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PNAS 104 (16): 6638-6643

Copyright © 2007 by the National Academy of Sciences.

BIOLOGICAL SCIENCES / BIOCHEMISTRY

SLP-76 mediates and maintains activation of the Tec family kinase ITK via the T cell antigen receptor-induced association between SLP-76 and ITK

Yaron Bogin, Carmit Ainey, Dvora Beach, and Deborah Yablonski*

The Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion–Israel Institute of Technology, P.O. Box 9649, Bat Galim, Haifa 31096, Israel

Edited by Arthur Weiss, University of California, San Francisco School of Medicine, San Francisco, CA, and approved February 21, 2007

Received for publication November 5, 2006.

Abstract: ITK (IL-2-inducible T cell kinase), a Tec family protein tyrosine kinase (PTK), is one of three PTKs required for T cell antigen receptor (TCR)-induced activation of phospholipase C-{gamma}1 (PLC-{gamma}1). Like Src and Abl family PTKs, ITK adopts an inactive, "closed" conformation, and its conversion to the active conformation is not well understood, nor have its direct substrates been identified. In a side-by-side comparison of ITK and ZAP-70 ({zeta} chain-associated protein kinase of 70 kDa), ITK efficiently phosphorylated Y783 and Y775 of PLC-{gamma}1, two phosphorylation sites that are critical for its activation, whereas ZAP-70 did not. SLP-76 (SH2-domain-containing leukocyte protein of 76 kDa), an adaptor required for TCR-induced activation of PLC-{gamma}1, was required for the phosphorylation of both PLC-{gamma}1 sites in intact cells. Furthermore, this event depended on the N-terminal tyrosines of SLP-76. Likewise, SLP-76, particularly its N-terminal tyrosines, was required for TCR-induced tyrosine phosphorylation and activation of ITK but was not required for the phosphorylation or activation of ZAP-70. Both ZAP-70 and ITK phosphorylated SLP-76 in vitro; thus, both PTKs are potential regulators of SLP-76, but only ITK is regulated by SLP-76. Upon TCR stimulation, a small fraction of ITK bound to SLP-76. This fraction, however, encompassed most of the catalytically active ITK. Catalytic activity was lost upon mild elution of ITK from the SLP-76-nucleated complex but was restored upon reconstitution of the complex. We propose that SLP-76 is required for ITK activation; furthermore, an ongoing physical interaction between SLP-76 and ITK is required to maintain ITK in an active conformation.

Key Words: adaptor protein • kinase regulation • phospholipase C-{gamma} • signal transduction

Author contributions: Y.B. and D.Y. designed research; Y.B. and D.B. performed research; C.A. contributed new reagents/analytic tools; Y.B. and D.Y. analyzed data; and Y.B. and D.Y. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

*To whom correspondence should be addressed. E-mail: debya{at}tx.technion.ac.il

© 2007 by The National Academy of Sciences of the USA

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