Our analyses have led to the discovery that several sites within conserved region 4 of C/EBPα are phosphorylated in vivo (Fig. ). Two sites, T222 and T226, are phosphorylated by GSK3, whereas S230 is phosphorylated by an unknown kinase. In addition, this work delineates a part of the signaling mechanism through which C/EBPα phosphorylation is regulated. Previous work (38
) has established that insulin, acting through signaling intermediates, including PI 3-kinase, causes inhibition of GSK3 in adipocytes. We report here that inactivation of GSK3 leads to dephosphorylation of two sites, T222 and T226 (Fig. ). Furthermore, since okadaic acid treatment of adipocytes blocks the lithium-induced dephosphorylation of C/EBPα, it is likely that PP1 or PP2A is responsible for dephosphorylation of the insulin-sensitive sites (Fig. D). Our work also shows that insulin and wortmannin alter the protease accessibility of C/EBPα, thereby implying that phosphorylation at T222 and T226 causes a conformational change in the structure of C/EBPα (Fig. ). Finally, we report here that lithium inhibits preadipocyte differentiation (Fig. ), suggesting that GSK3-mediated phosphorylation of C/EBPα and other transcription factors, such as C/EBPβ, is required for adipogenesis.
A hierarchical model of phosphorylation has been described for several GSK3 substrates (7
). If this model holds true for C/EBPα, then phosphorylation of S230 is required for subsequent phosphorylation of T226 and then T222. Results from two experiments indicate that phosphorylation by GSK3 need not occur in this requisite order. First, a glutathione S
-transferase–C/EBPα fusion protein purified from bacteria (and therefore not phosphorylated) is phosphorylated by GSK3 in vitro on threonine alone (not shown). Second, based on the hierarchical model, we would expect neither the p30TTA mutant nor the p30AAA mutant to be phosphorylated by GSK3. However, we observed that while p30AAA exists as a single dephosphorylated band upon SDS-PAGE, p30TTA migrates as a doublet whose low-mobility band is rapidly lost after lithium treatment (not shown). Together, these experiments suggest that T222 and T226 are substrates for GSK3 even when S230 is mutated to alanine. Despite this negative evidence, it remains possible that phosphorylation of S230 changes the affinity of C/EBPα for recognition by GSK3 and may therefore serve a modulatory role.
Phylogenetic analysis of C/EBPα has allowed us to define four highly conserved regions in the N-terminal transactivation domain, and we believe that the regions of greatest functional importance are likely to be found therein (18
). Each of conserved regions 1, 2, and 3 has intrinsic transactivation ability, suggesting that these regions may directly interact with coactivators or with the basal transcriptional machinery. In contrast, the fourth conserved region, although the most highly conserved, has no known function. Our finding that GSK3 phosphorylates three sites within conserved region 4 suggests that C/EBPα activity may be regulated through this mechanism. However, the precise role of this modification has proved difficult to uncover. Mutation of T222 and T226 to alanines does not change the ability of C/EBPα to transactivate leptin or C/EBPα promoters in reporter gene assays (data not shown). Furthermore, altering the phosphorylation status of C/EBPα by cotransfection of GSK3 also does not influence the ability of C/EBPα to transactivate in these experiments (data not shown). Finally, ectopic expression of the T222A, T226A mutant, like its wild-type counterpart, is sufficient to induce spontaneous differentiation of preadipocytes (data not shown), demonstrating that phosphorylation of these sites is not required for activation of chromatin-embedded genes. Thus, GSK3-mediated phosphorylation does not, in itself, dramatically alter the activity of C/EBPα in our assays. Since the protease accessibility of C/EBPα was determined in nuclei, the effects of phosphorylation on sensitivity of C/EBPα to trypsin (Fig. ) may be the result of conformational changes or differential interactions with nuclear proteins.
It has been reported that protein kinase C can phosphorylate specific sites within the basic region of C/EBPα in vitro (33
). This modification is an attractive mechanism for the regulation of C/EBPα activity, since addition of a negative charge to the basic region profoundly reduces its affinity for DNA. However, we have no evidence from labeling experiments that this phosphorylation occurs in vivo. Phosphate incorporation into His-p18AAA was undetectable in our studies, suggesting that T222, T226, and S230 are the only phosphorylated residues in the C-terminal 18 kDa of C/EBPα (Fig. C).
We have shown that phosphorylation of C/EBPα by GSK3 is regulated by insulin. In addition to its role in insulin signaling, GSK3 is a mediator in the Wnt signaling pathway, where it has been shown to be important in the specification of cell fate (reviewed in references 4
). Wnts are a family of secreted glycoproteins which, probably through activation of frizzled receptors, stimulate a signaling cascade resulting in the inactivation of GSK3. Improper expression of Wnts has severe developmental consequences. For instance, overexpression of Wnt-1 results in the formation of two-headed tadpoles (36
). Wnts are also important for the formation of mesodermal derivatives such as Xenopus
myoblasts, in which dominant-negative expression of Wnt blocks MyoD expression and impairs skeletal muscle formation (23
). It is tempting to speculate that Wnt signaling may also be important in the differentiation of mesodermal derivatives into adipocytes by regulating the activity of transcription factors such as the C/EBP family. As shown in Fig. B, C/EBPβ and C/EBPδ contain GSK3 consensus sequences and may, like C/EBPα, be phosphorylated by GSK3. Our finding that lithium prevents adipogenesis supports the hypothesis that GSK3 activity is required for the differentiation of 3T3-L1 preadipocytes. Although the presence of receptors for Wnt on 3T3-L1 preadipocytes has not been reported, other cell models with adipogenic potential, NIH 3T3 cells and CH310T1/2, respond to Wnts (1
). We propose that regulation by GSK3 of C/EBPα phosphorylation and possibly preadipocyte differentiation is controlled not only by insulin, but also by Wnts and other ligands.
Insulin has rapid effects on the flow of carbon through metabolic pathways by regulating the activity of metabolic enzymes through stimulating their phosphorylation or dephosphorylation. Insulin also has longer-term effects on metabolism by altering gene expression to regulate the amount of enzyme or regulatory proteins available for metabolism. GSK3 acts as a node through which insulin signals to regulate both carbohydrate metabolism and adipocyte gene expression. The best-characterized role of GSK3 is in mediating the effects of insulin on glycogen metabolism through its phosphorylation and inhibition of glycogen synthase (28
). It is now apparent that GSK3 also mediates the effects of insulin on adipocyte gene expression, since C/EBPα, a transcription factor required for acquisition of insulin sensitivity, is phosphorylated by GSK3. Dephosphorylation of the GSK3 sites in both glycogen synthase and C/EBPα appears to be mediated by PP1, since both activities are sensitive to okadaic acid and wortmannin. Thus, insulin appears to use reciprocal regulation of GSK3 and PP1 activities to coordinately regulate short- and long-term effects on adipocyte metabolism.