O-linked N-acetylglucosamine modification on CCAAT enhancer-binding protein β. Role during adipocyte differentiation

CCAAT enhancer-binding protein (C/EBP)β is a basic leucine zipper transcription factor family member, and can be phosphorylated, acetylated, and sumoylated. C/EBPβ undergoes sequential phosphorylation during 3T3-L1 adipocyte differentiation. Phosphorylation on Thr¹⁸⁸ by MAPK or cyclin A/cdk2 primes the phosphorylations on Ser¹⁸⁴/Thr¹⁷⁹ by GSK3β, and these phosphorylations are required for the acquisition of DNA binding activity of C/EBPβ. Here we show that C/EBPβ is modified by O-GlcNAc, a dynamic single sugar modification found on nucleocytoplasmic proteins. The GlcNAcylation sites are Ser¹⁸⁰ and Ser¹⁸¹, which are in the regulation domain and are very close to the phosphorylation sites (Thr¹⁸⁸, Ser¹⁸⁴, and Thr¹⁷⁹) required for the gain of DNA binding activity. Both in vitro and ex vivo experiments demonstrate that GlcNAcylation on Ser¹⁸⁰ and Ser¹⁸¹ prevents phosphorylation on Thr¹⁸⁸, Ser¹⁸⁴, and Thr¹⁷⁹, as indicated by the decreased relative phosphorylation and DNA binding activity of C/EBPβ delayed the adipocyte differentiation program. Mutation of both Ser¹⁸⁰ and Ser¹⁸¹ to Ala significantly increase the transcriptional activity of C/EBPβ. These data suggest that GlcNAcylation regulates both the phosphorylation and DNA binding activity of C/EBPβ.