Cbl-mediated ubiquitinylation and negative regulation of Vav.

The Cbl ubiquitin ligase has emerged as a negative regulator of receptor and non-receptor tyrosine kinases. Cbl is known to associate with the proto-oncogene product Vav, a hematopoietic-restricted Rac guanine nucleotide exchange factor, but the consequences of this interaction remain to be elucidated. Using immortalized T cell lines from Cbl(+/+) ...
and Cbl(-/-) mice, and transfection analyses in 293T cells, we demonstrate that Vav undergoes Cbl-dependent ubiquitinylation under conditions that promote Cbl and Vav phosphorylation. Interaction with Cbl also induced the loss of phosphorylated Vav. In addition, we show that an activated Vav mutant (Vav-Y174F) is more sensitive to Cbl-dependent ubiquitinylation. We demonstrate that the Cbl-dependent ubiquitinylation of Vav requires Cbl/Vav association through phosphorylated Tyr-700 on Cbl, and also requires an intact Cbl RING finger domain. Finally, using transfection analyses in the Jurkat T cell line, we show that Cbl, but not its ubiquitin ligase mutant, can inhibit Vav-dependent signaling. Thus, our findings strongly support the role of Cbl, via its ubiquitin ligase activity, as a negative regulator of activated Vav.
Mesh Terms:
Animals, Cell Line, Electrophoresis, Polyacrylamide Gel, Gene Expression Regulation, Humans, Immunoblotting, Jurkat Cells, Luciferases, Mice, Mice, Transgenic, Models, Genetic, Mutation, Oncogene Protein v-cbl, Oncogene Proteins, Phosphorylation, Plasmids, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Proto-Oncogene Proteins c-vav, Retroviridae Proteins, Oncogenic, Signal Transduction, Time Factors, Transfection, Tyrosine, Ubiquitin, Vanadates
J. Biol. Chem.
Date: Oct. 03, 2003
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