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

In chronic myeloid leukemia (CML), the BCR-ABL fusion oncoprotein activates multiple pathways involved in cell survival, growth promotion and disease progression. In this report, we show that the signal transducing adaptor protein-2 (STAP-2) is involved in BCR-ABL activity. We demonstrate that STAP-2 bound to BCR-ABL, and BCR and ABL proteins, depending on the STAP-2 Src homology 2-like domain. BCR-ABL phosphorylates STAP-2 Tyr250 and the phosphorylated STAP-2 in turn up-regulated BCR-ABL phosphorylation, leading to enhanced activation of downstream signaling molecules including ERK, STAT5, BCL-xL and BCL-2. In addition, STAP-2 interacts with BCR-ABL to alter chemokine receptor expression leading to downregulation of CXCR4 and upregulation of CCR7. The interaction between STAP-2 and BCR-ABL plays a crucial role in conferring a growth advantage and resistance to imatinib, a BCR-ABL inhibitor, as well as tumor progression. Notably, mice injected with BCR-ABL/STAP-2-expressing Ba/F3 cells developed lymph node enlargement and hepatosplenomegaly. Moreover, suppression of STAP-2 in K562 CML cells resulted in no tumor formation in mice. Our results demonstrate a critical contribution of STAP-2 in BCR-ABL activity, and suggest that STAP-2 might be an important candidate for drug development for patients with CML. Further, the expression of STAP-2 provides useful information for estimating the characteristics of individual CML clones

Edited by Giulio Superti-FurgaKeywords: Epstein-Barr virus Latent membrane protein 1 BS69 NF-jB TNFR-associated death domain protein a b s t r a c t Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates NF-jB signaling pathways through the two C-terminal regions, CTAR1 and CTAR2. BS69 has previously been shown to be involved in LMP1-induced c-Jun N-terminal kinase activation through CTAR2 by interacting with tumor necrosis factor (TNFR) receptor-associated factor 6. In the present study, our manipulation of BS69 expression clearly indicates that BS69 negatively regulates LMP1-mediated NF-jB activation and up-regulates IL-6 mRNA expression and IjB degradation. Our immunoprecipitation experiments suggest that BS69 decreases complex formation between LMP1 and TNFR-associated death domain protein (TRADD).

Signal-transducing adaptor protein (STAP)-2 is a recently identified adaptor protein that contains Pleckstrin homology and Src homology 2-like domains, and is also known to be a substrate of breast tumor kinase (Brk). In a previous study, we found that STAP-2 upregulated Brk-mediated activation of signal transducer and activator of transcription (STAT) 3 in breast cancer cells. Here, we examined the involvement of STAP-2 in Brk-mediated STAT5 activation in breast cancer cells. Ectopic expression of STAP-2 induced Brk-mediated transcriptional activity of STAT5. Furthermore, STAP-2-knockdown in T47D breast cancer cells induced a marked decrease in proliferation that was as strong as that after Brk-or STAT5b-knockdown. Regarding the mechanism, the Pleckstrin homology domain of STAP-2 is likely to participate in the process by which Brk phosphorylates and activates STAT5. Taken together, our findings provide insights toward the development of novel therapeutic strategies as well as novel prognostic values in breast carcinomas. (Cancer Sci 2011; 102: 756-761) A key step in the progression of the majority of breast cancers is the transition to steroid hormone-independent proliferation, and estrogen-independent tumors often have increased levels of total tyrosine kinase activity in both the cytosolic and membrane fractions.(1) Although breast tumor kinase (Brk), also known as protein tyrosine kinase 6, is undetectable in the normal mammary gland, it is overexpressed in more than 60% of human breast tumors and breast cancer cell lines, with the highest levels in advanced tumors.(2-4) Notably, small-interfering RNA (siRNA)-mediated downregulation of Brk expression in breast cancer cells results in a decrease in their growth capacity, (4) indicating that Brk is one of the major molecules causing excessive proliferation of breast cancer cells. Brk is a nonreceptor tyrosine kinase that activates signal transducer and activator of transcription (STAT)3 and STAT5.(5,6) Brk phosphorylates STAT3 and STAT5, leading to increases in their transcriptional activity. Furthermore, both STATs play fundamental roles in the normal growth and development of the mammary gland, (7) and are often overexpressed or constitutively activated in breast cancer tumors.(8) Thus, both the Brk ⁄ STAT3 and Brk ⁄ STAT5 axes are likely to be potential targets for breast cancer therapy.On the other hand, signal-transducing adaptor protein (STAP)-2, which we isolated as a c-fms-interacting protein, is another substrate whose tyrosine residues are phosphorylated by Brk.(9-11) STAP-2 shows high sequence and structural similarities to STAP-1, which we cloned as a c-kit-interacting protein.(12) Both STAP-1 and STAP-2 contain an N-terminal Pleckstrin homology (PH) domain and a region weakly related to a Src homology 2 (SH2) domain (overall amino acid identity, 33%). The N-terminal PH domains of STAP-2 and STAP-1 share 36% identity and 58% similarity. The central region of STAP-2 is distantly related to the SH2 domain. This region of STAP-2 shares 40% sequenc...