ULK1-dependent phosphorylation of OGT instructs the tumorigenicity of O-GlcNAcylation.

Investigations from the last four decades have correlated high O-linked N-acetylglucosamine (O-GlcNAc) levels with various cancer types, but it is not known how OGT responds to diverse nutrients to finetune cellular O-GlcNAcylation levels. Herein we identified a critical OGT phosphorylation site by unc-51 like autophagy activating kinase 1 (ULK1) under glucose depletion. First, we demonstrated that glucose levels modulate the interaction between OGT and ULK1 and cellular O-GlcNAcylation levels. Low glucose induces high O-GlcNAcylation, which could be reversed by ULK1 inhibition. Then, using mass spectrometry, we showed that ULK1 phosphorylates OGT at Ser576 and stabilizes OGT. Further biochemical experiments revealed that Ser576 phosphorylation inhibits Lys604 ubiquitination by stimulating OGT binding with BAP1, a de-ubiquitinase for OGT. Strikingly, using the OGT knock-in cells, we found that in mouse xenograft models OGT-S576A completely abolishes the tumorigenicity of OGT, probably due to low O-GlcNAcylation. In sum, we found that ULK1 phosphorylates OGT at Ser-576 under glucose deprivation, which stabilizes OGT by promoting OGT-BAP1 association and is pivotal for O-GlcNAcylation levels and tumorigenesis. As low glucose is often associated with tumor progression, our work not only unearths a key mechanism of how OGT is regulated by glucose levels, but also offers new therapeutic opportunities targeting OGT.