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.
Miura-Shimura
Yuko
Y
Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Duan
Lei
L
Rao
Navin L
NL
Reddi
Alagarsamy L
AL
Shimura
Hideki
H
Rottapel
Rob
R
Druker
Brain J
BJ
Tsygankov
Alexander
A
Band
Vimla
V
Band
Hamid
H
D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D009154:Mutation; D050722:Oncogene Protein v-cbl; D015513:Oncogene Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000090063:Proto-Oncogene Mas; D051567:Proto-Oncogene Proteins c-vav; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D025801:Ubiquitin / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
C000715470:MAS1 protein, human; D050722:Oncogene Protein v-cbl; D015513:Oncogene Proteins; D000090063:Proto-Oncogene Mas; D051567:Proto-Oncogene Proteins c-vav; D015682:Retroviridae Proteins, Oncogenic; D025801:Ubiquitin; C496618:VAV1 protein, human; C496619:Vav1 protein, mouse; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D008156:Luciferases