O-GlcNAc transferase (OGT) is an essential X-chromosome-encoded enzyme that catalyzes the addition of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of serine and threonine residues on many nuclear and cytosolic proteins. This posttranslational modification is reversible and is actively removed by the O-GlcNAcase OGA. It was shown more than two decades ago that OGT is essential for mammalian cell viability, but the underlying mechanisms are still enigmatic. Given the close association between OGT and human diseases, such as cancer, diabetes and cardiovascular disease, identification of the mechanisms by which OGT controls cell viability will facilitate the development of therapeutic strategies to manipulate OGT activity. Here, we employ a genome-wide CRISPR-Cas9 viability screen in mouse embryonic stem cells (mESCs) with inducible Ogt deletion to show that the block in cell viability induced by Ogt-deficiency stems from a deleterious increase in mitochondrial oxidative phosphorylation (OXPHOS). Mechanistically, we demonstrate that OGT safeguards mTOR (mechanistic target of rapamycin) activity to maintain mitochondrial fitness through modulation of proteasome activity and intracellular amino acid homeostasis. In the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR translocation and activation and increased oxidative phosphorylation. This mechanism also operates in CD8+ T cells, indicating its generality across mammalian cell types. Genome-wide proteomic and phosphoproteomic analyses show extensive changes in global signaling and confirm our finding of mTOR hyperactivation in OGT-deficient cells. In sum, our study highlights a novel function for OGT in regulating the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness and cell viability. Since many of the proteins involved in proteasome, mTOR and mitochondrial activity are aberrantly expressed in cancer, and since inhibitors for proteasome and mTOR have been used in cancer therapy, manipulating OGT activity may have therapeutic potential in diseases in which this signaling axis is impaired.
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