ORIGINAL ARTICLE Identification of STAT3 as a specific substrate of breast tumor kinase L Liu 1 , Y Gao 1 , H Qiu 2 , WT Miller 2 , V Poli 3 and NC Reich 1 1 Department of Microbiology and Molecular Genetics, Stony Brook University, Stony Brook, NY, USA; 2 Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY, USA and 3 Department of Genetics, Biology and Biochemistry, University of Turin, Turin, Italy Breast tumor kinase (Brk) is a non-receptor tyrosine kinase distantly related to the Src family kinase. It is expressed in more than 60% of breast tumors, but the biological role of this kinase remains to be determined. Only a limited number of substates have been identified for Brk, and the link of Brk to tumorigenesis remains largely unknown. In this study, we provide evidence that the signal transducer and activator of transcription 3, STAT3, is a physiological target of Brk. Activation of STAT3 previously has been linked to oncogenesis, and results in this study demonstrate that STAT3 is tyrosine phosphorylated and transcriptionally activated in cells expressing endogenous Brk. Signal transducer and activator of transcription 3 is specifically targeted since other STAT members are not responsive to Brk expres- sion. Signal transducer and activator of transcription 3 activation requires the catalytic activity of Brk, and expression of both STAT3 and Brk stimulate cellular proliferation. In addition, we have identified a negative regulator of Brk, the suppressor of cytokine signaling, SOCS3. The SOCS3 protein is known to block signaling mediated by cytokine receptors, and here we find that SOCS3 is able to repress the activity of the Brk non- receptor tyrosine kinase. Oncogene (2006) 25, 4904–4912. doi:10.1038/sj.onc.1209501; published online 27 March 2006 Keywords: tyrosine phosphorylation; transcription fac- tor; signal transduction Introduction Breast tumor kinase (Brk) is a non-receptor tyrosine kinase originally isolated from a metastatic human breast carcinoma (Mitchell et al., 1994). Subsequent characterization of Brk showed it to be present in approximately 60% of human breast tumors, yet absent in normal or fibrocystic mammary tissues. It has now been shown to be expressed in other cancers including metastatic melanomas, and colon and prostate tumors (Barker et al., 1997; Easty et al., 1997; Llor et al., 1999; Derry et al., 2003). The murine homolog of Brk has been found in normal differentiating epithelial cells of the skin, and epithelial cells lining the gastrointestinal tract and the prostate lumen. However, its expression is undetectable in mammary tissues at any stage of development (Siyanova et al., 1994; Vasioukhin et al., 1995). Overexpression of Brk was found to sensitize mammary epithelial cells to the mitogenic effect of epidermal growth factor (EGF) and enhance the coupling of EGF and PI3 kinase pathways via erbB3 phosphorylation (Kamalati et al., 1996, 2000). Breast tumor kinase has also been reported to associate with insulin receptor substrate 4 (IRS4), suggesting a potential role of Brk in insulin-like growth factor 1- induced proliferation (Qiu et al., 2005). Collectively, these findings indicate elevated expression of Brk may promote abnormal growth below the threshold that proliferative growth factors exert their mitogenic effects. Breast tumor kinase displays 46% sequence homol- ogy to c-Src kinase and a similar domain arrangement (Mitchell et al., 1994; Serfas & Tyner, 2003). Breast tumor kinase possesses an Src Homology 3 (SH3), an Src Homology 2 (SH2), and a tyrosine kinase catalytic domain, but it lacks an amino myristoylation site for membrane targeting. Molecular characterization con- firms that Brk is regulated by autophosphorylation and autoinhibition, involving both the SH3 and SH2 domains, whereas substrate recognition is mediated mainly by the SH3 domain (Qiu & Miller, 2002, 2004; Kim and Lee, 2005). To date, only five substrates have been identified for Brk, and the downstream events triggered by Brk largely remain to be elucidated. One Brk substrate is signal-transducing adaptor protein-2 (STAP-2) that has been implicated in modulating the activity of some members of the signal transducer and activator of transcription (STAT) family (Mitchell et al., 2000; Minoguchi et al., 2003). Signal transducers and activators of transcription are a family of transcription factors that were initially discovered as key mediators of cytokine signaling (Akira, 1999; Darnell et al., 1994; Decker, 1999; Ihle, 2001). Subsequently a few of the STATs were found to be activated by growth factors such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in addition to cytokines (Darnell, 1997; Levy & Darnell, 2002). The STAT proteins have been shown to play significant roles in immune function, and also in Received 1 December 2005; revised 8 February 2006; accepted 9 February 2006; published online 27 March 2006 Correspondence: Dr NC Reich, Department of Microbiology and Molecular Genetics, Stony Brook University, Nicolls Road, Life- sciences Rm 168, Stony Brook, New York 11794-8691, USA. E-mail: nreich@notes.cc.sunysb.edu Oncogene (2006) 25, 4904–4912 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc