T cell activation induces direct binding of the Crk adapter protein to theregulatory 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 activat ion because of their induced association with tyrosine-phosphorylated prote ins, such as ZAP-70 and Cbl, and with the phosphatidylinositol 3-kinase reg ulatory 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 p 85 interact with tyrosine-phosphorylated Cbl, which serves as a major scaff old protein in activated T lymphocytes. Our present results demonstrate a c ell 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 distin ct and mutually independent regions in each molecule. A relatively high aff inity 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 re sting or activated T cells. Direct physical interaction between the CrkII-S H3(N) and the p85-PBP domain was demonstrated using recombinant fusion prot eins and was further substantiated by binding competition studies. In addit ion, immobilized fusion proteins possessing the CrkII-SH2 and p85-SH3 domai ns were found to pull down p85 and CrkII, respectively, but only from lysat es of activated T cells. Nevertheless, the GST-CrkII-SH2 fusion protein was unable to mediate direct association with p85 from lysates of either resti ng or activated T cells. Our results support a model in which T cell activa tion dependent conformational changes in CrkII and/or p85 promote an initia l direct or indirect low affinity interaction between the two molecules, wh ich is then stabilized by a secondary high affinity interaction mediated by direct binding of the CrkII-SH3(N) to the p85-PBP domain.