Regulation of the association of p120cbl with Grb2 in Jurkat T cells.
The c-cbl protooncogene product (p120(cbl)) is a known substrate of multiple tyrosine kinases. It is found in complexes with critical signal transduction molecules, including the linker protein Grb2. Here, we demonstrate using an immobilized Grb2-binding peptide that the Grb2-p120(cbl) complex dissociates in vivo following engagement of the T-cell antigen receptor in Jurkat T-cells. The early kinetics of this dissociation correlate with the known time course of tyrosine phosphorylation of p120(cbl) and other substrates. This dissociation persists in vivo even when p120(cbl) becomes dephosphorylated to basal levels. However, this decreased association is not observed in protein overlay assays on nitrocellulose membranes in which a Grb2 fusion protein is used to detect p120(cbl) from stimulated or unstimulated cells. These data suggest that the tyrosine phosphorylation of p120(cbl) does not completely account for the regulation of its association with Grb2. Additionally, we used truncation mutations of p120(cbl) to map the p120(cbl)-Grb2 interaction to amino acids 481-528 of p120(cbl); this interaction is stronger in longer constructs that include additional proline-rich motifs. The in vivo regulation of the Grb2-p120(cbl) complex further supports the idea of a significant role for p120(cbl) in receptor-mediated signaling pathways.
Donovan
J A
JA
CBMB/NICHD, National Institutes of Health, Bethesda, Maryland, 20892, USA.
Ota
Y
Y
Langdon
W Y
WY
Samelson
L E
LE
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D051380:GRB2 Adaptor Protein; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008969:Molecular Sequence Data; D016853:Muromonab-CD3 / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D014638:Vanadates / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D016853:Muromonab-CD3; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C076715:pervanadate; D014638:Vanadates; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C067419:CBL protein, human