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A complex of Grb2 adaptor protein, Sos exchange factor, and a 36-kDa membrane-bound tyrosine phosphoprotein is
L Buday1, S E Egan, P Rodriguez Viciana
1Signal Transduction Laboratory, Imperial Cancer Research Fund, London, United Kingdom.
Abstract:
T lymphocytes contain both Grb2, an SH2 and SH3 domain containing adaptor protein, and Sos, a guanine nucleotide exchange factor for Ras. Immunoprecipitates of Sos from the lysates of T cells contain a 36-kDa protein which is phosphorylated on tyrosine residues in response to T cell receptor/CD3 cross-linking. In vitro studies using different bacterially synthesized GST-Sos fusion proteins confirm the formation of complexes containing p36 and the proline-rich COOH-terminal domain of Sos. The use of mutant GST-Grb2 proteins in which both SH3 domains have been mutationally inactivated shows that Grb2 binds to tyrosine phosphorylated p36 via its SH2 domain. In Jurkat cells phosphorylated p36 is localized exclusively in the particulate fraction. In addition, another SH2 domain-containing protein, p52Shc is tyrosine phosphorylated upon TCR.CD3 cross-linking and associates with a 150-kDa phosphotyrosine containing protein. Taken together these data suggest that activation of Ras in T cells via the TCR.CD3 complex might be controlled, at least in part, by mechanisms similar to those found in fibroblasts, involving in this case formation of a complex of Grb2, Sos, and a membrane-bound tyrosine phosphoprotein of molecular mass 36-kDa.
Insights
T cell receptor activation involves a 36-kDa tyrosine phosphoprotein binding to Grb2 (an adaptor protein) and Sos (a guanine nucleotide exchange factor). This complex formation suggests Ras activation mechanisms similar to those in fibroblasts.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- T lymphocytes utilize Grb2 (an SH2 and SH3 domain adaptor protein) and Sos (a guanine nucleotide exchange factor for Ras).
- T cell receptor/CD3 cross-linking triggers signaling pathways crucial for T cell activation.
Purpose of the Study:
- To investigate the molecular mechanisms of Ras activation in T cells following T cell receptor/CD3 stimulation.
- To identify proteins interacting with Sos and involved in T cell signaling.
Main Methods:
- Immunoprecipitation of Sos from T cell lysates.
- In vitro binding assays using bacterially synthesized GST-Sos fusion proteins.
- Analysis of mutant Grb2 proteins to determine binding domains.
- Cellular fractionation to localize phosphorylated proteins.
Main Results:
- A 36-kDa protein, phosphorylated on tyrosine residues, was identified in Sos immunoprecipitates after T cell receptor/CD3 cross-linking.
- Grb2 binds to this tyrosine-phosphorylated 36-kDa protein via its SH2 domain.
- The phosphorylated 36-kDa protein is localized in the particulate fraction of Jurkat cells.
- p52Shc also undergoes tyrosine phosphorylation and associates with a 150-kDa phosphotyrosine protein upon T cell receptor/CD3 cross-linking.
Conclusions:
- Ras activation in T cells via the T cell receptor/CD3 complex may involve mechanisms analogous to those in fibroblasts.
- Formation of a complex involving Grb2, Sos, and a membrane-bound 36-kDa tyrosine phosphoprotein is implicated in T cell signaling.