A critical role for the E3-ligase activity of c-Cbl in VEGFR-2-mediated PLCgamma1 activation and angiogenesis.
Activation of phospholipase Cgamma1 (PLCgamma1) 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 PLCgamma1 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 PLCgamma1 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 phospho-tyrosines 1052 and 1057 of VEGFR-2 via its TKB domain and indirect association with phospho-tyrosine 1173 of VEGFR-2 via PLCgamma1. The tertiary complex formation between VEGFR-2, PLCgamma1 and c-Cbl selectively promotes ubiquitylation and suppression of tyrosine phosphorylation of PLCgamma1 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 PLCgamma1 to VEGFR-2 serves as a mechanism to fine-tune the angiogenic signal relay of VEGFR-2.
Singh
Amrik J
AJ
Department of Periodontology, Boston University Medical School, Boston, MA 02118, USA.
Meyer
Rosana D
RD
Navruzbekov
Gyulmagomed
G
Shelke
Rajani
R
Duan
Lei
L
Band
Hamid
H
Leeman
Susan E
SE
Rahimi
Nader
N
D000818:Animals; D004789:Enzyme Activation; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D018919:Neovascularization, Physiologic; D051966:Phospholipase C gamma / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D025801:Ubiquitin; D014443:Tyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D040301:Vascular Endothelial Growth Factor Receptor-2; D051966:Phospholipase C gamma