2005, 25(13):5466. DOI:
Mol. Cell. Biol.
Daniel, Fatiha Moukdar, Xu Bai, Lisa M. Floering and Sheila
Jacques Robidoux, Wenhong Cao, Hui Quan, Kiefer W.
AMP-Dependent UCP1 Expression in
MAP Kinase Is Essential for Cyclic
Protein (MAP) Kinase Kinase 3 and p38
Selective Activation of Mitogen-Activated
Updated information and services can be found at:
This article cites 73 articles, 45 of which can be accessed free
more»articles cite this article),
Receive: RSS Feeds, eTOCs, free email alerts (when new
Information about commercial reprint orders:
To subscribe to to another ASM Journal go to:
on June 13, 2014 by guest
on June 13, 2014 by guest
MOLECULAR AND CELLULAR BIOLOGY, July 2005, p. 5466–5479
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 13
Selective Activation of Mitogen-Activated Protein (MAP) Kinase
Kinase 3 and p38? MAP Kinase Is Essential for Cyclic
AMP-Dependent UCP1 Expression in Adipocytes
Jacques Robidoux,1,3Wenhong Cao,2,3Hui Quan,3Kiefer W. Daniel,1,3Fatiha Moukdar,3
Xu Bai,1Lisa M. Floering,1,3and Sheila Collins1,2,3*
Department of Psychiatry and Behavioral Sciences1and Division of Endocrinology, Department of Medicine,2
Duke University Medical Center, Durham, North Carolina 27710, and Endocrine Biology Program,
Division of Biological Sciences, CIIT Centers for Health Research,
Research Triangle Park, North Carolina 277093
Received 25 November 2004/Returned for modification 21 December 2004/Accepted 1 April 2005
The sympathetic nervous system regulates the activity and expression of uncoupling protein 1 (UCP1)
through the three ?-adrenergic receptor subtypes and their ability to raise intracellular cyclic AMP (cAMP)
levels. Unexpectedly, we recently discovered that the cAMP-dependent regulation of multiple genes in brown
adipocytes, including Ucp1, occurred through the p38 mitogen-activated protein kinases (MAPK) (W. Cao,
K. W. Daniel, J. Robidoux, P. Puigserver, A. V. Medvedev, X. Bai, L. M. Floering, B. M. Spiegelman, and S.
Collins, Mol. Cell. Biol. 24:3057–3067, 2004). However, no well-defined pathway linking cAMP accumulation or
cAMP-dependent protein kinase (PKA) to p38 MAPK has been described. Therefore, in the present study using
both in vivo and in vitro models, we have initiated a retrograde approach to define the required components,
beginning with the p38 MAPK isoforms themselves and the MAP kinase kinase(s) that regulates them. Our
strategy included ectopic expression of wild-type and mutant kinases as well as targeted inhibition of gene
expression using small interfering RNA. The results indicate that the ?-adrenergic receptors and PKA lead to
a highly selective activation of the p38? isoform of MAPK, which in turn promotes Ucp1 gene transcription.
In addition, this specific activation of p38? relies solely on the presence of MAP kinase kinase 3, despite the
expression in brown fat of MKK3, -4, and -6. Finally, of the three scaffold proteins of the JIP family expressed
in brown adipocytes, only JIP2 coimmunoprecipitates p38? MAPK and MKK3. Therefore, in the brown
adipocyte the recently described scaffold protein JIP2 assembles the required factors MKK3 and p38? MAPK
linking PKA to the control of thermogenic gene expression.
Uncoupling protein 1 (UCP1) is essential for rodents and
other small mammals to maintain their body temperatures,
since it is the sole mediator of cold-induced nonshivering ther-
mogenesis (4, 6, 48); UCP1 is also a key contributor to the
regulation of diet-induced thermogenesis (6, 58). The UCP1
protein resides within the inner membrane of mitochondria,
where it serves as a portal for dissipation of the proton gradient
such that respiration is uncoupled from ATP production and
generates heat (35, 49, 54). The UCP1 mRNA and protein are
found in “brown” and to a lesser extent in “white” adipose
tissue; however, its expression is confined to brown adipocytes
(53). Similar brown adipocytes exist scattered within white
adipose depots in adult humans (22, 37), but their contribution
to thermogenesis is admittedly modest. Nevertheless, studies in
animals or humans exposed to high catecholamine levels or
treated with sympathomimetics show that brown adipocytes
expressing UCP1 can be recruited within white adipose depots
(10, 12, 13, 16, 29).
Brown adipose tissue (BAT) and white adipose tissue are
innervated by sympathetic noradrenergic nerves (2, 3, 42, 50,
63). In response to cold exposure or diet, sympathetic nervous
system activation leads to the release of norepinephrine to
interact with adrenergic receptors (AR); in particular the fam-
ily of ?ARs (39, 49, 55, 72). Catecholamine stimulation of the
three ?ARs present in adipocytes promotes a series of events
initiated by the production of cyclic AMP (cAMP) and the
activation of cAMP-dependent protein kinase (PKA) (20, 56,
64). These events result in lipolysis and liberation of free fatty
acids (FFA) from triglyceride stores (39). These FFA serve not
only as substrates for oxidative respiration but also as allosteric
activators of UCP1 function (24, 25, 60). ?AR-mediated in-
creases in cAMP also stimulate Ucp1 gene transcription. The
cAMP response of the Ucp1 gene is achieved predominantly
through an enhancer region (9, 15, 38). This enhancer, which is
well conserved among species (11), confers specificity of ex-
pression to brown adipocytes as well as the cAMP response
and contains at least two key elements: a peroxisome prolif-
erator response element (PPRE) and a cAMP response ele-
We have recently shown that the cAMP-dependent tran-
scription of the Ucp1 gene is regulated through these two el-
ements by p38 mitogen-activated protein kinase (MAPK) (7).
The effect of p38 MAPK on these elements occurs in a coor-
dinated fashion. First, p38 MAPK phosphorylates a protein
called PGC-1? (7), which is a transcriptional coactivator and
mediator of mitochondriogenesis (68), among other functions.
This modification of PGC-1? enhances its activity as a nuclear
coactivator of gene transcription in coordination with peroxi-
* Corresponding author. Mailing address: CIIT Centers for Health
Research, 6 Davis Drive, Box 12137, Research Triangle Park, NC 27709.
Phone: (919) 558-1378. Fax: (919) 558-1305. E-mail: firstname.lastname@example.org.
on June 13, 2014 by guest
some proliferator-activated receptor ? (PPAR?); PPAR? in
turn binds to the UCP1 PPRE (7). Second, p38 directly stim-
ulates expression of the Ucp1 gene through phosphorylation of
the transcription factor ATF-2; ATF-2 binds to the CRE2 (7).
Finally, the PGC-1? gene itself also possesses a CRE (28) but
in the brown adipocyte is a target of p38-activated ATF-2 and
not CREB (7). By increasing the overall amount of PGC-1?
protein over time, p38 MAPK primes the cell for a sustained
enhancement of UCP1 expression. Despite this new under-
standing of the role of p38 MAPK in the regulation of the Ucp1
and PGC-1? genes in brown fat, the cascade of signaling events
downstream of PKA by which p38 MAPK becomes activated is
To begin to unravel this new pathway, we realized that it was
necessary to tackle this problem in a “bottom-up” approach.
