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.
Gelkop
S
S
Department of Microbiology and Immunology, Faculty of Health Sciences, and the Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.
Babichev
Y
Y
Isakov
N
N
D015153:Blotting, Western; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000201:enzymology*; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D018909:src Homology Domains
D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; C067419:CBL protein, human