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

CCAAT enhancer-binding protein (C EBP) , C EBP , and peroxisome proliferator activated receptor (PPAR) act in a cascade where C EBP activates expression of C EBP and PPAR , 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 is rapidly expressed but still lacks DNA-binding activity. After a long (14-hour) lag, glycogen synthase kinase 3 enters the nucleus, which correlates with hyperphosphorylation of C EBP 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 show that phosphorylation of Thr-188 by mitogen-activating protein kinase ‘‘primes’’ C EBP for subsequent phosphorylation on Ser-184 and Thr-179 by glycogen synthase kinase 3 , acquisition of DNA-binding function, and transactivation of the C EBP and PPAR genes. The delayed transactivation of the C EBP and PPAR genes by C EBP appears necessary to allow mitotic clonal expansion, which would otherwise be prevented, because C EBP and PPAR are antimitotic.