Expression level and agonist-binding affect the turnover, ubiquitination and complex formation of peroxisome proliferator activated receptor b Markus Rieck*, Lena Wedeken*, Sabine Mu ¨ ller-Bru ¨ sselbach, Wolfgang Meissner and Rolf Mu ¨ ller Institute of Molecular Biology and Tumor Research (IMT), Philipps University, Marburg, Germany The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor super- family [1–6]. Although the DNA binding domains of the three subtypes PPARa, PPAR b ⁄ d and PPARc are 80% identical, their ligand-binding domains exhi- bit a higher degree of divergence (approximate 65% identity), which likely accounts for the differential activation of PPARs by fatty acid derivatives and synthetic compounds [6–9]. All PPARs bind to spe- cific DNA elements, the peroxisome proliferator responsive elements, as heterodimers with the reti- noid X receptor. Peroxisome proliferator responsive elements are found in many PPAR target genes involved in, for example, lipid metabolism and energy homeostasis [6]. PPARa is expressed at high levels in the liver, kid- ney, heart and muscle, where it plays a pivotal role in fatty acid catabolism, energy homeostasis and gluco- neogenesis [6,9–11]. PPARb is expressed ubiquitously, and is implicated in fatty acid oxidation and glucose homeostasis [6,12–14], but also in inflammation, pla- cental development, wound healing and keratinocyte differentiation and proliferation [15–20]. There are two tissue-specific PPARc isoforms generated by alterna- tive splicing [21,22]. PPARc1 is expressed in the liver and other tissues, whereas PPARc2 is expressed exclu- sively in adipose tissue, where it has essential functions Keywords GW501516; polyubiquitination; PPARb; ubiquitin Correspondence R. Mu ¨ ller, Institute of Molecular Biology and Tumor Research (IMT), Philipps University, Emil-Mannkopff-Strasse 2, 35032 Marburg, Germany E-mail: rmueller@imt.uni-marburg.de *These authors contributed equally to this work (Received 4 July 2007, revised 30 July 2007, accepted 1 August 2007) doi:10.1111/j.1742-4658.2007.06037.x Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to fatty acid ligands. Their regulation by post-translational modifications has been reported but is poorly understood. In the present study, we investigated whether ligand binding affects the turnover and ubiquitination of the PPARb subtype (also known as PPARd). Our data show that the ubiquitination and degradation of PPAR b is not significantly influenced by the synthetic agonist GW501516 under conditions of moder- ate PPARb expression. By contrast, the overexpression of PPARb dramati- cally enhanced its degradation concomitant with its polyubiquitination and the formation of high molecular mass complexes containing multiple, presumably oligomerized PPARb molecules that lacked stoichiometical amounts of the obligatory PPARb dimerization partner, retinoid X recep- tor. The formation of these apparently aberrant complexes, as well as the ubiquitination and destabilization of PPARb, were strongly inhibited by GW501516. Our findings suggest that PPARb is subject to complex post- translational regulatory mechanisms that partly may serve to safeguard the cell against deregulated PPARb expression. Furthermore, our data have important implications regarding the widespread use of overexpression sys- tems to evaluate the function and regulation of PPARs. Abbreviations PPAR, peroxisome proliferator-activated receptor; PSL, photo-stimulated luminescence. 