Therefore, we reasoned that a strategy that would best serve
this effort should first identify the actual p38 MAPK isoform(s)
involved and proceed in a retrograde manner. The p38 MAPK
group is composed of four isoforms: p38? (26, 41), p38? (32),
p38? (43), and p38? (66). Among them, p38? and -? are
sensitive to the pyrimidyl imidazoles SB202190 and SB205380
(14, 23). These two isoforms are expressed in adipocytes (36).
Depending on cell type and stimulus, p38 MAPK can be acti-
vated by MKK3 (17) or MKK6 (27, 46, 52, 61) or by both of
them. In some cell types MKK4 can activate p38 MAPK (17,
44). However, depending upon the stimulus or physiological
state, there are circumstances in which these MKKs can clearly
display substrate preferences or noninterchangeable roles (62,
69). For example, MKK3 tends to prefer p38? while MKK6 is
equally efficient at both p38? and p38? (18). We also em-
barked on the current series of studies because defining the
exact p38 isoform(s) and its immediate activator(s) in the con-
trol of UCP1 transcription may provide clear targets to mod-
ulate thermogenesis. Using a variety of experimental ap-
proaches, we show that p38? and its activator MKK3 are the
sole players in the control of Ucp1 gene transcription.
MATERIALS AND METHODS
Chemicals and plasmids. The plasmid BBCAT, containing the 3.74-kb mouse
UCP1 promoter in plasmid pBLCAT6, was a gift from Leslie P. Kozak (38). The
EN-tk-CAT vector containing the UCP1 enhancer (?2530 to ?2310), the mouse
?3AR expression plasmid, and the ?-actin-luciferase (?-actin-luc) control vector
for transfections were all previously described (8, 45). The expression vectors
pCDNA3-p38? and pCDNA3-p38? MAPK as well as their dominant-negative
forms pcDNA3-p38?AF and pCDNA3-p38?AF, all FLAG-tagged, were gifts
from Jiahuai Han (26, 32). The mouse pCMV-SPORT6-p38? MAPK vector was
purchased from Open Biosystems (Huntsville, AL). A collection of FLAG-
tagged expression vectors created in the plasmid pcDNA3 for the following genes
were all gifts from Roger J. Davis, University of Massachusetts Medical School:
MKK3, MKK4, MKK6, and its constitutively active form, MKK6E (17, 52). The
expression vector pCDNA3-MKK7 was a gift from Josef M. Penninger, Univer-
sity of Toronto (70). The S-protein-tagged JLP was a gift from E. Premkumar
Reddy, Temple University (5, 40). The full-length clones for JIP1, JIP2, and JIP3
were purchased from Open Biosystems (Huntsville, AL) and were subcloned in
pcDNA4/HisMax TOPO TA Expression vector from Invitrogen (Carlsbad,
Calif.). The ?3AR-selective agonist CL316,243 (CL) was a gift from Elliott Dan-
forth, Jr. (American Cyanamid Co., Pearl River, NY). The anti-FLAG M2-
agarose antibody, dobutamine, forskolin, H89, IGEPAL, isoproterenol, norepi-
nephrine, and salbutamol were from Sigma (St. Louis, MO). Rp-cAMPS was
from Biomol Research Laboratory (Plymouth Meeting, PA). SB202190 and
SB203580 were from Calbiochem (La Jolla, CA). The nonradioactive PKA
activity assay was from Promega (Madison, WI). The Rap1 activation assay was
from Upstate (Charlottesville, VA). Specific antibodies to the following epitopes
or individual proteins were from Cell Signaling Technologies Inc. (Beverly, MA):
6?-His, p38 MAPK, p38? MAPK, phospho-p38 MAPK, MKK3, phospho-
MKK3/6, MKK4, phospho-MKK4, MKK7, phospho-MKK7, and glutathione
S-transferase (GST)-ATF2. Antibodies specific for the p38? and p38? MAPK
isoforms and MKK6 were from Chemicon (Temecula, CA). For immunoprecipi-
tation experiments, MKK3, MKK6, and the S-tag antibodies were obtained from
Santa Cruz (Santa Cruz, CA). The S-protein agarose resin was from Santa Cruz.
An additional antibody to phospho-p38 MAPK was from Zymed (South San
Francisco, CA). The small interfering RNAs (siRNAs) presented in Table 1 and
related reagents such as siPORT-Lipid, RNAlater, RNAaquous4PCR, and
RNAaquous MIDI RNA extraction kit were from Ambion (Austin, TX). The
High Capacity cDNA Archive kit, reverse transcription-PCR (RT-PCR) primers,
FAM- and VIC-labeled probes, and the TaqMan enzyme were from Applied
Biosystems (Foster City, CA). Lipofectamine and precast Tris-glycine polyacryl-
amide gels were obtained from Invitrogen (Carlsbad, CA). The alkaline phos-
phatase-conjugated secondary antibodies, the alkaline phosphatase detection
reagents, and glutathione Sepharose 4B were from Amersham Biosciences (Pis-
cataway, NJ). Chloramphenicol acetyltransferase (CAT) assays, Complete Pro-
tease Inhibitor Cocktail (CPIC) tablets, and protein G-agarose were from Roche
Molecular Biochemicals (Indianapolis, IN). The ProQ Diamond Phosphoprotein
Gel Stain and destaining solution were from Molecular Probes (Eugene, OR).
Rosiglitazone was a gift from GlaxoWellcome Inc. (Research Triangle Park,
NC). All other reagents were from the best available sources.
Cell culture and transfection. The HIB-1B brown preadipocytes (57) were
maintained in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal
bovine serum. Cells in 6-well plates were transfected with a total amount of
plasmid DNA up to 2.2 ?g/well and 5 to 10 ?l Lipofectamine. As needed, these
DNA mixtures included pGL2-?3AR (0.5 ?g), pCDNA3-kinases (0.2 to 1 ?g),
UCP1 enhancer-TK-CAT (1.0 ?g), and ?-actin-luc (0.2 ?g) or cytomegalovirus–
?-galactosidase (CMV–?-GAL) (0.125 ?g). Transfection with siRNAs (20 to 60
nM) were performed using siPORT lipid (4 to 8 ?l). In cotransfection experi-
ments involving siRNAs, the siRNA was added to the well at the time of the
seeding the cells, and the plasmid transfection was performed 12 h later. In all
cases, the PPAR? agonist rosiglitazone (1 ?M) was added at the same time as the
serum following the serum-free period of the transfection protocols. Where
indicated, HIB-1B cells were treated for 1 h with 10 ?M H89, 0.5 mM Rp-
cAMPS, or 5 ?M SB prior to treatment with CL (10 ?M), Forsk (10 ?M),
dobutamine (10 ?M), salbutamol (10 ?M), isoproterenol (10 ?M), or norepi-
nephrine (1 ?M), for which the time of incubation varied in accord to the assay
needs. Also, for cells treated with norepinephrine, there was a preincubation
phase for 30 min with yohimbine (10 ?M) and prazosin (1 ?M) in order to inhibit
any activation of ?-adrenergic receptors.
