Cells need to accurately perceive the extracellular nutrient levels to modulate their metabolic processes and adjust their growth status. The mTORC1 complex serves as a crucial nutrient sensor, activating the mTORC1 signaling pathway when nutrients are plentiful to promote anabolic metabolism. Conversely, under nutrient deprivation, mTORC1 is inhibited, promoting catabolic metabolism and slowing down cell growth. Therefore, aberrant mTORC1 signaling may contribute to various conditions such as cancer, obesity, diabetes, neurodegenerative diseases, and aging, as it serves as a central regulator of cellular anabolic metabolism.
Amino acids serve as essential building blocks for protein synthesis and are sensed by cells through amino acid receptors, which then transmit signals to mTORC1. However, the molecular mechanisms by which individual amino acids regulate mTORC1, particularly whether a universal sensing mechanism for amino acids exists, remain unclear.
Case. Regulatory Mechanisms of mTOR Signaling Pathway (1)
Research Background
The study focuses on unraveling the intricate regulatory mechanisms governing the mTOR signaling pathway. mTOR, a central regulator of cell growth and metabolism, plays crucial roles in various physiological and pathological processes, making it a promising target for therapeutic interventions. Dysregulation of the mTOR pathway is implicated in numerous diseases, including cancer, metabolic disorders, and neurodegenerative diseases. Understanding the precise regulatory mechanisms controlling mTOR activity is essential for developing targeted therapies and improving disease management strategies.
Sample Sources:
The research utilized mouse models maintained in a C57BL/6 strain background. Mice were housed under controlled conditions with ad libitum access to regular chow diet and water. The study also involved the generation of genetically modified mouse models, including Mtor K2066R knockin mice. Additionally, various cell lines, such as 293T and HeLa cells, and their derivatives were employed in the experiments. These cell lines were cultured under specified conditions and authenticated to ensure reliability.
Technical Methods:
Firstly, researchers identified through immunoprecipitation (Co-IP) that FBXO22 (an F-box receptor subunit of the SCF E3 ligase) interacts with mTOR. To further investigate the function of FBXO22, researchers found that overexpression of FBXO22 promotes mTOR ubiquitination. Subsequent mutagenesis experiments (mutating seven lysine residues on the ubiquitin chain) revealed that FBXO22 mediates K27 ubiquitination of mTOR. Further identification of ubiquitination modification sites based on mass spectrometry confirmed that FBXO22 mediates ubiquitination modification of the K2066 residue of mTOR both in vitro and in cells.
FBXO22 mediates the K27-linked ubiquitination of mTOR.
Next, to further explore the function of mTOR ubiquitination, researchers investigated the impact of mTOR ubiquitination on the recruitment of three known mTORC1 substrates (S6K1, 4EBP1, and ULK1). The results revealed that overexpression of FBXO22 weakened the binding of mTORC1 substrates to mTOR or RAPTOR, but did not affect the interaction between mTOR and RAPTOR. Further mechanistic studies found that ubiquitination of mTOR at the K2066 site prevents the recruitment of mTORC1 substrates.
The ubiquitination of mTOR at the K2066 site mediated by FBXO22 inhibits the recruitment of mTORC1 substrates.
Previous studies have shown that changes in amino acids, growth factors, and energy reserve levels signal to mTORC1 through various pathways, dynamically regulating cellular activities. To determine whether FBXO22-mediated mTOR ubiquitination is associated with these processes, the authors depleted amino acids, serum, or glucose in cells (293T-Endo-HA-mTOR). The results showed that only amino acid deprivation induced substantial mTOR ubiquitination, which was absent in cells with the K2066 mutation (mTOR-K2066R-KI mutant cells). Furthermore, the researchers found that the elevation of mTOR ubiquitination levels was dependent on amino acid deprivation. These findings indicate that FBXO22-mediated mTOR ubiquitination is induced by amino acid deprivation.
The ubiquitination of mTOR mediated by FBXO22 is induced by amino acid deprivation.
