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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 21, 2002 as doi:10.1096/fj.01-0835fje. |
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Cardiology Division, Department of Medicine, and the Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA
2Correspondence: Cardiology Division, Department of Medicine, Forchheimer 715, 1300 Morris Park Ave., Bronx, NY 10461, USA. E-mail: Terman{at}aecom.yu.edu
SPECIFIC AIMS
The goal of this study was to test the hypothesis that the fibroblast growth factor receptor substrate 2 (FRS2) participates in vascular endothelial growth factor (VEGF) -induced signal transduction.
PRINCIPAL FINDINGS
1. VEGF treatment of human umbilical vein endothelial (HUVE) cells and KDR-transfected porcine aortic endothelial (PAE) cells leads to the rapid tyrosine phosphorylation of FRS2.
2. FRS2 and KDR coimmunoprecipitate in cells not incubated with VEGF indicating that they are bound to each other, and VEGF has no effect on this interaction.
3. VEGF treatment of HUVE and KDR-transfected PAE cells leads to the recruitment of Nck, the p21-activated kinase PAK, and Crk to FRS2.
4. VEGF treatment of these cells also leads to the recruitment of Grb2 to FRS2. We did not observe an enhanced interaction of Grb2 with Shc after VEGF treatment.
5. VEGF-treatment of the endothelial cells leads to an enhancement in PKC
catalytic activity, and the recruitment of PKC to FRS2.
CONCLUSIONS AND SIGNIFICANCE
The angiogenic growth factor VEGF binds to two high-affinity receptors, known as FLT1 and KDR, that are predominantly specific to endothelial cells. Although expression of both VEGF receptors occurs in adult endothelial cells, recent findings demonstrate that KDR, and not FLT1, is able to mediate the mitogenic, chemotactic, and survival responses to VEGF.
VEGF stimulates tyrosine phosphorylation and the activities of endothelial cell signaling proteins including PLC
, MAPK, PI3-kinase, Nck, Crk, FAK, and paxillin. As KDR is a receptor tyrosine kinase, it might be expected that VEGF-induced receptor autophosphorylation leads to the recruitment of signaling proteins to specific receptor autophosphorylation sites. To date, six sites have been identified; two of these have been confirmed as sites for direct interaction with SH2 domain containing signaling proteins.
We previously reported that the SH2 domain containing adaptor proteins Crk and Nck are tyrosine phosphorylated and immunoprecipitate with KDR after VEGF treatment. To determine whether Nck and Crk bind directly to tyrosine phosphorylated receptors, we tested the effect of VEGF on Nck and Crk activation in cell lines expressing receptors mutated at cytosolic tyrosines contained within an amino acid sequence showing resemblance to known Nck and Crk recognition motifs. We found that Nck and Crk are phosphorylated and immunoprecipitate with activated KDR for each mutant receptor, indicating that the interaction of Crk and Nck with KDR is indirect and mediated by another protein.
Some receptor tyrosine kinases use various docking proteins to expand their ability to activate downstream effectors. We decided to test whether FRS2 might mediate the interaction of Nck and Crk to KDR because we found that after VEGF treatment, Nck immunoprecipitates with 60 and 90 kDa tyrosine phosphorylated proteins, and it is known that phosphorylated FRS2 migrates on SDS-PAGE gels at these weights.
FRS2 was discovered partially based on the recognition that unlike other growth factor receptors, the FGF receptor is poorly phosphorylated after ligand binding. Instead, a 90 kDa FRS2 is phosphorylated at multiple tyrosine and serine/threonine sites. Phosphorylated FRS2 recruits Grb2 and Shp2 to the cell surface as well as other downstream effectors, including PKC, PI3-K, Src, and Crk. FRS2 plays a necessary role in FGF-induced cellular responses, including activation of MAPK and PI-3 kinase, and cell migration and cell proliferation. FRS2 serves as a docking protein for at least four other receptor tyrosine kinases: the FGFR, insulin receptor (IR), TRKA nerve growth factor receptor, and the RET subunit of the GDNF neurotrophin receptor.
To test the hypothesis that FRS2 participates in VEGF-induced signal transduction, we initially asked whether VEGF treatment of endothelial cells leads to an enhancement in FRS2 phosphorylation. HUVE cells were incubated with or without VEGF, the cells were lysed, the proteins immunoprecipitated using anti-FRS2 antibody, and Western blotting was done using anti-phosphotyrosine antibody. As shown in Fig. 1
, after VEGF treatment, tyrosine phosphorylated proteins of 90, 60, 47, and 20 kDa immunoprecipitated using the anti-FRS2 antibody.
