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Originally published In Press as doi:10.1074/jbc.M203877200 on July 16, 2002

J. Biol. Chem., Vol. 277, Issue 37, 34634-34641, September 13, 2002
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Regulation of the Nonreceptor Tyrosine Kinase Brk by Autophosphorylation and by Autoinhibition*

Haoqun Qiu and W. Todd MillerDagger

From the Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook, New York 11794-8661

Brk (breast tumor kinase) is a nonreceptor tyrosine kinase that is most closely related to the Frk family of kinases, and more distantly to Src family kinases. Brk was originally identified in a screen for tyrosine kinases that are overexpressed in human metastatic breast tumors. To shed light on the activity and regulation of Brk and related tyrosine kinases, we expressed and purified Brk using the Sf9/baculovirus system. We characterized the substrate specificity of Brk using synthetic peptides, and we show that the kinetic parameters Km and kcat both play a role in specificity. We carried out mass spectrometry experiments to show that Brk autophosphorylates within the predicted kinase activation loop and at additional sites in the N terminus. Autophosphorylation increases enzyme activity of wild-type Brk but not of a Y342A mutant form of Brk. We also carried out experiments to address the possible involvement of the Src homology (SH) 2 and SH3 domains of Brk in enzyme regulation. Mutation of a C-terminal tyrosine (Tyr-447) increases enzyme activity and SH2 domain accessibility, consistent with a role for this residue in autoinhibition. A proline-rich peptide activates Brk, suggesting that the SH3 domain is also involved in maintaining an inactive form of Brk. These biochemical results for Brk may aid in the understanding of other tyrosine kinases in the Frk family.

* This work was supported by National Institutes of Health Grant CA58530 (to W. T. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Basic Science Tower, T-6, School of Medicine, State University of New York, Stony Brook, NY 11794-8661. Tel.: 631-444-3533; Fax: 631-444-3432; E-mail: miller@physiology.pnb.sunysb.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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