Transcriptional Activation by Peroxisome Proliferator-activated Receptor gamma  Is Inhibited by Phosphorylation at a Consensus Mitogen-activated Protein Kinase Site*

(Received for publication, November 4, 1996, and in revised form, December 12, 1996)

Maria Adams Dagger §, Mauricio J. Reginato §par , Dalei Shao , Mitchell A. Lazar ** and V. Krishna Chatterjee Dagger Dagger Dagger

From the Dagger  Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom and the  Division of Endocrinology, Diabetes and Metabolism, Departments of Medicine, Genetics, and Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES


ABSTRACT

The nuclear receptor peroxisome proliferator-activated receptor gamma  (PPARgamma ) regulates transcription in response to prostanoid and thiazolidinedione ligands and promotes adipocyte differentiation. The amino-terminal A/B domain of this receptor contains a consensus mitogen-activated protein kinase site in a region common to PPARgamma 1 and -gamma 2 isoforms. The A/B domain of human PPARgamma 1 was phosphorylated in vivo, and this was abolished either by mutation of serine 84 to alanine (S84A) or coexpression of a phosphoprotein phosphatase. In vitro, this domain was phosphorylated by ERK2 and JNK, and this was markedly reduced in the S84A mutant. A wild type Gal4-PPARgamma (A/B) chimera exhibited weak constitutive transcriptional activity. Remarkably, this was significantly enhanced in the S84A mutant fusion. Ligand-dependent activation by full-length mouse PPARgamma 2 was also augmented by mutation of the homologous serine in the A/B domain to alanine. The nonphosphorylatable form of PPARgamma was also more adipogenic. Thus, phosphorylation of a mitogen-activated protein kinase site in the A/B region of PPARgamma inhibits both ligand-independent and ligand-dependent transactivation functions. This observation provides a potential mechanism whereby transcriptional activation by PPARgamma may be modulated by growth factor or cytokine-stimulated signal transduction pathways involved in adipogenesis.


INTRODUCTION

Adipocyte differentiation is a complex process regulated by extracellular hormone and cytokine stimulation (1). Cultured preadipocyte cell lines differentiate into lipid-laden adipocytes following exposure to insulin, glucocorticoid, and inducers of intracellular cyclic AMP (2). Conversely, epidermal growth factor (EGF)1 and transforming growth factor alpha  act via the EGF receptor to inhibit both primary and preadipocyte cell line differentiation (3, 4). Tumor necrosis factor alpha  (TNFalpha ) is also a potent inhibitor of differentiation. In addition, this cytokine promotes lipolysis and down-regulates adipocyte-specific gene expression in mature adipocytes (5, 6).

Adipocyte differentiation is driven by the coordinate expression of a range of transcription factors, including C/EBPalpha , -beta , and -delta (7) and ADD1 (8), which lead to the expression of adipocyte-specific genes. In addition, the peroxisome proliferator-activated receptor gamma  (PPARgamma ) has been shown to be selectively expressed in adipocytes (9, 10) and to modulate adipocyte-specific gene expression (10). Alternative splicing generates two receptor isoforms such that mouse PPARgamma 2 has a 28-residue extension at its amino terminus compared with human PPARgamma 1. PPARgamma 2 mRNA is highly expressed in murine adipocyte cell lines (10), whereas both receptor isoforms are abundant in freshly isolated mouse adipocytes (11). The early induction of PPARgamma mRNA expression during adipogenesis, combined with the ability of retrovirally overexpressed PPARgamma to induce lipid accumulation and the expression of adipocyte-specific genes (12), suggests that this receptor plays an important role in preadipocyte differentiation. This notion is strengthened by the observation that specific high affinity ligands for PPARgamma including thiazolidinediones (which act as insulin sensitizers in vivo), as well as eicosanoids, promote the differentiation of murine preadipocyte cell lines (13-15).

PPARgamma is an orphan member of the nuclear receptor family. These receptors share a conserved domain structure and modulate gene transcription through two transcription activation mechanisms: a hormone-dependent transcription activation function (AF-2) is located in the carboxyl-terminal hormone-binding domain, whereas the amino-terminal A/B domain contains a ligand-independent activation function (AF-1). Recently, the AF-1 activity of the estrogen receptor (ER), another member of this family, has been shown to be modulated following phosphorylation by mitogen-activated protein (MAP) kinase (16, 17). Three MAP kinase pathways have been identified in mammalian cells. Members of the extracellular signal-regulated kinases, ERK1 and ERK2, are activated predominantly by growth factor stimulation via a Ras-dependent signal transduction cascade (18). In contrast, activity of Jun NH2-terminal kinase (JNK, also known as SAPK) and p38 kinase is increased by exposure of cells to environmental stress or to cytokines including TNFalpha (19, 20). In turn, activated MAP kinases have been shown to regulate the activity of specific transcription factors including Elk-1, ATF-2, and c-Jun by phosphorylation of serine or threonine residues in the appropriate context (21).

