Role of cdk2 in the sequential phosphorylation/activation of C/EBPβ during adipocyte differentiation

Upon induction of differentiation, growth-arrested (G(1) phase) 3T3-L1 preadipocytes express CCAAT/enhancer binding protein-beta (C/EBP beta), initiating a transcriptional cascade. C/EBP beta immediately undergoes a priming phosphorylation (on Thr,88) by MAPK/ERK. However, the acquisition of DNA binding and transactivation capacity of C/EBP beta is delayed until further phosphorylation (on Ser,84 or Thr(179)) by GSK3 beta occurs. Phosphorylation by glycogen synthase kinase-3 beta (GSK3 beta) induces S phase entry and thereby mitotic clonal expansion (MCE), a requirement for terminal differentiation. Because MAPK activity is down-regulated before S phase is completed, we sought to identify the kinase that maintains C/EBP beta)3 in the primed phosphorylated state throughout S phase and MCE. We show here that cdk2/cyclinA, whose expression is activated at the onset of S phase, functions in this capacity. Ex vivo and in vitro experiments show that cdk2/cyclinA catalyzes this delayed priming phosphorylation. Mass spectrometric analysis revealed that cdk2/cyclinA phosphorylates C/EBP beta on Thr(188) and is required for phosphorylation (on Ser(184) or Thr(179)) of C/EBP beta by GSK3 beta and maintenance of DNA binding activity. Suppression of cdk2 activity by RNA interference or pharmacologic inhibitor disrupts subsequent events in the differentiation program. Thus, MAPK and cdk2/cyclinA act sequentially to maintain Thr,88 of C/EBP beta in the primed phosphorylated state during MCE and thereby progression of terminal differentiation.