Role of cdk2 in the sequential phosphorylation/activation of C/EBPbeta during adipocyte differentiation.
Upon induction of differentiation, growth-arrested (G(1) phase) 3T3-L1 preadipocytes express CCAAT/enhancer binding protein-beta (C/EBPbeta), initiating a transcriptional cascade. C/EBPbeta immediately undergoes a priming phosphorylation (on Thr(188)) by MAPK/ERK. However, the acquisition of DNA binding and transactivation capacity of C/EBPbeta is delayed until further phosphorylation (on Ser(184) or Thr(179)) by GSK3beta occurs. Phosphorylation by glycogen synthase kinase-3beta (GSK3beta) 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/EBPbeta 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/EBPbeta on Thr(188) and is required for phosphorylation (on Ser(184) or Thr(179)) of C/EBPbeta by GSK3beta 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(188) of C/EBPbeta in the primed phosphorylated state during MCE and thereby progression of terminal differentiation.
Li
Xi
X
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Kim
Jae Woo
JW
Grønborg
Mads
M
Urlaub
Henning
H
Lane
M Daniel
MD
Tang
Qi-Qun
QQ
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology* / Q000201:enzymology* / Q000378:metabolism; D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D051357:Cyclin-Dependent Kinase 2 / Q000502:physiology*; D019281:Dimerization; D051379:Mice; D010766:Phosphorylation; D011487:Protein Conformation; D016196:S Phase / Q000502:physiology; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D022782:CCAAT-Enhancer-Binding Protein-beta; D013912:Threonine; D051357:Cyclin-Dependent Kinase 2