INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

In view of the prevalence of obesity and obesity-related diseases, such as type II diabetes, it is important to understand the molecular basis for adipose tissue development and metabolism. Traditionally, adipocytes were known to play an important role in lipid homeostasis through their ability to store triacyglycerol and release free fatty acids and glycerol in response to changing energy needs. It is now recognized that adipocytes play a more central role in metabolism through their secretion of factors that regulate food intake and metabolic efficiency (46). The negative health consequences of excess white adipose tissue have been well documented (49), but the effects of an absence of fat have only been studied recently. Mice without white adipose tissue were created by directing expression to adipocytes of a dominant-negative protein that forms dimers with members of the C/EBP and AP-1 families, but which does not bind DNA (37). These mice have abnormal growth rates and enlarged internal organs and die prematurely. In addition, they exhibit a number of metabolic defects, including diabetes, with reduced leptin and increased serum glucose, insulin, free fatty acids, and triacylglycerols. The widespread effects of blocking C/EBP and AP-1 activities in adipose tissue highlight the importance of studying these transcription factors in adipocyte differentiation and metabolism.

The molecular events associated with preadipocyte differentiation have been most thoroughly studied in 3T3-L1 cells (reviewed in references 9, 13, 32, 34, and 46). Treatment of preadipocytes with inducers of differentiation stimulates a rapid and transient increase in C/EBP and C/EBP, which in turn mediates the transcriptional activation of peroxisomal proliferator-activated receptor  (PPAR) during the 2 days following stimulation of differentiation. Expression of PPAR, probably in conjunction with C/EBP, induces expression of C/EBP to its maximal level 4 to 5 days after initiation of differentiation. Together, C/EBP and PPAR activate the transcription of genes involved in creating and maintaining the adipocyte phenotype (e.g., adipocyte lipid binding protein [422/aP2], stearoyl coenzyme A desaturase I, insulin-responsive glucose transporter [GLUT4], and leptin) (6, 24, 25, 31). The expression of PPAR and C/EBP remains elevated in adipocytes through positive self- and cross-regulation.

C/EBP not only plays an important role in preadipocyte differentiation, but also regulates gene expression in fully differentiated adipocytes. Although C/EBP-deficient fibroblasts acquire the morphological characteristics of adipocytes upon ectopic expression and activation of PPAR, these C/EBP/ adipocytes are resistant to insulin. Further analyses revealed that these cells fail to increase glucose uptake in response to insulin because of impaired expression of insulin receptor and insulin receptor substrate 1 (50). Likewise, NIH-3T3 cells, which differentiate into adipocytes without expression of C/EBP, are insulin resistant. In this case, it was found that the cells do not acquire the ability to transport glucose in response to insulin because of impaired expression of GLUT4 (17). Thus, C/EBP is essential for the acquisition of insulin sensitivity by adipocytes. Given the requirement of C/EBP for insulin responsivity, it is not surprising that insulin feeds back to inhibit this transcription factor. In 3T3-L1 adipocytes, insulin suppresses transcription of the C/EBP gene and stimulates dephosphorylation of C/EBP protein (22, 31). These events closely correlate with suppression of GLUT4 gene expression, perhaps as part of the mechanism of desensitization to insulin. In this study, we investigate further the dephosphorylation of C/EBP by insulin.

