Effect of phosphorylation and S-S bond-induced dimerization on DNA binding and transcriptional activation by C/EBPβ

CCAAT enhancer binding protein beta (C/EBP beta) plays an essential role in the cascade that triggers adipocyte differentiation. C/EBP beta activates transcription of C/EBP alpha and peroxisome proliferatoractivated receptor-gamma, transcriptional activators of genes that give rise to the adipocyte phenotype. Sequential phosphorylation of C/EBP beta/liver activating protein (LAP) on Thr(188) by MAPK and on Ser(184) or Thr(179) by glycogen synthase kinase beta (GSK3 beta) is required for acquisition of DNA binding activity and transcriptional activation. To investigate how phosphorylation and dimerization of C/EBP beta/LAP alter these activities, wild-type (Wt) and mutant rC/EBP beta s were prepared and purified to assess DNA binding and transcription in cell-free systems. rC/EBP beta/LAP, phosphorylated by MAPK and GSK3 beta 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/EBP beta/LAP, only doubly phosphorylated rC/EBP beta/LAP (by MAPK and GSK3 beta) activated transcription driven by Wt C/EBP alpha, 422/aP2, and SCD1 promoters. Oxidation-induced dimerization of doubly phosphory- lated Wt rC/EBP beta/LAP increased DNA binding, whereas unphosphorylated Wt rC/EBP beta/LAP lacked DNA binding activity. Mutation of the C-terminal CYS296 adjacent to the leucine zipper and CYS143 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/EBP beta/LAP caused a conformational change that facilitates S-S bond formation and dimerization, rendering the basic region accessible to the C/EBP regulatory element.