The study further investigated whether the K2066 ubiquitination of mTOR is related to the inhibition of mTORC1 caused by amino acid deficiency. The results revealed that the K2066 mutation did not affect the inhibition of mTORC1 induced by specific deficiencies in three amino acids (Leu, Arg, and Gln), but it did weaken to varying degrees the inhibition of mTORC1 caused by deficiencies in the other ten amino acids. This indicates that the deficiency of the other ten amino acids inhibits mTORC1 through mTOR ubiquitination, whereas the deficiency of Leu, Arg, and Gln mainly inhibits mTORC1 through other mechanisms. Additionally, the authors confirmed the presence of at least two amino acid sensing modes in cells by knocking out DEPDC5 (a key component of the GATOR1 complex in the GATOR2-GATOR1-KICSTOR-Rags pathway), namely the GATOR2/GATOR1-KICSTOR-Rags pathway and the FBXO22-mediated mTOR ubiquitination pathway.
The ubiquitination of mTOR mediated by FBXO22 inhibits mTORC1 activity under amino acid deprivation.
The research team found that amino acid deprivation leads to the co-localization of FBXO22 and mTOR in the cytoplasm. Therefore, transient expression of GFP-tagged FBXO22 (GFP-FBXO22) in cells (293T or HeLa) showed an increased ratio of cytoplasmic/nuclear GFP-FBXO22 protein and accumulation of newly synthesized GFP-FBXO22 in the cytoplasm upon amino acid deprivation. This indicates that under conditions of amino acid deprivation, FBXO22 is retained in the cytoplasm. Consequently, the researchers overexpressed two protein mutants (eNLS-FBXO22 fusion protein with a nuclear localization sequence and DNLS-FBXO22 with the deletion of the nuclear localization sequence) in 293T cells lacking FBXO22 and assessed the ubiquitination level of mTOR. The results revealed that the abundance of cytoplasmic FBXO22 determines the level of mTOR ubiquitination. These findings suggest that amino acid deprivation leads to the retention of FBXO22 in the cytoplasm, thereby triggering mTOR ubiquitination.
Retention of FBXO22 in the cytoplasm during amino acid depletion leads to mTOR ubiquitination.
To further identify the upstream regulatory factor of FBXO22-mediated mTOR inhibition during amino acid depletion, researchers screened for interacting proteins of FBXO22 and found that the protein kinase GCN2 interacts with FBXO22. It was discovered that GCN2 induces mTOR ubiquitination in a FBXO22-dependent manner. Further mass spectrometry analysis revealed that GCN2 may be a specific kinase phosphorylating FBXO22 at threonine 127 (T127). This was further confirmed by custom-made phospho-specific antibodies targeting FBXO22 T127, showing GCN2 phosphorylates FBXO22 at T127 under amino acid deprivation. Moreover, mutation of the T127 site of FBXO22 prevented GCN2-CA mutant or amino acid deprivation from inducing mTOR ubiquitination. These results indicate that GCN2-mediated phosphorylation of FBXO22 at T127 is necessary for mTOR ubiquitination.
Finally, validation was performed in vivo. Detection of mTOR ubiquitination in the liver of mice fasted for 72 hours confirmed the existence of mTOR ubiquitination in vivo. Further construction of mTOR K2066R (mTORKR/KR) mice, and comparison with wild-type mice in terms of mTOR ubiquitination levels and mTORC1 activity after 12 hours of fasting, validated the critical role of mTOR ubiquitination in sensing amino acid deficiency in vivo.
In summary, this study identified a novel amino acid sensing mechanism of mTORC1, revealing that depletion of cellular amino acids leads to mTOR ubiquitination, thereby inhibiting the activity of mTORC1 kinase by preventing substrate recruitment. Mechanistically, amino acid deficiency leads to the accumulation of uncharged tRNAs, stimulating GCN2 phosphorylation of FBXO22. FBXO22, in turn, accumulates in the cytoplasm and ubiquitinates mTOR at Lys2066 with K27 ubiquitin chains. Overall, these data uncover a new mechanism by which mTORC1 senses amino acids through the tRNA-GCN2-FBXO22-mTOR pathway, providing new insights for the treatment of diseases related to mTORC1 dysregulation.
Reference
- Ge, Meng-Kai, et al. "The tRNA-GCN2-FBXO22-axis-mediated mTOR ubiquitination senses amino acid insufficiency." Cell Metabolism 35.12 (2023): 2216-2230.
