Proline cis/trans-Isomerase Pin1 Regulates PeroxisomeProliferator-activated Receptor � Activity through the DirectBinding to the Activation Function-1 Domain*□S

Received for publication, August 12, 2009, and in revised form, November 21, 2009 Published, JBC Papers in Press, December 7, 2009, DOI 10.1074/jbc.M109.055095

Yoshito Fujimoto‡, Takuma Shiraki‡§1, Yuji Horiuchi¶, Tsuyoshi Waku‡, Akira Shigenaga�, Akira Otaka�,Tsuyoshi Ikura§, Kazuhiko Igarashi§, Saburo Aimoto**, Shin-ichi Tate¶, and Kosuke Morikawa‡2

From ‡The Takara-Bio Endowed Division, Department of Biomolecular Recognition, Institute for Protein Research, OsakaUniversity, Open Laboratories of Advanced Bioscience and Biotechnology, 6-2-3, Furuedai, Suita, Osaka 565-0874,the §Department of Biochemistry, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai,Miyagi 980-8575, the ¶Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University,1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, the �Institute of Health Biosciences and Graduate School of PharmaceuticalSciences, The University of Tokushima, Tokushima 770-8505, and the **Laboratory of Protein Organic Chemistry, Division ofProtein Chemistry, Institute for Protein Research, Osaka University, 3-2, Yamadaoka, Suita, Osaka 565-0871, Japan

The important roles of a nuclear receptor peroxisome prolif-erator-activated receptor � (PPAR�) are widely accepted in var-ious biological processes as well as metabolic diseases. Despitethe worldwide quest for pharmaceutical manipulation ofPPAR� activity through the ligand-binding domain, very littleinformation about the activation mechanism of the N-terminalactivation function-1 (AF-1) domain. Here, we demonstrate themolecular and structural basis of the phosphorylation-depen-dent regulation of PPAR� activity by a peptidyl-prolyl isomer-ase, Pin1. Pin1 interacts with the phosphorylated AF-1 domain,thereby inhibiting the polyubiquitination of PPAR�. The inter-action and inhibition are dependent upon the WW domain ofPin1 but are independent of peptidyl-prolyl cis/trans-isomeraseactivity. Gene knockdown experiments revealed that Pin1inhibits the PPAR�-dependent gene expression in THP-1 mac-rophage-like cells. Thus, our results suggest that Pin1 regulatesmacrophage function through the direct binding to the phos-phorylated AF-1 domain of PPAR�.

Peroxisome proliferator-activated receptor � (PPAR�;NR1C3)3 is a key regulator of adipocyte differentiation, glucosehomeostasis, and macrophage function (1, 2). Alternative pro-moter utilization generates two major PPAR� isoforms: ashorter isoform, PPAR�1, is widely distributed, whereasPPAR�2 is restricted to adipose tissues (3). PPAR� ligands acton the ligand-binding domain and induce conformational

changes to recruit coactivators (4, 5). On the other hand,PPAR� activity is also regulated by the phosphorylation of itsligand-independent activation function-1 (AF-1) domain.Mitogen-activated protein kinase and c-Jun N-terminal kinasereportedly phosphorylate Ser84 in the AF-1 domain of PPAR�1(6, 7). Ser84 of human PPAR�1 corresponds to Ser112 in humanand mouse PPAR�2, and hereafter, we will refer to this residueby its numbering in human PPAR�1. In mice, prevention ofSer84 phosphorylation by mutating Ser84 to alanine increasesthe insulin sensitivity, suggesting the enhancement of PPAR�

function (8). In humans, the P85Q mutation was observed inGerman obese subjects with a high body mass index (9) or withsevere insulin resistance (10). This mutation may affect thephosphorylation status of Ser84, immediately preceding themutated residue. Thus, the phosphorylation of the AF-1domain has functional significance in PPAR�-mediated generegulation, but the molecular mechanism remains obscure.In this study, we examined the mechanism of phosphoryla-

