A single amino acid substitution in the activation loop defines the decoy characteristic of VEGFR-1/FLT-1

Abstract

VEGFR-1 is a kinase-defective receptor tyrosine kinase (RTK) and negatively modulates angiogenesis by acting as a decoy receptor. The decoy characteristic of VEGFR-1 is required for normal development and angiogenesis. To date, there is no molecular explanation for this unusual characteristic of VEGFR-1. Here we show that the molecular mechanisms underlying the decoy characteristic of VEGFR-1 is linked to the replacement of a highly conserved amino acid residue in the activation loop. This amino acid is highly conserved among all the type III RTKs and corresponds to aspartic acid, but in VEGFR-1 it is substituted to asparagine. Mutation of asparagine (Asn(1050)) within the activation loop to aspartic acid promoted enhanced ligand-dependent tyrosine autophosphorylation and kinase activation in vivo and in vitro. The mutant VEGFR-1 (Asp(1050)) promoted endothelial cell proliferation but not tubulogenesis. It also displayed an oncogenic phenotype as its expression in fibroblast cells elicited transformation and colony growth. Furthermore, mutation of the invariable aspartic acid to asparagine in VEGFR-2 lowered the autophosphorylation of activation loop tyrosines 1052 and 1057. We propose that the conserved aspartic acid in the activation loop favors the transphosphorylation of the activation loop tyrosines, and its absence renders RTK to a less potent enzyme by disfavoring transphosphorylation of activation loop tyrosines.

FIGURE 1. Amino acid sequence alignment of the activation loop of type III receptor tyrosine kinases.

A schematic representation of the VEGFR-1 (FLT-1) structure is shown. The extracellular region contains seven IgG loops, the transmembrane (TM), and the cytoplasmic region containing the kinase domain (KD) indicated. The location of Asn¹⁰⁵⁰ (N1050) in the activation loop of human VEGFR-1 is indicated by an arrow. Tyrosines 1048 and 1053 are putative autophosphorylation sites. Sequence alignment of the catalytic and activation loop regions of human VEGFR-1 and various receptor tyrosine kinases of type III family are shown. The residue corresponding to asparagine 1050 of VEGFR-1 is aspartic acid in type III kinases. This residue is highly conserved and is invariable among the type III family kinases except VEGFR-1. Asparagine 1050 of VEGFR-1 is conserved among humans (GenBank^™ accession number NM002019), mice (GenBank^™ accession number BAA24498), rats (GenBank^™ accession number P53767), and chickens (GenBank^™ accession number BAB84690).

FIGURE 2. Replacement of asparagine 1050 to aspartic acid unleashes the poor tyrosine autophosphorylation and kinase activation of VEGFR-1.

Serum-starved PAE cells individually expressing either wild type chimeric VEGFR-1 (CTR), mutant VEGFR-1 (D1050/CTR), or CKR (chimeric VEGFR-2) were stimulated with CSF-1 for the indicated periods of times. Cell lysates were immunoprecipitated (Ipt) with an anti-VEGFR-1 antibody or anti-VEGFR-2 antibody. The immunoprecipitated proteins were divided to two groups and were subjected to Western blot analysis using either an anti-phosphotyrosine antibody (A) or anti-VEGFR-1 and anti-VEGFR-2 antibodies for protein levels (B). Serum-starved PAE cells expressing CTR and D1050/CTR were left unstimulated or stimulated for 10 min with CSF-1, lysed, immunoprecipitated with anti-VEGFR-1 antibody, and subjected to in vitro kinase assay using poly-Glu peptide as a substrate as described under “Materials and Methods” (C). Results are expressed as arbitrary units (AU) corresponding to the dpm values obtained from counting of the phosphorylation of substrate with a scintillation counter.

FIGURE 3. In vitro kinase activation of CTR and D1050/CTR.

Serum-starved PAE cells expressing wild type chimeric VEGFR-1 (CTR), D1050/CTR, or CKR were lysed without stimulation, and cell lysates were immunoprecipitated (Ipt) with an anti-VEGFR-1 antibody or VEGFR-2 antibody. The immunoprecipitated proteins were subjected to an in vitro kinase assay using ATP at different concentrations as indicated. The reaction stopped after 15 min, and samples were analyzed by Western blot using an anti-phosphotyrosine antibody as described under “Materials and Methods” (A). The same membranes were reprobed with an anti-VEGFR-1 and anti-VEGFR-2 antibodies for protein levels (B). Quantitation of tyrosine phosphorylation of CTR and D1050/CTR in response to ATP stimulation is shown (C). The total protein level was used to normalize the tyrosine phosphorylation values. AU (arbitrary units). Serum-starved PAE cells expressing wild type chimeric VEGFR-1 (CTR), or D1050/CTR were lysed without stimulation, and cell lysates were immunoprecipitated with an anti-VEGFR-1 antibody and divided into two groups. One group was subjected to in vitro kinase assay using cold ATP (1 mm) and incubated for the indicated periods of time. Samples were analyzed by Western blot using an anti-phosphotyrosine antibody as described under “Materials and Methods” (D). The second group was blotted for protein level using anti-VEGFR-1 antibody (E). Quantitation of tyrosine phosphorylation of CTR and D1050/CTR in response to ATP stimulation is shown (F). The Eastman Kodak Co. ID Image analysis program was used to quantify the data. The total protein level was used to normalize the tyrosine phosphorylation values. AU, arbitrary units.

