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 - PubMed (original) (raw)

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

T Takahashi et al. EMBO J. 2001.

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-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.

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Figures

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Fig. 1. Wild-type (Wt) and mutant receptors of KDR/Flk-1. (A) Schematic representation of Wt and mutant of KDR/Flk-1s. D1 lacks 143 amino acids from the C-terminal region of Wt. Y1175F, Y1214F and Y801F contain a Tyr→Phe mutation at positions 1175, 1214 and 801, respectively. K868M is a kinase-inactive receptor mutant containing a Lys→Met mutation at position 868. The last amino acids of each protein are numbered. Open circles in the extracellular domain of KDR/Flk-1 indicate immunoglobulin-like domains; the transmembrane-spanning domain and the tyrosine kinase domains are represented by closed and open boxes, respectively. Amino acid alignments around Y801, Y1175 and Y1214 of KDR/Flk-1 are shown below. The putative phosphorylated tyrosines are presented in bold. The tyrosine residue and three amino acids to the C-terminal side are underlined. (B) An alignment of amino acid sequences for the PLC-γ-binding sites in several growth factor receptors.

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Fig. 2. The in vitro kinase assay, phosphoamino acid analysis and phosphopeptide mapping of KDR/Flk-1 overexpressed in NIH-3T3 and HUVE cells. (A) The in vitro kinase assay in KDR/Flk-1. The wild-type KDR/Flk-1 was immunoprecipitated from unstimulated or VEGF-A-stimulated NIH-3T3-KDR or HUVEC-KDR cells, and labeled in the in vitro kinase assay in the presence of [γ-32P]ATP. The radiolabeled KDR/Flk-1 was subjected to SDS–PAGE and autoradio graphy. (B) Phosphoamino acid analysis. The radiolabeled KDR/Flk-1 band was excised from the gel and subjected to trypsin digestion. The resulting phosphopeptides were then hydrolyzed and analyzed by two-dimensional chromatography (see Materials and methods). (C) Tryptic phosphopeptide mapping of KDR/Flk-1. The KDR/Flk-1 was labeled by the in vitro kinase assay and subjected to SDS–PAGE. The labeled KDR/Flk-1 band as shown in (A) was excised from the gel and subjected to trypsin digestion. The resultant phosphopeptides were resolved by electrophoresis at pH 8.9, followed by thin-layer chromatography (see Materials and methods).

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Fig. 3. Phosphopeptide mapping of KDR/Flk-1. Wild-type or mutant receptors were transiently expressed in 293 cells. NIH-3T3-KDR cells were used as the positive control. The KDR/Flk-1s were analyzed as indicated in the legend of Figure 2. The closed arrowheads (B and D) and the open arrowheads (E) indicate the phosphorylated peptide containing Y1175 and Y1214, respectively.

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Fig. 4. Analysis of KDR/Flk-1 mutants expressed in the adenovirus vector expression system. MSS31 cells were infected with adenovirus vectors containing wild-type or mutant KDR/Flk-1 receptors. Two days later, the cells were first starved for 12 h in DMEM–0.1% FCS and subsequently were not stimulated or stimulated with VEGF-A (10 ng/ml). Total cell lysates expressing the wild-type or various mutants of KDR/Flk-1 were analyzed by western blotting using anti-KDR/Flk-1 (A) or anti-phosphotyrosine (B) antibody. (C) The cell lysates were immunoprecipitated with anti-PLC-γ antibodies and blotted with anti-phosphotyrosine or anti-PLC-γ antibodies. (D) The same total cell lysates were blotted with anti-phosphoMAP kinase or anti-MAP kinase antibody. HUVE cells were used as the positive control.

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Fig. 5. Signal transduction from VEGF-A in BAE cells expressing LacZ, wild-type, Y1175F or K868M KDR/Flk-1. Intracellular signaling from various KDR/Flk-1s was analyzed as indicated in the legend of Figure 4, except for the in-gel kinase assay for MAP kinase (D) (see Materials and methods).

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Fig. 6. Mutation of Y1175F in KDR/Flk-1 blocks VEGF-A-induced initiation of DNA synthesis. BAE cells were infected with adenovirus vectors containing wild-type or mutant receptors and subsequently starved for 48 h in DMEM–0.1% FCS. The total cell lysates were blotted with anti-KDR/Flk-1 antibody. NIH-3T3-KDR cells were used as the positive control (A). Alternatively, BAE cells were stimulated with BSA or VEGF-A (10 ng/ml). After incubation for 16 h, cells were pulse-labeled with [3H]thymidine (1 mCi/ml) for 4 h. Subsequently, they were harvested on glass filters and incorporated radioactivity was measured. Data represent the average of triplicate samples. Fold induction was calculated relative to the values for the cells stimulated by BSA in each mutant (B).