Interestingly, it has been reported that PPARgamma 1 and PPARgamma 2 are similarly adipogenic but that a truncated receptor in which the amino-terminal domain of PPARgamma 2 is deleted is a more potent inducer of adipocyte differentiation (12). We noted that NH2-terminal domain of PPARgamma contains a consensus MAP kinase site in a region conserved between PPARgamma 1 and PPARgamma 2 isoforms. Furthermore, we and others (11, 22) have observed that PPARgamma proteins migrate on immunoblots as closely spaced doublets, a pattern suggestive of phosphorylation. In this report we show that this putative MAP kinase site is phosphorylated in vivo and also in vitro by ERK2 and JNK. Furthermore, we demonstrate that phosphorylation significantly inhibits both ligand-independent and ligand-dependent transcriptional activation by PPARgamma . Finally, we show that mutation of this MAP kinase site in PPARgamma increases its adipogenic activity. These findings provide a potential pathway by which extracellular hormones and cytokines might regulate adipocyte differentiation by phosphorylation-dependent modulation of PPARgamma activity.


EXPERIMENTAL PROCEDURES

Plasmid Constructs

The GAL4 UAS-TKLUC luciferase reporter contains two copies of the GAL4 17-mer binding site (23) and the BOSbeta -gal and CMVbeta -gal reference plasmids have been described previously (23, 24). The GAL4-hPPARgamma (A/B) wild type and S84A mutant expression vectors contain residues 1-108 of human PPARgamma 1 cloned into the EcoRI site of pSG424 (25). These domains were also cloned into pGEX4T for expression of glutathione S-transferase (GST) fusion proteins. pCMV-flag-p38, which contains the Flag epitope between codons 1 and 2 of p38, was obtained from J. Han (Dept. of Immunology, Scripps Research Institute, San Diego, CA). RSV-PP1 is an expression vector encoding protein phosphatase 1alpha (26). pSG-CL100 is an expression vector encoding a dual specificity MAP kinase phosphatase (27). pSPORT-mPPARgamma 2 encodes full-length mouse PPARgamma 2 (10), and the S112A point mutation was introduced into it by polymerase chain reaction-directed mutagenesis. Both PPARgamma 2 and PPARgamma 2-SA cDNAs were then subcloned into the SalI site of pCMX. All mutations and ligation junctions were confirmed by sequencing. The acyl-CoA × 3-TK-LUC construct contains three copies of the acyl-CoA oxidase PPAR response element (5'-GATCTGGACCAGGACAAAGGTCACGTTCA) in pTK-luciferase.

Cell Culture and Transfection Studies

For functional studies, JEG-3 cells were cultured in DMEM containing 10% fetal bovine serum and transferred to DMEM plus charcoal-stripped fetal calf serum immediately prior to transfection. Cells were transfected with luciferase reporter, receptor expression vector, beta -gal expression vector, and phosphatase expression vector where indicated using the calcium phosphate precipitation method. 5 µM BRL49653 (in Me2SO) or vehicle control was added 16 h after transfection. Cells were lysed 24 h later, and luciferase and beta -gal was measured as described (23, 28). Luciferase values were normalized to beta -gal activity and fold activation was calculated.

In Vivo Phospholabeling, Immunoprecipitation, and Western Blotting

JEG-3 cells, plated on 10-cm plates, were transfected with 30 µg of GAL4-hPPARgamma plus 10 µg of PP1 expression vector as indicated. 40 h later cells were incubated in phosphate-free DMEM for 30 min, followed by a 4-h incubation with 1 mCi/ml [32P]orthophosphate (DuPont NEN). Cells were lysed by a 30-min incubation at 4 °C in RIPA buffer (1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M NaPO4, pH 7.2, 2 mM EDTA, 50 mM NaF, 0.2 mM sodium vanadate, 1 µg/ml leupeptin, and 1 µg/ml aprotinin). Extracts were cleared by centrifuging at 26,000 × g for 30 min at 4 °C.Samples were precleared with 10 µl of whole rabbit serum (Cappel) in a 50% slurry of protein A-agarose (Life Technologies, Inc.). The resulting supernatant was incubated with 10 µl of GAL4 DBD rabbit polyclonal antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. Immune complexes were then precipitated with a 50% slurry of protein A-agarose and washed five times with RIPA buffer. The immunoprecipitate was analyzed by 12% SDS-PAGE and autoradiography.