tion-dependent regulation of PPAR� and revealed that thephosphorylated AF-1 domain targets only the WW domain ofpeptidyl-prolyl cis/trans-isomerase (PPIase), Pin1, and was nota substrate for the PPIase of Pin1. The direct binding of Pin1 tothe AF-1 domain resulted in inhibiting the polyubiquitinationand the transcriptional activity of PPAR� revealed by overex-pression and knockdownexperiments using cell lines.Data pre-sented here suggest that the phosphorylation-dependent regu-lation of PPAR� activity is mediated by the direct binding toPin1 without proline isomerization.

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—HEK293T cells were main-tained in Dulbecco’s modified Eagle’s medium (Wako) supple-mented with 10% fetal bovine serum and penicillin/streptomy-cin mix (Nacalai Tesque). Cells were transfected by thecalcium-phosphate method with a CellPhect transfection kit(GE Healthcare). HEK293P cells were maintained in the samemanner as HEK293T cells. Cells were transfected by GeneJuicetransfection reagent (Takara Bio) for packaging the retrovirus.

* This work was supported by a donation from Takara Bio, Inc. and by a Grant-in-aid for Creative Scientific Research Program (18GS0316) from the JapanSociety for the Promotion of Science.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental “Experimental Procedures,” Equations 1– 4, additional refer-ences, and Figs. S1–S3.

1 To whom correspondence may be addressed. E-mail: tshiraki@m.tains.tohoku.ac.jp.

2 To whom correspondence may be addressed. E-mail: morikako@protein.osaka-u.ac.jp.

3 The abbreviations used are: PPAR�, peroxisome proliferator-activatedreceptor �; PPIase, peptidyl-prolyl cis/trans-isomerase; AF-1, activationfunction-1; HA, hemagglutinin; GST, glutathione S-transferase; PPRE, PPARresponse element; SPR, surface plasmon resonance; sh, short hairpin.

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THP-1 cells were maintained in RPMI containing 10% fetalbovine serum and penicillin/streptomycin mix (NacalaiTesque).Coimmunoprecipitation and Immunoblot Analysis—Tran-

siently transfected cells plated on 60-mmdisheswere harvestedwith 350 �l of lysis buffer (50 mM Tris-Hcl, pH 8.0, 150 mM

NaCl, 1% Triton X-100, 10% glycerol, 50 mM NaF, 100 �M

Na3VO4, and protease inhibitor mixture (Nacalai Tesque)).After centrifugation, the lysates were incubated with anti-FLAGM2 agarose affinity Gel (Sigma) at 4 °C for 2 h. After theincubation, the beads were washed 5� with 1-ml aliquots oflysis buffer. Samples were subjected to SDS-PAGE. The immu-noblot analysis was performed using anti-FLAG M2-peroxi-dase (Sigma) or anti-hemagglutinin (HA)-peroxidase, highaffinity (3F10) (Roche). Chemiluminescence was detected withECL Plus Western blotting detection reagents and HyperfilmECL (GE Healthcare).Purification of Recombinant Pin1—Human Pin1 and its

mutant forms were cloned into the pGEX-4T-1 expressionplasmid (GE Healthcare). The plasmids were transformed intoEscherichia coli BL21 (DE3) cells (Novagen). The cells weregrown in LB medium with 100 �g/ml ampicillin at 37 °C to anA600 of 0.7–0.8. The expression of glutathione S-transferase(GST)-fusion proteins was induced by the addition of isopropyl�-D-thiogalactopyranoside to a final concentration of 0.5 mM,and the cells were further incubated for 16 h at 18 °C. Cells froma 1-liter culture were harvested and resuspended in 100 ml of