C/EBP is a member of the basic region-leucine zipper (bZIP) family of transcription factors (reviewed in reference 35). The C-terminal zipper domain mediates the formation of homodimers as well as heterodimers with family members and perhaps a subset of other bZIP transcription factors (e.g., ATF2) (44). The basic region is adjacent to the zipper domain and binds specific DNA sequences. The N terminus of C/EBP contains multiple transactivation domains that work synergistically to transactivate C/EBP-dependent promoters. The region N terminal to bZIP contains four regions that are highly conserved throughout evolution and that are separated by linker regions enriched in glycines and prolines (18). Conserved regions 1, 2, and 3 loosely correspond to transactivation domains identified previously (19, 39-41). Each conserved region is capable of transactivating the leptin promoter when fused to the C/EBP bZIP domain (18). Conserved region 4 is the most highly conserved region outside the bZIP and probably plays a regulatory role in C/EBP function. In the present study, three sites of phosphorylation were identified within this region, T222, T226, and S230, two of which are regulated by insulin. We present evidence that glycogen synthase kinase 3 (GSK3) phosphorylates T222 and T226, causing a conformational change in C/EBP. Upon treatment with insulin, T222 and T226 are dephosphorylated through inactivation of GSK3 and activation of protein phosphatase 1 (PP1) or PP2A. Finally, we present evidence that GSK3 is required for adipocyte development, since inhibition of GSK3 activity with lithium blocks, not only C/EBP phosphorylation, but also preadipocyte differentiation.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cell culture and transfection. Mouse 3T3-L1 preadipocytes (20) and human embryonic kidney 293T cells (a kind gift from Mitchell Lazar) were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL) supplemented with 10% calf serum, as described previously (22). Cells were transfected by calcium phosphate coprecipitation, as described previously (24). Expression plasmids, at the amounts indicated, were supplemented with pcDNA3.1() (Invitrogen), such that the total DNA was 20 µg/10-cm-diameter plate. Cells were lysed 24 to 48 h following transfection. Constitutively active GSK3 (S9A) was obtained from Peter J. Roach (Indiana University School of Medicine) (16).

C/EBP expression plasmids. To increase the ease of manipulating the mouse C/EBP gene (5), four unique restriction sites were created through introduction of silent mutations. C/EBP from the NruI site (+5 nucleotide [nt]) to the EcoRV site (+2111 nt) was subcloned into pBluescript (KS+) [pBS(KS+)] (Stratagene), and the following silent mutations were made by site-directed mutagenesis (QuickChange; Stratagene): a KpnI site was generated by mutation of C531 to G, a PstI site was generated by mutation of A774 to G, an SphI site was generated by mutation of C822 to T, and an XhoI site was generated by mutation of G1074 to C. In addition, the translational start site for C/EBP was optimized, as described previously (29), to maximize expression of full-length p42C/EBP, while minimizing translation from internal methionines. A p30C/EBP expression vector was constructed by excising the first 180 nt of the gene, which includes the start sites of translation for p42C/EBP and p40C/EBP, but not the start site for p30C/EBP.

View this table: [in this window] [in a new window]   TABLE 1.   Oligonucleotides synthesized and then subcloned into newly created PstI and SphI sites of C/EBP

In vivo phosphorylation. 293T cell monolayers were preincubated in phosphate-free DMEM (Gibco-BRL) for 30 min. These cells were then incubated in phosphate-free DMEM that was supplemented with ³²P_i (Amersham; 0.5 mCi/ml; 4 ml/10-cm-diameter plate) for 3 h. After being rinsed twice with phosphate-buffered saline, the cells were lysed in urea lysis buffer (8 M urea, 0.5 M NaCl, 45 mM Na₂HPO₄, 5 mM NaH₂PO₄, 10 mM imidazole [pH 8.0]) for protein purification.

Purification of His-tagged proteins. To identify sites of phosphorylation in C/EBP, 100 10-cm-diameter plates of 293T cells were transiently transfected with expression plasmids encoding either His-p42C/EBP or His-p18C/EBP. These plates were rinsed with phosphate-buffered saline, and then each was lysed in 0.5 ml of urea lysis buffer and sonicated. Pooled lysates were incubated with 2 ml of ProBond nickel resin (Invitrogen) for 2 h at room temperature. Nickel resin was washed three times with 10 bed volumes of wash buffer (8 M urea, 0.4 M NaCl, 17.6 mM Na₂HPO₄, 32.4 mM NaH₂PO₄, 10 mM imidazole [pH 6.75]), and bound proteins were eluted by being washed two times in 5 bed volumes of elution buffer (8 M urea, 0.4 M NaCl, 6 mM Na₂HPO₄, 44 mM NaH₂HPO₄, 10 mM imidazole [pH 5.30]). The solution containing these eluted proteins was adjusted to pH 8.0, and the proteins were incubated a second time with nickel resin. This purification process was repeated twice with the following changes: for each of the subsequent rounds, the bed volume was decreased by half, and for the final wash, the stringency was increased by raising the concentration of imidazole from 10 to 50 mM. This protocol was also used in a scaled-down version to purify His-C/EBP from 12 plates of transfected 293T cells labeled with ³²P_i. Unlabeled and labeled products from this purification procedure were pooled and concentrated by centrifugation (Centricon-10; Amicon). Purification products were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To optimize protein purification and monitor protein purity, proteins were visualized by silver staining (2), and C/EBP was detected by immunoblot analysis. For protein recovery, SDS-PAGE gels were zinc stained according to the manufacturer's instructions (Pierce). His-C/EBP was identified by its mobility and excised from the gel. Samples were then sent to the Protein Structure Core Facility at the University of Nebraska Medical Center for analyses. In-gel cleavage was performed by using vapor cyanogen bromide followed by trypsin. Peptides were separated on a Vydac C₁₈ column and sequenced with an ABI 477 protein sequencer, as described previously (48).

Immunoblot analysis. Cell lysis and immunoblotting for C/EBP were performed as described previously (22) with a polyclonal C/EBP antibody that was raised against a synthetic peptide (amino acids 253 to 265) (29).

Phosphoamino acid analysis. Purified His-tagged forms of truncated or full-length C/EBP that had been phosphorylated either in vitro or in vivo were separated by SDS-PAGE. After transfer onto PVDF (polyvinylidene difluoride)-Plus membrane (Micron Separations, Inc.) and visualization by autoradiography, His-tagged C/EBP was excised and hydrolyzed in 6 M HCl for 60 min at 110°C. Acid was removed by evaporation in a Speedvac and with two 0.5-ml washes of water. Samples were resuspended in 10 µl of water containing 1 ng of phosphoserine, phosphotyrosine, and phosphothreonine (Sigma) and were spotted onto cellulose plates (Kodak). Amino acids were separated by thin-layer electrophoresis for 45 min at 20 mA in pH 2.5 buffer (5.9% [vol/vol] glacial acetic acid, 0.8% [vol/vol] formic acid [88%], 0.3% [vol/vol] pyridine, 0.3 mM EDTA) (21). Phosphoamino acid standards were visualized by ninhydrin staining, and ³²P-labeled phosphoamino acids were detected by autoradiography.

In vitro kinase reactions. Protein purification for in vitro kinase assays of His-p42C/EBP, His-p18C/EBP, His-p12C/EBP, and His-p10C/EBP was performed as described above, except that His-tagged forms of C/EBP were not eluted from nickel resin following the last purification. Rather, purified C/EBP proteins were washed with kinase buffer and then incubated in kinase buffer with or without 5 U of GSK3 (New England Biolabs) for 60 min at 37°C. The nickel resin was washed extensively with wash buffer, and proteins were eluted in elution buffer and separated by SDS-PAGE as described above. Proteins were transferred to PVDF-Plus membrane (22), and the blot was subjected to autoradiography and immunoblot analyses.

Preparation of nuclei. Nuclei were purified from 3T3-L1 preadipocytes or adipocytes by a procedure modified from that of Dignam et al. (15). Briefly, 3T3-L1 cells were washed with 5 ml of phosphate-buffered saline, and then 2 ml of hypotonic lysis buffer (20 mM Tris [pH 7.5], 10 mM NaCl, 3 mM MgCl₂, 1 mM dithiothreitol [DTT]) with 2-µl/ml protease inhibitors (PIC 1, which is 1-mg/ml leupeptin, 1-mg/ml antipain, and 10-mg/ml benzamidine in aprotinin; and PIC 2, which is 1-mg/ml chymostatin and 1-mg/ml pepstatin A in dimethyl sulfoxide [DMSO]), and phosphatase inhibitors (30 mM -glycerophosphate, 1 mM sodium orthovanadate, and 1-mg/ml p-nitrophenylphosphate). Cells were scraped in 1 ml of hypotonic lysis buffer, and Igepal CA-630 was added (1:100 [vol/vol] for preadipocytes, 1:67 [vol/vol] for adipocytes) prior to Dounce homogenization (25 strokes). Disruption of plasma membranes was verified by trypan blue staining and light microscopy. Nuclei were pelleted in a microcentrifuge at 5,000 × g for 1 min. The supernatant was removed, the nuclei-containing pellet was resuspended in 1 ml of hypotonic lysis buffer, and the sample was centrifuged as described above. The process of resuspension and centrifugation was repeated. For quantification of nuclei, 1 5-µl aliquot was lysed in 0.5% SDS and vortexed, and A₂₆₀ was measured. Nuclear pellets were lysed in isoelectric focusing buffer or immunoblot lysis buffer or used for the protease accessibility assay.

