A critical role for the E3-ligase activity of c-CbI in VEGFR-2-mediated PLCγ1 activation and angiogenesis

Activation of phospholipase C gamma 1 (PLC gamma 1) by vascular endothelial growth factor receptor-2 (VEGFR-2) in endothelial cells in part is responsible for angiogenesis in vivo. The cellular mechanisms exerting negative control over PLC gamma 1 activation, however, remain unaddressed. Here by using in vitro and in vivo binding assays, we show that the Casitas B-lineage lymphoma (c-Cbl) E3 ubiquitin ligase constitutively associates with PLC gamma 1 via its C-terminal domain and conditionally interacts with VEGFR-2 via the N-terminal/ TKB domain. Site-directed mutagenesis of VEGFR-2 showed that full activation of c-Cbl requires its direct association with phosphotyrosines 1052 and 1057 of VEGFR-2 via its TKB domain and indirect association with phospho-tyrosine 1173 of VEGFR-2 via PLC gamma 1. The tertiary complex formation between VEGFR-2, PLC gamma 1 and c-Cbl selectively promotes ubiquitylation and suppression of tyrosine phosphorylation of PLC gamma 1 by a proteolysis-independent mechanism. Further analysis showed that association of c-Cbl with VEGFR-2 does not impact ubiquitylation, down-regulation, or tyrosine phosphorylation of VEGFR-2. Silencing of c-Cbl by siRNA revealed that endogenous c-Cbl plays an inhibitory role in angiogenesis. Our data demonstrate that corecruitment of c-Cbl and PLC gamma 1 to VEGFR-2 serves as a mechanism to fine-tune the angiogenic signal relay of VEGFR-2.