JBC Ideal method for primary cell transfection

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Originally published In Press as doi:10.1074/jbc.M100731200 on June 20, 2001

J. Biol. Chem., Vol. 276, Issue 39, 36174-36182, September 28, 2001
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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*

Sigal Gelkop, Yael Babichev, and Noah IsakovDagger

From the Department of Microbiology and Immunology, Faculty of Health Sciences, and the Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84105, Israel

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.

* The work reported herein was supported in part by grants from the Israel Science Foundation, the Israel Academy of Sciences and Humanities, the United States-Israel Binational Science Foundation, the Chief Scientist's office, Israel Ministry of Health, the Israel Cancer Association (ICA) through the ICA friends in Brazil, and the Israel Cancer Research Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, P. O. Box 653, Beer Sheva 84105, Israel. Tel.: 972-7-647-7267; Fax: 972-7-647-7626; E-mail: noah@bgumail.bgu.ac.il.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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