5068 FEBS Journal 274 (2007) 5068–5076 ª 2007 The Authors Journal compilation ª 2007 FEBS in adipocyte differentiation, lipid storage and energy dissipation [6,9,23]. All three PPAR subtypes have also been implicated in macrophage activation, immune modulation, atherosclerosis and other metabolic dis- eases, and cancer [3,11,13,24–28]. The activity of PPARs is regulated not only by the binding of ligands, but also appears to be influenced by post-translational modifications. For example, PPARc activity is regulated by sumoylation at differ- ent sites [29–31], and there is evidence that phosphory- lation may regulate PPARc and PPARa activity [32,33]. Furthermore, both PPARa and PPARc have been reported to be ubiquitinated in a ligand-regulated fashion [34,35]. However, although the agonist-medi- ated activation of PPARa resulted in decreased ubiqui- tination and increased stability [35], the opposite was reported for PPARc [34]. To date, no post-transla- tional modifications have been described for PPARb. Likewise, the effect of PPARb ligands on protein turn- over has not been analyzed. We addressed these ques- tions in the present study. We show that: (a) the turnover of PPARb is not affected by its synthetic ago- nist GW501516 under conditions of moderate PPARb expression; (b) the overexpression of PPARb dramati- cally enhances its degradation, which is inhibited by GW501516; and (c) this increased turnover correlates with the ubiquitination of PPARb and the formation of apparently aberrant high molecular mass complexes. Our results point to a new regulatory mechanism impinging on PPARb that could be relevant, for exam- ple, in protecting the cell against the overexpression of PPARb in pathophysiological conditions. Further- more, our findings indicate that the experimental data obtained by the overexpression of PPARs have to be considered with great caution, and suggest that previ- ously published studies making use of overexpressed PPARs may have to be re-evaluated. Results and Discussion Agonist and protein level influence PPARb turnover The stability of PPARb protein was determined by pulse-chase labeling under different experimental con- ditions. First, we measured PPARb turnover in tran- siently transfected cells (i.e. an approach previously used with other PPAR subtypes). The expression vec- tor pCMX-mPPARb was transfected into HEK293 cells and, after 24 h in either the presence or absence of 1 lm GW501516, the cells were metabolically labeled for 2 h with [ 35 S]methionine and [ 35 S]cysteine. Cell extracts were analyzed by immunoprecipitaton after different times of chase in normal growth med- ium containing unlabeled methionine and cysteine. The autoradiographs in Fig. 1 show long-term kinetics 0481216A B C 20 24 36 48 no GW +GW * 5.3 2.2 1.6 1.6 1.3 0.6 0.6 0.3 0.01 100 41 31 30 25 11 11 4.6 1.6 signal intensity % of t=0 signal intensity % of t=0 11 15 8.6 7.4 4.3 2.7 2.8 1.3 0.4 100 136 78 67 39 24 25 11 3.4 x10 3 x10 3 0 60 80 100 0246 40 20 120 untreated GW501516 untreated GW501516 Time (h) Time (h) IntensityIntensity 0 60 80 100 40 20 120 140 0 5 10 15 20 25 Fig. 1. Ligand-dependent turnover of overexpressed PPARb in transiently transfected cells. HEK293 cells were transfected with pCMX-mPPARb and either treated with 1 l M GW501516 for 24 h post-transfection or left untreated. The cells were metabolically pulse-labeled with [ 35 S]methionine and [ 35 S]cysteine for the final 2 h in methionine- and cysteine-free medium. The medium was exchanged with normal growth medium and cells were harvested after different times (chase). PPARb protein was immunoprecipitat- ed and analyzed by PAGE followed by phosphorimaging. (A) Auto- radiograph showing a 48 h chase (*nonspecific band). The amount of labeled PPARb in the GW501516-treated cells is higher due its greater stability under these conditions. (B) Quantitative evaluation by phosphorimaging of pulse chase experiments (24 h chase) per- formed as in (A). (C) Short-term pulse chase experiment (6 h chase) performed as in (A). Exposure times were 48 h for autoradiography and 26 h for phosphorimaging. Signal intensities represent phospho- stimulated luminescence (PSL) ⁄ mm 2 ⁄ 1000 (PSL values generated by a Phospoimager; Fuji, Du ¨ sseldorf, Germany). Values represent the mean of three independent experiments; error bars indicate SD. M. Rieck