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Papers In Press, published online ahead of print June 20, 2001
Microbiolog and Immunology, Ben Gurion University of the Negev, Beer Sheva 84105
Corresponding Author: noah{at}bgumail.bgu.ac.il
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 3?-kinase regulatory subunit, p85, following engagement of the T cell antigen receptor (TCR). 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-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 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 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.
J. Biol. Chem, 10.1074/jbc.M100731200
Submitted on January 25, 2001
Revised on June 14, 2001
Accepted on June 20, 2001
T cell activation induces direct binding of the Crk adapter protein to the regulatory subunit of PI3K (p85) via a complex mechanism involving the Cbl protein
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