RNA Interference Screen Identifies Usp18 as a Regulator of Epidermal Growth Factor Receptor Synthesis
RNA Interference Screen Identifies Usp18 as a Regulator of Epidermal Growth Factor Receptor Synthesis
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DOI：
10.1091/mbc.e08-08-0880
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发表时间：
2009-03-15
影响因子：
3.3
通讯作者：
Sorkin, Alexander
中科院分区：
文献类型：
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作者：
Duex, Jason E.;Sorkin, Alexander
表皮生长因子受体(EGFR)的高表达有助于多种类型癌症的进展。因此，我们开发了一种高通量筛选来识别调节鳞状细胞癌中EGFR水平的蛋白质。用小干扰RNA敲除各种泛素化相关基因，导致了几个参与这一过程的新基因的鉴定。其中一个基因，Usp18，是泛素特定的蛋白水解酶家族的成员。我们发现，在几种细胞系中，Usp18的敲除使EGFR的表达水平降低了50%-80%，而其他受体酪氨酸激酶的水平保持不变。Usp18的过表达以一种需要Usp18催化半胱氨酸的方式增加了EGFR的水平。对代谢性放射性标记细胞的分析表明，在没有Usp18的情况下，EGFR蛋白质合成的速度最多减少了四倍。有趣的是，尽管EGFR信使核糖核酸水平没有变化，这种显著的减少还是发生了。这表明Usp18的缺失抑制了EGFR mRNA的翻译。事实上，这种抑制需要在EGFR mRNA上存在天然的5‘和3’非翻译区序列。总之，我们的数据为EGFR在受体合成的翻译步骤中调节的新机制提供了证据。
Elevated expression of epidermal growth factor receptor ( EGFR) contributes to the progression of many types of cancer. Therefore, we developed a high-throughput screen to identify proteins that regulate the levels of EGFR in squamous cell carcinoma. Knocking down various ubiquitination-related genes with small interfering RNAs led to the identification of several novel genes involved in this process. One of these genes, Usp18, is a member of the ubiquitin-specific protease family. We found that knockdown of Usp18 in several cell lines reduced expression levels of EGFR by 50-80%, whereas the levels of other receptor tyrosine kinases remained unchanged. Overexpression of Usp18 elevated EGFR levels in a manner requiring the catalytic cysteine of Usp18. Analysis of metabolically radiolabeled cells showed that the rate of EGFR protein synthesis was reduced up to fourfold in the absence of Usp18. Interestingly, this dramatic reduction occurred despite no change in the levels of EGFR mRNA. This suggests that depletion of Usp18 inhibited EGFR mRNA translation. In fact, this inhibition required the presence of native 5 ' and 3 ' untranslated region sequences on EGFR mRNA. Together, our data provide evidence for the novel mechanism of EGFR regulation at the translational step of receptor synthesis.