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Generation of a Novel System for Studying Spleen Tyrosine Kinase Function in Macrophages and B Cells¹

Allison L. Miller^*, Chao Zhang, Kevan M. Shokat and Clifford A. Lowell^*,2

^* Department of Laboratory Medicine, and Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143

Spleen tyrosine kinase (Syk) is a nonreceptor tyrosine kinase that is expressed primarily in hematopoietic cells. Because this protein has been implicated in processes such as Fc-mediated phagocytosis, BCR signaling, oxidative burst, degranulation, cytokine secretion, and integrin-mediated outside-in signaling, it is hypothesized that Syk may be a viable target in the treatment of a variety of autoimmune and inflammatory diseases. Because efforts to design a small-molecule therapeutic that specifically inhibits Syk have been largely unsuccessful, and genetic studies of Syk have been hampered by the fact that syk^–/– mice die in utero, we have taken a chemical genetic approach to study the function of Syk. Specifically, we have created a mutant form of Syk that retains its wild-type function, but is susceptible to inhibition by enlarged derivatives of the tyrosine kinase inhibitor, PP1. We report in this study that Syk M442A S505A reconstituted wild-type function when introduced into murine syk^–/– bone marrow-derived macrophages and syk^–/– DT40 chicken B cells, as determined by functional and biochemical assays. Furthermore, after screening a series of PP1 derivatives, we identified one compound, namely 2,3-DMB-PP1, that specifically inhibited Syk M442A S505A, but not wild-type Syk. This system provides us with the power to characterize immune functions that are Syk specific, and furthermore, it provides us with a tool to assess how inhibition of Syk may alter an immune response and influence disease pathogenesis and/or progression.

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 This work was supported by Grants R01AI68150 and R01AI65495.

² Address correspondence and reprint requests to Dr. Clifford A. Lowell, Department of Laboratory Medicine, University of California, 513 Parnassus Avenue, Box 0451, San Francisco, CA 94143.

³ Abbreviations used in this paper: ASKA, analog-sensitive kinase allele; pERK, phospho-ERK; ptyr, phosphotyrosine; Syk, spleen tyrosine kinase.