Related Experiment Videos
Cbl-mediated ubiquitinylation and negative regulation of Vav
Yuko Miura-Shimura1, Lei Duan, Navin L Rao
1Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
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.
Insights
The Cbl ubiquitin ligase negatively regulates Vav signaling by promoting Vav ubiquitylation and degradation. This interaction is crucial for controlling T cell activation and preventing aberrant signaling pathways.
Area of Science:
- Molecular Biology
- Cell Signaling
- Immunology
Background:
- The Cbl ubiquitin ligase is a key negative regulator of receptor and non-receptor tyrosine kinases.
- Vav is a hematopoietic-restricted Rac guanine nucleotide exchange factor that plays a role in T cell signaling.
Purpose of the Study:
- To elucidate the consequences of the interaction between Cbl and Vav.
- To investigate the role of Cbl's ubiquitin ligase activity in regulating Vav function.
Main Methods:
- Utilized immortalized T cell lines from Cbl(+/+) and Cbl(-/-) mice.
- Performed transfection analyses in 293T and Jurkat T cell lines.
- Investigated Vav ubiquitylation, phosphorylation, and signaling in response to Cbl.
Main Results:
- Vav undergoes Cbl-dependent ubiquitylation and loss of phosphorylation upon Cbl interaction.
- An activated Vav mutant (Vav-Y174F) showed increased sensitivity to Cbl-dependent ubiquitylation.
- Cbl-dependent Vav ubiquitylation requires Cbl/Vav association via phosphorylated Tyr-700 on Cbl and an intact Cbl RING finger domain.
- Cbl, but not its ligase mutant, inhibited Vav-dependent signaling in Jurkat T cells.
Conclusions:
- Cbl acts as a negative regulator of activated Vav through its ubiquitin ligase activity.
- Cbl-mediated ubiquitylation of Vav leads to its degradation and inhibition of downstream signaling.
- These findings highlight a critical mechanism for controlling T cell activation and signaling pathways.