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

Abstract

CCAAT enhancer-binding protein (C͞EBP)␤, C͞EBP␣, and peroxisome proliferator activated receptor (PPAR)␥ act in a cascade where C͞EBP␤ activates expression of C͞EBP␣ 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, C͞EBP␤ 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 C͞EBP␤ 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 C͞EBP␤ show that phosphorylation of Thr-188 by mitogen-activating protein kinase ''primes'' C͞EBP␤ for subsequent phosphorylation on Ser-184 and Thr-179 by glycogen synthase kinase 3␤, acquisition of DNA-binding function, and transactivation of the C͞EBP␣ and PPAR␥ genes. The delayed transactivation of the C͞EBP␣ and PPAR␥ genes by C͞EBP␤ appears necessary to allow mitotic clonal expansion, which would otherwise be prevented, because C͞EBP␣ and PPAR␥ are antimitotic.3T3-L1 preadipocyte ͉ cell cycle ͉ differentiation ͉ mitotic clonal expansion C CAAT enhancer-binding protein ␤ (C͞EBP␤) is expressed early in the adipocyte differentiation program, first initiating mitotic clonal expansion (MCE) (1, 2) and later activating expression of C͞EBP␣ and peroxisome proliferator-activated receptor ␥ (PPAR␥) (3-6), pleiotropic activators of adipocyte genes (3,4,7,8). Both MCE and expression of C͞EBP␣ and PPAR␥ are required for differentiation (1, 2, 9). When treated with differentiation inducers, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle, undergo approximately two rounds of mitosis, then exit the cell cycle and enter the terminal stages of differentiation. Transcriptional activation of the C͞EBP␣ and PPAR␥ genes is induced by the interaction of C͞EBP␤ with C͞EBP regulatory elements in these gene promoters (3-6).Although expression of C͞EBP␤ occurs within 2 h of induction of differentiation, acquisition of DNA-binding activity and thus transcription of the C͞EBP␣ and PPAR␥ genes are delayed (10). Acquisition of DNA-binding activity begins after a long lag (Ϸ14 h), concurrent with the entry of S phase at the onset of MCE and transcription of the C͞EBP␣ and PPAR␥ genes (ref. 10 and Fig. 1). This lag appears necessary, because C͞EBP␣ and PPAR␥ are antimitotic (11-15), and their premature expression would otherwise prevent the MCE required for differentiation. To elucidate the mechanism by which C͞EBP␤ acquires DNAbinding activity, we considered the possibility that covalent modification of C͞EBP␤ occurs during this time window.Several lines of evidence indicated that C͞EBP␤ can be phosphorylated in vitro by a variety of kinases, including PKA (16), PKC (16), mitogen-activated protein kinase (MAPK) (17), and Ca 2ϩ -calmodulin-dependent kinase II (18). ...

Effect of phosphorylation and S–S bond-induced dimerization on DNA binding and transcriptional activation by C/EBPβ

“…We recently showed that C/EBP␤ must undergo sequential phosphorylation to acquire DNA binding ability (6). Immediately after the expression of C/EBP␤, a ''priming'' phosphorylation occurs on Thr 188 catalyzed by MAP kinase, which is required but is insufficient for acquisition of DNA binding activity.…”

“…C/EBP␤ activates the expression of the genes for two pleiotropic transcription factors, C/EBP␣ and PPAR␥ (4,5), which in turn activate the expression of numerous adipogenic genes. Although maximal expression of C/EBP␤ occurs rapidly (within 2-4 h) on induction of differentiation and remains at this level over the next 48 h, the acquisition of DNA binding activity by C/EBP␤ required for the expression of C/EBP␣ and PPAR␥ only occurs after a long lag period of Ϸ12-14 h (1,2,6). At the end of the lag period, growth-arrested preadipocytes synchronously enter the cell cycle and undergo several rounds of mitotic clonal expansion (2, 3), a process required for subsequent differentiation.…”

G0/G1 switch gene 2 has a critical role in adipocyte differentiation

“…7 Upon hormonal stimulation, cells begin to express C/EBPb; this protein is then serially phosphorylated by mitogen-activated protein kinase and glycogen synthase kinase b, resulting in active C/EBPb with full DNA-binding capacity. 14 Activation of C/EBPb is clearly associated with MCE, as cells without C/EBPb cannot complete clonal expansion. 15 Those studies revealed a role of MCE in adipogenesis, and they explain why C/EBPb is needed for entering the terminal differentiation process.…”

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

“…MAPK/ERK itself is rapidly (Յ1 h) phosphorylated/activated after induction of differentiation (Fig. 1B) (3). Although phosphorylation of C/EBP␤ on Thr 188 persists at a high level for Ͼ24 h ( Fig.…”

“…Blocking these steps disrupts MCE and progression of the differentiation program. The synchrony by which 3T3-L1 preadipocytes proceed through MCE has been invaluable in delineating the sequence of events that occur early in the adipocyte differentiation program (2,3).…”