phosphate-buffered saline. The cellswere sonicated, and the solublefraction was isolated by centrifuga-tion at 12,000 rpm and 4 °C for 15min. The supernatants were appliedto phosphate-buffered saline-equil-ibrated glutathione-Sepharose 4Bresin (GE Healthcare). After a thor-oughwashwith phosphate-bufferedsaline, the fusion proteins wereeluted with 80 ml of 10 mM reducedglutathione in 50 mM Tris-HCl, pH8.0/100 mM NaCl. The collectedproteins were cleaved with throm-bin protease at 4 °C for 12 � 15 hduring dialysis against 20 mM Tris-HCl, pH 8.0/1 mM TCEP. Thedigested solution was passedthrough a 5-ml HiTrap Q HP col-umn (GE Healthcare). The flowthrough was collected and concen-trated to 7ml, using a 30,000molec-ular weight cut-off Amicon Ultra 15centrifugal concentrator. The sam-ple was fractionated on a HiLoad26/60 Superdex 75 prep grade col-umn (GE Healthcare), and theappropriate fractions were concen-trated using a 10,000 molecularweight cut-off AmiconUltra 15 cen-trifugal concentrator. The protein

sample was frozen at �80 °C until use.Surface Plasmon Resonance (SPR) Analysis—SPR measure-

ments were performed using Biacore 2000 at 24 °C. The syn-thetic, biotinylated PPAR�1 AF-1 peptides were immobilizedon the Sensor chip SA (Biacore). Both the phosphorylated andnonphosphorylated peptides were immobilized on the samechip in different lanes. Experiments were performed by inject-ing the wild type recombinant Pin1 or its mutants at a flow rateof 50 �l/min. Binding was measured in the following order:blank, the nonphosphorylated peptide, and the phosphorylatedpeptide.NMR Spectra Acquisition—The standard set of triple reso-

nance experiments, including HNCO, HNCOCA, HNCA,HNCACB, and CBCA(CO)NH, was performed to obtain thebackbone 1H, 15N, 13CA, 13CB, and 13C� resonance assignmentsfor human Pin1 under the present experimental conditions(11); the proteinwas dissolved in a buffer solution containing 50mM Tris-HCl, pH 7.5, with 150 mMNaCl, 5 mM EDTA, and 0.1mM pefabloc, and all of the NMR spectra were collected at 293K. All NMR experiments were performed with a Bruker DMX-600 spectrometer equippedwith shielded XYZ gradients. NMRdata were processed by the program NMRPipe (12). The pro-grams NMRView version 5.0.4 (B.A. Johnson, Merck ResearchLaboratories) and Smart Notebook (13) were used for the res-onance assignment. The Pin1 protein used in these experi-ments contained all 163 residues of the native protein, with 20

FIGURE 1. Phosphorylation-dependent binding of Pin1 to the AF-1 domain of PPAR�. A, schematic repre-sentation of the domain structures of PPAR� and Pin1. The amino acid sequence indicates the AF-1 peptidesequence used for in vitro experiments in this study. The mutated residues in PPAR� are denoted above thesequence. Bold letters indicate the recognition motif of Pin1, in which pS denotes the phosphorylated serine.B, effects of coexpression of RasV12G or Pin1 on the intrinsic activity of the AF-1 domain. GAL4-fused AF-1domains with the indicated mutations were transfected with or without RasV12G or Pin1 (left and right panels,respectively). Data are presented as the means � S.D. DBD, DNA-binding domain. C, coimmunoprecipitation ofPin1 with full-length PPAR� in HEK293T cells. Immunoprecipitations (IP) were analyzed by Western blots(WB) probed with either an anti-FLAG or anti-HA antibody (WB:flag and WB:HA, respectively). D, Pin1 interactswith PPAR� in vitro. The cell lysate was incubated with GST or GST-Pin1, and the interaction between Pin1 andPPAR� was detected by Western blotting, using an anti-PPAR� antibody. Cell lysates were also probed withanti-FLAG antibody. LBD, ligand-binding domain.