Nonradioactive PKA assay. HIB-1B cells were preincubated for 50 min with
H89 (10 ?M), Rp-cAMPS (0.1 mM), or SB (5 ?M), followed by 10 min with 0.2
mM isobutylmethylxanthine in order to inhibit phosphodiesterase activity. They
TABLE 1. Sequences for the siRNAs used in these studiesa
Targeted geneSense sequence 5?–3?
Antisense sequence 5?–3?
aNote that for some of the targeted genes, two (p38? MAPK) or three (MKK3) siRNA duplexes were used because they achieved ?80% specific gene knockdown.
VOL. 25, 2005PKA ACTIVATION OF MKK3 AND p38? MAPK 5467
on June 13, 2014 by guest
were then incubated for 5 min with either CL (10 ?M) or Forsk (10 ?M). Cells
were then washed once with phosphate-buffered saline (PBS) containing 5 mM
?-glycerophosphate and 1 mM sodium orthovanadate, followed by a lysis buffer
(25 mM Tris-HEPES, 150 mM NaCl, 10 mM ?-mercaptoethanol, 5 mM ?-glyc-
erophosphate, 1 mM sodium orthovanadate, 0.5 mM EDTA, 0.5 mM EGTA, 1%
triton X-100, 0.1% IGEPAL, and 1 CPIC tablet per 10 ml) for 15 min. Five
microliters of this cell lysate was incubated for 30 min at 30°C with 5 ?l of the 5?
PepTag PKA reaction buffer, 2 ?g of PepTag A1 peptide (fluorescent kemptide),
and 1 ml of the peptide protection solution (all parts of the PKA assay kit were
from Promega) in a 25-?l total volume. For the positive control, the sample has
been replaced by 10 ng of PKA catalytic subunit, and for the negative only lysing
buffer is added to the reaction mixture. The reaction was stopped by boiling the
sample for 10 min, a final concentration of 3.2% glycerol was added, and 3.5 ?l
sample was loaded and resolved on a 0.8% agarose gel. Image acquisition was
performed on a typhoon 9410 variable modes imager and analyzed using Image-
Quant TL v2003.03 software.
Rap1 activation assay using RalGDS-RBD. Rap1 pull-down assays were per-
formed essentially as described previously (19) using the reagents from Upstate
(Charlottesville, VA). HIB-1B cells were seeded in 10-cm-diameter dishes. Cells
were washed twice in cold PBS and lysed on ice in 1 ml of lysis buffer (50 mM
Tris-HCl [pH 7.5], 500 mM NaCl, 2.5 mM MgCl2, 1% NP-40, 10% glycerol, and
1 Complete Mini Antiprotease tablet per 10 ml of lysis buffer). Cell debris was
removed by centrifugation at 13,200 ? g for 10 min at 4°C. Fifty microliters of the
GST-RalGDS-RBD-agarose slurry was added to each supernatant, and the mix-
ture was incubated at 4°C for 45 min on a rotating wheel. Beads were washed
three times with lysis buffer. Samples were denatured for 3 min at 95°C and
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE). Western blotting was performed using the anti-Rap1 antibody included
in the kit and an alkaline phosphatase-conjugated anti-rabbit secondary antibody
and ECF detection kit from Amersham Biosciences. Image acquisition was
performed on a typhoon 9410 variable modes imager and analyzed using image-
Quant TL v2003.03 software, both from GE healthcare (Piscataway, NJ).
Protein kinase assay for p38 MAPK activity. HIB-1B cells were transfected or
not with FLAG-tagged p38 MAPK, and total cell p38 MAPK activity was as-
sessed from cellular lysate while isoform-specific activity was assessed following
immunoprecipitation using M2 anti-FLAG agarose antibody. The cells were
washed twice with PBS containing 5 mM ?-glycerophosphate and 1 mM sodium
orthovanadate and then lysed for 30 min in a 25 mM Tris-HEPES buffer con-
taining 150 mM NaCl, 5 mM ?-glycerophosphate, 1 mM sodium orthovanadate,
5 mM EDTA, 5 mM EGTA, 1% Triton X-100, 0.1% IGEPAL, and 1 CPIC tablet
per 10 ml. Kinase assays in vitro were performed either on whole-cell lysates or
after immunoprecipitation. In the latter case, the lysate was incubated overnight
with 40 ?l of M2 anti-FLAG agarose antibody. Cell lysate or immune complexes
were incubated at 30°C for 45 min with 2 ?g GST-ATF-2(1–109), 250 ?M ATP
(containing or not 10 ?Ci [?-32P]ATP) in 40 ?l of kinase reaction buffer (43). For
the radioactive version of the protocol, an equal amount of 2? Laemmli sample
buffer was added to terminate the reactions. In the nonradioactive version, 40 ?l
of glutathione Sepharose 4B was used to pull down the substrate. Proteins were
resolved with 4 to 20% acrylamide gradient Tris-glycine gels. Protein phosphor-
ylation was visualized either by autoradiography for the radioisotopic protocol or
with the Pro-Q Diamond phosphor-protein stain for the nonradioactive method.
In both cases image acquisition was performed on a Typhoon 9410 variable-
mode imager and analyzed using ImageQuant TL v2003.03 software.
Western blot for MAPK phosphorylation. MAPK phosphorylation was eval-
uated by Western blot using specific anti-phospho-MAPK and total MAPK
antibodies (1:1,000 dilution) and secondary antibodies (1:10,000 for the Amer-
sham antibody and 1:25,000 for the Sigma antibodies). The alkaline phosphatase
activity was determined using the ECF detection kit from Amersham Bio-
sciences. Image acquisition was performed on a Typhoon 9410 variable modes
imager and analyzed using ImageQuant TL v2003.03 software.
CAT and luciferase assays. Cells were harvested to assay UCP1 enhancer
activities 48 h after transfection. CL316,243 or Forsk was added for the last 6 to
8 h of the transfection to stimulate cAMP production, after which cell extracts
were prepared in lysis buffer from a CAT enzyme-linked immunosorbent assay
(ELISA) kit (Roche Molecular Biochemicals). CAT and luciferase assays were
performed as previously described (8).
RNA isolation, reverse transcription-PCR, and real-time PCR. Total mRNA
was extracted from cultured cells using RNAaquous4PCR and from tissue using
the RNAaquous MIDI RNA purification kits. For tissues, the samples were
submerged in “RNAlater” prior to the extraction. These RNA reagents were
from Ambion. cDNA was generated using the High Capacity cDNA Archive kit
from Applied Biosystems exactly as described in the kit (although scaled down to
a 50-?l total volume). Real-time PCR was performed using TaqMan probes from
Applied Biosystems (Foster City, CA) on an ABI PRISM 7700 Sequence De-
tector from Perkin Elmer (Boston, MA) exactly as indicated by Applied Biosys-
tems. For the quantification of the p38? and p38? MAPK mRNA, standard
curves (0 to 4,000 amol) were generated using plasmids containing the cDNAs of
the mouse genes. GAPDH was used as internal standard.
