Amino acids are essential for the activation of the mechanistic target of rapamycin (mTOR), but the corresponding molecular mechanism is not yet fully understood. We previously found that Met stimulated eukaryotic elongation factor α (eEF1Bα) nuclear localization in bovine mammary epithelial cells (MECs). Herein, we explored the role and molecular mechanism of eEF1Bα in methionine (Met)- and leucine (Leu)-stimulated mTOR gene transcription and milk synthesis in MECs. eEF1Bα knockdown decreased milk protein and fat synthesis, cell proliferation, and mTOR mRNA expression and phosphorylation, whereas eEF1Bα overexpression had the opposite effects. QE-MS analysis detected that eEF1Bα was phosphorylated at Ser106 in the nucleus and Met and Leu stimulated p-eEF1Bα nuclear localization. eEF1Bα knockdown abrogated the stimulation of Met and Leu by mTOR mRNA expression and phosphorylation, and this regulatory role was dependent on its phosphorylation. Akt knockdown blocked the stimulation of Met and Leu by eEF1Bα and p-eEF1Bα expression. ChIP-PCR detected that p-eEF1Bα bound only to the −548 to −793 nt site in the mTOR promoter, and ChIP-qPCR further detected that Met and Leu stimulated this binding. eEF1Bα mediated Met and Leu’ stimulation on mTOR mRNA expression and phosphorylation through inducing AT-rich interaction domain 1A (ARID1A) ubiquitination degradation, and this process depended on eEF1Bα phosphorylation. p-eEF1Bα interacted with ARID1A and ubiquitin protein ligase E3 module N-recognition 5 (UBR5), and UBR5 knockdown rescued the decrease of the ARID1A protein level by eEF1Bα overexpression. Both eEF1Bα and p-eEF1Bα were highly expressed in mouse mammary gland tissues during the lactating period. In summary, we reveal that Met and Leu stimulate mTOR transcriptional activation and milk protein and fat synthesis in MECs through eEF1Bα-UBR5-ARID1A signaling.

中文翻译：

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蛋氨酸和亮氨酸通过 eEF1Bα-UBR5-ARID1A 信号传导促进乳腺上皮细胞中 mTOR 基因转录和乳汁合成


氨基酸对于雷帕霉素（mTOR）机制靶点的激活至关重要，但相应的分子机制尚未完全了解。我们之前发现 Met 刺激牛乳腺上皮细胞 (MEC) 中的真核延伸因子 α (eEF1Bα) 核定位。在此，我们探讨了 eEF1Bα 在 MEC 中蛋氨酸 (Met) 和亮氨酸 (Leu) 刺激的 mTOR 基因转录和乳汁合成中的作用和分子机制。 eEF1Bα 敲低降低了乳蛋白和脂肪合成、细胞增殖以及 mTOR mRNA 表达和磷酸化，而 eEF1Bα 过表达则具有相反的效果。 QE-MS 分析检测到 eEF1Bα 在细胞核中的 Ser106 位点被磷酸化，Met 和 Leu 刺激 p-eEF1Bα 核定位。 eEF1Bα 敲除消除了 mTOR mRNA 表达和磷酸化对 Met 和 Leu 的刺激，并且这种调节作用依赖于其磷酸化。 Akt 敲低可阻断 eEF1Bα 和 p-eEF1Bα 表达对 Met 和 Leu 的刺激。 ChIP-PCR 检测到 p-eEF1Bα 仅与 mTOR 启动子中的 -548 至 -793 nt 位点结合，ChIP-qPCR 进一步检测到 Met 和 Leu 刺激了这种结合。 eEF1Bα 通过诱导富含 AT 的相互作用域 1A (ARID1A) 泛素化降解来介导 Met 和 Leu’ 对 mTOR mRNA 表达和磷酸化的刺激，而该过程依赖于 eEF1Bα 磷酸化。 p-eEF1Bα 与 ARID1A 和泛素蛋白连接酶 E3 模块 N-识别 5 (UBR5) 相互作用，UBR5 敲低可挽救 eEF1Bα 过表达导致的 ARID1A 蛋白水平下降。 eEF1Bα和p-eEF1Bα在哺乳期小鼠乳腺组织中均高表达。 总之，我们发现 Met 和 Leu 通过 eEF1Bα-UBR5-ARID1A 信号传导刺激 MEC 中的 mTOR 转录激活以及乳蛋白和脂肪合成。