T cell activation induces direct binding of the Crk adapter protein to the regulatory subunit of phosphatidylinositol 3-kinase (p85) via a complex mechanism involving the Cbl protein.

The Crk adapter proteins are assumed to play a role in T lymphocyte activation because of their induced association with tyrosine-phosphorylated proteins, such as ZAP-70 and Cbl, and with the phosphatidylinositol 3kinase regulatory subunit, p85, following engagement of the T cell antigen receptor. Although the exact mechanism of interaction between ...
these molecules has not been fully defined, it has been generally accepted that Crk, ZAP-70, and p85 interact with tyrosine-phosphorylated Cbl, which serves as a major scaffold protein in activated T lymphocytes. Our present results demonstrate a cell activation-dependent reciprocal co-immunoprecipitation of CrkII and p85 from lysates of Jurkat T cells and a direct binding of CrkII to p85 in an overlay assay. The use of bead-immobilized GST fusion proteins indicated a complex mechanism of interaction between CrkII and p85 involving two distinct and mutually independent regions in each molecule. A relatively high affinity binding of the CrkII-SH3(N) domain to p85 and the p85-proline-B cell receptor-proline (PBP) region to CrkII was observed in lysates of either resting or activated T cells. Direct physical interaction between the CrkII-SH3(N) and the p85-PBP domain was demonstrated using recombinant fusion proteins and was further substantiated by binding competition studies. In addition, immobilized fusion proteins possessing the CrkII-SH2 and p85-SH3 domains were found to pull down p85 and CrkII, respectively, but only from lysates of activated T cells. Nevertheless, the GST-CrkII-SH2 fusion protein was unable to mediate direct association with p85 from lysates of either resting or activated T cells. Our results support a model in which T cell activation dependent conformational changes in CrkII and/or p85 promote an initial direct or indirect low affinity interaction between the two molecules, which is then stabilized by a secondary high affinity interaction mediated by direct binding of the CrkII-SH3(N) to the p85-PBP domain.
Mesh Terms:
Blotting, Western, Cell Line, Cytosol, Glutathione Transferase, Humans, Immunoblotting, Jurkat Cells, Lymphocyte Activation, Models, Biological, Phosphatidylinositol 3-Kinases, Phosphorylation, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-cbl, Proto-Oncogene Proteins c-crk, Recombinant Fusion Proteins, T-Lymphocytes, Time Factors, Tyrosine, Ubiquitin-Protein Ligases, src Homology Domains
J. Biol. Chem.
Date: Sep. 28, 2001
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