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J. Biol. Chem., Vol. 282, Issue 46, 33760-33768, November 16, 2007

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Generation of an Analog-sensitive Syk Tyrosine Kinase for the Study of Signaling Dynamics from the B Cell Antigen Receptor^*

Hyunju Oh, Elif Ozkirimli¹, Kavita Shah^¶, Marietta L. Harrison, and Robert L. Geahlen²

From the Departments of Medicinal Chemistry and Molecular Pharmacology and ^¶Chemistry and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907

The Syk protein-tyrosine kinase is an essential component of the signaling machinery that couples the B cell receptor for antigen to multiple downstream signal transduction pathways. Syk is phosphorylated and activated rapidly and transiently following receptor engagement, but many signaling events, such as the activation of transcription factors occur over the course of several minutes or hours. To investigate a role for the continued activation of Syk in these processes, we generated an analog-sensitive mutant with an engineered ATP-binding pocket to render the kinase uniquely sensitive to an orthogonal inhibitor. Mutation of the gatekeeper residue in Syk yielded an enzyme with very low activity. Second-site mutations, selected based on structural comparisons between Syk and Src, were introduced that restored catalytic activity to the mutant Syk. Syk-deficient DT40 B cells were prepared expressing the analog-sensitive Syk (Syk-AQL). Inhibition of the activity of Syk prior to, concomitant with or shortly following receptor engagement led to the rapid inhibition of receptor-mediated tyrosine phosphorylation and blocked the activation of extracellular signal-regulated kinase, NF-B, and NFAT. The receptor-mediated activation of NF-B required active Syk for a relatively short period of time, whereas the activation of NFAT required active kinase for a prolonged (>1 h) period. Receptor cross-linking led to the recruitment of Syk to the clustered receptor. Retention of these receptor-kinase complexes on the cell surface was dependent on the continued activity of Syk. Thus, despite the apparent transient nature of the activation of Syk, the catalytic activity of the Syk was required for sustained signaling from ligated receptors.

Received for publication, June 12, 2007 , and in revised form, September 17, 2007.

^* This work was supported by National Institutes of Health Grant CA37372 awarded by the National Cancer Institute (to R. L. G. and M. L. H.). 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.

¹ Current address: Dept. of Chemical Engineering, Bogazici University, Bebek 34342, Istanbul, Turkey.

² To whom correspondence should be addressed: Dept. of Medicinal Chemistry and Molecular Pharmacology, Purdue University, Hansen Life Sciences Research Bldg., 201 S. University St., West Lafayette, IN 47907-2064. E-mail: geahlen{at}purdue.edu.

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