O-Linked N-Acetylglucosamine Modification on CCAAT Enhancer-binding Protein β: ROLE DURING ADIPOCYTE DIFFERENTIATION

Xi Li,Henrik Molina,Haiyan Huang,You-You Zhang,Mei Liu,Shu-Wen Qian,Chad Slawson,Wagner B Dias,Akhilesh Pandey,Gerald W Hart,M Daniel Lane,Qi-Qun Tang

doi:10.1074/jbc.m109.005678

Abstract

CCAAT enhancer-binding protein (C/EBP)beta is a basic leucine zipper transcription factor family member, and can be phosphorylated, acetylated, and sumoylated. C/EBPbeta 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 GSK3beta, and these phosphorylations are required for the acquisition of DNA binding activity of C/EBPbeta. Here we show that C/EBPbeta 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/EBPbeta delayed the adipocyte differentiation program. Mutation of both Ser(180) and Ser(181) to Ala significantly increase the transcriptional activity of C/EBPbeta. These data suggest that GlcNAcylation regulates both the phosphorylation and DNA binding activity of C/EBPbeta.

Highlights

CCAAT enhancer-binding protein (C/EBP)␤ is a basic leucine zipper transcription factor family member, and can be phosphorylated, acetylated, and sumoylated
We find that C/EBP␤ is GlcNAcylated on Ser180 and Ser181, which is in the regulation domain (RD) region and adjacent to the phosphorylation sites (Thr188, Ser184, and Thr179) required for the gain of DNA binding activity
These data indicate that C/EBP␤ can be GlcNAcylated and that the residue or residues very likely are located in the DNA-binding and/or regulation domain of C/EBP␤, which is shared by both LAP and LIP

Summary

ROLE DURING ADIPOCYTE DIFFERENTIATION*

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/EBP␤ delayed the adipocyte differentiation program Mutation of both Ser180 and Ser181 to Ala significantly increase the transcriptional activity of C/EBP␤. Three phosphorylation sites (Thr188, Ser184, and Thr179) have been identified in this RD region of C/EBP␤ during 3T3-L1 adipocyte differentiation [14] Both ex vivo and in vitro experiments demonstrate that phosphorylation on Thr188 (first by MAPK during G1 phase and late by CyclinA/cdk2 [15]) primes C/EBP␤ for the phosphorylation on Ser184 or Thr179 by GSK3␤, and these phosphorylations are required for the gain of DNA binding activity of C/EBP␤ [14, 16]. This suggests that GlcNAcylation regulates both the phosphorylation and DNA binding activity of C/EBP␤, and GlcNAcylation and phosphorylation regulate the transcriptional activities of C/EBP␤ in a competitive way and by alternative occupancy at adjacent sites

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION