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J. Biol. Chem. 280 (9): 8364-8370

© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

T Cell Receptor-induced Activation of Phospholipase C-{gamma}1 Depends on a Sequence-independent Function of the P-I Region of SLP-76*

Ronnie Gonen, Dvora Beach, Carmit Ainey, , and Deborah Yablonski{ddagger}

Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P. O. Box 9649, Bat Galim, Haifa 31096, Israel

Abstract: SLP-76 forms part of a hematopoietic-specific adaptor protein complex, and is absolutely required for T cell development and activation. T cell receptor (TCR)-induced activation of phospholipase C-{gamma}1 (PLC-{gamma}1) depends on three features of SLP-76: the N-terminal tyrosine phosphorylation sites, the Gads-binding site, and an intervening sequence, denoted the P-I region, which binds to the SH3 domain of PLC-{gamma}1 (SH3PLC) via a low affinity interaction. Despite extensive research, the mechanism whereby SLP-76 regulates PLC-{gamma}1 remains uncertain. In this study, we uncover and explore an apparent paradox: whereas the P-I region as a whole is essential for TCR-induced activation of PLC-{gamma}1 and nuclear factor of activated T cells (NFAT), no particular part of this region is absolutely required. To better understand the contribution of the P-I region to PLC-{gamma}1 activation, we mapped the PLC-{gamma}1-binding site within the region, and created a SLP-76 mutant that fails to bind SH3PLC, but is fully functional, mediating TCR-induced phosphorylation of PLC-{gamma}1 at tyrosine 783, calcium flux, and nuclear factor of activated T cells activation. Unexpectedly, full functionality of this mutant was maintained even under less than optimal stimulation conditions, such as a low concentration of the anti-TCR antibody. Another SLP-76 mutant, in which the P-I region was scrambled to abolish any sequence-dependent protein-binding motifs, also retained significant functionality. Our results demonstrate that SLP-76 need not interact with SH3PLC to activate PLC-{gamma}1, and further suggest that the P-I region of SLP-76 serves a structural role that is sequence-independent and is not directly related to protein-protein interactions.

Received for publication August 17, 2004. Revision received December 13, 2004.

* This work was supported by German-Israeli Foundation for Scientific Research and Development Grant I-2022-1045.11/2000, the Matilda Barnett Revocable Trust, the Technion V.P.R.-Charles Crown Research Fund, and the Fund for the Promotion of Research at the Technion. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{ddagger} To whom correspondence should be addressed: Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P. O. Box 9649, Bat Galim, Haifa 31096, Israel. Tel.: 972-4-829-5393; Fax: 972-4-829-5255; E-mail: debya{at}

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