Protease accessibility assay. To assess the sensitivity of nuclear C/EBP to protease, nuclei from 3T3-L1 adipocytes were resuspended in hypotonic lysis buffer at 60 A₂₆₀/ml. Increasing amounts of trypsin in 20 µl of phosphate-buffered saline were added to 30-µl aliquots of nuclei on ice for 30 min. The final concentrations of trypsin were 0, 8, 24, 80, and 240 µg/ml. Reactions were terminated with 50 µl of 2× immunoblot lysis buffer (2% SDS and 120 mM Tris [pH 6.8]) and heated to 100°C for 10 min. Samples were subjected to SDS-PAGE and immunoblot analysis for C/EBP.

Alkaline phosphatase treatment of samples. Nuclei were lysed in an SDS-containing buffer (1% SDS, 60 mM Tris [pH 6.8]) at a concentration of approximately 20 mg of protein/ml. Nuclear proteins were incubated with alkaline phosphatase (Boehringer Mannheim) at a concentration of approximately 1 U/mg of protein in the SDS-containing buffer for 1 h at 37°C. Samples were mixed with an equal volume of isoelectric focusing buffer prior to electrophoretic separation.

Isoelectric focusing. Nuclei were lysed in isoelectric focusing buffer (9 M urea, 1% Igepal CA-630, 1% DTT) at a concentration of approximately 10 mg of protein/ml. This nuclear lysate was sonicated and then centrifuged at 4°C for 15 min, and the supernatant was collected for further analysis. Polyacrylamide-urea mini gels were made according to the manufacturer's instructions (Bio-Rad) with a 1:1 mixture of pH 3 to 10 and pH 5 to 8 ampholytes (Bio-Rad). Protein samples (approximately 20 µg) were loaded and focused for 15 min at 100 V, 15 min at 200 V, and then 1 h at 450 V. Proteins were then transferred onto PVDF-plus membrane for immunoblot analysis.