For immunoprecipitation of p38 kinase, JEG-3 cells were maintained on 10-cm plates and transfected with pCMV-flag-p38 by a 4-h exposure to calcium phosphate. After 48 h cells were UV irradiated (40 J/m2 for 30 s) and lysed by a 5-min incubation at 4 °C in buffer containing 20 mM Hepes, 2 mM EGTA, 50 mM beta -glycerophosphate, pH 7.4, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM orthovanadate, 2 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 0.1 µg/ml okadaic acid. Lysates were cleared by centrifugation at 10000 rpm for 10 min at 4 °C. Flag-p38 was immunoprecipitated by a 1-h incubation of cleared cell lysates with anti-FLAG M2 monoclonal antibody (Scientific Imaging Systems, New Haven, CT) followed by a 1-h incubation with protein G-Sepharose. Precipitates were washed three times with lysis buffer followed by three washes in lysis buffer containing 150 mM NaCl and a further three washes in kinase buffer (20 mM MOPS, pH 7.0, 1 mM EDTA, 5% glycerol, 0.1% beta -mercaptoethanol, and 0.1 µg/ml okadaic acid).

Whole cell extracts were prepared from JEG-3 cells transfected with wild type or mutant mPPARgamma 2 as described above and analyzed by SDS-PAGE and Western blotting followed by chemiluminescent detection (Amersham Corp.) with anti-PPARgamma IgG at a dilution of 1:1000.

Expression of GST Fusion Proteins

GST fusion proteins were expressed in Escherichia coli, induced with 1 mM isopropylthio-beta -D-galactosidase, and purified using glutathione-Sepharose 4B affinity resin (Pharmacia Biotech Inc.) but not removed from the matrix. Following purification, proteins were resuspended in NETN buffer (20 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 0.5% Nonidet P-40, pH 8.0), and their concentrations were determined.

Protein Kinase Assays

Phosphorylation of 2 µg of GST fusion proteins by ERK2 (New England Biolabs, Bishop's Stortford, UK) or JNK (Calbiochem-Novabiochem, Nottingham, UK) were carried out as recommended by the manufacturers. p38 kinase assays were performed with 2 µg of substrate at 30 °C for 30 min in kinase buffer containing 18 mM magnesium acetate, 90 µM ATP, and 2 µCi of [gamma -32P]ATP. Myelin basic protein (Life Technologies, Inc.) was used as a control substrate for ERK2, and a GST fusion protein containing a truncated form of activating transcription factor-2 (GST-ATF2, residues 1-96, Santa Cruz Biotechnology, Wembley, UK) provided positive controls for JNK and p38. 32P incorporation was determined following SDS-PAGE fractionation. Gels were stained with Coomassie Blue to check equal loading of GST fusion proteins and autoradiographed.

Retroviral Infection and Adipocyte Differentiation of 3T3-L1 Cells

3T3-L1 cells were made to ectopically express PPARgamma 2 or PPARgamma 2-S112A using retroviral gene transduction as described previously (22). Infected 3T3-L1 cells were selected in G418 and grown to confluence in DMEM containing 10% iron-enriched fetal bovine serum. 2 days after reaching confluency, cells were treated with BRL49653 dissolved in Me2SO or Me2SO alone. After 7 days, cells were washed three times with phosphate-buffered saline, fixed by 10% formalin in phosphate buffer for 1 h at room temperature, washed once again with phosphate-buffered saline, then stained with 60% filtered Oil Red O stock solution (0.5 g of Oil Red O (Sigma) in 100 of ml isopropanol) for 15 min, washed four times with water, and photographed.


RESULTS

Sequence alignment of the amino-terminal A/B region that is common to PPARgamma 1 and PPARgamma 2 isoforms indicated that a consensus MAP kinase site is conserved between species (Fig. 1A). To determine whether the NH2-terminal region of PPARgamma is phosphorylated in vivo, an expression vector encoding the the A/B domain of human PPARgamma 1 (residues 1-108) fused to the heterologous DBD of GAL4 (residues 1-147) was transfected into JEG-3 cells. Orthophosphate labeling followed by immunoprecipitation with an antibody directed against GAL4 revealed that the GAL4 PPARgamma (A/B) fusion protein was highly phosphorylated, whereas Gal4 DBD alone was not (Fig. 1B, lanes 2 and 3). In comparison, phosphorylation of transfected GAL4 PPARgamma (A/B S84A), in which a putative phosphoserine at position 84 has been mutated to alanine (S84A) was markedly reduced (Fig. 1B, lane 4), although this fusion is expressed and even more transcriptionally active than wild type Gal4-PPARgamma (A/B) (see below). Furthermore, cotransfection of PP1, an activated form of a serine phosphatase, led to near complete loss of phosphorylation of the wild type PPARgamma construct (Fig. 1B, compare lanes 3 and 6).


