Vascular endothelial growth factor-dependent down-regulation of Flk-1/KDR involves Cbl-mediated ubiquitination. Consequences on nitric oxide production from endothelial cells.
Ligand-stimulated degradation of receptor tyrosine kinase (RTK) is an important regulatory step of signal transduction. The vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is responsible for the VEGF-stimulated nitric oxide (NO) production from endothelial cells. Cellular mechanisms mediating the negative regulation of Flk-1 signaling in endothelial cells have not been investigated. Here we show that Flk-1 is rapidly down-regulated following VEGF stimulation of bovine aortic endothelial cells (BAECs). Consequently, VEGF pretreatment of endothelial cells prevents any further stimulation of Flk-1, resulting in decreased NO production from subsequent VEGF challenges. Ubiquitination of RTKs targets them for degradation; we demonstrate that activation of Flk-1 by VEGF leads to its polyubiquitination in BAECs. Furthermore, VEGF stimulation of BAECs or COS-7 cells transiently transfected with Flk-1 results in the phosphorylation of the ubiquitin ligase Cbl, the enhanced association of Cbl with Flk-1, and the relocalization of Cbl to vesicular structures in BAECs. Overexpression of Cbl in COS-7 cells enhances VEGF-induced ubiquitination of Flk-1, whereas a Cbl mutant lacking the ubiquitin ligase RING finger domain, 70Z/3-Cbl, does not. Moreover, expression of Cbl in contrast to 70Z/3-Cbl inhibits the Flk-1-dependent activation of eNOS and, thus, NO release. In BAEC overexpressing Cbl, the degradation of Flk-1 upon VEGF stimulation is accelerated compared with cells transfected with a control vector (green fluorescent protein). Our findings demonstrate that Flk-1 is rapidly down-regulated following sustained VEGF stimulation and identify Cbl as a negative regulator of Flk-1 signaling to eNOS. Cbl thus plays a role in the regulation of VEGF signaling by mediating the stimulated ubiquitination and, consequently, degradation of Flk-1 in endothelial cells.
Duval
Martine
M
Laboratory of Endothelial Cell Biology, Institut de Recherches Cliniques de Montréal (IRCM), Quebec H2W 1R7, Canada.
Bédard-Goulet
Sara
S
Delisle
Chantal
C
Gratton
Jean-Philippe
JP
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D015536:Down-Regulation / Q000502:physiology*; D016228:Endothelial Growth Factors / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000502:physiology*; D008222:Lymphokines / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D050722:Oncogene Protein v-cbl; D015682:Retroviridae Proteins, Oncogenic / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000502:physiology*; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D050722:Oncogene Protein v-cbl; D015682:Retroviridae Proteins, Oncogenic; D025801:Ubiquitin; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D009569:Nitric Oxide; D040301:Vascular Endothelial Growth Factor Receptor-2