Preadipocyte differentiation and Oil Red-O staining. 3T3-L1 preadipocytes were induced to differentiate as described previously (47), except that the medium was not supplemented with insulin on day 4 or thereafter (30). Oil Red-O staining was performed essentially as described previously (42). Briefly, cell monolayers were washed with phosphate-buffered saline, fixed in 3.7% formaldehyde for 2 min, washed with H₂O, incubated with Oil Red-O solution for 1 h at room temperature, and then washed with H₂O.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Identification of T222, T226, and S230 as phosphoamino acids. We have shown previously that C/EBP contains amino acids that become dephosphorylated in response to insulin (22, 31). To determine which amino acids in C/EBP are phosphorylated, we labeled the cellular phosphate pool and purified ³²P-labeled C/EBP for analyses. In these experiments, 293T cells were transiently transfected with expression vectors encoding His-tagged C/EBP constructs. Transfected cells were incubated with ³²P_i, and His-tagged forms of C/EBP were purified on a nickel resin. Eluted proteins were separated by SDS-PAGE, and C/EBP was excised from the gel. C/EBP was subjected to in-gel cleavage by treatment with cyanogen bromide and trypsin. The C/EBP peptides were separated by high-performance liquid chromatography. Following this separation, labeled peptides were sequenced by Edman degradation to identify the phosphoamino acids. Using this approach, in three independent experiments, the peptide spanning amino acids H215 through K250 was identified as a phosphopeptide (solid bar in Fig. 1A). Amino acid analysis of this phosphopeptide revealed that it contains both phosphoserine and phosphothreonine. Edman degradation demonstrated that this peptide is phosphorylated on T222 and S230.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Identification of phosphorylation sites in C/EBP. (A) A schematic diagram of C/EBP. Regions that are highly conserved across species (conserved regions 1 to 4 and bZIP domain) are shaded. Bent arrows denote translational start sites for two predominant C/EBP species (p42C/EBP and p30C/EBP). A phosphopeptide identified in three independent labeling experiments is indicated by the solid bar. Amino acids within a region of this phosphopeptide are shown for mouse, human, chicken, Xenopus, and Aplysia proteins. The consensus sequence for GSK3 is given. (B) Schematic representation of the His-tagged C/EBP fusion proteins used in subsequent labeling experiments. The C/EBP amino acid sequence from T222 to P231 is given, and the C/EBP mutants in which T222, T226, and S230 were mutated to either alanines or serines are illustrated. (C) His-p18TTS (lane 1), His-p18AAA (lane 2), His-p18SSS (lane 3), His-p18STS (lane 4), His-p18ATS (lane 5), and His-p18AAS (lane 6) were expressed in 293T cells, labeled with 32P in vivo, purified, and separated by SDS-PAGE. These samples were subjected to immunoblot analysis for C/EBP (top panel; IB) and autoradiography (middle panel; 32P). Phosphoamino acid analysis was performed with the remainder of the His-tagged C/EBP mutants (bottom panels). The positions of phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) are indicated. His-p18AAA contained no detectable 32P.

GSK3 phosphorylates C/EBP in vitro. To investigate further whether GSK3 phosphorylates C/EBP, we used purified C/EBP as a substrate for GSK3 in vitro. For these assays, His-tagged versions of full-length or truncated forms of C/EBP (Fig. 2A) were purified from 293T cells. Wild-type p42C/EBP and p18C/EBP contain the GSK3 consensus sites of phosphorylation, whereas p12C/EBP and p10C/EBP do not. Purified His-tagged forms of C/EBP were combined, and duplicate kinase assays were performed in either the absence or presence of GSK3 prior to separation by SDS-PAGE. Immunoblot analysis revealed that the amounts of C/EBP proteins in each reaction varied by less than a factor of 2 (Fig. 2B). Autoradiography revealed that p42C/EBP and p18C/EBP, but not p12C/EBP and p10C/EBP, are substrates for phosphorylation by GSK3 (Fig. 2C). Phosphoamino acid analysis demonstrated that phosphorylation of p42C/EBP by GSK3 in vitro occurs only on threonine (Fig. 2D). These findings indicate that GSK3 phosphorylates threonine specifically in a region between amino acids R192 and G246 in vitro and are consistent with GSK3 phosphorylating T222 and T226.

View larger version (43K): [in this window] [in a new window]   FIG. 2.   GSK3 phosphorylates C/EBP in vitro. (A) Schematic representation of His-tagged C/EBP fusion proteins used as substrates in kinase assays in vitro. A putative region of GSK3 phosphorylation is denoted by the solid bar. (B) His-tagged p42, p18, p12, and p10 C/EBP were expressed in 293T cells and purified. These proteins were mixed together, and kinase reactions were performed in the absence () or presence (+) of recombinant GSK3. Proteins were separated by SDS-PAGE (15% polyacrylamide gel) and subjected to immunoblot analysis with antibody specific for C/EBP. The migration of His-tagged p42, p18, p12, and p10 C/EBP is indicated. (C) The immunoblot in panel B was subjected to autoradiography. (D) 32P-labeled His-p42C/EBP was excised, and phosphoamino acid analysis was performed. Phosphoamino acid standards (pS, pT, and pY) are indicated. Similar results were obtained in three independent experiments.

