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 VEGFR2/KDR/Flk-1/VEGFR2/KDR/Flk-1 is responsible for the VEGF-stimulated nitric oxide (NO) production from endothelial cells. Cellular mechanisms mediating the negative regulation of VEGFR2/KDR/Flk-1 signaling in endothelial cells have not been investigated. Here we show that VEGFR2/KDR/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 VEGFR2/KDR/Flk-1, resulting in decreased NO production from subsequent VEGF challenges. Ubiquitination of RTKs targets them for degradation; we demonstrate that activation of VEGFR2/KDR/Flk-1 by VEGF leads to its polyubiquitination in BAECs. Furthermore, VEGF stimulation of BAECs or COS-7 cells transiently transfected with VEGFR2/KDR/Flk-1 results in the phosphorylation of the ubiquitin Ligase Cbl, the enhanced association of Cbl with VEGFR2/KDR/Flk-1, and the relocalization of Cbl to vesicular structures in BAECs. Overexpression of Cbl in COS-7 cells enhances VEGF-induced ubiquitination of VEGFR2/KDR/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 VEGFR2/KDR/Flk-1 upon VEGF stimulation is accelerated compared with cells transfected with a control vector (green fluorescent protein). Our findings demonstrate that VEGFR2/KDR/Flk-1 is rapidly down-regulated following sustained VEGF stimulation and identify Cbl as a negative regulator of VEGFR2/KDR/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 VEGFR2/KDR/Flk-1 in endothelial cells.