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additional residues from the expression vector pET-28a; theHis6 tag was not cleaved in the present NMR experiments.Luciferase Assay—The cell-based transcription assay was

described previously (14). In this study, we usedHEK293T cellsinstead of COS-7 cells. Transient transfections of reportergenes were performed by using CellPhect (GE Healthcare).Data are represented as mean � S.D.Flow Cytometry—THP-1 cells were detached from dishes by

a treatment with 1 mM EDTA/phosphate-buffered saline. Cellswere first treated with mouse BD Fc Block (BD BiosciencePharmingen) and thenwere treatedwith an fluorescein isothio-cyanate-labeled anti-CD36 antibody (MCA772F, Serotec Ltd.).Fluorescent intensities of individual cells were analyzed byusing FACSCalibur (BD Biosciences).Knockdown Experiments with Short Hairpin (sh) RNA

Expression by a Retrovirus—The procedure for the retroviruspreparation was described previously (15). The sequences usedfor PPAR� knockdown were: shPPAR�599, gatccccGCTTAT-CTATGACAGATGTGATCTTttcaagagaAAGATCACATC-TGTCATAGATAAGCttttta and shPPAR�1210, gatccccGCT-TCATGACAAGGGAGTTTCTAAAttcaagagaTTTAGAAA-CTCCCTTGTCATGAAGCttttta. (Capital letters indicate thetarget sequences for the human PPAR� mRNA.) The DNAsequence used for Pin1 knockdown was shPin1, gatccccGAG-ACCTGGGTGCCTTCAGCA ttcaagagaTGCTGAAGGCAC-CCAGGTCTCttttta. These oligonucleotideDNAswere clonedinto pSUPER.puro (OligoEngine).

RESULTS

Due to the overlapped recognition sequences between theproline-directed kinases and Pin1 (16), we investigated the pos-sibility of the Pin1-mediated regulation of AF-1 activity. Coex-pression of the constitutively active form of the Ras protein(RasV12G), to induce the phosphorylation of PPAR� throughthe Ras-mitogen-activated protein kinase (MAPK) pathway,inhibited AF-1 activity, but the S84A mutation in the AF-1domain abolished the inhibitory effects of RasV12G (Fig. 1B, leftpanel). Coexpression of Pin1 reduced AF-1 activity, but notsignificantly, in the S84A, S84E, and S85Q mutants (Fig. 1B,right panel). We then analyzed the interaction between Pin1and the AF-1 domain of PPAR� by coimmunoprecipitation.The interaction of Pin1 with PPAR� was observed for the wildtype (Fig. 1C, lane 2) but not for the P85Qmutatnt (Fig. 1C, lane5). The amount of Pin1 copurified with PPAR� was reduced bythe S84A mutation (Fig. 1C, lane 3). Another PPAR� mutant,S84E, which mimics the phosphorylated state of the AF-1domain, displayed significant affinity for Pin1 (Fig. 1C, lane 4).Using a recombinant GST-Pin1 protein, we observed theenhancement of the interaction betweenGST-Pin1 and PPAR�by the coexpression of RasV12G (Fig. 1D, lanes 5 and 6). Bycontrast, the S84A mutation completely inhibited the binding(Fig. 1D, lane 7). Thus, we concluded that the phosphorylationof the AF-1 domain induces the binding of Pin1 to PPAR�,which concomitantly inhibits the transcriptional activity. How-ever, we noticed that the interaction was slightly retained in thepresence of RasV12G (Fig. 1D, lane 8). In addition to Ser84, thereare two other possible phosphorylation sites by proline-di-rected kinases at Ser245 and Thr268 in the PPAR� ligand-bind-

ing domain. In fact, slowermigration of the PPAR� protein wasstill observed in the S84A mutant coexpressed with RasV12G(Fig. 1D, lower panel). Although we do not have direct evidencefor the phosphorylation of these residues, we do not exclude thepossibility that Pin1 also interacts with the ligand-bindingdomain in addition to the AF-1 domain.Further characterization of the interaction, using SPR,

revealed that the phosphorylated AF-1 (pAF-1) peptide but notthe nonphosphorylated (AF-1) peptide, was specifically boundto Pin1 in a dose-responsive manner (Fig. 2A). The Pin1 WWdomain, classified as group IV, preferentially binds to the phos-phorylated serine/threonine-proline residues, while the PPIase