GSK3 phosphorylates C/EBP in vivo. Since the steady-state stoichiometry of protein phosphorylation depends upon the balance of kinase and phosphatase activities, we reasoned that overexpression of a C/EBP kinase would increase the proportion of C/EBP that is phosphorylated. To assess whether GSK3 is a C/EBP kinase in a cellular context, 293T cells were transfected with expression vectors encoding either p30C/EBP alone (Fig. 3, lanes 1 to 4) or p30C/EBP and constitutively active GSK3 (Fig. 3, lanes 5 to 8). p30C/EBP is an alternate translation product which arises from initiation at the third start codon (29). This form was used because phosphorylation-induced shifts in p30C/EBP can be visualized by SDS-PAGE (22). Two days after transfection, cells were not treated or treated with the GSK3 inhibitor lithium for 1 h prior to lysis. Samples were separated by SDS-PAGE, and C/EBP was detected by immunoblot analysis. When expressed alone, p30C/EBP exists predominantly as a single high-mobility form (Fig. 3, lanes 1 and 3). Inhibition of GSK3 activity by treatment of these cells with lithium results in the loss of the weak band that crowns the p30C/EBP species (Fig. 3, lanes 2 and 4). This observation suggests that, under these conditions, a small proportion of p30C/EBP is phosphorylated due to endogenous GSK3 activity in 293T cells. Overexpression of constitutively active GSK3 dramatically increases the proportion of the upper species (Fig. 3, lanes 5 and 7). Inhibition of GSK3 activity by 1 h of lithium treatment results in the loss of this upper species. These data suggest that GSK3 phosphorylates C/EBP in vivo.

View larger version (11K): [in this window] [in a new window]   FIG. 3.   p30C/EBP is a substrate for GSK3 in vivo. 293T cells were transfected with p30C/EBP alone or both p30C/EBP and constitutively active GSK3. Forty-eight hours later, these cells were not treated () or treated with 25 mM LiCl (+) for 1 h prior to lysis. Samples were separated by SDS-PAGE (11.5% polyacrylamide gel) and subjected to immunoblot analyses for C/EBP. Similar results were obtained in three independent experiments.

Phosphorylation of C/EBP correlates with the regulation of GSK3 activity by insulin, wortmannin, and lithium. We have demonstrated that GSK3 phosphorylates C/EBP in vitro and in vivo. We next tested whether the ability of insulin to inhibit GSK3 (38) is responsible for insulin-dependent dephosphorylation of C/EBP in 3T3-L1 adipocytes. In many cell types, including adipocytes, activation of the insulin receptor is known to stimulate phosphatidylinositol (PI) 3-kinase activity, which, through the actions of other signaling molecules (possibly Akt [10] or PDK1 [14]), causes phosphorylation and inhibition of GSK3 (11). If GSK3 is the insulin-sensitive C/EBP kinase, then we would expect inhibition of PI 3-kinase with wortmannin to stimulate GSK3 and therefore cause phosphorylation of C/EBP. Conversely, we would expect inhibition of GSK3 activity with lithium to result in dephosphorylation of C/EBP.

View larger version (27K): [in this window] [in a new window]   FIG. 4.   Regulation of C/EBP phosphorylation by pharmacological agents. (A) 3T3-L1 adipocytes were not treated (Con, lanes 1 and 2) or were treated with 167 nM insulin for 60 min (Ins; lanes 3 and 4), 200 nM wortmannin for 60 min (Wort; lanes 5 and 6), or 25 mM LiCl for 30 min (Li; lanes 7 and 8). Whole-cell lysates were separated by SDS-PAGE (11.5% polyacrylamide gel) and subjected to immunoblot analysis for C/EBP. The migration of p42C/EBP and p30C/EBP is indicated. p30C/EBP migrates as three bands (a to c). (B) 3T3-L1 adipocytes were treated as in panel A, except that nuclei were prepared. Nuclear proteins were separated by isoelectric focusing and subjected to immunoblot analysis for C/EBP. Acidic (+) and basic () ends of the blot are indicated. Similar results were obtained in three independent experiments. Since p30C/EBP is more basic (predicted pI of 9.7) than p42C/EBP (predicted pI of 7.6), p30C/EBP is not resolved into discreet bands when focused to equilibrium with standard ampholytes (pH range of 3 to 10). p42C/EBP can be resolved into five bands (a to 3) by this technique. (C) 3T3-L1 adipocytes were not treated (Con; lane 1) or were treated with 25 mM LiCl (Li; lane 2), 1.5 µM okadaic acid (OA; lane 3), or both 25 mM LiCl and 1.5 µM okadaic acid (lane 4) for 1 h. Whole-cell lysates were separated by SDS-PAGE (11.5% polyacrylamide gel) and subjected to immunoblot analysis for C/EBP. (D) Nuclear lysates were prepared from 3T3-L1 adipocytes and either not treated (Con; lane 1) or treated with alkaline phosphatase (AP; lane 2) prior to isoelectric focusing and immunoblot analysis for C/EBP. (E) Model of signaling pathway through which insulin, wortmannin, lithium, and okadaic acid regulate the phosphorylation of C/EBP.