Immunoprecipitation. For immunoprecipitation experiments, the tissue or the
cells were lysed with the same buffer as for the kinase assays. The lysate (1 to 2
mg of total protein) was precleared by a preincubation of 2 h with 40 ?l protein
G-agarose. The cleared lysate was then incubated for 3 h with the antibody
(kinase assay) or overnight (coimmunoprecipitation) with antibodies. The G-
protein agarose was added, and the mixture was incubated for an additional 3 h
and washed once with the lysing buffer and five times with the washing buffer
composed of 50 mM Tris-HCl, 150 mM NaCl, and antiproteases. The proteins
are eluted from the resin in an ultrafree-mc 5-?m centrifugal filtration device by
exposing the resin to sample buffer without reducing agent at room temperature
for 10 min.
In previous studies we demonstrated that p38 MAPK activity
is involved in the ?AR- and cAMP-dependent induction of the
Ucp1 gene in brown adipocytes (7, 8). However, neither the
identity of the p38 MAPK nor the molecular intermediaries
linking PKA to the activation of p38 MAPK are known. There-
fore, we used a series of experiments designed to define the
p38 MAPK isoforms and immediate upstream activators in-
volved. In our earlier studies, we presumed that the elevated
cAMP levels generated in response to ?-agonist stimulation
are activating PKA, concluding that this kinase is solely re-
sponsible for conveying the cAMP signal that leads to p38
MAPK activation and Ucp1 gene expression. This conclusion
was based on the ability of two mechanistically different “in-
hibitors” of cAMP, the competitive antagonist Rp-cAMPS and
the catalytic inhibitor H89, to suppress both p38 MAPK acti-
vation and transcription of the Ucp1 gene. This “signature”
typically indicates involvement of PKA. However, in a variety
of cell types, cAMP has been shown to activate the small
G-protein Rap1 through its interaction with a family of gua-
nine nucleotide exchange factors (GEFs) that include Epac
(exchange protein directly activated by cAMP), cAMP–GEF-I,
and cAMP–GEF-II (1, 33, 65). The activities of these mole-
cules are blocked by Rp-cAMPS but are unaffected by H89.
Importantly, Rap1 has been shown to be an activator of p38
MAPK (30, 59). Therefore, as we began this series of studies it
was necessary to unequivocally determine whether PKA or a
GEF (or some combination of both) leads to stimulation of
p38 MAPK and Ucp1 gene expression. We treated HIB-1B
brown adipocytes with the ?3AR agonist, CL316,243 (CL), or
the adenylyl cyclase stimulator, forskolin (Forsk), in the pres-
ence of H89 or Rp-cAMPS or the p38 MAPK inhibitor
SB202190 (SB). As shown in Fig. 1A, PKA was activated by
either CL or Forsk. The response to both activators was blocked
by H89 or Rp-cAMPS but not by SB. Therefore, these results
indicate activation of PKA and additionally show that inhibi-
tion of p38 MAPK does not affect PKA. As shown in Fig. 1B,
neither CL nor Forsk was able to elicit GTP loading to Rap1.
Together these results strongly support the conclusion that p38
MAPK activation by cAMP does not depend upon a cAMP-
GEF and Rap1 activation but, rather, solely requires PKA.
To confirm the role of both PKA and p38 MAPK in UCP1
induction, HIB-1B cells were pretreated with H89, Rp-cAMPS,
or SB, followed by stimulation with either CL or Forsk. Acti-
vation of p38 MAPK was measured using glutathione-S-trans-
5468ROBIDOUX ET AL.MOL. CELL. BIOL.
on June 13, 2014 by guest
ferase (GST)-tagged ATF-2 as a substrate. As shown in Fig.
1C, p38 MAPK enzyme activity stimulated by CL or Forsk was
abrogated by H89, Rp-cAMPS, and SB. These inhibitors sim-
ilarly blocked the transactivation of the UPC1 enhancer in re-
sponse to CL and Forsk (Fig. 1D). These results indicate that,
irrespective of the stimulus, PKA is necessary for p38 activa-
tion and that, in turn, induction of UCP1 enhancer activity
requires p38 MAPK activity.
FIG. 1. ?-Adrenergic receptor (?AR) agonists or Forskolin (Forsk) promotes uncoupling protein 1 (UCP1) enhancer activity through protein
kinase A (PKA) and p38 MAPK. HIB-1B cells were transfected (A to D) or not (E and F) with the mouse ?3AR. (A to D) Cells were preincubated
1 h with the solvent only (Cont), 10 ?M H89, 0.5 mM Rp-cAMPS, or 5 ?M SB202190, and then the cells were incubated with the solvent only (Basal),
10 ?M CL316,243 (CL), or 10 ?M Forskolin (Forsk). (A) After 5 min, the cells were lysed and PKA activity was measured in the whole-cell lysate using
kemptide as substrate. (B) Also after 5 min, Rap1 activation was evaluated using a Ral-GDS pull-down assay. (C) After 20 min, p38 MAPK activity was
measured using GST-ATF2 as substrate. (D) After 6 h, UCP1 enhancer activity was evaluated using a UCP1 enhancer-chloramphenicol acetyltransferase
(CAT) assay: Basal (white bars), CL (oblique bars), and Forsk (black bars). (E and F) Cells were preincubated 1 h with the solvent only (Cont), 10 ?M
H89, or 5 ?M SB202190, and then the cells were preincubated an additional 15 min with 10 ?M yohimbine and 1 ?M prazosin (for the norepinephrine
treated cells only) or the solvent, and then the cells were incubated with the solvent only (Basal), 10 ?M dobutamine (Dobu), 10 ?M salbutamol (Salbu),
10 ?M isoproterenol (Iso), or 1 ?M norepinephrine (NE). (E) After 20 min, p38 MAPK activity was measured using GST-ATF2 as substrate. (F)
After 6 h, UCP1 enhancer activity was evaluated by CAT assays: Basal (white bars), Dobu (horizontal bars), Salbu (oblique bars), Iso (black bars),
and NE (reverse oblique bars). The blots shown are the results of one of two (E), three (A and B), or five (C) experiments. The results presented
in graphs are the means ? standard deviations of two (F) or three (D) independent experiments, each performed in duplicate.
VOL. 25, 2005PKA ACTIVATION OF MKK3 AND p38? MAPK5469
on June 13, 2014 by guest
Since all three ?ARs are expressed in brown adipocytes and
can stimulate cAMP production (56), we proposed that all of
them can activate p38 MAPK and UCP1 transcription. To test
this hypothesis, we treated HIB-1B cells with specific agonists
of these receptors. As shown in Fig. 1E, the ?1AR-selective
agonist, dobutamine, and the ?2AR-selective agonist, salbuta-
mol, both activated p38 MAPK in a PKA-dependent manner.
The nonselective ?AR activator isoproterenol and the natural
adrenergic agonist norepinephrine also activated p38 MAPK
in a PKA-dependent fashion (Fig. 1E). Furthermore, these
four ?AR agonists also induced UCP1 enhancer activation,
which was blocked by p38 MAPK inhibition (Fig. 1F). These
results show that all three ?AR subtypes can stimulate p38
MAPK activity and subsequently Ucp1 gene transcription.