Fig. 1. A, sequence alignment of PPARgamma 1 and PPARgamma 2 isoforms showing conservation of a putative MAP kinase site. Comparison of the amino-terminal regions of PPARgamma with a consensus MAP kinase sequence (where X is one or two basic or neutral residues; Ref. 43). The putative phosphoserine is boxed. This residue is conserved between the A/B domains of human PPARgamma 1(44), mouse PPARgamma 1 and PPARgamma 2 (45, 10), hamster PPARgamma 1 (46), and Xenopus PPARgamma (29) isoforms. The codon nomenclature reflects two additional residues at the NH2 terminus of hPPARgamma 1 that are not represented in hamster or mouse PPARgamma 1. B, the amino-terminal A/B domain of hPPARgamma 1 is phosphorylated in vivo. JEG-3 cells were transfected with 30 µg of GAL4-DBD or wild type GAL4-PPARgamma (A/B) or mutant (S84A) expression vectors. 10 µg of PP1 protein phosphatase expression vector was also cotransfected where indicated. Following labeling with [32P]orthophosphate, cell lysates were immunoprecipitated using an antibody directed against the GAL4-DBD and 32P-labeled products were analyzed by SDS-PAGE and autoradiography. The predicted size of the GAL4 PPARgamma (A/B) fusion protein is approximately 29 kDa.
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Several MAP kinase cascades have been defined, each with a slightly different substrate specificity and activation pathway (21). In view of this, we tested the ability of three kinases, ERK2, JNK, and p38, to phosphorylate the isolated A/B domains of wild type and mutant hPPARgamma 1 expressed as GST fusion proteins in E. coli. Purified recombinant ERK2 was able to phosphorylate the wild type GST-PPARgamma 1 fusion protein (Fig. 2A). This phosphorylation was abolished by mutation of the serine at position 84 in hPPARgamma 1, which corresponds to the MAP kinase site to alanine (S84A) under conditions in which myelin basic protein was a good substrate for this kinase. Similarly, purified recombinant JNK was also able to phosphorylate wild type GST-PPARgamma (A/B) in addition to a control GST-ATF2 fusion protein (Fig. 2B). Mutation of serine 84 to alanine markedly reduced but did not abolish phosphorylation by JNK, suggesting weak phosphorylation of a second kinase site in vitro, which is not phosphorylated in vivo (Fig. 1B). Similar experiments have been performed with p38 kinase and phosphorylation of wild type or mutant GST PPARgamma was not detected under conditions in which GST-ATF2 was a good substrate for this enzyme (data not shown).


Fig. 2. Phosphorylation of the A/B domain of PPARgamma 1 by MAP kinases. In vitro phosphorylation of GST PPARgamma (A/B) by ERK2 (A) and JNK (B). Bacterially expressed and purified GST, wild type PPARgamma (A/B), or mutant PPARgamma (A/B S84A) fusion proteins were incubated with recombinant ERK2 or JNK as detailed under "Experimental Procedures" (myelin basic protein (MBP) and ATF2 used as positive controls, respectively). Phosphorylation was analyzed by autoradiography following SDS-PAGE (upper panels). Lower panels indicate the same gels after staining with Coomassie Blue to show total protein. The predicted positions of full-length fusion proteins are indicated with arrowheads, and lower molecular weight species are presumed to be degradation products.
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These results suggested that phosphorylation of a MAP kinase site by either ERK2 or JNK might regulate the ligand-independent transcriptional activity (AF1) of hPPARgamma . Transient transfection assays in JEG-3 cells showed that the A/B domain of hPPARgamma acts as a weak, ligand-independent transcriptional activator when fused to the DBD of GAL4 (Fig. 3). Mutation of serine 84 to alanine, which abolished phosphorylation of the GAL4-PPARgamma fusion protein (see above and Fig. 1B), markedly enhanced the AF1 transactivation function of the PPARgamma A/B domain. Similar results were obtained following transfection of wild type and mutant GAL4-PPARgamma fusions in COS-7 cells (data not shown). Furthermore, coexpression of CL100, the human homologue of murine MAP kinase phosphatase (MKP-1), also augmented the transcriptional activity of wild type Gal4-PPARgamma A/B (Fig. 3) but had no effect on the mutant GAL4-PPAR A/B S84A fusion. Thus, two independent paradigms that either prevent (S84A mutation) or reduce (CL-100 coexpression) phosphorylation of the PPARgamma A/B domain markedly enhance AF1 activity.


