Sun Yanan, Jia Xiaopeng, Gao Qiang, Liu Xing, Hou Lianguo
Department of Obstetrics and Gynecology, Bethune International Peace Hospital of the People's Liberation Army (PLA), Shijiazhuang, Hebei, China.
Department of Urology, The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China.
IUBMB Life. 2017 Jan;69(1):16-21. doi: 10.1002/iub.1585. Epub 2016 Nov 10.
Epithelial to mesenchymal transition (EMT) is an important prerequisite for metastasis to secondary organs. Interleukin-like EMT inducer (ILEI) protein has been shown to translationally upregulated during EMT and metastatic progression as a consequence of aberrant TGF-β signaling. Our initial evaluation of FAM3C (encoding ILEI) and ILEI expression in normal prostate (PCS-440-010) and prostate cancer cell lines (DU145, LNCaP, and PC3) revealed detectable protein expression in only LNCaP cell line even though all cell lines tested had comparable FAM3C expression. Given that PC3 and DU145 cell lines did not have detectable ILEI expression hinted at additional level of regulation of ILEI expression. Treatment with MG-132 resulted in robust detection of ILEI in the PCS-440-010, PC3 and DU145 cell lines, suggesting that at least in these cell lines, ILEI is actively degraded by the proteasome. Mass spectrometric analysis of FLAG immunoprecipitates of untreated and MG-132 treated FLAG-ILEI transfected cells indicated that UBE4A and UBE3C ubiquitin ligases were interacting with ILEI. Ectopic overexpression of UBE4A, but not UBE3C, resulted in destabilization of ILEI in LNCaP cells, whereas RNAi-mediated silencing of UBE4A in PCS-440-010, PC3 and DU145 cell lines resulted in robust accumulation of ILEI, indicating UBE4A as the cognate ubiquitin ligase for ILEI. Co-immunoprecipitation experiments established direct interaction of endogenous ILEI and UBE4A. Furthermore, co-immunoprecipitation of FLAG-tagged ILEI in cells co-transfected with either HA-UBE4A or HA-UBE3C revealed robust polyubiquitinated smear of ILEI in cells transfected with UBE4A, but not UBE3C, thus confirming UBE4A as the ubiquitin ligase for ILEI degradation. Ectopic overexpression of UBE4A, but not UBE3C, in cells was downregulated in vitro migration and invasion in these cells. Cumulatively, our data reveals a novel post-translational regulatory mechanism of regulating ILEI1 expression, a protein required for metastatic progression in prostate cancer cells. © 2016 IUBMB Life, 69(1):16-21, 2017.
上皮-间质转化（EMT）是转移至继发器官的重要前提条件。白细胞介素样EMT诱导剂（ILEI）蛋白已被证明在EMT和转移进展过程中由于异常的TGF-β信号传导而在翻译水平上上调。我们对正常前列腺（PCS-440-010）和前列腺癌细胞系（DU145、LNCaP和PC3）中FAM3C（编码ILEI）和ILEI表达的初步评估显示，尽管所有测试的细胞系具有相当的FAM3C表达，但仅在LNCaP细胞系中检测到可检测的蛋白表达。鉴于PC3和DU145细胞系没有可检测到的ILEI表达，这暗示了ILEI表达存在额外的调控水平。用MG-132处理导致在PCS-440-010、PC3和DU145细胞系中强烈检测到ILEI，这表明至少在这些细胞系中，ILEI被蛋白酶体积极降解。对未处理和MG-132处理的FLAG-ILEI转染细胞的FLAG免疫沉淀物进行质谱分析表明，UBE4A和UBE3C泛素连接酶与ILEI相互作用。UBE4A而非UBE3C的异位过表达导致LNCaP细胞中ILEI的不稳定，而在PCS-440-010、PC3和DU145细胞系中通过RNAi介导沉默UBE4A导致ILEI的强烈积累，表明UBE4A是ILEI的同源泛素连接酶。免疫共沉淀实验证实了内源性ILEI和UBE4A的直接相互作用。此外，在与HA-UBE4A或HA-UBE3C共转染的细胞中对FLAG标记的ILEI进行免疫共沉淀，发现在用UBE4A转染的细胞中ILEI有强烈的多聚泛素化条带，而用UBE3C转染的细胞中没有，从而证实UBE4A是降解ILEI的泛素连接酶。UBE4A而非UBE3C在细胞中的异位过表达下调了这些细胞的体外迁移和侵袭能力。累积而言，我们的数据揭示了一种调节ILEI1表达的新的翻译后调控机制，ILEI1是前列腺癌细胞转移进展所需的一种蛋白。© 2016国际生物化学与分子生物学联盟生命科学，69(（1）：16 - 21，2017。