et al. Regulation of PPARb turnover FEBS Journal 274 (2007) 5068–5076 ª 2007 The Authors Journal compilation ª 2007 FEBS 5069 (48 h and 24 h) (Fig. 1A,B) and a short-term chase (6 h) (Fig. 1C). The quantitative evaluation by phos- phorimaging revealed clear differences between untreated and GW501516-treated cells with respect to both protein levels (upper rows: signal intensity) and degradation (bottom rows: percent of t ¼ 0). Thus, the levels of labeled PPARb protein were approximately two-fold higher already at the beginning of the chase period (t ¼ 0), and remained higher throughout the time course. Differences in protein stability were, how- ever, only evident during the initial chase period: in untreated cells, PPARb protein levels dropped to less than 50% at 4 h whereas, in the presence of GW501516, no decrease was detectable. Both these observations are consistent with a drastically increased stability of PPAR b in the presence of GW501516. Very similar results were obtained with transiently transfect- ed NIH3T3 cells (data not shown), indicating that the observed effects are not cell line specific. At later time points of the chase, differences in stability between untreated and GW501516-treated cells became basically undetectable, indicating that the PPARb protein levels may have an impact on the kinetics of degradation. To address this question, we established a cell line (3Fb1) stably expressing 3xFLAG-tagged PPARb in a PPARb null background at less than 1% of the PPARb level observed in tran- siently transfected cells. These cells were analyzed in a pulse-chase experiment similar to the one described above (Fig. 2A,B). In addition, we used a FLAG- 1 2 4 8 12 24 31 48 * no GW +GW * 0 27 20 31 30 15 16 12 8.1 4.7 29 29 32 15 13 10 12 6.1 4.2 0 Con signal intensity %oft=0 100 signal intensity %oft=0 74 114 111 56 61 44 30 18 100 100 112 51 45 36 42 21 15 chase (h) FLAG-PPAR β A B C D FLAG-PPAR β 0 5 10 15 20 25 30 35 01020304050 Chase (h) no GW +GW 481220 untreated +GW 0 rel. signal intensity 100 42 16 15 11 11 chase (h) 24 rel. signal intensity 100 80 71 56 30 36 U D no GW L no GW L - MG132 + MG132 Fig. 2. Turnover of FLAG-PPARb expressed at moderate levels in retrovirally transduced mouse fibroblasts. (A) Pparb null cells were infected with a 3xFLAG-PPARb expressing retrovirus and a stable clone (3Fb1 cells) was analyzed in a pulse-chase experiment as in Fig. 1A, except that the cells were labeled for 30 min. The experiment was also repeated with cells labeled for 2 h with basically results (the 4 h value shown for GW501516-treated cells is an outlier). The autoradiograph exposed for 6 days. Signal intensities represent PSL ⁄ mm 2 and indicate a more than 100-fold lower expression of PPARb compared to Fig. 1 (*nonspecific band). (B) Quantitative evaluation by phosphorimaging (exposure time 24 h) of the experiment shown in (A). (C) Pulse-chase experiment as in Fig. 1A, except that the same expression vector for 3xFLAG-PPARb as in (A) was used for transient transfection (high expression). (D) Immunoblot analysis of 3xFLAG-PPARb in 3Fb1 cells; moderate expression, see (A). Cells were either untreated, or treated with the PPARb agonists GW501516 (GW) or L165 041 (L) either alone or in combination with the proteasome inhibitor MG132. Agonist treatment was for 48 h. MG132 was included during the final 6 h of the experiment. U, presumably polyubiquitinated high-molecular mass 3xFLAG-PPARb forms; D, presumably a 3xFLAG-PPARb protein fragment stabilized by MG132. The agonist function of GW501516 and L165 041 was verified in transient reporter gene assays performed in parallel (not shown). Regulation of PPARb turnover M. Rieck et al. 5070 FEBS Journal 274 (2007) 5068–5076 ª 2007 The Authors Journal compilation ª 2007 FEBS specific antibody because none of the available PPARb-specific antibodies are suitable for a quantifi- able detection of PPARb at low expression