Fig. 3. Phosphorylation of hPPARgamma 1 at S84 inhibits ligand-independent transcriptional activation. Cells were grown in 24-well plates and transfected with 10 ng of either GAL 4-PPARgamma (A/B) wild type or mutant S84A expression vectors, 500ngUAS-TKLUC, and 200 ng Bosbeta -gal. 50 ng of CL100 expression vector was added where indicated. Luciferase activity was normalized for transfection efficiency using beta -gal values and fold activation is expressed relative to cells transfected with the Gal4 DBD expression vector alone.
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In addition to the constitutive AF1 function in the amino-terminal domain, PPARgamma also contains a ligand-dependent transcription activation function (AF2). We therefore investigated whether phosphorylation of the MAP kinase site in the A/B domain could influence the ligand-dependent AF2 activity of full-length PPARgamma . Because PPARgamma is conserved between species and the residues encompassing the amino-terminal MAP kinase site are identical in PPARgamma 1 and PPARgamma 2 isoforms (Fig. 1A), murine PPARgamma 2, which is induced specifically during adipocyte differentiation, was used in these studies. Transfection of wild type mPPARgamma 2 expression vector together with a reporter construct containing three copies of the PPAR-response element from the acyl-CoA oxidase gene and the thiazolidenedione ligand BRL49653 was associated with significant transcriptional activation (Fig. 4). Mutation of the homologous serine within the putative MAP kinase site in mPPARgamma 2 to alanine (S112A) significantly increased ligand-dependent transactivation to approximately double that of the wild type receptor. These observations indicate that in addition to inhibiting the inherent AF1 activity of PPARgamma , phosphorylation of the MAP kinase site within the A/B domain also attenuates ligand-induced transcription activation by this receptor.


Fig. 4. Phosphorylation inhibits ligand-dependent transcriptional activation of full-length PPARgamma 2. 6-cm plates of cells were transfected with 2 µg of wild type or mutant pCMX PPARgamma 2 or pCMX vector alone plus 2 µg of acyl-CoA × 3-TK-LUC and 0.5 µg of beta -gal expression vector. Luciferase activity was determined following incubation with 5 µM BRL49653 and normalized for transfection efficiency using beta -gal values. Ligand-dependent activation is expressed relative to the vector control. The inset shows the expression levels of wild type (WT) and mutant mPPARgamma 1 and PPARgamma 2 protein, determined by Western blotting, following transfection as above.
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We next tested whether the enhanced transactivation by PPARgamma 2-S112A resulted in increased adipogenic activity. Wild type PPARgamma 2 or PPARgamma 2-S112A were ectopically expressed in 3T3-L1 preadipocytes using a retroviral expression strategy that we have previously described (22). In the absence of adipogenic stimulation, confluent preadipocytes differentiate into adipocytes at a very low frequency (<1%). Ectopic expression of PPARgamma 2 resulted in ~10% adipose conversion, as shown by Oil Red O staining of accumulated intracellular lipid (Fig. 5). This effect is presumably mediated by low levels of an endogenous PPARgamma 2 ligand, although the possibility that this was a ligand-independent effect of PPARgamma overexpression cannot be discounted. Remarkably, ectopic expression of PPARgamma 2-S112A at levels similar to those of the ectopically expressed wild type PPARgamma 2 (data not shown) induced adipocyte differentiation much more dramatically. In fact, Fig. 5 shows that the degree of adipocyte differentiation due to ectopic PPARgamma 2-S112A expression was comparable with that achieved by cells expressing wild type PPARgamma and treated with the ligand BRL49653 (50 nM). At this concentration BRL49653 alone is a relatively weak adipogenic stimulus for control cells (13, 15, 30), resulting in ~10% adipose conversion. These results show clearly that mutation of the MAP kinase consensus site produces a more adipogenic form of PPARgamma 2, consistent with the increased transcriptional activity of this mutant receptor.


