Amino Acids and mTOR Mediate Distinct Metabolic Checkpoints in Mammalian G1 Cell Cycle

Abstract

ObjectiveIn multicellular organisms, cell division is regulated by growth factors (GFs). In the absence of GFs, cells exit the cell cycle at a site in G1 referred to as the restriction point (R) and enter a state of quiescence known as G0. Additionally, nutrient availability impacts on G1 cell cycle progression. While there is a vast literature on G1 cell cycle progression, confusion remains – especially with regard to the temporal location of R relative to nutrient-mediated checkpoints. In this report, we have investigated the relationship between R and a series of metabolic cell cycle checkpoints that regulate passage into S-phase.MethodsWe used double-block experiments to order G1 checkpoints that monitor the presence of GFs, essential amino acids (EEAs), the conditionally essential amino acid glutamine, and inhibition of mTOR. Cell cycle progression was monitored by uptake of [3H]-thymidine and flow cytometry, and analysis of cell cycle regulatory proteins was by Western-blot.ResultsWe report here that the GF-mediated R can be temporally distinguished from a series of late G1 metabolic checkpoints mediated by EAAs, glutamine, and mTOR – the mammalian/mechanistic target of rapamycin. R is clearly upstream from an EAA checkpoint, which is upstream from a glutamine checkpoint. mTOR is downstream from both the amino acid checkpoints, close to S-phase. Significantly, in addition to GF autonomy, we find human cancer cells also have dysregulated metabolic checkpoints.ConclusionThe data provided here are consistent with a GF-dependent mid-G1 R where cells determine whether it is appropriate to divide, followed by a series of late-G1 metabolic checkpoints mediated by amino acids and mTOR where cells determine whether they have sufficient nutrients to accomplish the task. Since mTOR inhibition arrests cells the latest in G1, it is likely the final arbiter for nutrient sufficiency prior to committing to replicating the genome.

Phospholipase D and the Maintenance of Phosphatidic Acid Levels for Regulation of Mammalian Target of Rapamycin (mTOR)

“…As indicated in Fig. 2, an mTOR-dependent cell cycle checkpoint maps late in G 1 downstream of essential amino acid and Gln checkpoints (25). Because PA interacts directly with mTOR (29) and is required for the stability of both mTORC1 and mTORC2 complexes (30), PA likely works in concert with essential amino acids and possibly Gln to promote cell cycle progression through the late mTOR-dependent checkpoint.…”

“…There must be a supply of essential amino acids, Gln, and glucose available to generate the biological molecules needed to produce two daughter cells. Notably, there are a series of cell cycle checkpoints that sense the presence of essential amino acids and Gln in late G 1 that must be passed before cells commit to enter S-phase and replicate the genome (25) (Fig. 2).…”

“…2). Suppression of mTOR, like amino acid deprivation, also leads to late G 1 arrest (25,26). Because essential amino acids activate mTOR via Rag GTPases at the lysosomal membrane (27), it was surprising that suppression of mTOR blocked cell cycle progression at a site later in G 1 than the checkpoints that monitor the presence of essential amino acids and Gln (25) (Fig.…”

“…Gln is designated as a "conditionally" essential amino acid because although it is synthesized under non-proliferative conditions, it becomes essential during proliferation. Of significance, there is a Gln-sensitive G 1 cell cycle checkpoint that can be distinguished from an essential amino acid checkpoint in mammalian cells (25). Thus, it may be important for mTOR to sense this critical nutrient input.…”

Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in Dividing Human Cells

“…Conversely, experimental knockout of a number of genes involved in PI3K activation and its downstream effectors causes organ hypoplasia and reduced body size in mice (Liu et al, 1993; Dummler et al, 2006). mTORC‐1 is a pivotal sensor of nutrient availability and stress conditions (Ellisen, 2005; Gwinn et al, 2008; Saqcena et al, 2013). In stress conditions the cell is generally under a dominant metabolic checkpoint that follows different mechanisms according to the severity and duration of such stimuli.…”