FIGURE 2. Phosphorylation-dependent binding of Pin1 to the AF-1 pep-tide of PPAR�. A, SPR analyses of the interaction between Pin1 and the AF-1peptide. Traces of the titration of Pin1 on sensor chips immobilized with thenon-phosphorylated (AF-1) and phosphorylated AF-1 (pAF-1) peptide (leftand right panels, respectively). Concentrations of analyte were 0, 0.04, 0.2, 1, 5,and 25 �M. Data are presented as the response difference in resonance units.B, concentration dependence of the interaction between Pin1 and pAF-1,plotted for each Pin1 protein with the indicated mutation. C, overlay of part ofthe amide region of the 1H-15N heteronuclear-single-quantum coherence(HSQC) spectra for Pin1 with various amounts of pAF-1 peptide. In the spectrafor the pAF-1 titration to Pin1, the overlaid spectra were collected for sampleswith pAF-1 to Pin1 ratios of 0.0, 0.2, 0.5, 0.7, 1.0, and 1.3. The correspondingspectra for the nonphosphorylated AF-1 with Pin1 and with AF-1 to Pin1ratios of 0.0, 0.40, and 1.12, are displayed. WT, wild type.

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domain catalyzes the isomerization of the pSer/Thr-Pro seg-ment in the target protein (16). The mutations of Y23A orW34A in theWWdomain andK63A in the PPIase domain (Fig.1A) abolished the binding activity to the phospho-peptides andthe PPIase activity of Pin1, respectively (17). The K63A muta-tionwithin the PPIase domain only slightly reduced the binding(Fig. 2B). By contrast, the W34A or Y23A mutation within theWW domain completely abolished the interaction. In agree-ment with the SPR experiments, NMR titration measurementsrevealed that the pAF-1 peptide mainly induced chemical shiftperturbations in the WW domain, whereas the nonphosphor-

ylated counterpart caused no signif-icant spectral change (Fig. 2C andsupplemental Fig. S1). A conven-tional PPIase assay (see supplemen-tal “Experimental Procedures”)revealed that an excess amount ofeither AF-1 or the pAF-1 peptidedid not inhibit the Pin1-inducedisomerization of the substrate pep-tide (supplemental Fig. S2). Thus,we concluded that the pAF-1 pep-tide is not a substrate for the PPIaseactivity of Pin1.We noticed a PPxY motif imme-

diately after the Pin1-recognitionmotif. The PPxYmotif is recognizedby several ubiquitin ligases contain-ing a group IWWdomain. Then,wedetermined the possible cross-talkbetween phosphorylation andpolyubiquitination of the PPAR�protein through Pin1 binding. Wefound that the polyubiquitination ofPPAR� in the absence of ligandswasinhibited by coexpressed Pin1 (Fig.3A, lanes 7 and 8). The Y23Amutant of Pin1 lost the ability toinhibit polyubiquitination, whereasthe K63A mutation retained it (Fig.3A, lanes 9 and 10, respectively).Pin1-mediated inhibition of poly-ubiquitination was not observedin PPAR� lacking the AF-1 domain(Fig. 3A, lanes 11 and 12). Thepolyubiquitination of PPAR� wasalso inhibited by the Pin1 C113Amutant and the WW domain alonebut not by the PPIase domain alone(Fig. 3B, lanes 4, 5, and 6, respec-tively). Pin1-mediated inhibition ofpolyubiquitination was also ob-served even in the presence of a pro-teasome inhibitor, MG132 (Fig. 3C,lanes 3 and 4), suggesting that Pin1regulates the ubiquitination step,rather than the degradation step.Collectively, these results indicated

that the inhibition of the PPAR� polyubiquitination by Pin1requires the interaction between theWWdomain and theAF-1domain, whereas PPIase activity is not essential.To explore the physiological importance of the Pin1-PPAR�

interaction, we investigated the involvement of Pin1 in thePPAR�-mediated activation of macrophage function. Fattyacid binding protein 4 (FABP4) is one of the target genes ofPPAR� in macrophage cells (18), and it has strong connectionswith the development of metabolic diseases, such as type 2 dia-betes and atherosclerosis (19). The PPAR-response element(PPRE) for the mouse Fabp4 gene has been extensively ana-

