Nakashima Akio, Yoshino Ken-ichi, Miyamoto Takafumi, Eguchi Satoshi, Oshiro Noriko, Kikkawa Ushio, Yonezawa Kazuyoshi
Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
Biochem Biophys Res Commun. 2007 Sep 14;361(1):218-23. doi: 10.1016/j.bbrc.2007.07.011. Epub 2007 Jul 16.
TBC7, a TBC (Tre-2/Bub2/Cdc16) 1 domain protein, was identified as a novel binding protein to the TSC1-TSC2 tumor suppressor complex by peptide mass fingerprinting analysis of the proteins immunoprecipitated with FLAG-epitope tagged TSC1 and TSC2 from the transfected mammalian cells. The in vivo and in vitro association of TBC7 and the TSC1-TSC2 complex was confirmed by the co-immunoprecipitation and pull-down analysis, respectively, and TBC7 was revealed to bind to the C-terminal half region of TSC1, which is distinct from the binding site with TSC2. The immunofluorescence microscopy and subcellular fractionation showed that TBC7 co-localizes with the tumor suppressor complex in the endomembrane. Overexpression of TBC7 enhanced ubiquitination of TSC1 and increased phosphorylation of S6 protein by S6 kinase, that is located in the mTOR-signaling pathway. These results indicate TBC7 could take a part in the negative regulation of the tumor suppressor complex through facilitating the downregulation of TSC1.
TBC7是一种含TBC（Tre-2/Bub2/Cdc16）1结构域的蛋白质，通过对用FLAG表位标记的TSC1和TSC2从转染的哺乳动物细胞中免疫沉淀的蛋白质进行肽质量指纹分析，被鉴定为一种与TSC1-TSC2肿瘤抑制复合物结合的新型蛋白质。分别通过免疫共沉淀和下拉分析证实了TBC7与TSC1-TSC2复合物在体内和体外的结合，并且发现TBC7与TSC1的C端半区结合，这与它和TSC2的结合位点不同。免疫荧光显微镜和亚细胞分级分离显示，TBC7与肿瘤抑制复合物在内膜中共定位。TBC7的过表达增强了TSC1的泛素化，并增加了位于mTOR信号通路中的S6激酶对S6蛋白的磷酸化。这些结果表明，TBC7可能通过促进TSC1的下调参与肿瘤抑制复合物的负调控。