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Fig. 7. In vivo Y1175 phosphorylation in VEGF-A-stimulated cells. MSS cells were infected with adenovirus vectors containing wild-type, Y1175F, Y1214F or Y801F. After 2 days, cells were stimulated with or without VEGF-A (A). NIH-3T3-KDR, NIH-3T3-Flt or HeLa cells were stimulated with VEGF-A, bFGF, PDGF or EGF as indicated (B). Primary endothelial cells, HUVE cells or rat SE cells were stimulated with VEGF-A, bFGF or HGF (C). Total cell lysates were blotted with anti-PY1175 (upper panel), anti-phosphotyrosine (middle panel) or anti-KDR/Flk-1 (lower panel) antibody. Histocytochemistry of HUVE cells using anti-PY1175. HUVE cells were starved and stimulated with VEGF-A, bFGF or PDGF for the indicated times. Subsequently, the cells were fixed and immunostained with anti-PY1175. Alternatively, PY1175 peptide was added as a competitor. The cells were examined with a fluorescent microscope (D). Bar, 50 µm.

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Fig. 7. In vivo Y1175 phosphorylation in VEGF-A-stimulated cells. MSS cells were infected with adenovirus vectors containing wild-type, Y1175F, Y1214F or Y801F. After 2 days, cells were stimulated with or without VEGF-A (A). NIH-3T3-KDR, NIH-3T3-Flt or HeLa cells were stimulated with VEGF-A, bFGF, PDGF or EGF as indicated (B). Primary endothelial cells, HUVE cells or rat SE cells were stimulated with VEGF-A, bFGF or HGF (C). Total cell lysates were blotted with anti-PY1175 (upper panel), anti-phosphotyrosine (middle panel) or anti-KDR/Flk-1 (lower panel) antibody. Histocytochemistry of HUVE cells using anti-PY1175. HUVE cells were starved and stimulated with VEGF-A, bFGF or PDGF for the indicated times. Subsequently, the cells were fixed and immunostained with anti-PY1175. Alternatively, PY1175 peptide was added as a competitor. The cells were examined with a fluorescent microscope (D). Bar, 50 µm.

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Fig. 8. The C-terminal SH2 domain of PLC-γ associates with KDR/Flk-1 through phosphorylated Y1175. (A) An in vivo association of KDR/Flk-1 with PLC-γ. BAE cells were infected with adenovirus vectors containing wild-type or Y1175F. After stimulation with VEGF-A (10 ng/ml), cell lysates were prepared and immunoprecipitated with anti-KDR/Flk-1 antibody. The samples were resolved by SDS–PAGE and immunoblotted with anti-PLC-γ antibodies or anti-KDR/Flk-1 as indicated. (B–E) In vitro association of KDR/Flk-1 with PLC-γ. NIH-3T3-KDR cell lysates unstimulated or stimulated with VEGF-A (10 ng/ml) were incubated with 1.5 µg of GST, GST–PLC-γ SH2–SH2, GST–PLC-γ N-SH2 or GST–PLC-γ C-SH2 protein pre-bound to glutathione–Sepharose beads for 2 h at 4°C (B). The same experiments were carried out using GST, GST–PLC-γ C-SH2, GST–Grb2 SH2 or GST–PI3 kinase N-SH2 protein (C). The same cell lysates as in (A) were incubated with 1.5 µg of GST–PLC-γ C-SH2 protein pre-bound to glutathione–Sepharose beads. During the incubation, antibody (D) or peptide (E) was added as a competitor. The beads were then washed three times with cold lysis buffer. The proteins bound to the beads or total cell lysate (TCL) were resolved by SDS–PAGE and immunoblotted with anti-KDR/Flk-1 or anti-GST antibody as shown.

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Fig. 9. Inhibition of VEGF-A-induced DNA synthesis in SE cells. SE cells were starved in modified HuMedia-EG2 medium without VEGF-A for 6–7 h. The cells were either not injected (A), injected with control normal rabbit IgG or injected with anti-PY1175 antibody (B). After 1–3 h, the injected cells were stimulated with VEGF-A and simultaneously labeled with BrdU for 20 h. The injected cells were identified by staining with FITC-labeled anti-rabbit antibody and BrdU-incorporated cells by staining with anti-BrdU antibody. Results were expressed as the percentage of stained cells exhibiting nuclear fluorescence (C).

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