To determine whether ?3AR stimulation leads to p38 MAPK
activation and to a p38 MAPK-dependent Ucp1 gene expres-
sion in brown fat in vivo, SB (12.5 mg/kg of body weight) and
CL (1 mg/kg) were administered to mice. Phosphorylation and
activation of p38 MAPK and JNK was assessed by Western
blotting and kinase assays and Ucp1 gene expression by real-
time PCR. As shown in Fig. 2A, CL treatment induced phos-
phorylation of p38 MAPK by 2.5- ? 0.2-fold and p38 MAPK
enzyme activity by 2.4- ? 0.1-fold. In contrast, following CL
treatment, phosphorylation of JNK could not be detected (Fig.
2B). The ability of antibody to recognize phospho-JNK was
confirmed by treating HIB-1B cells with 5 ?g/ml anisomycin
for 15 min (Fig. 2B). Under these same treatment conditions,
CL injection stimulated Ucp1 gene expression, and this stim-
ulation was largely prevented (70%) by prior p38 MAPK in-
hibition (Fig. 2C). Consistent with what we have previously
reported (7), these results clearly show that selective ?3AR
agonist stimulation in vivo triggers p38 MAPK activity to reg-
ulation of Ucp1 gene transcription.
To identify the p38 MAPK isoforms(s) responsible for
UCP1 enhancer activation, we first assessed which SB-sensitive
isoforms were expressed in BAT and in the brown adipocyte
cell line used to dissect the molecular pathway between PKA
and Ucp1 gene expression. As shown in Fig. 3A, both p38? and
-? mRNAs were expressed in BAT as well as in HIB-1B cells.
Consistent with this finding, both proteins were detected by
Western blot (Fig. 3B). We next overexpressed the p38? or -?
isoforms in HIB-1B cells and measured UCP1 promoter activ-
ity. As shown in Fig. 3C, both isoforms could stimulate UCP1
enhancer activation equally (with a slight preference for the ?
isoform). Next, we coexpressed MKK6E (a constitutively ac-
tive form of this kinase that can phosphorylate and activate p38
MAPK) with either p38? or -? in HIB-1B cells, followed by
measurements of UCP1 enhancer activity. As shown in Fig.
3D, MKK6E could activate either of the p38 isoforms as mea-
sured by significant amplification of UCP1 enhancer activity,
but there was a greater preference for p38? MAPK. Together,
these results indicate that both p38? and -? isoforms are ca-
pable of stimulating UCP1 transcription and that under con-
ditions of maximal stimulation p38? MAPK might couple
more efficiently to UCP1 induction. However, these data do
not indicate whether either or both isoforms play a role under
adrenergic stimulation. To address this issue, we introduced
FLAG-tagged p38? or -? MAPK into HIB-1B cells and sub-
sequently treated the cells with CL or Forsk. As clearly shown
in Fig. 4A and B, p38? MAPK but not p38? was activated by
CL or Forsk. We also performed immunoprecipitation exper-
iments of the endogenous p38 MAPK isoforms and confirmed
that Forsk-induced p38 MAPK activity could be recovered
only from the p38? MAPK immunoprecipitate (Fig. 4C). In-
terestingly, using our brown adipocyte model, transactivation
of the UCP1 enhancer by CL or Forsk was potentiated only by
p38? but not by the p38? isoform (Fig. 4D). In order to
validate this selectivity in vivo, mice were either injected with 1
mg/kg CL or exposed to a 4°C environment. As shown in Fig.
4E and F, both manipulations led to the sole activation of the
FIG. 2. ?3AR agonist CL316,243 promotes uncoupling protein 1
(Ucp1) gene expression in vivo through p38 MAPK. Mice were pre-
treated with two injections of saline (Control) or 12.5 mg/kg SB203580.
The first injection was 25 h and the second 1 h prior to the saline
(Basal) or CL injection (1 mg/kg). (A) After 30 min, the brown adipose
p38 MAPK activation was evaluated by Western blotting using an
anti-phospho-p38 MAPK antibody (top blot) or using GST-ATF2 as a
substrate (middle blot). (B) After 30 min, JNK activation was evalu-
ated by Western blot. (C) After 6 h, Ucp1 gene expression was evalu-
ated by real-time PCR (triplicate samples per group) using Taqman
probe: Basal (white bars) and CL (gray bars). The blots shown are the
results of one of two experiments using two mice per each treatment
group. For panels A and B, HIB-1B cells treated with anisomycin (5
?g/ml) were used as a positive control. The results presented in graphs
are the means ? standard deviations of two independent experiments
using two mice per treatment group.
5470 ROBIDOUX ET AL.MOL. CELL. BIOL.
on June 13, 2014 by guest
p38? MAPK isoform in interscapular BAT. Altogether, these
data establish that p38? MAPK but not p38? is activated dur-
ing sympathetic nervous system stimulation of the thermogenic
program and following exposure of BAT and brown adipocytes
to sympathomimetic drugs.
Establishing that the ? isoform of p38 MAPK is the one that
is activated, we used siRNA gene silencing to demonstrate that
p38? MAPK and not p38? MAPK was responsible for the in-
duction of UCP1 expression. In these studies it was first
necessary to demonstrate the efficacy of the siRNAs direct-
ed against either the p38? or -? isoforms. This was examined
in HIB-1B cells. As shown in Fig. 5A, the siRNA against either
form of p38 MAPK reduced the targeted protein level by more
than 80% without affecting the other isoform. More impor-
tantly, as shown in Fig. 5B, using this approach we found that
essentially all CL- and Forsk-promoted p38 activity can be
attributed to the p38? MAPK isoform. Figure 5C further
shows that the siRNA against p38? MAPK completely inhib-
ited UCP1 enhancer activation, while the siRNA against p38?
failed to do so. Similar results were obtained in experiments
FIG. 3. p38? or p38? MAPK can promote UCP1 enhancer activity. The expression of p38? and p38? MAPKs was measured in BAT and
HIB-1B cells by quantitative real-time PCR (A) and Western blotting with selective antibodies for each isoform (B). (C) HIB-1B cells were
transfected with increasing concentration of FLAG-tagged p38? MAPK. Two days later, the kinases were immunoprecipitated and their expression
was evaluated by Western blot using anti-p38 MAPK antibody (second and fourth blots); we measured their activity using GST-ATF2 as a substrate
(first and third blots), and we evaluated UCP1 enhancer activity using a CAT assay (graph). (D) HIB-1B cells were transfected with FLAG-tagged
p38 MAPK and/or FLAG-tagged MKK6E. Two days later, theirs kinases were immunoprecipitated and their expression was measured by Western
blot using anti-p38 MAPK and anti-MKK6 antibodies (second, third, and fourth blots), p38 MAPK activity was measured using GST-ATF2 as
substrate (first blot), and UCP1 enhancer activity was evaluated by CAT assay (graph). The results shown are means ? standard deviations of three
independent experiments, each performed in triplicate, while the blots are from one of three experiments.
