Interactions of STAP-2 with Brk and STAT3 participate in cell growth of human breast cancer cells

STAP-2 (signal transducing adaptor protein-2) is a recently identified adaptor protein that contains pleckstrin homology (PH) and Src homology 2-like domains, as well as a STAT3-binding motif in its C-terminal region. STAP-2 is also a substrate of breast tumor kinase (Brk). In breast cancers, Brk expression is deregulated and promotes STAT3-dependent cell proliferation. In the present study, manipulated STAP-2 expression demonstrated essential roles of STAP-2 in Brk-mediated STAT3 activation. STAP-2 interacts with both Brk and STAT3. In addition, small interfering RNA-mediated reduction of endogenous STAP-2 expression strongly decreased Brk-mediated STAT3 activation in T47D breast cancer cells. The PH domain of STAP-2 is involved in multiple steps: the binding between Brk and STAP-2, the activation and tyrosine phosphorylation of STAT3, and the activation of Brk. Notably, a STAP-2 PH-Brk fusion protein exhibited robust kinase activity and increased activation and tyrosine phosphorylation of STAT3. Finally, STAP-2 knockdown in T47D cells induced a significant decrease of proliferation, as strong as that of Brk or STAT3 knockdown. Taken together, our findings are likely to inform the development of a novel therapeutic strategy, as well as the determination of novel prognostic values, in breast carcinomas.     

Discovery of novel imidazo[1,2-a]pyrazin-8-amines as Brk/PTK6 inhibitors

A series of substituted imidazo[1,2-a]pyrazin-8-amines were discovered as novel breast tumor kinase (Brk)/protein tyrosine kinase 6 (PTK6) inhibitors. Tool compounds with low-nanomolar Brk inhibition activity, high selectivity towards other kinases and desirable DMPK properties were achieved to enable the exploration of Brk as an oncology target.     

Discovery of 4-anilino α-carbolines as novel Brk inhibitors

Dysregulation of cell signalling processes caused by an enhanced activity of protein kinases mainly contributes to cancer progression. Protein kinase inhibitors have been established as promising drugs that inhibit such overactive protein kinases in cancer cells. The formation of metastases, which makes a therapy difficult, remains a great challenge for cancer treatment. Recently, breast tumor kinase (Brk) was discovered as novel and interesting target for a cancer therapy because Brk participates in both cell dysregulation and metastasis. We discovered 4-anilino substituted α-carboline compounds as a novel class of highly active Brk inhibitors. In the current work, structure–activity relationships are discussed including docking results obtained for 4-anilino α-carbolines. A first profiling of selective kinase inhibition and a proof of concept for the antiproliferative effects is demonstrated. These results qualify the compounds as a promising class of novel antitumor agents.     

微丝相关蛋白HSPC300/hHBRK1与肌球蛋白Ⅵ相互作用的鉴定

为鉴定微丝相关蛋白HSPC300/hHBRK1在肝脏组织的功能,采用GST pull-down结合质 谱技术,检测该蛋白在肝脏中的结合蛋白,结果提示,肌球蛋白Ⅵ与HSPC300/hHBRK1共沉降,Western 印迹杂交证实了质谱的结果．构建HSPC300/hHBRK1原核表达载体,诱导并获得了His-hHBRK1融合蛋白．利用免疫共沉淀证实hHBRK1与肌球蛋白Ⅵ存在相互作用,激光共聚焦检测显示hHBRK1与肌球蛋白Ⅵ在肺癌95D细胞的胞浆共定位,提示其相互作用可能是直接结合．肌球蛋白Ⅵ参与细胞迁移、高尔基分泌泡的运输和维持高尔基体稳定性等作用．hHBRK1与肌球蛋白Ⅵ相互作用,为微丝相关蛋白HSPC300/hHBRK1参与细胞迁移和胞内物质运输提供了进一步佐证．     

DNA recognition by the brinker repressor--an extreme case of coupling between binding and folding

The Brinker (Brk) nuclear repressor is a major element of the Drosophila Decapentaplegic morphogen signaling pathway. Its N-terminal part has weak homology to the Antennapedia homeodomain and binds to GC-rich DNA sequences. We have investigated the conformation and dynamics of the N-terminal 101 amino acid residues of Brk in the absence and in the presence of cognate DNA by solution NMR spectroscopy. In the absence of DNA, Brk is unfolded and highly flexible throughout the entire backbone. Addition of cognate DNA induces the formation of a well-folded structure for residues R46 to R95. This structure consists of four helices forming a helix-turn-helix motif that differs from homeodomains, but has similarities to the Tc3 transposase, the Pax-6 Paired domain, and the human centromere-binding protein. The GC-rich DNA recognition can be explained by specific major groove hydrogen bonds from the N-terminal end of helix alpha3. The transition from a highly flexible, completely unfolded conformation in the absence of DNA to a well-formed structure in the complex presents a very extreme case of the "coupling of binding and folding" phenomenon.     

微丝相关新基因hHBrk1的克隆及功能鉴定

微丝相关新基因hHBrk1被克隆 .hHBrk1基因位于 3p2 5 3 2 4 1区 ,由 3个外显子和 2个内含子组成 .Northern印迹杂交结果表明hHBrk1基因有 2个转录本 ,在人体 12种组织中均有表达 ,尤以心肌和骨骼肌为著 .hHBRK1蛋白含 75个氨基酸 ,分子量约 9kD ,与动植物界相关蛋白的同源性达 98%～ 35 % .hHBRK1蛋白定位于细胞浆 ,在细胞运动的最前沿富集 ;在有丝分裂期 ,hHBRK1蛋白定位于细胞膜皮质区和缢缩环 .实验结果提示 ,hHBRK1蛋白可能通过调控F 肌动蛋白的聚合而参与调控细胞运动、分化等基础生命活动 .