FIGURE 3. Inhibition of polyubiquitination of PPAR� by Pin1. A, Pin1 inhibits the polyubiquitination ofPPAR� in a WW domain-dependent manner. Immunoprecipitations (IP) were analyzed by Western blot (WB)probed with an anti-HA antibody to detect polyubiquitination (bottom panel, WB:HA). Cell lysates were alsoprobed with an anti-FLAG or anti-green fluorescent protein (GFP) antibody (top panel, WB:flag and WB:GFP,respectively). B, Pin1 inhibits the polyubiquitination of PPAR� in a WW domain-dependent manner. Experi-mental condition was same as A. C, effects of proteasome inhibitor, MG132, on the polyubiquitination ofPPAR�. Cells were treated with 10 �M MG132 for 5 h before harvest. Immunoprecipitation condition was thesame as in A. wt, wild type; Ub, ubiquitin.

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lyzed (20), but that of the humanFABP4 gene has not. Therefore, weanalyzed the human FAPB4 pro-moter and found that one PPRE islocated at �5216 bp (Fig. 4A). Toinvestigate the function of the PPREat �5216 bp, we made a series ofdeletion andmutation constructs ofthe FABP4 5� upstream region (Fig.4A). PPAR�-dependent activationof the FABP4 promoter was totallydependent on the PPRE sequence(Fig. 4B). A highly conserved se-quence around this PPRE amongmammals suggests that this regionis involved in the PPAR�-depen-dent regulation of FABP4 expression.Pin1 inhibited the PPAR�-activatedhuman FABP4 promoter in both thepresence and absence of a syntheticPPAR� agonist, BRL49653 (Fig. 4C).Ubiquitin-dependent degradationis required for efficient transactiva-tion by nuclear receptors (21), andtherefore, Pin1 might inhibit thePPAR� activity by inhibiting thereceptor turnover. In fact, a protea-some inhibitor, MG132, reducedthe activity of PPAR� (Fig. 4D). Pin1mutation in the WW domain suchas Y23A and W34A abolished theinhibitory effect of Pin1 on thePPAR�-dependent transcription(Fig. 4E). On the contrary, muta-tions in the PPIase domain such asK63A and C113A showed the sameinhibitory effect on PPAR� as thewild type (Fig. 4E), suggesting thatthe Pin1-mediated regulation ofPPAR� is dependent on the WWdomain rather than PPIase activity.We next examined the effects ofPin1 knockdown on the expressionof the endogenous PPAR�-regu-lated proteins. The efficiency of theknockdown by short hairpin RNAexpression were determined byWestern blot and RT-PCR (supple-mental Fig. S3). In THP-1 macro-phage-like cells differentiated byphorbol 12-myristate 13-acetatetreatment, the PPAR� agonistincreased the expression of FABP4and another PPAR�-target gene, heme oxygenase-1 (22) (Fig.4F, lanes 1 and 2). The expression levels of both the FABP4 andHO-1 proteins were decreased in the PPAR� knockdown cells(Fig. 4F, lanes 3–6), indicating that both proteins are regulatedby PPAR�. On the other hand, the knockdown of Pin1 aug-

mented the expression of these proteins by PPAR� (Fig. 4F,lanes 7 and 8). Therefore, we concluded that Pin1 reduces theactivity of PPAR� in THP-1 cells. Lastly, we analyzed theexpression of CD36, which is one of the surface markers foractivated macrophages in atherosclerosis (23). The PPAR�

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knockdown abolished the agonist-induced up-regulation ofCD36 (Fig. 4G, middle). In contrast, the Pin1 knockdownincreased the basal level of CD36 and also sensitized the induc-tion against the addition of the PPAR� agonist (Fig. 4G, right).Thus, we concluded that Pin1 modulates macrophage activa-tion through an interaction with PPAR�.

