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 enhanc
Trang 1ubiquitination 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, PPARb⁄ 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 PPARb 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.
Trang 2in 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 [35S]methionine and [35S]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
0 4 8 12 16
A
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
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
x10 3
x10 3
0
60 80 100
40 20 120
untreated GW501516
untreated GW501516
Time (h)
Time (h)
0
60 80 100
40 20
120 140
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 analyzimmunoprecipitat-ed by PAGE followimmunoprecipitat-ed 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.
Trang 3(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 t¼ 0) Thus, the
levels of labeled PPARb protein were approximately
two-fold higher already at the beginning of the chase
period (t¼ 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 PPARb 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
signal intensity
% of t=0
74 114 111 56 61 44 30 18
100 100 112 51 45 36 42 21 15
chase (h)
FLAG-PPARβ
A
B
C
D
FLAG-PPARβ
0
5
10
15
20
25
30
35
Chase (h)
no GW + GW
4 8 12 20
untreated
+ GW
0
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).
Trang 4specific 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 Mrcomplexes 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 Mr 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.
Trang 5by 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 RxRa 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 PPARb,
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 Mrcomplex formation
To investigate the nature of the high Mr 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 coprecipitaQuantita-tion 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, 1 lm 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 Mr 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 Mr 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 Mr 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 GW501516 + GW501516
+ GW501516
fraction 14 16 18 20 22 24 26 28 30 32 34 36 38
fraction
A
B
C
14 16 18 20 22 24 26 28 30 32 34 36 38
1
10
100
Fraction
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.
Trang 6polyubiquitination, 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 Mr 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 Mr 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, 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
PPARββ
PPARβ
Ubi-PPARβ
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 untreatrea-ted (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 MrPPARb forms.
Trang 7DMEM supplemented with 10% fetal bovine serum,
100 UÆmL)1 penicillin and 100 lgÆmL)1 streptomycin in a
humidified incubator at 37C and 5% CO2
Plasmids pCMX-mPPARaˆ [7] was kindly provided by Dr
R Evans (The Salk Institute, La Jolla, CA, USA)
3xFlag-PPARb (pCDNA3.1 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 mass¼ 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⁄ Pifor 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/group/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)1for 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 4C 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
4C 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)1bovine 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⁄ Pi 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
Trang 810 min with a Pico Biofuge and transferred to a 1.5 mL
tube Immunoprecipitation was carried out with 150 lL of
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⁄ Pi
-Tween The membrane was incubated with the first
anti-body (1 : 2000–1 : 4000) overnight at 4C Membranes
were washed three times for 10 min in NaCl⁄ Pi-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 This work was supported by grants from the
Deutsche Forschungsgemeinschaft (SFB-TR17) and
the Deutsche Krebshilfe
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