Cbl-mediated ubiquitinylation and negative regulation of Vav

J Biol Chem. 2003 Oct 3;278(40):38495-504. doi: 10.1074/jbc.M305656200. Epub 2003 Jul 24.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression Regulation*
  • Humans
  • Immunoblotting
  • Jurkat Cells
  • Luciferases / metabolism
  • Mice
  • Mice, Transgenic
  • Models, Genetic
  • Mutation
  • Oncogene Protein v-cbl
  • Oncogene Proteins / metabolism*
  • Phosphorylation
  • Plasmids / metabolism
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Proto-Oncogene Mas
  • Proto-Oncogene Proteins c-vav
  • Retroviridae Proteins, Oncogenic / metabolism
  • Retroviridae Proteins, Oncogenic / physiology*
  • Signal Transduction
  • Time Factors
  • Transfection
  • Tyrosine / chemistry
  • Ubiquitin / metabolism*
  • Vanadates / pharmacology

Substances

  • MAS1 protein, human
  • Oncogene Protein v-cbl
  • Oncogene Proteins
  • Proto-Oncogene Mas
  • Proto-Oncogene Proteins c-vav
  • Retroviridae Proteins, Oncogenic
  • Ubiquitin
  • VAV1 protein, human
  • Vav1 protein, mouse
  • pervanadate
  • Vanadates
  • Tyrosine
  • Luciferases