Department of Surgery (RMH), The University of Melbourne, The Royal Melbourne Hospital, Parkville, VIC, 3010, Australia.
Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, VIC, Australia.
Cell Commun Signal. 2018 Mar 13;16(1):10. doi: 10.1186/s12964-018-0223-4.
Transformation by oncogene Ras overcomes TGF-β mediated growth inhibition in epithelial cells. However, it cooperates with each other to mediate epithelial to mesenchymal transition (EMT). The mechanism of how these two pathways interact with each other is controversial.
Molecular techniques were used to engineer expression plasmids for Ras, SPRY, TGF-β receptors, type I and II and ubiquitin. Immunoprecipitation and western blots were employed to determine protein-protein interactions, preotein levels, protein phosphorylation while immunofluorecesent staining for molecular co-localization. TGF-β signalling activities is also determined by its luciferase reporter assay. Trans-well assays were used to measure cell migration and invasion.
Ras interacts with the SPSB1's SPRY domain to enhance TGF-β signaling. Ras interacts and colocalizes with the TGF-β type II receptor's (TβRII) negative regulator SPSB1 on the cell membrane, consequently promoting SPSB1 protein degradation via enhanced mono- and di-ubiquitination. Reduced SPSB1 levels result in the stablization of TβRII, in turn the increase of receptor levels significantly enhance Smad2/3 phosphorylation and signaling. Importantly, forced expression of SPSB1 in Ras transformed cells suppresses TGF-β signaling and its mediated migration and invasion.
Ras positively cooperates with TGF-β signaling by reducing the cellular protein levels of TβRII negative regualtor SPSB1.
致癌基因 Ras 的转化作用克服了 TGF-β 对上皮细胞生长的抑制作用。然而，它与 TGF-β 一起介导上皮细胞向间充质转化（EMT）。这两种途径相互作用的机制存在争议。
采用分子技术构建 Ras、SPRY、TGF-β 受体、I 型和 II 型以及泛素的表达质粒。免疫沉淀和 Western blot 用于确定蛋白质-蛋白质相互作用、蛋白水平、蛋白磷酸化，而免疫荧光染色用于分子共定位。TGF-β 信号转导活性也通过其荧光素酶报告基因测定来确定。Trans-well 测定用于测量细胞迁移和侵袭。
Ras 与 SPSB1 的 SPRY 结构域相互作用，以增强 TGF-β 信号。Ras 与 TGF-β 型 II 受体（TβRII）的负调节剂 SPSB1 在细胞膜上相互作用并共定位，从而通过增强单泛素化和二泛素化促进 SPSB1 蛋白降解。SPSB1 水平降低导致 TβRII 稳定，进而受体水平的增加显著增强 Smad2/3 磷酸化和信号转导。重要的是，在 Ras 转化细胞中强制表达 SPSB1 可抑制 TGF-β 信号及其介导的迁移和侵袭。
Ras 通过降低 TGF-β 信号的细胞蛋白水平 TβRII 负调节因子 SPSB1 与 TGF-β 信号正向合作。
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