Interactions of FLT-1 and KDR with Phospholipase C γ: Identification of the Phosphotyrosine Binding Sites

Cunningham, Sonia; Arrate, Maria Pia; Brock, Tommy A.; Waxham, M. Neal

doi:10.1006/bbrc.1997.7719

Cited by 81 publications

(62 citation statements)

References 18 publications

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“…PLC␥ binding to both KDR (40,41) and Flt-1 (19,42,43) has been reported. To determine which VEGF receptor(s) are involved in PLC␥ activation in primary endothelial cells, HUVEC were treated with VEGF or VEGF receptor-selective mutants, and PLC␥ phosphorylation was assessed after immunoprecipitation (Fig.…”

Section: Resultsmentioning

confidence: 99%

Analysis of Biological Effects and Signaling Properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2)

Gille

Kowalski

Li³

et al. 2001

Journal of Biological Chemistry

537

177

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Section: Resultsmentioning

confidence: 99%

Analysis of Biological Effects and Signaling Properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2)

Gille

Kowalski

Li³

et al. 2001

Journal of Biological Chemistry

537

177

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“…Dougher- Vermazen et al (15) reported that in a bacteria expression system, Y951, Y996, Y1054, and Y1059 on KDR were phosphorylated. On the other hand, Cunningham et al (16) reported that in the yeast system, Y801 and Y1175 on KDR were phosphorylated. The role of these tyrosine residues for Flk-1͞KDR-mediated signal transduction remains controversial.…”

Section: Discussionmentioning

confidence: 99%

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

Sakurai

Ohgimoto²,

Kataoka³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

286

250

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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 ...

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“…In order to identify the phosphorylated tyrosine residues of the VEGFR-2, essential for the activation of intracellular signaling pathways leading to eNOS activation and NO production, we generated punctual substitutions on the receptor. Tyrosine residues 801, 1175, and 1214, proposed in the literature as being potential autophosphorylation sites of the VEGFR-2 receptor, were changed to phenylalanine (8,27,28,(35)(36)(37)41). In addition, a kinase-inactive receptor that has a lysine to arginine mutation at residue 868 in the ATP binding site of the tyrosine kinase domain was generated as a negative control.…”

Section: Vegfr-2 Tyrosinementioning

confidence: 99%

“…Many of these ligand-induced autophosphorylated tyrosines have been directly identified through systematic phosphomapping of activated receptors or have been proposed to be phosphorylated based on their essential role in the activation of the defined signaling pathway by VEGF. Some of the proposed phosphorylated tyrosines residues are 801, 951, 996, 1008, 1054, 1059, 1175, and 1214 (8,(25)(26)(27)(28). Although some slight discrepancies are present in the literature on the implication of certain residues in VEGFR-2 signaling, a consensus emerged on the role of some.…”

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confidence: 99%

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Phosphorylation of Tyrosine 801 of Vascular Endothelial Growth Factor Receptor-2 Is Necessary for Akt-dependent Endothelial Nitric-oxide Synthase Activation and Nitric Oxide Release from Endothelial Cells

Blanes¹,

Oubaha

Rautureau

et al. 2007

Journal of Biological Chemistry

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Vascular endothelial growth factor (VEGF)-stimulated nitric oxide (NO) release from endothelial cells is mediated through the activation of VEGF receptor-2 (VEGFR-2). Herein, we have attempted to determine which autophosphorylated tyrosine residue on the VEGFR-2 is essential for VEGF-mediated endothelial nitric-oxide synthase (eNOS) activation and NO production from endothelial cells. Tyrosine residues 801, 1175, and 1214 of the VEGFR-2 were mutated to phenylalanine, and the mutated receptors were analyzed for their ability to stimulate NO production. We show, both in COS-7 cells cotransfected with the VEGFR-2 mutants and eNOS and in bovine aortic endothelial cells, that the Y801F-VEGFR-2 mutant is unable to stimulate NO synthesis and eNOS activation in contrast to the wild type, Y1175F-VEGFR-2, and Y1214F-VEGFR-2. However, the Y801F mutant retains the capacity to activate phospholipase C-␥ in contrast to the Y1175F-VEGFR-2. Interestingly, the Y801F-VEGFR-2, in contrast to the wild type receptor, does not fully activate phosphatidylinositol 3-kinase or recruit the p85 subunit upon receptor activation. This results in a complete incapacity of the Y801F-VEGFR-2 to stimulate Akt activation and eNOS phosphorylation on serine 1179 in endothelial cells. In addition, constitutive activation of Akt or a phosphomimetic mutant of eNOS (S1179D) fully rescues the inability of the Y801F-VEGFR-2 to induce NO release. Finally, we generated an antibody that specifically recognizes the phosphorylated form of tyrosine 801 of the VEGFR-2 and demonstrate that this residue is actively phosphorylated in response to VEGF stimulation of endothelial cells. We thus conclude that autophosphorylation of tyrosine residue 801 of the VEGFR-2 is essential for VEGFstimulated NO production from endothelial cells, and this is primarily accomplished via the activation of phosphatidylinositol 3-kinase and Akt signaling to eNOS.

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Interactions of FLT-1 and KDR with Phospholipase C γ: Identification of the Phosphotyrosine Binding Sites

Cited by 81 publications

References 18 publications

Analysis of Biological Effects and Signaling Properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2)

Analysis of Biological Effects and Signaling Properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2)

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

Phosphorylation of Tyrosine 801 of Vascular Endothelial Growth Factor Receptor-2 Is Necessary for Akt-dependent Endothelial Nitric-oxide Synthase Activation and Nitric Oxide Release from Endothelial Cells

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