STAP-2 (signal transducing adaptor protein-2) is a recently identified adaptor protein that contains pleckstrin homology (PH) andThe nonreceptor tyrosine kinase breast tumor kinase (Brk) 2 was originally isolated from a human breast carcinoma (1). Brk is also known as PTK6, having been identified as a highly expressed protein-tyrosine kinase in human melanocytes (2). In addition, a cDNA for the mouse orthologue, Ptk6 (previously termed Sik), which has 80% amino acid identity to Brk/ PTK6, was cloned from mouse intestinal crypt cells (3). Brk contains an Src homology (SH) 3 domain, an SH2 domain, and a tyrosine kinase catalytic domain, but it lacks an N-terminal myristoylation site for membrane targeting (1). Brk is expressed in many malignancies, such as metastatic melanomas and colon and prostate tumors (4 -6). Brk expression is also detected in a large proportion of human mammary gland tumors, whereas it is not expressed in the normal mammary gland (1, 7). It is noteworthy that small interfering RNA-mediated down-regulation of Brk expression in breast cancer cells results in their decreased growth capacity (8). Furthermore, it has been demonstrated that overexpression of Brk sensitizes human mammary epithelial cells to EGF and/or heregulin stimuli and increases anchorage-independent growth (9, 10). Down-regulation of Brk can also influence EGF-and heregulin-induced cell proliferation, suggesting a contribution of Brk to signaling induced by members of the EGF receptor family (11). However, the molecular mechanism by which Brk participates in tumorigenesis remains poorly characterized.STAT3 and STAT5, which play crucial roles in cell proliferation and differentiation, are believed to be activated by Brk (12,13). Another Brk substrate is STAP-2 (signal transducing adaptor protein-2), whose tyrosine residues are phosphorylated by Brk (14, 15). STAP-2, which we isolated as a c-Fmsinteracting protein, contains an N-terminal pleckstrin homology (PH) domain and a region distantly related to the SH2 domain (16). In its C-terminal region, a proline-rich region and a STAT3-binding YXXQ motif are also present (16). Our previous experiments have suggested that STAP-2 interacts with and influences several signaling molecules, including STAT3 and STAT5 (16,17). The dysregulated activation of STAT3 is a possible mechanism of tumorigenesis in breast cancers; therefore, it would be very informative to analyze the interactions among Brk, STAP-2, and STAT3.In the present study, we found that STAP-2 acts as an endogenous positive regulator of breast cancer cell growth. Manipulation of STAP-2 expression also indicated that STAP-2 is essential for Brk-mediated STAT3 activation. In addition, we investigate the domains responsible for the effects of STAP-2. EXPERIMENTAL PROCEDURESReagents and Antibodies-Expression vectors, STAP-2, STAT3, and their mutants were described previously (15)(16)(17)(18)

Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. STAP-2 is also known as breast tumor kinase (Brk) substrate (BKS). Our previous studies revealed that STAP-2 binds to signal transducer and activator of transcription 3 (STAT3) and STAT5, and regulates the signaling pathways downstream of them. In the present study, we identified tyrosine-250 (Tyr250) in STAP-2 as a major site of phosphorylation by Brk, using a series of STAP-2 YF mutants and anti-phospho-STAP-2 Tyr250 antibody. Furthermore, overexpression of the STAP-2 Y250F mutant protein affected Brk-mediated STAT3 activation.Importantly, small-interfering RNA-mediated reduction of endogenous STAP-2 expression decreased Brk-mediated STAT3 activation. Taken together, our findings demonstrate that STAP-2 is phosphorylated at Tyr250 by Brk, and plays an important role in Brk-mediated STAT3 activation.3

Edited by Gianni CesareniKeywords: BS69 EBV LMP1 TRAF3 NF-jB Transcription a b s t r a c t Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-jB signaling pathways through two C-terminal regions, CTAR1 and CTAR2. Previous studies have demonstrated that BS69, a multidomain cellular protein, regulates LMP1/CTAR2-mediated NF-jB activation by interfering with the complex formation between TRADD and LMP1/CTAR2. Here, we found that BS69 directly interacted with the LMP1/CTAR1 domain and regulated LMP1/CTAR1-mediated NF-jB activation and subsequent IL-6 production. Regarding the mechanisms involved, we found that BS69 directly interacted with TRAF3, a negative regulator of NF-jB activation. Furthermore, small-interfering RNA-mediated knockdown experiments revealed that TRAF3 was involved in the BS69-mediated suppression of LMP1/CTAR1-induced NF-jB activation.