Fig. 5. Phosphorylation inhibits the adipogenic activity of PPARgamma 2. 3T3-L1 preadipocytes were infected with control, PPARgamma 2, or PPARgamma 2-S112A mutant expressing retroviruses. 100-mm dishes of confluent cells were treated with BRL49653 (50 nM) or vehicle for 7 days and then stained with Oil Red O and photographed.
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DISCUSSION

Our studies indicate that a consensus MAP kinase site located within the conserved amino-terminal A/B domains of PPARgamma 1 and PPARgamma 2 regulates the transcriptional activity of these adipogenic nuclear receptor isoforms. Phosphorylation of this site reduces the inherent transcriptional activity of the AF1 transactivation domain. In addition, ligand-dependent transactivation by the full-length receptor is also inhibited. This observation contrasts with previous studies of the ER showing that MAP kinase-mediated phosphorylation of the amino-terminal A/B domain enhanced the transcriptional activity of this receptor (16, 17) and indicates that constitutive activity of the NH2-terminal domains of nuclear receptors can be modulated in response to other signal transduction pathways. Furthermore, the divergent transcriptional effects of phosphorylation in PPARgamma versus ER suggest that the different sequences around the consensus MAP kinase site in each A/B domain may also influence the way AF1 activity is modulated.

Although the A/B domain sequence motif that we have identified represents a consensus MAP kinase site, we have demonstrated that this residue is amenable to phosphorylation by both ERK2 and JNK. Interestingly, both TNFalpha and EGF, which are potent inhibitors of adipocyte differentiation (3, 4, 5, 6) are known to activate these pathways. In addition to enhancing JNK activity, TNFalpha stimulation has also been reported to activate MAP kinase (31, 32), and EGF stimulation enhances MAP kinase activity in a variety of cell types (18). The present work suggests that activation of either pathway could phosphorylate and hence inhibit PPARgamma activity, thus contributing to the anti-adipogenic effects of these agents.

It is also possible that other signal transduction pathways may be involved. For example, it is interesting to note that adipocyte differentiation is promoted by culture in conditions that raise intracellular cAMP (33, 34), and elevated cAMP levels have been reported to inhibit ERK activity in rat adipocytes (35, 36). In some cell types, cAMP-dependent stimulation of protein kinase A leads to direct inhibition of Raf-1, which in turn inhibits EGF-stimulated MAP kinase activity (37). Given the convergence of these pathways, it is tempting to speculate that altered PPARgamma phosphorylation might contribute to the abnormal adipogenesis seen in mice harboring a mutant protein kinase A regulatory subunit (38).

Phosphorylation has been shown to regulate the activity of transcription factors by a number of different mechanisms (21). Because equal levels of mutant and wild type PPARgamma 2 were detected in transfected cells (Fig. 4), phosphorylation does not appear to alter PPARgamma protein stability, as has been shown for the transcription factor Fos (39). Furthermore, because the inhibitory effect of NH2-terminal PPARgamma phosphorylation is transferable to the heterologous DNA binding domain of GAL4, we also consider it unlikely that phosphorylation alters the ability of PPARgamma to bind to DNA. A third possibility is that phosphorylation inhibits the activity of PPARgamma by altering receptor interaction with other transcription intermediary proteins. For example, an interaction between the A/B domain of thyroid hormone receptors TRbeta 2 and TRalpha 1 and TFIIB, which might influence transcription by altering preinitiation complex formation or stability, has recently been described (40, 41). This raises the possibility of analogous interactions between basal transcription factors and the A/B region of PPARgamma that are phosphorylation-sensitive. Alternatively, phosphorylation at this site might influence PPARgamma interaction with specific coactivator or corepressor proteins, in the same way that phosphorylation of c-Jun enhances recruitment of the CREB-binding coactivator protein (42).

Our finding of increased adipogenicity of a PPARgamma 2 point mutant that is not phosphorylated by ERK and related MAP kinases in its A/B domain strongly suggests that NH2-terminal phosphorylation inhibits the adipogenic activity of the wild type receptor. This also provides a molecular basis for an earlier observation that retroviral expression of NH2-terminally truncated PPARgamma 2 induces murine preadipocyte differentiation more strongly than its wild type counterpart (12). The precise role of PPARgamma phosphorylation in regulation of adipocyte differentiation remains to be elucidated. However, the ability to modulate the activity of this adipogenic transcription factor by altering its phosphorylation state might provide a novel strategy for development of anti-adipogenic therapeutic agents to treat obesity.