DISCUSSION

It was reported that the two domains of Pin1 both target tothe same phosphorylated serine/threonine-proline sequence(16). However, we found that the phospho-AF-1 domain inPPAR� was recognized by the WW domain but not the PPIasedomain of Pin1. These results allow us to propose that Pin1affects the function of PPAR� through the interaction betweenthe AF-1 region of PPAR� and theWWdomain in Pin1, ratherthan the proline isomerization by the PPIase domain.Among themany reports about the interaction between Pin1

and phosphorylated proteins, several studies described the dis-tinct usage of two domains for regulation of the target proteins(24, 25) in agreement with our results. For example, Pin1 pro-motes the Stat3 transcriptional activity in a Ser727 phosphory-lation-dependent manner. However, the WW domain muta-tion (Y23A) but not the catalytic domain mutations abolishedthis effect (24), suggesting that the PPIase activity is dispensablefor transcriptional activation. In the case of the Tau protein,both the phosphorylated Thr212 and phosphorylated Thr231sites are recognized by the WW domain of Pin1, but only thephosphorylated Thr212–Pro bond is isomerized by the PPIasedomain (25), suggesting that the phosphorylated Ser/Thr-Pro isnot always a substrate for PPIase activity.How does Pin1 affect the function of PPAR� independently

of its catalytic activity? TheAF-1 sequence, adjacent to the Pin1target motif, overlaps with a motif (PPxY) for the group I WWdomain (26). The binding of Pin1 to the pSer-Pro sequence,sterically blocks the PPxY sequence, thereby prohibiting thebinding of a protein harboring the group IWWdomains. Inter-estingly, the Nedd4 family of E3 ubiquitin ligases contains sev-eral group I WW domains (27), and thus, it is possible that theinteraction with Pin1 is involved in the regulation of the polyu-biquitination-mediated PPAR� degradation, in a “binaryswitch”manner (Fig. 5). Such competitionmay not be observedin other PPARs, because they lack themotif for the group IWWdomain near the phosphorylation site. Therefore, it will beinteresting to determine whether this difference between the

subtypes is connected with the AF-1-mediated isotype-selec-tive regulation of gene expression (28). Identification of thephysiological ubiquitin ligase for PPAR� will greatly advanceour understanding for the regulation of the PPAR� activity.In this study, we showed that Pin1-mediated negative reg-

ulation of PPAR� has an important role for macrophagefunction. FABP4 is one of the most important downstreamtargets among many PPAR�-regulated genes, becauseFABP4 in macrophages is involved in atherosclerosis (19). Inthe future, it is important to investigate the possible involve-ment of Pin1 in atherosclerosis. There are several Pin1-tar-geted drugs, but most of them bind to the PPIase domain ofPin1 (29, 30). Tomanipulate the Pin1-mediated regulation ofPPAR�, the WW domain, rather than the PPIase domain,must be targeted by drugs. Structural information providedby our NMR study will help the design and screening of suchnovel Pin1 inhibitors.

Acknowledgments—We thank Drs. Hiroto Yamaguchi, TakujiOyama, and Akira Kakizuka for helpful comments and Ms. KanakoMaebara and Ms. Sayaka Shiki for technical support. We also thankDrs. Kozo Tanaka and Akihiko Muto for critical reading of themanuscript.

