diacylglycerol and increased concentrations of intracellular calcium. A Tyr1173Phe mutation of VEGFR-2 causes embryonic lethality due to vascular defects, mimicking the defects of VEGFR-2 −/− mice (Sakurai et al, 2005). These data demonstrate an essential function of the Tyr1173 residue during vascular development.…”

“…Mice that express a Tyr1212Phe (corresponding to the human Tyr1214) VEGFR-2 mutant are viable and fertile (Sakurai et al, 2005). However, phosphorylation of Tyr1212/1214 has been implicated in VEGF-induced actin remodeling through the sequential activation of CDC42 and p38 MAPK (Lamalice et al, 2004).…”

Purinergic regulation of vascular endothelial growth factor signaling in angiogenesis

“…Tyr1175 of VEGFR-2 and its phosphorylation have been shown to be the important regulators of VEGF-dependent angiogenic signaling (Holmqvist et al, 2004;Sakurai et al, 2005;Shibuya, 2006). This P2YR-mediated transphosphorylation and transactivation of VEGFR-2 provides further evidence that P2YR uses VEGF signaling to stimulate angiogenesis.…”

A critical role for the E3-ligase activity of c-Cbl in VEGFR-2-mediated PLCγ1 activation and angiogenesis

“…Activation of VEGF receptor-2 (VEGFR-2/Flk-1/KDR) in endothelial cells orchestrates a wide variety of biological responses that emanate from several key autophosphorylation sites within the cytoplasmic domain of VEGFR-2. Recruitment, tyrosine phosphorylation, and subsequent activation of phospholipase C␥1 (PLC␥1) by VEGFR-2 are essential for VEGFR-2-induced angiogenesis in vivo and in vitro (3)(4)(5). Indeed, a recent gene-targeting study using a knock-in strategy to replace the wild-type Flk-1 allele with a mutant Flk-1 allele harboring a point mutation in PLC␥1 binding site has demonstrated the physiological significance and essential role for PLC␥1 in angiogenesis (5).…”

“…Recruitment, tyrosine phosphorylation, and subsequent activation of phospholipase C␥1 (PLC␥1) by VEGFR-2 are essential for VEGFR-2-induced angiogenesis in vivo and in vitro (3)(4)(5). Indeed, a recent gene-targeting study using a knock-in strategy to replace the wild-type Flk-1 allele with a mutant Flk-1 allele harboring a point mutation in PLC␥1 binding site has demonstrated the physiological significance and essential role for PLC␥1 in angiogenesis (5). In earlier studies, mice nullizygous for Plc␥1 were subject to embryonic lethality due to significantly impaired vasculogenesis and erythrogenesis (6,7).…”