Requirement of the mTOR Kinase for the Regulation of Maf1 Phosphorylation and Control of RNA Polymerase III-dependent Transcription in Cancer Cells
Requirement of the mTOR Kinase for the Regulation of Maf1 Phosphorylation and Control of RNA Polymerase III-dependent Transcription in Cancer Cells
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DOI：
10.1074/jbc.m109.071639
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发表时间：
2010-05-14
影响因子：
4.8
通讯作者：
Yu, Ker
中科院分区：
文献类型：
--
作者：
Shor, Boris;Wu, Jiang;Yu, Ker
哺乳动物雷帕霉素靶标 (mTOR) 通过促进翻译和转录来调节生长。在这里，我们使用 mTOR 活性位点抑制剂 WYE-125132 (WYE-132) 进行了定量磷酸化蛋白质组学，并从 Maf1（一种已知的 RNA 聚合酶 III (Pol III) 转录抑制因子）中鉴定了一种含有 Ser-75 的磷酸肽。用 WYE-132 或雷帕霉素类似物 CCI-779 处理癌细胞会导致 Ser-75 磷酸化快速丧失，而在用细胞毒剂或不相关抑制剂处理的细胞中未观察到这种效应。 WYE-132 诱导的 Maf1 去磷酸化与其在细胞核中的积累以及前 tRNA 的细胞水平显着下降相关。通过小干扰 RNA 消除细胞 Maf1 会增加基础前 tRNA 并使 tRNA 合成对 mTOR 抑制剂产生耐药性。单独携带 S75A 或与 S60A、T64A 和 S68A (Maf1-S75A、Maf1-4A) 一起携带的 Maf1 突变蛋白逐渐增强活跃增殖细胞中 tRNA 的基础抑制，并减弱氨基酸诱导的 tRNA 转录。基因比对揭示了高等真核生物中所有四个 Ser/Thr 位点的保守性，进一步支持了这些残基在 Maf1 功能中的关键作用。有趣的是，mTOR 抑制导致 Maf1 在一组 Pol III 依赖性基因上的占据增加，同时 Pol III 和 Brf1 的结合减少。出乎意料的是，mTORC1 本身也在同一组 Pol III 模板中富集，但这种关联不受 mTOR 抑制剂治疗的影响。我们的结果强调了 mTOR 调节 Pol III 转录的一种新的、独特的模式，并表明 Pol III 活性的正常化可能有助于 mTOR 抑制剂的治疗效果。
The mammalian target of rapamycin (mTOR) regulates growth via promoting translation and transcription. Here, employing an mTOR active-site inhibitor WYE-125132 (WYE-132), we have performed quantitative phospho-proteomics and identified a Ser-75-containing phosphopeptide from Maf1, a known repressor of RNA polymerase III (Pol III) transcription. Treatment of cancer cells with WYE-132 or the rapamycin analog CCI-779 led to a rapid loss of the phosphorylation at Ser-75, whereas this effect was not seen in cells treated with cytotoxic agents or unrelated inhibitors. WYE-132-induced Maf1 dephosphorylation correlated with its accumulation in the nucleus and a marked decline in the cellular levels of pre-tRNAs. Depletion of cellular Maf1 via small interfering RNA increased basal pre-tRNA and rendered tRNA synthesis refractory to mTOR inhibitors. Maf1 mutant proteins carrying S75A alone or with S60A, T64A, and S68A (Maf1-S75A, Maf1-4A) progressively enhanced basal repression of tRNA in actively proliferating cells and attenuated amino acid-induced tRNA transcription. Gene alignment revealed conservation of all four Ser/Thr sites in high eukaryotes, further supporting a critical role of these residues in Maf1 function. Interestingly, mTOR inhibition led to an increase in the occupancy of Maf1 on a set of Pol III-dependent genes, with concomitant reduction in the binding of Pol III and Brf1. Unexpectedly, mTORC1 itself was also enriched at the same set of Pol III templates, but this association was not influenced by mTOR inhibitor treatment. Our results highlight a new and unique mode of regulation of Pol III transcription by mTOR and suggest that normalization of Pol III activity may contribute to the therapeutic efficacy of mTOR inhibitors.