levels. In these experiments, no significant differences were detectable between untreated and GW501516-treated cells with respect to either the initial level of labeled FLAG-PPARb or the turnover FLAG-PPARb. This turnover of FLAG-PPARb is similar to that of PPARb in transiently transfected GW501516-treated cells (Fig. 1), indicating that overexpressed PPARb protein is subject to an enhanced degradation that is prevented by GW501516. To exclude the possibility that the FLAG tag influenced the results obtained with the 3Fb1 cells, we also analyzed 3xFLAG-tagged PPARb in transiently transfected cells with virtually identical results compared to untagged PPARb (Figs 1B and 2C). Finally, we analyzed steady-state 3xFLAG-PPARb levels in 3Fb1 cells by immunoblotting either untreated, treated with the PPARb agonists GW501516 or L165 041 and in combination with the proteasome inhibitor MG132 (Fig. 2D). In agreement with the pulse-chase experiments, the immunoblot data clearly show that, in 3Fb1 cells expressing PPARb at moderate levels, neither agonist had any detectable effect on pro- tein levels in spite of a clear stabilization by MG132 (visible as strongly increased protein levels and the presence of presumably polyubiquitinated 3xFLAG- PPARb). Formation of high M r complexes in PPARb overexpressing cells We next sought to elucidate the biochemical basis of the enhanced degradation of overexpressed PPARb protein. Expression plasmids for normal PPARb (pCMX-PPARb) and FLAG-tagged PPARb (3xFlag- PPARb) were cotransfected into HEK293 cells, and cell extracts were investigated by immunoblot analysis of immunoprecipitated PPARb (Fig. 3A). Three dif- ferent antibodies were used for immunoprecipitation: polyclonal-antibody directed against the subtype- specific N-terminus of PPARb (lane 2), polyclonal antibody against FLAG (lane 3) and monoclonal antibody against FLAG (M2, lane 4). PPARb pro- teins were visualized on immunoblots with either the PPARb-specific antibody (upper panel) or the M2 antibody (lower panel). This experiment clearly showed that FLAG-PPARb was precipitated by the PPARb-specific antibody (lane 2), and vice versa, that PPARb was coprecipitated by both FLAG-directed antibodies (lanes 3 and 4), suggesting the formation of PPARb oligomers. This conclusion was confirmed Fig. 3. Effect of PPARb protein levels and GW501516 on oligo- merization of PPARb. (A) Co-immunoprecipitation of FLAP-PPARb and PPARb. HEK293 cells were cotransfected with pCDNA3.1- zeo-3xFlag-mPPARb and pCMX-mPPARb. Cells were harvested after 24 h and RIPA extracts were immunoprecipitated using anti-mPPARb serum (lane 2), polyclonal (pc) antibody against FLAG (lane 3), monoclonal antibody against FLAG M2 (lane 4) or no antibody (mock, lane 5). One third of the immunoprecipitate was analyzed by immunoblotting using antibodies specific for PPARb (upper panel) and FLAG (lower panel), respectively (*immunoglobulin heavy chain). The indicated molecular masses are based on a calibration curve using molecular mass standards. The 3xFlag-mPPARb protein shows a higher M r as calculated due the highly charged nature of the tag (DYKDDDDK). (B) Effect of PPARb protein levels on oligomerization. Decreasing amounts of pCMX-mPPARb and pCDNA3.1zeo-3xFlag-mPPARb were trans- fected into HEK293 cells as in (A). All samples contained a total amount of 10 lg plasmid DNA. RIPA extracts were immunopre- cipitated and analyzed by immunoblotting using antibodies spe- cific for PPARb as in (A). (C) Reduction of PPARb oligomerization by GW501516. HEK293 cells were transfected as in (A), and subsequently cultured in the presence of different concentrations of GW501516 for 24 h. RIPA extracts were immunoprecipitated with antibody against FLAG M2. One third of the immunoprecipi- tate was analyzed by immunoblotting using PPARb specific antibodies. M. Rieck et al. Regulation of PPARb turnover FEBS Journal 274 (2007) 5068–5076 ª 2007 