Src kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells.
BACKGROUND
The cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor) - dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1) and Flt-1 (Fms-like tyrosine kinase-1). However, to date, it has not been determined which VEGF receptor (VEGFR) is involved in binding to and activating Src kinase following VEGF stimulation of the receptors.


RESULTS
In this report, we demonstrate that Src preferentially associates with KDR/Flk-1 rather than Flt-1 in human umbilical vein endothelial cells (HUVECs), and that VEGF stimulation resulted in an increase of Src activity associated with activated KDR/Flk-1. These findings were determined through immunoprecipitation-kinase experiments and coimmunoprecipitation studies, and were further confirmed by GST-pull-down assays and Far Western studies. However, Fyn and Yes, unlike Src, were found to associate preferentially with Flt-1.


CONCLUSIONS
Thus, Src preferentially associates with KDR/Flk-1, rather than with Flt-1, upon VEGF stimulation in endothelial cells. Our findings further highlight the potential significance of upregulated KDR/Flk-1-associated Src activity in the process of angiogenesis, and help to elucidate more clearly the specific roles and mechanisms involving Src family tyrosine kinase in VEGF-stimulated signal transduction events.
Chou
Mary T
MT
Department of Biochemistry and Molecular Biology, University of Calgary Health Sciences Center, 3330 Hospital Dr, N,W, Calgary, AB, Canada T2N 4N1. mthchou@ucalgary.ca
Wang
Jing
J
Fujita
Donald J
DJ
D000081247:CSK Tyrosine-Protein Kinase; D002478:Cells, Cultured; D003593:Cytoplasm / Q000201:enzymology; D016228:Endothelial Growth Factors / Q000302:isolation & purification / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008222:Lymphokines / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013997:Time Factors; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D042442:Vascular Endothelial Growth Factors; D018909:src Homology Domains / Q000502:physiology; D019061:src-Family Kinases
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D000081247:CSK Tyrosine-Protein Kinase; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; D019061:src-Family Kinases; C000631346:CSK protein, human