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The predicted molecular mass of FRS2, based on its amino acid sequence is 60 kDa, but depending on its degree of phosphorylation, FRS2 electrophoreses as multiple proteins up to 90 kDa by SDS-PAGE. To determine whether the 60 and 90 kDa tyrosine phosphorylated proteins observed in Fig. 1
are FRS2, we immunoprecipitated VEGF-treated cell lysates using the anti-phosphotyrosine antibody and performed Western blotting using the anti-FRS2 antibody. Phosphorylated proteins of 60 and 90 kDa were detected. The relative amounts of the 60 and 90 kDa FRS2 species varied between experiments and intermediate molecular weight species were often observed.
It is known that FRS2 participates in bFGF-induced signal transduction, and we observed an enhanced FRS2 tyrosine phosphorylation in response to VEGF and bFGF in the KDR-transfected PAE cells. To rule out the possibility that VEGF augments FRS2 phosphorylation not through binding and signaling through KDR, but by a mechanism by which VEGF stimulates the secretion of bFGF, we used a commercially available ELISA kit to quantitate the amount of bFGF secretion in cells treated or not with VEGF; it was found that VEGF has no effect on bFGF secretion (data not shown).
The molecular interactions by which FRS2 participates in FGF signaling are different from that for the NGF and GDNF in that FRS2 binds to the FGFR in quiescent cells whereas the interaction of FRS2 with TRKA and RET is growth factor dependent. To gain insight as to the molecular interactions by which FRS2 participates in VEGF-induced signaling, we immunoprecipitated lysates prepared from VEGF-treated and control cells using anti-FRS2 antibody and did Western blotting using anti-KDR antibody. It was found that the amount of KDR was the same in both samples, indicating similarity to what is observed for FGF and its receptor.
As shown in Fig. , 1a
47 kDa tyrosine-phosphorylated protein immunoprecipitates with FRS2 after VEGF treatment. This is the size of Nck, a cell signaling adaptor protein that shows enhanced tyrosine phosphorylation after VEGF treatment. To test the hypothesis that VEGF treatment leads to a recruitment of Nck to FRS2, an experiment was performed where HUVE cell lysates were immunoprecipitated using the FRS2 antibody; Western blotting was done using a Nck antibody. An enhanced level of Nck was found in the FRS2 immunoprecipitates after VEGF treatment (Fig. 2
A).
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We have previously documented that Nck interacts with PAK in VEGF-treated and control cells. After VEGF treatment, the Nck/Pak complex is recruited to the cell surface where PAK is activated, presumably by Rac and cdc42. Since VEGF treatment leads to an interaction of Nck with FRS2, it would be expected that PAK would also interact with FRS2. This was confirmed in an experiment that showed that VEGF treatment enhanced the amount of PAK in FRS2 immunoprecipitates (Fig. 2C
).
We have also obtained experimental results demonstrating that VEGF treatment leads to the recruitment of Crk (Fig. 2E
), Grb2, and PKC to FRS2. These results are similar to that found for other growth factors which signal through FRS2.
The described results imply that VEGF is similar to some other growth factors that use docking proteins as part of the signaling pathways participating in cellular activation. FRS2 participates in the signaling pathways activated by at least four other growth factors. Other growth factors use docking proteins distinct from FRS2; for example, members of the insulin receptor substrate family of docking proteins have an amino-terminal PH domain, followed by a PTB domain and a large carboxyl-terminal sequence containing numerous tyrosine phosphorylation sites. Dok has a modular structure similar to that of the IRS proteins. p130CAS (Crk-associated substrate) and its structurally related proteins Efs/Sin and Hef 1) contains an SH3 domain, two proline-rich regions, and a substrate domain consisting of 15 potential SH-2 binding motifs. Another example would be the Gab family of proteins, which contain one amino-terminal PH domain and multiple SH2 domains.
The utilization of FRS2 in VEGF and other growth factor-induced signaling offers several advantages. First, docking proteins provide additional recruitment sites for signaling proteins that might allow for an increased number of signaling pathways that are activated by growth factor. Second, docking proteins could potentially localize interacting signaling proteins in close proximity to each other, and thus provide coordination in the signaling cascade. Evidence supporting this notion has been documented for FRS2’s role in bFGF signaling.
The fact that FRS2 participates in VEGF and bFGF signal transduction may imply cross-talk between these two signaling pathways. This would be similar to that observed during neuronal differentiation, where it has been suggested that FRS2 serves as a molecular switch that mediates the interplay between FGFR and TRK. VEGF and bFGF are angiogenic growth factors and stimulate both similar and distinct cellular responses. One potential mechanism by which FRS2 might mediate cross-talk between KDR and the FGFR would be that since FRS2 interacts constitutively with both receptors, the enhanced expression of one receptor, say KDR, might recruit the cellular complement of FRS2 to KDR, thus blocking the signaling by the FGFR.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0835fje; to cite this article, use FASEB J. (June 21, 2002) 10.1096/fj.01-0835fje 
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