Abstract
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.
Highlights
We recently showed that p120cbl, the product of the c-cbl protooncogene, is a prominent tyrosine-phosphorylated substrate in T-cells [35]. p120cbl is one of several substrates that binds to Grb2 in vitro and in vivo through the amino-terminal SH3 domain of Grb2 [35,36,37]
We verify that the Grb2-p120cbl interaction in Jurkat cells is mediated through the amino-terminal SH3 domain of Grb2 (N-SH3-Grb2), and we show that this interaction decreases in vitro and in vivo following T-cell antigen receptor (TCR)-induced stimulation
On protein overlay assays, we show that Grb2 and p120cbl can interact in a manner independent of the tyrosine phosphorylation of p120cbl
Summary
Several investigators report that T-cell activation results in a number of tyrosine-phosphorylated proteins binding to Grb2 [28, 33, 34]. The functions of these proteins are presently unknown. P120cbl is one of several substrates that binds to Grb in vitro and in vivo through the amino-terminal SH3 domain of Grb2 [35,36,37]. We verify that the Grb2-p120cbl interaction in Jurkat cells is mediated through the amino-terminal SH3 domain of Grb (N-SH3-Grb2), and we show that this interaction decreases in vitro and in vivo following TCR-induced stimulation. This region of p120cbl was found to be between amino acids 480 and 563 and contains several binding motifs that are required for Grb2-p120cbl interaction
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