VOL. 25, 2005PKA ACTIVATION OF MKK3 AND p38? MAPK5471
on June 13, 2014 by guest
FIG. 4. p38? MAPK is selectively activated following ?-adrenergic or forskolin stimulation. (A and B) HIB-1B cells were transfected with the
?3AR and with FLAG-tagged p38? MAPK (A) or FLAG-tagged p38? MAPK (B). (A and B) Cells were treated as detailed in Fig. 1A. Cells were
lysed, the kinase was immunoprecipitated (Western blot, bottom blots in A and B), and activity was measured using GST-ATF2 as a substrate (top
blots in A and B). The results shown are from one of three independent experiments. (C) HIB-1B cells were treated as shown, and p38? and p38?
MAPKs were immunoprecipitated and kinase activity measured using GST-ATF2 as a substrate. (D) HIB-1B cells were transfected with the ?3AR
and with FLAG-tagged p38? MAPK or FLAG-tagged p38? MAPK. Cells were treated as follows: Basal (white bars), CL (gray bars), and Forsk
(black bar). For measurement of UCP1 enhancer activity cells were harvested after 6 h. The results shown are means ? standard deviations of three
independent experiments, each performed in duplicate. For measurement of kinase activity, 20 min posttreatment the kinases were immunopre-
cipitated and activity was measured using GST-ATF2 as a substrate (upper blot). Relative amounts of the kinases in the assay are shown by
Western blot using anti-FLAG antibody (lower blot). (E and F) Mice were either treated with CL (1 mg/kg intraperitoneally) for 30 min (E) or
placed at 4°C for 1 h (F), and BAT was excised and processed for immunoprecipitation of p38? MAPK or p38? MAPK. Kinase activity and the
protein levels of the p38 MAPK isoforms was measured as above.
5472 ROBIDOUX ET AL.MOL. CELL. BIOL.
on June 13, 2014 by guest
employing dominant-negative constructs of p38? and p38?
MAPK (not shown). Finally, it was rather remarkable to find
that even the 60- to 80-fold induction of the endogenous Ucp1
gene in HIB-1B cells was totally eliminated by the p38? MAPK
siRNA (Fig. 5C). Altogether these data leave little doubt about
the highly specific activation of p38? MAPK by ?AR agonists
and PKA and its essential role in the activation of the Ucp1
The next objective was to address the origin of p38? MAPK
selectivity. For this purpose we explored which of the MKKs
were activated by CL and Forsk. In Fig. 6A (middle panel),
MKK3 and/or MKK6 was phosphorylated in a PKA-dependent
manner following CL or Forsk. However, neither CL nor Forsk
was able to promote the phosphorylation of MKK4 or MKK7
(Fig. 6B). These results are consistent with the fact that the
JNK pathway is not activated in brown adipose tissue in vivo
(Fig. 3B) or in brown adipocytes in vitro (Fig. 6B). However,
the nonisoform selective nature of the MKK3/6 phospho-anti-
body required additional experimentation in order to address
whether one (or both) of these two MKK isoforms was acti-
vated. We performed selective immunoprecipitation of MKK3
and MKK6 under basal and cAMP-stimulated conditions. As
shown in Fig. 6C, Forsk-induced phospho-MKK3/6 immuno-
reactivity was detected only upon immunoprecipitation of
MKK3. Confirmation of this exclusive activation of MKK3
versus MKK6 in vivo was obtained from BAT samples of mice
treated with CL or placed in a 4°C environment. Collectively,
these findings demonstrate a selective activation at the level of
the direct upstream kinase within the p38 MAPK module,
which might be an underlying mechanism of the above-de-
scribed specific p38? MAPK activation.
MKK6 is a universal p38 MAPK activator, but the ability of
MKK3 to activate p38? MAPK is modest at best (18). It was
tempting to speculate that the specific involvement of p38?
MAPK would be recapitulated at the level of MKK3. We
therefore tested the functional impact of overexpressing
MKK3, MKK4, MKK6, and MKK7 individually on UCP1 en-
hancer activity and p38 MAPK activity. The results clearly
show that MKK3, and to a much lower extent MKK6, can
potentiate the effects of CL and Forsk on both UCP1 expres-
FIG. 5. p38? MAPK is essential for ?3AR agonist or Forsk induction of Ucp1 gene expression. (A and B) HIB-1B cells were transfected with
siRNA targeting p38? and p38? MAPK, and 48 h later kinases were immunoprecipitated with specific anti-p38? MAPK (top blot) or p38? MAPK
(bottom blot). (A) Presence of the kinases was measured by Western blot using isoform nonselective p38 MAPK antibody (both blots). The results
shown are from one of two independent experiments. (B) Kinase activity was measured using GST-ATF2 as a substrate. (C) HIB-1B cells were
transfected with siRNA targeting p38? and p38? MAPK and 12 h later with the ?3AR, UCP1 enhancer, and ?-actin-luciferase plasmids.
Forty-eight hours later, the cells were incubated with the solvent (Basal), and 10 ?M CL or 10 ?M Forsk was used for 6 h for the measurement
UCP1 enhancer activity a CAT reporter system: Basal (white bars), CL (gray bars), Forsk (black bars). The results shown are means ? standard
deviations of two independent experiments, each performed in triplicate. (D) HIB-1B cells were transfected with siRNA targeting p38? and p38?
MAPK and expression vector for ?3AR. The cells were incubated with the solvent (Basal), 10 ?M CL, or 10 ?M Forsk for 6 h, the RNA was
extracted, and the measurement of UCP1 was evaluated by real-time PCR using a TaqMan probe: Basal (white bars), CL (gray bars), and Fork
(black bars). The results shown are means ? standard deviations of two determinations, each performed in triplicate.
VOL. 25, 2005PKA ACTIVATION OF MKK3 AND p38? MAPK5473
on June 13, 2014 by guest
sion (Fig. 7A) and p38 MAPK activity (Fig. 7B). The reciprocal
experiments, using siRNAs that specifically downregulated
MKK3 and MKK6 by more than 80% (Fig. 7C), show that only
the selective siRNA directed against MKK3 could block CL
and Forsk induction of p38 MAPK activity (Fig. 7D) as well as
UCP1 expression (Fig. 7E). Finally, the siRNA that specifically
targets MKK3 was the only one capable of completely inter-
fering with the expression of the endogenous Ucp1 gene (Fig.
FIG. 6. ?-Adrenergic agonists and cAMP promotes activation of MKK3. (A and B) HIB-1B cells were treated as described in Fig. 1A and then
lysed for measurement of the activity and amounts of the indicated kinases by Western blotting as detailed in Materials and Methods, and MKK3/6
phosphorylation was evaluated by Western blot (middle blot). Each blot shown is one of three experiments. (C) HIB-1B cells were treated or not
with H89 and forskolin as described in Fig. 1A. The cells were lysed, MKK3 and MKK6 were immunoprecipitated, and their phosphorylation state
was measured by Western blotting. (D and E) Mice were either treated with CL (1 mg/kg intraperitoneally) for 30 min (E) or placed at 4°C for
1 h (F), and BAT was excised and processed for immunoprecipitation of MKK3 or MKK6. The ability to phosphorylate GST-tagged p38? MAPK
and the protein levels of the MKKs were measured by Western blotting.
