Activation of phospholipase Cγ1 (PLCγ1) 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 PLCγ1 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 PLCγ1 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 PLCγ1. The tertiary complex formation between VEGFR-2, PLCγ1 and c-Cbl selectively promotes ubiquitylation and suppression of tyrosine phosphorylation of PLCγ1 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 PLCγ1 to VEGFR-2 serves as a mechanism to fine-tune the angiogenic signal relay of VEGFR-2.
|Original language||English (US)|
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Mar 27 2007|
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