FIGURE 4. Inhibitory effect of Pin1 on PPAR�-dependent transcription. A, schematic representation of the human FABP4 5� upstream region and a series ofDNA constructs used for the reporter assay. PPRE was found at �5216 bp (arrowhead) within the mammalian conserved region. The figure was drawn using theENCODE web server. B, PPAR�-dependent activation of the human FABP4 enhancer. The indicated reporter plasmids, PPAR�1 and RXR� genes were cotrans-fected into HEK293T cells, and the transcriptional activities were determined by the luciferase activity in the presence or absence of 0.5 �M BRL49653. C, effectof Pin1 on the PPAR�-dependent activation of the FABP4 promoter. The indicated reporter plasmids, PPAR�1 and RXR� genes were cotransfected into HEK293Tcells with or without Pin1. D, effects of Pin1 mutations on the Pin1-mediated inhibition of the PPAR�-dependent transcription. The indicated reporter plasmids,PPAR�1 and RXR� genes were cotransfected into HEK293T cells with or without Pin1 carrying indicated mutations. E, effects of MG132 on the PPAR�-depen-dent transcription. The indicated reporter plasmids and PPAR�1 and RXR� genes were co-transfected into HEK293T cells. After 12 h, cells were treated with orwithout 10 �M MG132 for another 6 h. Luciferase activity was not normalized by any internal control because MG132 might change the stability of the controlprotein. F, effect of the Pin1 knockdown on the PPAR�-dependent up-regulation of endogenous proteins in THP-1 cells. Cells were differentiated intomacrophage-like cells by 5 nM phorbol 12-myristate 13-acetate. After 24 h, cells were treated with 0.5 �M BRL49653 for additional 24 h. Cellular lysates including50 �g proteins were separated by SDS-PAGE, and protein expression was analyzed by Western blots probed with either an anti-FABP4, anti-HO-1 or anti-�-Tubulin antibody. G, fluorescence-activated cell sorter analysis of the effect of the Pin1 knockdown on the PPAR�-dependent CD36 expression in THP-1 cells.Cells were differentiated into macrophage-like cells by 5 nM phorbol 12-myristate 13-acetate. After 24 h, cells were treated with 0.5 �M BRL49653 for additional24 h. 0.5 � 106 cells were incubated with fluorescein isothiocyanate (FITC)-labeled anti-CD36 antibody, and the fluorescent intensity was analyzed byfluorescence-activated cell sorter. wt, wild type; DMSO, dimethyl sulfoxide.

FIGURE 5. Model of the Pin1-mediated regulation of PPAR� activity.Binary switch model of the Pin1-mediated regulation of PPAR�. In theabsence of phosphorylation signals, PPAR� is polyubiquitinated. Ras-medi-ated kinase signals induce the phosphorylation of Ser84. Binding of Pin1 to thephosphorylated AF-1 prevents the polyubiquitination of PPAR�, resulting inslow turnover of the PPAR� protein. Because the proteasome inhibitor,MG132, reduces PPAR� activity, the efficient transcription seems to requirecontinuous turnover of PPAR� through the ubiquitin-proteasome pathway.Then, Pin1-mediated inhibition of polyubiquitination results in the reductionof the PPAR� activity. In this regulation, binding through the WW domain issufficient for the inhibition of polyubiquitination, and the PPIase activity ofPin1 is dispensable. LBD, ligand-binding domain; DBD, DNA-binding domain;(Ub)n, polyubiquitination.

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AF-1-mediated Regulation of PPAR�

3132 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 5 • JANUARY 29, 2010

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Kosuke MorikawaAkira Otaka, Tsuyoshi Ikura, Kazuhiko Igarashi, Saburo Aimoto, Shin-ichi Tate and

Yoshito Fujimoto, Takuma Shiraki, Yuji Horiuchi, Tsuyoshi Waku, Akira Shigenaga,Domain

Activity through the Direct Binding to the Activation Function-1γReceptor -Isomerase Pin1 Regulates Peroxisome Proliferator-activatedcis/transProline

doi: 10.1074/jbc.M109.055095 originally published online December 7, 20092010, 285:3126-3132.J. Biol. Chem. 

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