Phosphorylation of C/EBP correlates with the regulation of PP1 and PP2A by okadaic acid. Dephosphorylation of C/EBP requires not only inhibition of GSK3, but also activity of a C/EBP phosphatase. PP1 is a good candidate, since the activity of this phosphatase is induced by insulin in 3T3-L1 adipocytes, and since treatment of adipocytes with okadaic acid, which inhibits PP1 and PP2A, results in accumulation of hyperphosphorylated C/EBP (31). To determine whether PP1 or PP2A is directly responsible for dephosphorylation of the sites within p30C/EBP, 3T3-L1 adipocytes were not treated or treated with lithium, okadaic acid, or lithium and okadaic acid (Fig. 4C, lanes 1 to 4). Since lithium stimulates only minimal dephosphorylation in the presence of okadaic acid, PP1 or PP2A is likely responsible for dephosphorylation of C/EBP at these sites. Moreover, since induction of PP1 activity by insulin is inhibited by wortmannin (3), our finding that wortmannin stimulates phosphorylation of C/EBP in the presence of lithium is consistent with wortmannin also inhibiting C/EBP phosphatase activity (not shown). Taken together, these data are consistent with the model proposed in Fig. 4E, in which stimulation of PI 3-kinase by insulin results in simultaneous inhibition of the C/EBP kinase (GSK3) and activation of the C/EBP phosphatase (PP1 or PP2A) to cause dephosphorylation of C/EBP.

Phosphorylation alters C/EBP conformation. To determine whether phosphorylation alters the three-dimensional structure of C/EBP, we compared the accessibilities to protease of specific C/EBP residues after treatment of adipocytes with insulin or wortmannin. The sensitivity to proteolysis depends on the tertiary structure of the protein and the extent to which the protease-sensitive sites are protected or exposed through protein-protein or protein-DNA interactions. For instance, interaction of C/EBP with DNA protects the bZIP domain from tryptic digestion (data not shown and reference 45). Trypsin cleaves R-X and K-X and is predicted to cut C/EBP at 36 sites, of which 24 are in the bZIP domain and are therefore resistant to proteolysis. Although partial digestion of the 10 sites outside the bZIP domain by trypsin likely gives a large number of different C/EBP fragments, the anti-peptide C/EBP antibody used in our experiments only recognizes those that contain amino acids 253 to 265, a region just N terminal to the bZIP domain. Thus, only C-terminal proteolytic fragments of C/EBP are observed in this protease accessibility assay.

View larger version (31K): [in this window] [in a new window]   FIG. 5.   Insulin and wortmannin alter the trypsin sensitivity of C/EBP. 3T3-L1 adipocytes were treated for 1 h with 167 nM insulin or 250 nM wortmannin, as indicated. Nuclei were purified from cells and incubated for 30 min on ice in the absence (lanes 1 and 2) or presence of various concentrations of trypsin (lanes 3 and 4, 8 µg/ml; lanes 5 and 6, 24 µg/ml; lanes 7 and 8; 80 µg/ml; lanes 9 and 10, 240 µg/ml). Samples were separated by SDS-PAGE (15% polyacrylamide gel) and subjected to immunoblot analysis for C/EBP. Because the C/EBP antibody used in these experiments was raised against an epitope located near the bZIP domain, which is protected from trypsinization, these partially digested C/EBP fragments are N-terminal truncation products.