FOOTNOTES

*   This work was supported by Grant DK49780 from the National Institutes of Health (to M. A. L.) and a grant from the Wellcome Trust (to V. K. C.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   These authors contributed equally to this work.
par    Supported in part by National Institutes of Health Predoctoral Training Grant 5F31 GM15677 and by a minority predoctoral award from Dupont-Merck Pharmaceutical Company.
**   To whom correspondence can be addressed: University of Pennsylvania School of Medicine, CRB 611, 415 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-0198; Fax: 215-898-5408.
Dagger Dagger    To whom correspondence can be addressed: Dept. of Medicine, University of Cambridge, Level 5, Addenbrooke's Hospital, Hills Rd., Cambridge CB2 2QQ, UK. Tel.: 44-1223-336842; Fax: 44-1223-336846.
1    The abbreviations used are: EGF, epidermal growth factor; TNFalpha , tumor necrosis factor alpha ; PPARgamma , peroxisome proliferator-activated receptor gamma ; mPPAR, murine PPAR; hPPAR, human PPAR; ER, estrogen receptor; MAP, mitogen-activated protein kinase; JNK, Jun NH2-terminal kinase; beta -gal, beta -galactosidase; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; DBD, DNA binding domain; ERK, extracellular signal-regulated kinase.

Acknowledgment

We thank Sam Krakow for technical assistance.