5474ROBIDOUX ET AL.MOL. CELL. BIOL.
on June 13, 2014 by guest
7F). All together, these results unequivocally define the prox-
imal steps in the cAMP- and PKA-dependent activation of the
Ucp1 gene in brown fat as being mediated solely by MKK3 and
The MAP kinases are usually assembled together with their
upstream MKKs into a signaling module that is coordinated by
large scaffolding molecules such as the JIPs (JNK-interacting
proteins) (21, 47). As a result, these scaffold proteins can
FIG. 7. MKK3 is essential for ?3AR agonist or Forsk induction of Ucp1 gene expression. (A) HIB-1B cells were transfected with the ?3AR,
with FLAG-tagged MKK3, MKK4, MKK6, or MKK7, and with the enhancer-CAT plasmid. Two days later the cells were treated with the solvent
(Basal), 10 ?M CL, or 10 ?M Forsk. The treatment was stopped after 6 h for the measurement UCP1 enhancer activity in a CAT reporter system:
Basal (white bars), CL (gray bars), and Forsk (black bars). (B) HIB-1B cells were transfected with siRNA targeting MKK3 or MKK6, and 2 days
later its expression was evaluated by Western blots. (C) HIB-1B cells were transfected with siRNA targeting MKK3 or MKK6 and 12 h later with
the ?3AR, the UCP1 enhancer, and the ?-actin-luciferase plasmids. Forty hours later, the cells were incubated with the solvent (Basal), 10 ?M
CL, or 10 ?M Forsk for 6 h for the measurement UCP1 enhancer activity in a CAT reporter system: Basal (white bars) and CL (gray bars). (D)
HIB-1B cells were transfected with siRNA targeting p38? and p38? MAPK and 12 h later with the ?3AR. The cells were incubated with the solvent
(Basal), 10 ?M CL, or 10 ?M Forsk for 6 h, the RNA was extracted, and the measurement of UCP1 was evaluated by real-time PCR using a
TaqMan probe. The results shown are means ? standard deviations of two determinations, each performed in triplicate.
VOL. 25, 2005 PKA ACTIVATION OF MKK3 AND p38? MAPK5475
on June 13, 2014 by guest
concentrate interacting signaling partners in the vicinity of an
upstream activator in order to favor a particular pathway.
Since there is no established link between PKA and p38
MAPK, we attempted to determine which, if any, of the known
JIP family members in brown adipocytes might serve as the
scaffold to specifically bind p38? MAPK and MKK3. The first
objective was to determine the relative expression levels of the
four known JIPs: JIP1, JIP2, JIP3, and JLP. As shown in Fig.
8A, there was no detectable expression of JIP1 in either BAT
or the HIB-1B cell line (RT-PCR cycle, ?45). However, JIP2,
JIP3, and JLP were all found in both samples, with JIP2 being
the least abundant at the mRNA level (Fig. 8A). Based on
these results, each of these three JIPs was analyzed for its
ability to interact specifically with MKK3 and p38? MAPK.
His-tagged or S-protein-tagged constructs of JIP2, JIP3, and
JLP were transfected into HIB-1B cells, followed by their im-
munoprecipitation in order to assess the identity of any inter-
acting kinases. Figure 8B clearly shows that only JIP2 was able
to specifically recover both MKK3 and p38? MAPK. It is
noteworthy that neither MKK6 nor p38? MAPK was found
under any circumstance, although all these molecules are
clearly present as shown in the lysate.
Catecholamine regulation of brown fat thermogenesis has
been clearly shown to involve increased transcription of the
Ucp1 gene as a result of stimulation of the ?ARs and cAMP
production (see references 48 and 51 for reviews). Despite this,
the details of the signaling cascade beyond this point have been
ambiguous. It has been generally assumed that the elevated
levels of cAMP lead to activation of PKA, which in turn phos-
phorylates the nuclear factor CREB, resulting in transcription
of the various target genes in brown fat, including UCP1. In the
last few years we have reported results from studies in white
and brown adipocyte cell models and in vivo manipulations to
show that p38 MAPK is activated in response to ?-adrenergic
stimuli (7, 8). This was a novel and unexpected link between
cAMP and p38 MAPK, but one that relied heavily on the use
of chemical inhibitors of p38 MAPK and PKA. Since there is
no clearly delineated series of steps linking PKA to p38 MAPK
in the literature, unequivocal identification of the individual
molecules in this cascade requires the use of more stringent
The p38 MAPK group is composed of four isoforms, p38?
(26, 41), p38? (32), p38? (43), and p38? (66). Among them,
p38? and -? are sensitive to the pyrimidyl imidazoles
SB202190 and SB205380 (14, 23). Our earlier reports indicated
that ?AR and PKA stimulation of UCP1 expression in brown
adipocytes was sensitive to SB (7, 8) and thus narrowed the
scope of inquiry. The pyridinyl imidazole-sensitive p38 MAPK
isoforms are often considered to be redundant enzymes, as
their substrate specificities overlap significantly. However, in
some cases these two isoforms have been shown to be able to
discriminate between substrates, at least under conditions of
forced overexpression of dominant-negative mutants (67, 73).
In the studies that we report here, we used a combination of
in vivo and in vitro approaches designed to distinguish between
the p38 MAPK isoforms and to identify the upstream MKK
enzyme(s) responsible for mediating the PKA signal to activate
Ucp1 gene expression. Our results clearly establish that p38?
MAPK is a central obligatory component of this signaling
cascade, with arguably no contribution from p38? MAPK, de-
spite its presence. We do not at this point rule out the possi-
bility that p38? MAPK might regulate other genes in the
brown adipocyte that might be triggered by different stimuli.
FIG. 8. p38? MAPK and MKK3 are present in a complex with
JIP2. The identification and quantification of the JIPs at the mRNA
level was performed by quantitative real-time PCR (A). JIP1 was
undetectable in these assays (not shown). (B) HIB-1B cells were trans-
fected with pcDNA3 (first lane), His-tagged JIP2 (second lane), His-
tagged JIP3 (third lane), or S-protein-tagged JLP (fourth lane). Tagged
proteins were immunoprecipitated (top two gels), and MKK3, MKK6,
p38?, and p38? MAPK were detected by Western blotting in the
recovered immunoprecipitate (upper panel) and the lysate (bottom
5476 ROBIDOUX ET AL.MOL. CELL. BIOL.
on June 13, 2014 by guest
For example, insulin has been reported to selectively activate
p38? MAPK but not p38? in brown adipocytes, and this effect
was associated with increased glucose transport (36).
