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)beta, C/EBP alpha, and peroxisome proliferator activated receptor (PPAR)gamma act in a cascade where C/EBP beta activates expression of C/EBP alpha and PPAR gamma, which then function as pleiotropic activators of genes that produce the adipocyte phenotype. When growth-arrested 3T3-L1 preadipocytes are induced to differentiate, C/EBP beta is rapidly expressed but still lacks DNA-binding activity. After a long (14-hour) lag, glycogen synthase kinase 313 enters the nucleus, which correlates with hyperphosphorylation of C/EBP beta 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/EBP beta show that phosphorylation of Thr-188 by mitogen-activating protein kinase "primes" C/EBP beta for subsequent phosphorylation on Ser-184 and Thr-179 by glycogen synthase kinase 30, acquisition of DNA-binding function, and transactivation of the C/EBP alpha and PPAR gamma genes. The delayed transactivation of the C/EBP alpha and PPAR gamma genes by C/EBP beta appears necessary to allow mitotic clonal expansion, which would otherwise be prevented, because C/EBP alpha and PPAR-gamma are antimitotic.