REFERENCES

  1. Smas, C. M., and Sul, H. S. (1995) Biochem. J. 309, 697-710 [Medline] [Order article via Infotrieve]
  2. Green, H., and Kehinde, O. (1975) Cell 5, 19-27 [Medline] [Order article via Infotrieve]
  3. Luetteke, N. C., Lee, D. C., Palmiter, R. D., Brinster, R. L., and Sandgren, E. P. (1993) Cell Growth & Differ. 4, 203-213 [Abstract]
  4. Serrero, G., and Mills, D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3912-3916 [Abstract]
  5. Torti, F. M., Diekmann, B., Beutler, B., Cerami, A., and Ringold, G. M. (1985) Science 229, 867-869 [Medline] [Order article via Infotrieve]
  6. Petrusenke, T., and Hauner, H. (1993) J. Clin. Endocrinol. & Metab. 76, 742-747 [Abstract]
  7. Cao, Z., Umek, R. M., and McKnight, S. L. (1991) Genes & Dev. 5, 1538-1552 [Abstract]
  8. Kim, J. B., and Spiegelman, B. M. (1996) Genes & Dev. 10, 1096-1107 [Abstract]
  9. Chawla, A., Schwarz, E. J., Dimaculangan, D. D., and Lazar, M. A. (1994) Endocrinology 135, 798-800 [Abstract]
  10. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I., and Spiegelman, B. M. (1994) Genes & Dev. 8, 1224-1234 [Abstract]
  11. Vidal-Puig, A., Jimenez-Linan, M., Lowell, B. B., Hamann, A., Hu, E., Spiegelman, B., Flier, J. S., and Moller, D. E. (1996) J. Clin. Invest. 97, 2553-2561 [Abstract/Free Full Text]
  12. Tontonoz, P., Hu, E, and Spiegelman, B. M. (1994) Cell 79, 1147-1156 [Medline] [Order article via Infotrieve]
  13. Kliewer, S. A., Lenhard, J. M., Willson, T. M., Patel, I., Morris, D. C., and Lehmann, J. M. (1995) Cell 83, 813-819 [Medline] [Order article via Infotrieve]
  14. Yu, K., Bayona, W., Kallen, C. B., Harding, H. P., Ravera, C. P., McMahon, G., Brown, M., and Lazar, M. A. (1995) J. Biol. Chem. 270, 23975-23983 [Abstract/Free Full Text]
  15. Forman, B. M., Tontonoz, P., Chen, J., Brun, R. P., Spiegelman, B. M., and Evans, R. M. (1995) Cell 83, 803-812 [Medline] [Order article via Infotrieve]
  16. Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., Metzger, D., and Chambon, P. (1995) Science 270, 1491-1494 [Abstract]
  17. Bunone, G., Briand, P.-A., Miksicek, R. J., and Picard, D. (1996) EMBO J. 15, 2174-2183 [Abstract]
  18. Marshall, C. J. (1995) Cell 80, 179-185 [Medline] [Order article via Infotrieve]
  19. Davis, R. J. (1994) Trends Biochem. Sci. 19, 470-473 [CrossRef][Medline] [Order article via Infotrieve]
  20. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) J. Biol. Chem. 270, 7420-7426 [Abstract/Free Full Text]
  21. Treisman, R. (1996) Curr. Opin. Cell Biol. 8, 205-215 [CrossRef][Medline] [Order article via Infotrieve]
  22. Xue, J.-C., Schwarz, E. J., Chawla, A., and Lazar, M. A. (1996) Mol. Cell. Biol. 16, 1567-1575 [Abstract]
  23. Tone, Y., Collingwood, T. N., Adams, M., and Chatterjee, V. K. (1994) J. Biol. Chem. 269, 31157-31161 [Abstract/Free Full Text]
  24. Wu, G. D., Wang, W., and Traber, P. G. (1992) J. Biol. Chem. 267, 7863-7870 [Abstract/Free Full Text]
  25. Sadowski, I., and Ptashne, M. (1989) Nucleic Acids Res. 17, 7539 [Medline] [Order article via Infotrieve]
  26. Hagiwara, M., Alberts, A., Brindle, P., Meinkoth, J., Feramisco, J., Deng, T., Karin, M., Shenolikar, S., and Montminy, M. (1992) Cell 70, 105-114 [Medline] [Order article via Infotrieve]
  27. Groom, L. A., Sneddon, A. A., Alessi, D. R., Dowd, S., and Keyse, S. M. (1996) EMBO J. 15, 3621-3632 [Abstract]
  28. Ausubel, F. M., Brent, R., Kingston, D. D., Smith, J. A., Seidman, J. G., and Struhl, K. (1987) Current Protocols in Molecular Biology, Greene Publishing-Wiley Interscience, New York
  29. Dreyer, C., Krey, G., Keller, H., Givel, F., Helftenbein, G., and Wahli, W. (1992) Cell 68, 879-887 [Medline] [Order article via Infotrieve]
  30. Kallen, C. B., and Lazar, M. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5793-5796 [Abstract/Free Full Text]
  31. Van Lint, J., Agostinis, P., Vandervoorde, V., Haegeman, G., Fiers, W., Merlevede, W., and Vandenheede, J. R. (1992) J. Biol. Chem. 267, 25916-25921 [Abstract/Free Full Text]
  32. Vietor, I., Schwenger, P., Li, W., Schlessinger, J., and Vilcek, J. (1993) J. Biol. Chem. 268, 18994-18999 [Abstract/Free Full Text]
  33. Schramm, K., Neihof, M., Radziwill, G., Rommel, C., and Moelling, K. (1994) Biochem. Biophys. Res. Commun. 201, 740-747 [CrossRef][Medline] [Order article via Infotrieve]
  34. Whitehurst, C. E., Owaki, H., Bruder, J. T., Rapp, U. R., and Geppert, T. D. (1995) J. Biol. Chem. 270, 5594-5599 [Abstract/Free Full Text]
  35. Sevetson, B. R., Kong, X., and Lawrence, J. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 305-310
  36. Cook, S. J., and McCormick, F. (1993) Science 262, 1069-1072 [Medline] [Order article via Infotrieve]
  37. Wu, J., Dent, P., Jelinek, T., Wolfman, A., Weber, M. J., and Sturgill, T. W. (1993) Science 262, 1065-1069 [Medline] [Order article via Infotrieve]
  38. Cummings, D. E., Brandon, E. P., Planas, J. V., Motamen, K., Idzerda, R. L., and McKnight, G. S. (1996) Nature 382, 622-626 [CrossRef][Medline] [Order article via Infotrieve]
  39. Okazaki, K., and Sagata, N. (1995) EMBO J. 14, 5048-5059 [Abstract]
  40. Tomura, H., Lazar, J., Phyllaier, M., and Nikodem, V. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5600-5604 [Abstract]
  41. Hadzic, E., Desai-Yajnik, V., Helmer, E., Guo, S., Wu, S., Koudinova, N., Casanova, J., Raaka, B., and Samuels, H. H. (1995) Mol. Cell. Biol. 15, 4507-4517 [Abstract]
  42. Arias, J., Alberts, A. S., Brindle, P., Claret, F. X., Smeal, T., Karin, M., Feramisco, J., and Montminy, M. (1994) Nature 370, 226-229 [CrossRef][Medline] [Order article via Infotrieve]
  43. Gonzalez, F. A., Raden, D. L., and Davies, R. J. (1991) J. Biol. Chem. 266, 22159-22163 [Abstract/Free Full Text]
  44. Greene, M. E., Blumberg, B., McBride, O. W., Yi, H. F., Kronquist, K., Kwan, K., Hsieh, L., Greene, G., and Nimer, S. D. (1995) Gene Exp. 4, 281-299 [Medline] [Order article via Infotrieve]
  45. Zhu, Y., Alvares, K., Huang, Q., Rao, M. S., and Reddy, J. K. (1993) J. Biol. Chem. 286, 26817-26820
  46. Aperlo, C., Pogonec, P., Saladin, R., Auwerx, J., and Boulukos, K. E. (1995) Gene (Amst.) 162, 297-302 [CrossRef][Medline] [Order article via Infotrieve]

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