Combination of amino acids necessary and sufficient for the optimal activation of mTORC1

抄録

Background: mTORC1 (mechanistic target of rapamycin complex 1) is a serine/threonine kinase protein complex that functions as a master regulator of cellular metabolism and growth. Various extra- and intracellular signals including growth factors, nutrients such as amino acids, and energy state, are integrated by mTORC1 to control the downstream metabolic processes. Among amino acids, leucine, arginine and glutamine are known to strongly affect the mTORC1 activity. However, stimulation with each of the amino acids alone cannot efficiently activate mTORC1. Even though such a requirement for multiple amino acids has been well documented, the amino acids involved in the activation of mTORC1 are still unclear. In this study, we tried to reveal the specific combination of amino acids that is necessary and sufficient to activate mTORC1.

Methods: Human embryonic kidney HEK293T cells were starved for amino acids to inactivate mTORC1, and then stimulated with amino acids to re-activate mTORC1. The activity of mTORC1 was monitored by immunoblot analysis of the phosphorylation of mTORC1 substrates, 4EBP1 and p70S6K. For the screening of amino acids involved in the activation of mTORC1, each amino acid was depleted from the stimulation media. The identified amino acids were further tested for their function to activate mTORC1, by adding them into the stimulation media one by one or in combinations.

Results: Among 15 amino acids included in the DMEM medium, 5 amino acids including leucine, arginine and glutamine significantly reduced mTORC1 re-activation when depleted from the stimulation media. Mixture of the 5 amino acids was potent enough to activate mTORC1 to the same level as that of 15 amino acids does. As expected, when applied alone, none of the 5 amino acids was able to strongly activate mTORC1. The depletion of any single amino acid from the 5 amino acids significantly reduced mTORC1 activity, which was not compensated by increasing the concentration of other 4 amino acids.

Conclusions: We found the combination of amino acids that is necessary and sufficient for the optimal activation of mTORC1. This finding would contribute to the understanding of molecular mechanisms for amino acid sensing and signaling to activate mTORC1.