The Authors Journal compilation ª 2007 FEBS 5071 by superose 6 size exclusion chromatography followed by immunoblot analysis of the collected fractions (Fig. 4A). As expected, RxRa specific antibodies detected proteins that presumably represent mono- meric RxRa (55 kDa) and, to a lesser extent, higher order RxR a complexes. By contrast, PPARb occurred mainly in protein complexes of approximately 2 MDa (fraction 16). The same fraction contained only very low levels of RxRa in comparison to PPAR b, indicat- ing that these complexes are not composed of stoi- chiometric amounts of PPARb and its obligatory RxR heterodimerization partner. Agonist and protein level influence the degree of high M r complex formation To investigate the nature of the high M r PPARb com- plexes, we analyzed the effects of PPARb protein concentration and binding of GW501516. For this pur- pose, we performed the same analyses as above, but after transfection of different amounts of plasmid DNA into HEK293 cells. As can be seen in Fig. 3B, there was a clear reduction on the coprecipitation of PPARb by the FLAG-specific M2 antibody. Quantita- tion of the data showed a coprecipitation of PPARb of 98% relative to FLAG-PPARb after transfection of 2 lg of plasmid DNA, which was reduced to 82%, 52% and 14% when the amounts of transfected plas- mids were decreased to 0.2 lg, 0.05 lg and 0.02 lg, respectively. A clear reduction of coprecipitated PPARb was also seen when the transfected cells were treated with GW501516 (Fig. 3C). Although, in untreated cells (lane 1), coprecipitation of PPARb rela- tive to FLAG-PPARb was 87%, this was decreased to 55%, 37% and 35% in the presence of 0.5 lm,1lm and 2 lm GW501516, respectively. Likewise, the incu- bation with 0.1 lm GW501516 of a PPARb immuno- precipitate from untreated transfected cells resulted in the release of PPARb protein (data not shown). Con- sistent with these results, we observed a strong increase in the relative levels of lower M r complexes (frac- tions 22–30; corresponding to a molecular mass of approximately 800–100 kDa) after transfection of reduced amounts of plasmids or treatment with 1 lm GW501516 (Fig. 4B,C). Taken together, these findings clear suggest that the high M r complexes form selec- tively under conditions of PPARb overexpression. Ligand-inhibitable polyubiquitination of PPARb The results described above suggest that overexpres- sion of PPARb leads to the formation of aberrant complexes that are subject to an enhanced degrada- tion. We therefore investigated whether this would correlate with an enhanced ubiquitination of PPARb. HEK293 cells were transiently transfected with pCMX-PPARb or cotransfected with pCMX-PPARb and an expression vector for histidine-tagged ubiquitin (Ubi-His) [36]. The immunoblot in Fig. 5 clearly shows the presence of high M r PPARb forms in pCMX- PPARb transfected cells (lane 1). These occur at increased levels in the cotransfected cells (lane 3), strongly suggesting that these proteins represent poly- ubiquitinated PPARb. In both cases, ubiquitination was strongly inhibited by GW501516 (lanes 2 and 4). In spite of the readily detectable agonist effect on 10ng plasmid 40ng plasmid no GW501516 no GW50151 6 + GW501516 + GW501516 fraction 14 16 18 20 22 24 26 28 30 32 34 36 38 2 MDa 1MDa 60 kDa fraction A B C 14 16 18 20 22 24 26 28 30 32 34 36 38 2MDa 1 MDa 60 kDa 1 10 100 14 16 18 20 22 24 26 28 30 32 34 Fraction Relative units 10ng DNA, no GW 10ng DNA +GW 40ng DNA, no GW 40ng DNA +GW Fig. 4. Effect of GW501516 and protein levels on the native molec- ular mass of PPARb complexes. (A) High M r complexes in PPARb overexpressing cells. RIPA extract from HEK293 cells transiently transfected with pCMX-mPPARb (as in Fig. 1) was loaded on a su- perose 6 column. Forty-five 500 lL fractions were collected. Frac- tions were analyzed by immunoblotting using PPARb and RxRa specific antibodies. Cells were transfected