CCAAT enhancer binding protein β (C/EBPβ) plays an essential role in the cascade that triggers adipocyte differentiation. C/EBPβ activates transcription of C/EBPα and peroxisome proliferator-activated receptor-γ, transcriptional activators of genes that give rise to the adipocyte phenotype. Sequential phosphorylation of C/EBPβ/liver activating protein (LAP) on Thr188 by MAPK and on Ser184 or Thr 179 by glycogen synthase kinase β (GSK3β) is required for acquisition of DNA binding activity and transcriptional activation. To investigate how phosphorylation and dimerization of C/EBPβ/LAP alter these activities, wild-type (Wt) and mutant rC/EBPβs were prepared and purified to assess DNA binding and transcription in cell-free systems. rC/EBPβ/LAP, phosphorylated by MAPK and GSK3β in vitro, produced a >100-fold increase in DNA binding activity. Mutation of the phosphorylation to Glu increased DNA binding activity. Using a cell-free transcription system with nuclear extract from 3T3-L1 preadipocytes and rC/EBPβ/LAP, only doubly phosphorylated rC/EBPβ/LAP (by MAPK and GSK3β) activated transcription driven by Wt C/EBPα, 422/aP2, and SCD1 promoters. Oxidation-induced dimerization of doubly phosphorylated Wt rC/EBPβ/LAP increased DNA binding, whereas unphosphorylated Wt rC/EBPβ/LAP lacked DNA binding activity. Mutation of the C-terminal Cys296 adjacent to the leucine zipper and Cys 143 just upstream of the DNA binding domain eliminated phosphorylation-, oxidation-, and dimerization-dependent DNA binding activity, whereas mutation of Cys201 within the basic DNA binding domain had little effect on DNA binding. These findings indicate that dual phosphorylation of C/EBPβ/LAP caused a conformational change that facilitates S-S bond formation and dimerization, rendering the basic region accessible to the C/EBP regulatory element.
|Original language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Feb 6 2007|
- Adipocyte differentiation
- Disulfide bond
ASJC Scopus subject areas