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Interactions of Cbl with two adapter proteins, Grb2 and Crk, upon T cell activation
11st Institute of Biochemistry, Semmelweis University Medical School, Budapest, Hungary.
Abstract:
Several recent studies have demonstrated that Grb2, composed entirely of SH2 and SH3 domains, serves as an adaptor protein in tyrosine kinase signaling pathways. Cb1, the protein product of c-cbl proto-oncogene, has been reported to be phosphorylated on tyrosine residues upon T cell receptor (TCR) engagement. Here we show that in unstimulated Jurkat cells Cbl is co-immunoprecipitated with monoclonal antibody against Grb2. However, in lymphocytes activated through the TCR, Cbl loses its ability to bind to Grb2 precipitated either with anti-Grb2 antibody or with an immobilized tyrosine phosphopeptide, Y1068-P, derived from the epidermal growth factor receptor. In vitro studies confirm that the ability of Cb1 to bind to both SH3 domains of Grb2 is strongly reduced in activated T lymphocytes. Investigation of the time course of Cbl dissociation from Grb2 reveals that it is transient and correlates with the kinetics of tyrosine phosphorylation of Cbl. Moreover, Cb1 is co-immunoprecipitated with Crk, another SH2/SH3 domain-containing protein, upon TCR stimulation. Tyrosine-phosphorylated Cbl binds exclusively to the SH2 domain of Crk. These results suggest that different adaptor proteins may have different roles in the regulation of c-cbl proto-oncogene product.
Insights
The c-cbl proto-oncogene product (Cbl) interaction with Grb2 adaptor protein is disrupted upon T cell receptor (TCR) activation. Tyrosine-phosphorylated Cbl then binds to Crk, suggesting distinct adaptor roles in regulating Cbl function.
Area of Science:
- Cellular signaling
- Molecular biology
- Immunology
Background:
- Grb2 acts as an adaptor protein in tyrosine kinase signaling pathways.
- Cbl, a proto-oncogene product, is tyrosine phosphorylated upon T cell receptor (TCR) engagement.
Purpose of the Study:
- To investigate the interaction between Cbl and adaptor proteins Grb2 and Crk.
- To elucidate the role of Cbl phosphorylation in its binding to these adaptors during TCR signaling.
Main Methods:
- Co-immunoprecipitation assays using anti-Grb2 and anti-Crk antibodies.
- In vitro binding studies with activated and unstimulated T lymphocytes.
- Analysis of Cbl tyrosine phosphorylation kinetics.
Main Results:
- Cbl co-immunoprecipitates with Grb2 in unstimulated cells but loses this binding upon TCR stimulation.
- The dissociation of Cbl from Grb2 is transient and correlates with Cbl tyrosine phosphorylation.
- Tyrosine-phosphorylated Cbl specifically binds to the SH2 domain of Crk upon TCR stimulation.
Conclusions:
- TCR signaling dynamically regulates the interaction of Cbl with different SH2/SH3 domain-containing adaptor proteins.
- Cbl phosphorylation alters its binding preference from Grb2 to Crk, indicating distinct regulatory roles.
- These findings shed light on the complex mechanisms governing c-cbl proto-oncogene product function in T cell activation.