Endothelial cell adhesion to the extracellular matrix induces c-Src-dependent VEGFR-3 phosphorylation without the activation of the receptor intrinsic kinase activity.
BACKGROUND
Integrins cooperate with growth factor receptors to promote downstream signaling for cell proliferation and migration. However, the mechanism of receptor activation is still unknown.


OBJECTIVE
To analyze the mechanism of phosphorylation of the vascular endothelial growth factor receptor (VEGFR)-3 by cell adhesion.


RESULTS
We show that VEGFR-3 phosphorylation, induced by cell attachment to the extracellular matrix, is independent from the intrinsic kinase activity of the receptor, as evidenced from phosphorylation cell adhesion experiments with a mutant kinase dead receptor or in the presence of the specific kinase inhibitor MAZ 51. Cell adhesion experiments in the presence of the c-Src inhibitor PP2 or in fibroblast triple knockout for c-Src, Yes, and Fyn (SYF) demonstrate that VEGFR-3 phosphorylation, induced by extracellular matrix, is mediated by c-Src. Kinase assays in vitro with recombinant c-Src show that VEGFR-3 is a direct c-Src target and mass spectrometry analysis identified the sites phosphorylated by c-Src as tyrosine 830, 833, 853, 1063, 1333, and 1337, demonstrating that integrin-mediated receptor phosphorylation induces a phosphorylation pattern that is distinct from that induced by growth factors. Furthermore, pull-down assays show that integrin-mediated VEGFR-3 phosphorylation activates the recruitment to the receptor of the adaptor proteins CRKI/II and SHC inducing activation of JNK.


CONCLUSIONS
These data suggest that cell adhesion to extracellular matrix induces a downstream signaling using the tyrosine kinase receptor VEGFR-3 as scaffold.
Galvagni
Federico
F
Dipartimento di Biologia Molecolare, Università degli Studi di Siena via Fiorentina 1-53100 Siena, Italy.
Pennacchini
Susanna
S
Salameh
Ahmad
A
Rocchigiani
Marina
M
Neri
Francesco
F
Orlandini
Maurizio
M
Petraglia
Felice
F
Gotta
Stefano
S
Sardone
Gian Luca
GL
Matteucci
Giacomo
G
Terstappen
Georg C
GC
Oliviero
Salvatore
S
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002448:Cell Adhesion; D002460:Cell Line; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D023421:Models, Animal; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism; D051575:Proto-Oncogene Proteins c-yes / Q000235:genetics / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000378:metabolism; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000378:metabolism*; D019061:src-Family Kinases
D024042:Collagen Type I; D050737:Proto-Oncogene Proteins c-crk; D055765:Shc Signaling Adaptor Proteins; D011505:Protein-Tyrosine Kinases; D040321:Vascular Endothelial Growth Factor Receptor-3; D000081247:CSK Tyrosine-Protein Kinase; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; D019061:src-Family Kinases; C000631346:CSK protein, human; D048670:MAP Kinase Kinase 4