Ras enhances TGF-β signaling by decreasing cellular protein levels of its type II receptor negative regulator SPSB1.
Ras enhances TGF-β signaling by decreasing cellular protein levels of its type II receptor negative regulator SPSB1.
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DOI：
10.1186/s12964-018-0223-4
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发表时间：
2018-03-13
期刊：
影响因子：
--
通讯作者：
Zhu HJ
中科院分区：
文献类型：
--
作者：
Liu S;Iaria J;Simpson RJ;Zhu HJ
癌基因RAS的转化克服了转化生长因子-β介导的上皮细胞生长抑制。然而，它们相互协作，介导上皮向间充质转化(EMT)。这两条途径如何相互作用的机制存在争议。利用分子技术设计RAS、SPRY、转化生长因子-β受体、I型和II型及泛素的表达载体。免疫沉淀法和免疫印迹法检测蛋白-蛋白相互作用、蛋白原水平、蛋白磷酸化水平，免疫荧光染色进行分子共定位。转化生长因子-β的信号活性也是由其荧光素酶报告实验确定的。采用细胞迁移和侵袭实验测定细胞迁移和侵袭能力。RAS与SPSB1的S SPRY结构域相互作用，增强转化生长因子-β信号转导。RAS与细胞膜上的转化生长因子-β-II型受体(T-βRII)负调控因子SPSB1相互作用并共同定位，从而通过增强的单泛素化和双泛素化促进SPSB1蛋白的降解。Smad1水平的降低导致TβRII的稳定，而受体水平的升高又显著增强了Smad2/3的磷酸化和信号转导。重要的是，在RAS转化的细胞中强制表达SPSB1抑制了转化生长因子-β信号及其介导的迁移和侵袭。RAS与转化生长因子-β信号通路具有正向协同作用，可降低细胞内T-β、RII负性调节因子SPSB1的蛋白水平。本文的在线版本(10.1186/s12964-0180223-4)包含向授权用户提供的补充材料。
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. The online version of this article (10.1186/s12964-018-0223-4) contains supplementary material, which is available to authorized users.
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