Abstract
Vascular endothelial growth factors (VEGFs) regulate the development and growth of the blood and lymphatic vascular systems. Of the three VEGF receptors (VEGFR), VEGFR-1 and -2 are expressed on blood vessels; VEGFR-2 is found also on lymphatic vessels. VEGFR-3 is expressed mainly on lymphatic vessels but it is also up-regulated in tumor angiogenesis. Although VEGFR-3 is essential for proper lymphatic development, its signal transduction mechanisms are still incompletely understood. Trans-phosphorylation of activated, dimerized receptor tyrosine kinases is known to be critical for the regulation of kinase activity and for receptor interaction with signal transduction molecules. In this study, we have identified five tyrosyl phosphorylation sites in the VEGFR-3 carboxyl-terminal tail. These sites were used both in VEGFR-3 overexpressed in 293 cells and when the endogenous VEGFR-3 was activated in lymphatic endothelial cells. Interestingly, VEGF-C stimulation of lymphatic endothelial cells also induced the formation of VEGFR-3/VEGFR-2 heterodimers, in which VEGFR-3 was phosphorylated only at three of the five sites while the two most carboxyl-terminal tyrosine residues appeared not to be accessible for the VEGFR-2 kinase. Our data suggest that the carboxyl-terminal tail of VEGFR-3 provides important regulatory tyrosine phosphorylation sites with potential signal transduction capacity and that these sites are differentially used in ligand-induced homo- and heterodimeric receptor complexes.
Highlights
The receptor tyrosine kinase vascular endothelial growth factor receptor 3 (VEGFR-3,1 previously denoted fms-like tyrosine kinase-4 or Flt-4) is essential for the development of the
Stimulation of PAE cells expressing VEGF receptors (VEGFR)-3 with Vascular endothelial growth factors (VEGFs)-C resulted in strong induction of VEGFR-3 phosphorylation, as estimated in an in vitro immunocomplex kinase assay (Fig. 2A)
Trypsin-digested peptides of the receptor were separated by electrophoresis according to the charge/mass ratio (x-axis) and by liquid chromatography according to hydrophobicity (y-axis), creating a phosphopeptide map
Summary
Growth Factors and Antibodies—The growth factors used were epidermal growth factor (EGF, no. 100 –15; Peprotech, Rockyhill, NJ), human VEGF (no. 100 –20; Peprotech, Rockyhill, NJ), and human VEGF-C (Thr-103-Leu-215; Ref. 22). Human 293T cells were used for transient expression of VEGFR-3 and were maintained in Dulbecco’s modified Eagle’s medium/10% fetal calf serum. The phosphorylated bands on the membrane from the in vitro complex assay were localized using the BioImager scan, cut out and treated for 30 min at 37 °C in 0.5% polyvinylpyrrolidone in 100 mM acetic acid, washed four times in H2O, and digested by 1 g of trypsin (V542A; Promega, Madison, WI) in 200 l of 50 mM NH4CO3 at 37 °C overnight. The samples were dissolved in 7 l of pH 1.9 buffer supplemented with non-radioactive phosphorylated serine, threonine, and tyrosine as markers and spotted onto a thin-layer chromatography plate. By overlaying the resulting image with the ninhydrin pattern of the marker amino acids, the identity of the amino acid residues with incorporated 32P was determined
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