FIGURE 4. Replacement of asparagine 1050 to aspartic acid allows VEGFR-1 to stimulate cell proliferation but not tubulogenesis.

Serum-starved PAE cells expressing wild type chimeric VEGFR-1 (CTR), mutant chimeric VEGFR-1 (D1050/CTR), or chimeric VEGFR-2 (CKR) were stimulated with different concentrations of CSF-1, and DNA synthesis was measured by [³H]thymidine uptake method as described under “Materials and Methods.” The results are expressed as the mean (cpm/well) ±S.D. of quadruplicates (A). PAE cells expressing wild type CTR or D1050/CTR were prepared as spheroids and subjected to in vitro angiogenesis with or without CSF-1. In addition, PAE cells expressing chimeric VEGFR-2 (CKR) were prepared and used in a similar manner as a positive control. Sprouting and tubulogenesis was observed after 24 h under an inverted phase-contrast microscope (Leica), and pictures were taken using a Leica digital camera (B). Serum-starved PAE cells individually expressing either wild type chimeric VEGFR-1 (CTR), mutant VEGFR-1 (D1050/CTR), or CKR (chimeric VEGFR-2) were stimulated with CSF-1 for the indicated periods of time. Total cell lysates were subjected to Western blot analysis using an anti-pospho-PLCγ1 antibody (C) or an anti-PLCγ1 antibody (D).

FIGURE 5. D1050 mutant VEGFR-1 promotes transformation of fibroblast cells.

NIH-3T3 cells expressing CTR and D1050/CTR were plated in 10% FBS plus CSF-1 (20 μg/ml) and maintained for 3 weeks. Additional CSF-1 was added into medium every 2 days, and the foci formation of cells was evaluated. The foci formation of cells was viewed under an inverted phase-contrast microscope (Leica) and pictures were taken using a Leica digital camera (A). NIH-3T3 cells expressing CTR and D1050/CTR were subjected to soft agar colony formation assay as described under “Materials and Methods.” After 22 days, colonies were counted. NIH-3T3 cells and NIH-3T3 cells expressing CTR did not form large colonies. NIH-3T3 cells expressing D1050/CTR formed 145 large colonies. The representative colony formations of NIH-3T3, NIH-3T3 cells expressing CTR, and NIH-3T3 cells expressing D1050/CTR are shown (B). Expression of CTR and D0150/CTR in NIH-3T3 is shown (C).

FIGURE 6. Replacement of asparagine 1050 to aspartic acid in nonchimeric VEGFR-1 enhances tyrosine autophosphorylation of VEGFR-1.

Serum-starved PAE cells expressing wild type VEGFR-1 or D1050/VEGFR-1 were lysed without stimulation, and cell lysates were immunoprecipitated with an anti-VEGFR-1 antibody and divided into two groups. One group was subjected to an in vitro kinase assay using cold ATP with different concentrations of ATP as indicated. Samples were analyzed by Western blot using an anti-phosphotyrosine antibody as described under “Materials and Methods” (A). The second group was blotted for protein level using anti-VEGFR-1 antibody (B). The graph was generated by quantitation of tyrosine phosphorylation of VEGFR-1 and D1050/VEGFR-1 in response to ATP stimulation using the Kodak ID Image analysis program (C). The total protein levels were used to normalize the tyrosine phosphorylation values. AU, arbitrary units.

FIGURE 7. Mutation of conserved aspartic acid to asparagine in VEGFR-2/FLK-1 alters auto-phosphorylation of activation loop tyrosines.

Equal numbers of serum-starved PAE cells expressing chimeric VEGFR-2 (CKR) and N1054/CKR were stimulated with CSF-1 for the indicated periods of time and lysed, and total cell lysates were subjected to Western blot analysis using anti-phospho-Tyr^1052/1057 VEGFR-2 antibody (A). The same membrane was reprobed for protein levels (B). Phosphorylation of the activation loop tyrosines (Tyr¹⁰⁵² and Tyr¹⁰⁵⁷) was quantitated, and results are expressed as arbitrary units (AU). The total protein levels were used to normalize the tyrosine phosphorylation values (C). The same cell lysates were subjected to Western blot analysis using anti-phospho-Tyr¹¹⁷³ VEGFR-2 antibody (D). The same membrane was reprobed for protein level using anti-VEGFR-2 antibody (E). PAE cells expressing wild type CKR or D1050/CKR were prepared as spheroid and subjected to in vitro angiogenesis with or without CSF-1 as described under “Materials and Methods.” Sprouting and tubulogenesis was observed after 24 h under an inverted phase-contrast microscope (Leica), and pictures were taken using a Leica digital camera (F).