Validation, Identification, and Biological Consequences of the Site-specific O -GlcNAcylation Dynamics of Carbohydrate-responsive Element-binding Protein (ChREBP).

O -GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O -GlcNAcylation on ChREBP and no exact O -GlcNAcylation sites have been reported so far. Here, we validate O -GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O -GlcNAcylation on Ser614 in the C -terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O -GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O -GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O -GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O -GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O -GlcNAcylation and highlights the biological consequences of the site-specific O -GlcNAcylation dynamics of ChREBP.