Phosphorylations Of C/EBPβ Help Keep Its Stability In3T3-L1Preadipocyte Differentiation

C/EBPβ is an important transcription factor that belongs to C/EBPs (CCAAT enhancer binding proteins) family, whose C-terminal region contains the highly conserved DNA-binding domain and dimerization domain. Our recent studies demonstrated that C/EBPβ (LAP) plays an important role in proliferation and differentiation of3T3-L1preadipocytes. The function of C/EBPP (LAP) not only depends on the enhancing of transcription and translation, but also is regulated by proteolysis, phosphorylation, and the interaction between proteins, etc. In the progress of differentiation of3T3-L1preadipocytes, at the beginning of differentiation, MAPK induces phosphorylation of C/EBPβ (LAP) on Thr188,12hours later, the activation of MAPK decreases, but cdk2increases and keeps phosphorylation of C/EBPβ (LAP) on Thr188. Simultaneously, GSK3β transferred from cytoplasm to nucleus, and then induces phosphorylation of C/EBPβ (LAP) on Ser184or Thr-179basing on phosphorylation on Thr188. The double phosphorylations of C/EBPβ (LAP) on Thrl88and Ser184or on Thr188and Thr-179are required for the DNA binding activity and transcription activity of C/EBPP (LAP). Generally, proteins will be degraded through proteasome or calpain-mediated pathways after their functioning, and phosphorylations often regulate the degradation rate of these proteins. In this paper, in order to find whether phosphorylations by MAPK (cdk2) and GSK3β could regulate β-calpain-mediated degaradtion of C/EBPβ (LAP), we did the degradation assay with recombinant C/EBPβ (LAP) by μ-calpain in vitro. We found that singly phosphorylated C/EBPβ (LAP) by MAPK or doubly phosphorylated C/EBPβ (LAP) by MAPK and GSK3β was more stable than unphosphorylated C/EBPβ (LAP). Treatment of3T3-L1preadipocytes with U0126(a MAPK inhibitor)/roscovitine (a cdk2inhibitor) or lithium chloride (a GSK3P inhibitor) decreased the turnover rate of C/EBPβ (LAP). These findings demonstrated that phosphorylations of serine/threonine residues of C/EBPβ (LAP) by MAPK (cdk2) and GSK3β prevent μ-calpain-mediated degradation of C/EBPβ in3T3-L1preadipocytes.