with 4 lg of pCMXmP- PARb per 10 cm dish. (B) Effect of GW501516 and protein levels. The experiment was performed as in (A), except that cells were transfected with 10 ng and 40 ng of expression plasmid, respec- tively, in the presence or absence of 1 l M GW501516. (C) Quantita- tion by densitometric analysis of the gels shown in (B). Data are expressed as arbitrary units normalized to 1.0 for fraction 16. Regulation of PPARb turnover M. Rieck et al. 5072 FEBS Journal 274 (2007) 5068–5076 ª 2007 The Authors Journal compilation ª 2007 FEBS polyubiquitination, no significant differences in protein levels are visible between untreated and GW501516- treated cells, although the pulse-chase experiments in Fig. 1 showed a clear effect of the agonist on protein stability ⁄ degradation. We attribute this difference to the fact that the experiment in Fig. 5 analyzes steady- state levels, where the high rate of de novo synthesis presumably outweighs protein degradation. Consistent with this interpretation, we did not observe any change in protein levels in the PPARb overexpressing cells after treatment with the proteasome inhibitor MG132 (data not shown), in contrast to 3Fb1 cells expressing moderate levels of PPARb (Fig. 2D). Conclusions Our data show that the PPARb is a relatively stable protein when expressed at moderate levels in fibroblasts and that, under these conditions, its turnover is not sig- nificantly affected by the synthetic agonist GW501516. Transient transfection, on the other hand, leads to a more than 100-fold increased expression concomitant with a clearly accelerated degradation, which in turn can be prevented by GW501516. This influence of pro- tein levels and agonist binding on PPARb stability correlate with the formation of high M r PPARb complexes that consist predominantly of PPARb, and may even represent homooligomers. Such complexes have never been observed, and are unlikely to exist under physiological conditions. The correlation of their formation with high expression levels indeed strongly suggests that they occur specifically under conditions of overexpression. It is likely that overexpressed PPARb forms high M r complexes consisting at least in part of oligomerized PPARb, and that these complexes are polyubiquitinated and rapidly degraded. This possibly serves as a safeguard mechanism protecting the cell from deregulated PPARb expression that could poten- tially occur under certain pathological conditions. Such a safeguard mechanisms may be of particular impor- tance in view of the fact that, unlike steroid hormone receptors, PPARs do not require the interaction with a specific ligand for transcriptional activity [37,38] and figure in cancer-associated biological processes [26–28]. Our observations are also relevant in view of the fact that the modification, regulation and function of PPARs are commonly studied in transiently transfected cells (i.e. under conditions of PPAR overexpression), as is the case, for example, for the ligand-regulated turn- over and ubiquitination of PPARa [35] and PPARc [34]. Agonist-regulated PPARb ubiquitination and turnover has also been described in a recent study [39] published after the submission of this manuscript. However, because most experiments were performed with overexpressed tagged PPARb, the physiological relevance of these findings remains to be seen. In light of our results, it may be important to revaluate any conclusions derived from transient PPAR transfection and overexpression experiments. Experimental procedures Chemicals and antibodies GW501516 was purchased from Axxora (Lo ¨ rrach, Ger- many), MG132 was obtained from Sigma (Taufkirchen, Ger- many) and the protease inhibitor cocktail (PIC) was from Roche (Mannheim, Germany). The following sera were used in this study: polyclonal goat-anti-PPARb (sc-1987; Santa Cruz, Heidelberg, Germany), monoclonal anti-FLAG (M2, Sigma), polyclonal rabbit-anti-FLAG (sc-807; Santa Cruz) and polyclonal rabbit-anti-RxRa (sc-553; Santa Cruz). Ben- zonase was obtained from Merck (Darmstadt, Germany). Cell culture HEK293, NIH3T3 (provided by D. Lowy, NIH, Bethesda, MD, USA) and 3Fb1 cells (see below) were cultured in PPARβ β GW501516 - + - + PPAR β β +Ubi-His 1234 Ubi-PPAR β Fig. 5. Ligand-regulated ubiquitination of overexpressed PPARb HEK293 cells were transfected with pCMX-mPPARb plus either an empty vector (lanes 1 and 2) or an expression vector for histidine- tagged ubiquitin (Ubi-His; lanes 3 and 4). The cells were either trea- ted with GW501516 (lanes 2 and 4) or left untreated (lanes 1 and 3). Cells were harvested after 24 h and analyzed by immunoblotting using PPARb specific antibodies. The picture shows an overexpo- sure to visualize the ubiquitinated high M r PPARb forms. M. Rieck et al. Regulation of PPARb turnover FEBS Journal 274 (2007) 5068–5076 ª 2007 The Authors Journal compilation ª 2007 FEBS 5073 DMEM supplemented with 10% fetal bovine serum, 100 UÆmL )1 penicillin and 100 lgÆmL )1 streptomycin in a humidified incubator at 37 °C and 5% CO 2 . Plasmids pCMX-mPPARaˆ [7] was kindly provided by Dr R. Evans (The Salk Institute, La Jolla, CA, USA). 3xFlag- PPARb (pCDNA 3.1 zeo) was generated by cloning the coding sequence of mPPARb N-terminally fused to a triple FLAG tag (Sigma) into pcDNA3.1(+) zeo (Invitrogen, Karlsruhe, Germany). pCMX-empty has been described previously [40]. The Ubi-His expression vector [36] was a gift from Dr M. Eilers (Marburg, Germany). Transfections Transfections were performed with polyethylenimine (aver- age molecular mass ¼ 25 000 kDa; Sigma-Aldrich, Munich, Germany). Cells were transfected on 60 mm dishes at 70– 80% confluence in DMEM plus 2% fetal bovine serum with 10 lg of plasmid DNA and 10 lL of polyethylenimine (1 : 1000 dilution, adjusted to pH 7.0 and preincubated for 15 min in 200 lL of NaCl ⁄ P i for complex formation). Four hours after transfection, the medium was changed and cells were incubated in normal growth medium for 24–48 h. Retrovirally transduced cells expressing FLAG-PPARb 3xFLAG-PPARb was cloned into the retroviral vector pLPCX (Clontech, Heidelberg, Germany). Phoenix cells expressing ecotropic env were transfected with 3xFLAG- mPPARb-pLPCX (http://www.stanford.edu/g roup/nolan/ retroviral_systems/retsys .html). Culture supernatant was used to infect PPARb null fetal mouse lung fibroblasts that had previously been established from PPARb knockout mice by standard procedures. Cells were selected with puro- mycin (2 lgÆmL )1 ; Sigma), and a clone expressing 3xFLAG-mPPARb (3Fb1 cells) at moderate levels, compa- rable to endogenous PPARb in mouse fibroblasts, was used in the present study. Preparation of denatured whole cell extract Cells (60 mm dishes) were lysed with 400 lL of SDS sample buffer containing 125 U benzonaseÆmL )1 for 5 min at room temperature. The lysed cells were scraped with a rubber policeman and transferred to a 1.5 mL tube. After boiling for 5 min, the lysate was centrifuged for 10 min at 13 000 g with a Pico Biofuge (Heraeus, Osterode, Germany), and the supernatant was used for immunoblot analysis. Preparation of native whole cell extract Cells were lysed on 60 mm dishes with 400 lL of RIPA buffer containing 10 mm Tris (pH 7.5), 150 mm NaCl, 1% NP-40, 0.25% SDS, 1% sodium desoxycholate, 5 mm dithiothreitol, 0.2 mm phenylmethanesulfonyl fluoride, 0.5 · PIC and 125 U benzonaseÆmL )1 . Cells were scraped with a rubber policeman, and the lysate was incubated for 20 min on ice. Samples were centrifuged for 10 min at 13 000 g and 4 °C with a Pico Biofuge. The supernatant was transferred to a fresh 1.5 mL tube; 100 lL were used for size exclusion chromatography (see below) and 150 lL for immunoprecipitation. Size exclusion chromatography One hundred microlitres of native whole cell extract were loaded onto a HR10 ⁄ 30 column containing superose 6 (Amersham-Biosciences, Freiburg, Germany) using an A ¨ kta-purifier (Amersham-Biosciences). The running buffer consisted of 20 mm Tris ⁄ HCl pH 7.9, 5% (v ⁄ v) glycerol, 150 mm NaCl, 3 mm dithiothreitol and 0.2 phenylmethane- sulfonyl fluoride. Five-hundred microliter fractions were collected and 160 lL of each fraction were analyzed by immunoblotting. Immunoprecipitation 150 lL of the native whole cell extract were precleared with 20 lL of a 50% Protein A ⁄ G Plus agarose (Santa Cruz) for 3 h. The lysate was centrifuged for 1 min at 13 000 g and 4 °C with a Pico Biofuge, and the supernatant was subse- quently incubated overnight with 1 lg antibody. After the addition of 50 lL of protein A ⁄ G Plus agarose (preblocked with 50 lgÆmL )1 bovine serum albumin) the incubation was continued for another 4 h. The precipitate was washed three times with RIPA buffer, bound proteins were eluted with 100 lL of SDS sample buffer and analyzed by immu- noblotting as described below. Pulse-chase experiments Pulse chase experiments were carried out according to the Tansey Laboratory Protocol (http://tanseylab.cshl.edu/ protocols.html). After transfection, cells were starved for 45 min in methionine ⁄ cysteine-free DMEM (Invitrogen, Karlsruhe, Germany) containing 1% glutamine and 5% dialyzed fetal bovine serum, and incubated with 430 lCi of Redivue ProMix (14.3 lCiÆlL )1 ; Amersham-Biosciences, Freiburg, Germany). After labeling for 2 h or 30 min, cells were washed and subsequently incubated with standard growth medium (DMEM plus 10% fetal bovine serum). Cells were collected at different time points in ice-cold NaCl ⁄ P i with a rubber policeman and centrifuged at 13 000 g for 1 min with a Pico Biofuge. For storage, the cell pellet was frozen in liquid nitrogen. Prior to immuno- precipitation, the frozen cells were lysed in 400 lL of ice- cold RIPA buffer for 20 min, centrifuged at 13 000 g for Regulation of PPARb turnover M. Rieck et al. 5074 FEBS Journal 274 (2007) 5068–5076 ª 2007 The Authors Journal compilation ª 2007 FEBS 10 min with a Pico Biofuge and transferred to a 1.5 mL tube. Immunoprecipitation was carried out with 150 lLof the lysate, as described above. Kinetics were performed with the same pool of transfected cells to avoid the problem of variable transfection efficiencies. Immunoblot analysis Protein samples were separated by 12.5% SDS ⁄ PAGE, and proteins were transferred by semidry blotting to a poly(vinylidene difluoride) membrane (Millipore, Schwal- bach, Germany), stained with Ponceau S solution, destained and blocked with 5% skimmed milk in NaCl ⁄ P i - Tween. The membrane was incubated with the first anti- body (1 : 2000–1 : 4000) overnight at 4 °C. Membranes were washed three times for 10 min in NaCl ⁄ P i -Tween and then incubated with an peroxidase-coupled second antibody (1 : 4000) for 2 h at room temperature. Membranes were washed and bands were visualized on X-ray film (Fuji, Du ¨ s- seldorf, Germany) using the enhanced chemiluminescent method (Amersham-Biosciences, Freiburg, Germany). Acknowledgements We are grateful to Drs Ronald Evans (Salk Institute, La Jolla, CA, USA) and Martin Eilers (IMT Marburg, Germany) for plasmid vectors, and to Margitta Alt and Bernard Wilke for their excellent technical assis- tance. 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Expression level and agonist-binding affect the turnover, ubiquitination and complex formation of peroxisome proliferator activated receptor b Markus Rieck*, Lena Wedeken*, Sabine Mu ¨ ller-Bru ¨ sselbach,. stoichiometical amounts of the obligatory PPARb dimerization partner, retinoid X recep- tor. The formation of these apparently aberrant complexes, as well as the ubiquitination and destabilization of PPARb, were. the ubiquitination and degradation of PPAR b is not significantly influenced by the synthetic agonist GW501516 under conditions of moder- ate PPARb expression. By contrast, the overexpression of