Methionine and Leucine Promote mTOR Gene Transcription and Milk Synthesis in Mammary Epithelial Cells through the eEF1Bα-UBR5-ARID1A Signaling

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 alpha (eEF1B alpha) nuclear localization in bovine mammary epithelial cells (MECs). Herein, we explored the role and molecular mechanism of eEF1B alpha in methionine (Met)- and leucine (Leu)-stimulated mTOR gene transcription and milk synthesis in MECs. eEF1B alpha knockdown decreased milk protein and fat synthesis, cell proliferation, and mTOR mRNA expression and phosphorylation, whereas eEF1B alpha overexpression had the opposite effects. QE-MS analysis detected that eEF1B alpha was phosphorylated at Ser106 in the nucleus and Met and Leu stimulated p-eEF1B alpha nuclear localization. eEF1B alpha 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 eEF1B alpha and p-eEF1B alpha expression. ChIP-PCR detected that p-eEF1B alpha 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. eEF1B alpha 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 eEF1B alpha phosphorylation. p-eEF1B alpha 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 eEF1B alpha overexpression. Both eEF1B alpha and p-eEF1B alpha 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 eEF1B alpha-UBR5-ARID1A signaling.