O-Linked N-Acetylglucosamine Modification on CCAAT Enhancer-binding Protein Î

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 Thr 188 by MAPK or cyclin A/cdk2 primes the phosphorylations on Ser 184 /Thr 179 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 Ser 180 and Ser 181 , which are in the regulation domain and are very close to the phosphorylation sites (Thr 188 , Ser 184 , and Thr 179 ) required for the gain of DNA binding activity. Both in vitro and ex vivo experiments demonstrate that GlcNAcylation on Ser 180 and Ser 181 prevents phosphorylation on Thr 188 , Ser 184 , and Thr 179 , as indicated by the decreased relative phosphorylation and DNA binding activity of C/EBPβ delayed the adipocyte differentiation program. Mutation of both Ser 180 and Ser 181 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.