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J. Biol. Chem., Vol. 280, Issue 32, 29053-29059, August 12, 2005

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A Cell-permeable, Activity-based Probe for Protein and Lipid Kinases^*

Muh-ching Yee, Stefanie C. Fas, Michelle M. Stohlmeyer, Thomas J. Wandless, and Karlene A. Cimprich, A Leukemia and Lymphoma Society Scholar

From the Department of Molecular Pharmacology, Stanford University, Stanford, California 94305-5441

Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.

Received for publication, April 29, 2005 , and in revised form, June 21, 2005.

^* This work was supported by National Institutes of Health Grants GM062193 and GM068589 (the latter to T. J. W.), American Cancer Society Grant 99-241-01-CCG, and a Burroughs-Welcome New Investigator Award in Toxicology (to K. A. C.). 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 material that provides detailed experimental procedures for the preparation of wortmannin derivatives.

To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Clark Center, 318 Campus Dr., Stanford, CA 94305-5441. Tel.: 650-498-4720; Fax: 650-725-4665; E-mail: cimprich{at}stanford.edu.

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