Lithium inhibits preadipocyte differentiation. Given the importance of C/EBP in preadipocyte differentiation (13) and the established role of GSK3 in the development of tissues in other species (4), we considered the possibility that GSK3 activity is required for adipogenesis. To assess this putative role for GSK3, we investigated whether the GSK3 inhibitor lithium could block preadipocyte differentiation (Fig. 6A). 3T3-L1 preadipocytes were induced to differentiate under standard conditions, with isobutylmethylxanthine, dexamethasone, insulin, and fetal calf serum, in the presence of either 25 mM LiCl or 25 mM NaCl as a control. Thereafter, cells were continuously incubated in the presence of LiCl or NaCl over the course of differentiation. Eight days after the induction of differentiation, the degree of adipogenesis was assessed qualitatively by staining cellular lipid droplets with Oil Red-O. Upon induction, 3T3-L1 preadipocytes differentiated almost completely, irrespective of the presence of NaCl (Fig. 6A, top). In contrast, lithium treatment almost completely inhibited adipocyte differentiation (Fig. 6A, bottom). Expression of adipocyte markers C/EBP and 422/aP2 was also inhibited by lithium (data not shown). In addition to GSK3, lithium is known to inhibit inositol monophosphatase (27) and may inhibit other enzymatic activities as well. Nevertheless, the observed inhibition of adipogenesis by lithium is consistent with the hypothesis that GSK3 activity is required for adipocyte differentiation.

View larger version (61K): [in this window] [in a new window]   FIG. 6.   (A) Lithium inhibits preadipocyte differentiation. 3T3-L1 preadipocytes were induced to differentiate as described in Methods and Materials, except that on days 1, 2, 4, and 6, 25 mM NaCl or LiCl was added to the medium. On day 8, cells were fixed and stained with Oil Red-O to visually assess the accumulation of lipid droplets. (B) Schematic representation of a generalized C/EBP transcription factor. The region of C/EBP that is phosphorylated by GSK3 and putative GSK3 recognition sequences in C/EBP and C/EBP are given.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Our analyses have led to the discovery that several sites within conserved region 4 of C/EBP are phosphorylated in vivo (Fig. 1). 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/EBP 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. 4). Furthermore, since okadaic acid treatment of adipocytes blocks the lithium-induced dephosphorylation of C/EBP, it is likely that PP1 or PP2A is responsible for dephosphorylation of the insulin-sensitive sites (Fig. 4D). Our work also shows that insulin and wortmannin alter the protease accessibility of C/EBP, thereby implying that phosphorylation at T222 and T226 causes a conformational change in the structure of C/EBP (Fig. 5). Finally, we report here that lithium inhibits preadipocyte differentiation (Fig. 6), suggesting that GSK3-mediated phosphorylation of C/EBP and other transcription factors, such as C/EBP, is required for adipogenesis.

A hierarchical model of phosphorylation has been described for several GSK3 substrates (7, 43). If this model holds true for C/EBP, 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/EBP 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/EBP for recognition by GSK3 and may therefore serve a modulatory role.

Phylogenetic analysis of C/EBP 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/EBP 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/EBP to transactivate leptin or C/EBP promoters in reporter gene assays (data not shown). Furthermore, altering the phosphorylation status of C/EBP by cotransfection of GSK3 also does not influence the ability of C/EBP 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/EBP in our assays. Since the protease accessibility of C/EBP was determined in nuclei, the effects of phosphorylation on sensitivity of C/EBP to trypsin (Fig. 5) 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/EBP in vitro (33). This modification is an attractive mechanism for the regulation of C/EBP 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/EBP (Fig. 1C).

We have shown that phosphorylation of C/EBP 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 and 12). 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. 6B, C/EBP and C/EBP contain GSK3 consensus sequences and may, like C/EBP, 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, 8). We propose that regulation by GSK3 of C/EBP 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/EBP, a transcription factor required for acquisition of insulin sensitivity, is phosphorylated by GSK3. Dephosphorylation of the GSK3 sites in both glycogen synthase and C/EBP 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.