Brk is coamplified with ErbB2 to promote proliferation in breast cancer.

Amplification of the receptor tyrosine kinase ErbB2 is frequently observed in breast cancer. Amplification of erbB2 is also associated with multiple genomic gains and losses; however, the importance of these associated changes is largely unknown. We demonstrate that Brk, a cytoplasmic tyrosine kinase, is coamplified and coexpressed with ErbB2 in human breast cancers. ErbB2 interacts with Brk and increases its intrinsic kinase activity. Expression of Brk enhances the ErbB2-induced activation of Ras/MAPK signaling and cyclin E/cdk2 activity to induce cell proliferation of mammary 3-dimensional acini in culture. In a murine model of breast cancer, expression of Brk was found to shorten the latency of ErbB2-induced tumors by promoting cell proliferation, with no effect on protection from apoptosis. Furthermore, overexpression of Brk conferred resistance to the ability of Lapatinib, an ErbB2 kinase inhibitor, to inhibit ErbB2-induced proliferation. Thus, we identified Brk as a drug target for ErbB2-positive cancers.

Interaction between Brk kinase and insulin receptor substrate-4.

Breast tumor kinase (Brk) is a member of the Frk family of nonreceptor tyrosine kinases that is overexpressed in a high percentage of human breast tumors. The downstream substrates and effectors of Brk remain largely unidentified. In this study, we carried out immunoprecipitation and mass spectrometry experiments to identify new Brk binding partners. One interacting protein was insulin receptor substrate 4 (IRS-4), a member of the IRS family. We confirmed that Brk associates with IRS-4 in resting and insulin-like growth factor 1 (IGF-1)-stimulated HEK 293 cells. The SH3 and SH2 domains of Brk are both involved in the association. The tyrosine phosphorylation of Brk increases after stimulation with IGF-1, and in MCF-7 breast cancer cells we show that the presence of IRS-4 enhances this effect. Finally, we demonstrate that endogenous Brk and IRS-4 interact in A431 human epidermoid carcinoma cells.

Role of the Brk SH3 domain in substrate recognition.

Breast tumor kinase (Brk) is a nonreceptor tyrosine kinase that is overexpressed in a high percentage of breast carcinomas. Brk contains SH3, SH2, and tyrosine kinase catalytic domains in a similar arrangement as Src family kinases. In this study, we explored the roles of the SH3 and SH2 domains in Brk regulation and substrate binding. We introduced a series of mutations into Brk that were predicted to disrupt the intramolecular interactions involving the SH3 and SH2 domains. These mutant forms of Brk displayed higher activity than wild-type Brk when expressed in human embryonic kidney HEK293 cells. These studies also allowed us to pinpoint the intramolecular binding site for the SH3 domain. To examine substrate binding, we compared binding and phosphorylation of Sam68, a physiological substrate of Brk. These experiments showed that the SH3 domain plays a particularly important role in substrate recognition. We confirmed this conclusion using a series of synthetic peptides in which a substrate sequence was coupled to an SH3 or SH2 ligand. The SH3-binding substrate had a significantly lower K(m) than a control, while no difference was observed between an SH2-binding substrate and a control. Taken together, our data suggest that SH3 interactions will govern phosphorylation of many substrates by Brk.

Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition.

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 K(m) and k(cat) 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.