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J. Biol. Chem., Vol. 283, Issue 22, 15419-15430, May 30, 2008

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Inhibition of ZAP-70 Kinase Activity via an Analog-sensitive Allele Blocks T Cell Receptor and CD28 Superagonist Signaling^*

Susan E. Levin¹, Chao Zhang^¶, Theresa A. Kadlecek, Kevan M. Shokat^¶, and Arthur Weiss^||2

From the Departments of Medicine and Microbiology & Immunology, Biomedical Sciences Graduate Program, the ^||Rosalind Russell Medical Research Center for Arthritis, the ^¶Department of Cellular and Molecular Pharmacology, and the Howard Hughes Medical Institute, University of California, San Francisco, California 94143

ZAP-70 is a cytoplasmic protein tyrosine kinase that is required for T cell antigen receptor (TCR) signaling. Both mice and humans deficient in ZAP-70 fail to develop functional T cells, thus demonstrating its necessity for T cell development and function. There is currently no highly specific, cell-permeable, small molecule inhibitor for ZAP-70; therefore, we generated a mutant ZAP-70 allele that retains kinase activity but is sensitive to inhibition by a mutant-specific inhibitor. We validated the chemical genetic inhibitor system in Jurkat T cell lines, where the inhibitor blocked ZAP-70-dependent TCR signaling in cells expressing the analog-sensitive allele. Interestingly, the inhibitor also ablated CD28 superagonist signaling, thereby demonstrating the utility of this system in dissecting the requirement for ZAP-70 in alternative mechanisms of T cell activation. Thus, we have developed the first specific chemical means of inhibiting ZAP-70 in cells, which serves as a valuable tool for studying the function of ZAP-70 in T cells.

Received for publication, November 1, 2007 , and in revised form, March 21, 2008.

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^* These studies were supported in part by the Howard Hughes Medical Institute and the Rosalind Russell Medical Research Center for Arthritis. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Supported in part by a National Science Foundation predoctoral fellowship.

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² To whom correspondence should be addressed: 513 Parnassus Ave., Box 0795, San Francisco, CA 94143. Fax: 415-502-5081; E-mail: aweiss{at}medicine.ucsf.edu.

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