Sequential phosphorylation of CCAAT enhancer-binding protein β by MAPK and glycogen synthase kinase 3β is required for adipogenesis

CCAAT enhancer-binding protein (C/EBP)β, C/EBPα, and peroxisome proliferator activated receptor (PPAR)γ act in a cascade where C/EBPβ activates expression of C/EBPα and PPARγ, which then function as pleiotropic activators of genes that produce the adipocyte phenotype. When growth-arrested 3T3-L1 preadipocytes are induced to differentiate, C/EBPβ is rapidly expressed but still lacks DNA-binding activity. After a long (14-hour) lag, glycogen synthase kinase 3β enters the nucleus, which correlates with hyperphosphorylation of C/EBPβ and acquisition of DNA-binding activity. Concurrently, 3T3-L1 preadipocytes synchronously enter S phase and undergo mitotic clonal expansion, a prerequisite for terminal differentiation. Ex vivo and in vitro experiments with C/EBPβ show that phosphorylation of Thr-188 by mitogen-activating protein kinase "primes" C/EBPβ for subsequent phosphorylation on Ser-184 and Thr-179 by glycogen synthase kinase 3β, acquisition of DNA-binding function, and transactivation of the C/EBPα and PPARγ genes. The delayed transactivation of the C/EBPα and PPARγ genes by C/EBPβ appears necessary to allow mitotic clonal expansion, which would otherwise be prevented, because C/EBPα and PPARγ are antimitotic.