Our studies also indicate that there is a unique requirement
for MKK3 as the immediate upstream kinase for p38?. The
selectivity of these two elements to be activated in succession is
not particularly surprising, but nevertheless it does occur in
spite of the existence in brown adipocytes of at least four
MKKs, three of which have been demonstrated to activate
p38? in other settings (17, 27, 44, 46, 52, 61). This tight cou-
pling is most likely indicative of their existence in a multimo-
lecular signaling complex, as is known to exist for the stress-
activated kinases from yeast to mammals (21, 47). These
signaling modules are maintained together by scaffolding pro-
teins, such as KSR for the ERK pathway and the JIP proteins
for the JNK pathway. A recent estimate of the size of this
“functional family” of scaffold proteins suggests more than 19
members (47). Clearly, this complexity necessitates more ex-
tensive investigation in order to assign individual scaffolds to
specific kinase members but which will probably also depend
upon the stimuli and the cell types in which they exist. Much is
known about the role of these JIP scaffolding molecules for the
JNK pathway (for which they were named), while in the case of
p38 MAPK three members of the family (JIP2, JLP, and JIP4)
have been proposed to be able to interact with p38 MAPK
and/or MKK3 (5, 34, 40). When considering adipocytes it must
be acknowledged that relatively little is known about these
molecules aside from a recent report concerning a role for JIP1
in insulin-induced JNK activation (31). Nevertheless, since we
have now established that PKA specifically utilizes MKK3 and
p38? MAPK to regulate genes in the brown adipocyte, this
information directed our quest for the scaffolding protein in-
volved. Our results clearly show that at least three putative p38
MAPK scaffolds are expressed in brown adipose tissue. The
absence of JIP1 and the presence of JIP2 was somewhat sur-
prising, since JIP1 is clearly expressed at low levels in white adi-
pose tissue (31) and JIP2 is expressed predominantly in neu-
ronal tissue (71). However, as usual, adipose tissue was absent
from the panel of tissue surveyed for JIP2 expression in this
latter study. Since JIP3 and JLP are more widely expressed,
their existence in adipose tissue was not unexpected. Interest-
ingly, when JIP2 was immunoprecipitated, only MKK3 and p38?
MAPK were recovered within the coprecipitate. Although not a
definitive proof of the necessity of JIP2 for the PKA-depen-
dent activation of p38? MAPK, it at least places these com-
ponents within close proximity to each other. Also, during the
revision of the manuscript JIP4 was cloned (34), therefore the
role for JIP4 in parallel with JIP2 cannot be excluded.
From a signaling perspective, the adipocyte is unique in that
all three known ?AR subtypes are expressed there and each is
coupled to the production of cAMP (56). Here we have also
shown that all three ?ARs can stimulate p38 MAPK in the
adipocyte to increase Ucp1 gene expression. Based on the
present results and our earlier findings that cAMP-dependent
activation of p38 MAPK elicits an orchestrated response to
increase the thermogenic capacity of brown adipocytes by in-
creasing the expression of PGC-1?, a master regulator of mi-
tochondriogenesis (68), an interesting speculation arises. Since
human adipocytes also express multiple ?AR subtypes and the
critical region of the Ucp1 gene that responds to cAMP and
p38 MAPK is conserved between rodents and humans, there
may well be a similar regulation of p38 MAPK and activation
of UCP1 and PGC-1? expression in human adipocytes. This
prospect will require careful examination from human visceral
adipose samples, since this depot is the main location of brown
adipocytes in adult humans and may potentially be a reservoir
of quiescent cells capable of thermogenic activity upon appro-
We thank Leslie P. Kozak, Jiahuai Han, Roger J. Davis, Josef M.
Penninger, and E. Premkumar Reddy for their invaluable gifts of
plasmids (listed in Materials and Methods). We also thank Alexander
Medvedev for helpful discussions in the initial phase of the project.
This work was supported by NIH awards R01 DK57698 and R01
DK53092 (S.C.) and a fellowship from Fonds de la Recherche en Sante ´
du Que ´bec (J.R.).
1. Altschuler, D. L., S. N. Peterson, M. C. Ostrowski, and E. G. Lapetina. 1995.
Cyclic AMP-dependent activation of Rap1b. J. Biol. Chem. 270:10373–
2. Bamshad, M., V. T. Aoki, M. G. Adkison, W. S. Warren, and T. J. Bartness.
1998. Central nervous system origins of the sympathetic nervous system
outflow to white adipose tissue. Am. J. Physiol. 275:R291–R299.
3. Bamshad, M., C. K. Song, and T. J. Bartness. 1999. CNS origins of the
sympathetic nervous system outflow to brown adipose tissue. Am. J. Physiol.
4. Bouillaud, F., D. Ricquier, J. Thibault, and J. Weissenbach. 1985. Molecular
approach to thermogenesis in brown adipose tissue: cDNA cloning of the
mitochondrial uncoupling protein. Proc. Natl. Acad. Sci. USA 82:445–448.
5. Buchsbaum, R. J., B. A. Connolly, and L. A. Feig. 2002. Interaction of Rac
exchange factors Tiam1 and Ras-GRF1 with a scaffold for the p38 mitogen-
activated protein kinase cascade. Mol. Cell. Biol. 22:4073–4085.
6. Cannon, B., and J. Nedergaard. 2004. Brown adipose tissue: function and
physiological significance. Physiol. Rev. 84:277–359.
7. Cao, W., K. W. Daniel, J. Robidoux, P. Puigserver, A. V. Medvedev, X. Bai,
L. M. Floering, B. M. Spiegelman, and S. Collins. 2004. p38 mitogen-
activated protein kinase is the central regulator of cyclic AMP-dependent
transcription of the brown fat uncoupling protein 1 gene. Mol. Cell. Biol. 24:
8. Cao, W., A. V. Medvedev, K. W. Daniel, and S. Collins. 2001. ?-Adrenergic
activation of p38 MAP kinase in adipocytes: cAMP induction of the uncou-
pling protein-1 (UCP1) gene requires p38 MAP kinase. J. Biol. Chem. 276:
9. Cassard-Doulcier, A. M., C. Gelly, F. Bouillaud, and D. Ricquier. 1998. A
211-bp enhancer of the rat uncoupling protein-1 (UCP-1) gene controls
specific and regulated expression in brown adipose tissue. Biochem. J. 333:
10. Champigny, O., D. Ricquier, O. Blondel, R. M. Mayers, M. G. Briscoe, and
B. R. Holloway. 1991. ?3-Adrenergic receptor stimulation restores message
and expression of brown-fat mitochondrial uncoupling protein in adult dogs.
Proc. Natl. Acad. Sci. USA 88:10774–10777.
11. Collins, S., W. Cao, and J. Robidoux. 2004. Learning new tricks from old
dogs: beta-adrenergic receptors teach new lessons on firing up adipose tissue
metabolism. Mol. Endocrinol. 18:2123–2131.
12. Collins, S., K. W. Daniel, A. E. Petro, and R. S. Surwit. 1997. Strain-specific
response to ?3-adrenergic receptor agonist treatment of diet-induced obesity
in mice. Endocrinology 138:405–413.
13. Cousin, B., S. Cinti, M. Morrone, S. Raimbault, D. Ricquier, L. Penicaud,
and L. Casteilla. 1992. Occurrence of brown adipocytes in rat white adipose
tissue: molecular and morphological characterization. J. Cell Sci. 103:931–
14. Cuenda, A., P. Cohen, V. Buee-Scherrer, and M. Goedert. 1997. Activation of
stress-activated protein kinase-3 (SAPK3) by cytokines and cellular stresses
is mediated via SAPKK3 (MKK6); comparison of the specificities of SAPK3
and SAPK2 (RK/p38). EMBO J. 16:295–305.
15. del Mar Gonzalez-Barroso, M., C. Pecqueur, C. Gelly, D. Sanchis, M. C.
Alves-Guerra, F. Bouillaud, D. Ricquier, and A. M. Cassard-Doulcier. 2000.
Transcriptional activation of the human ucp1 gene in a rodent cell line.
Synergism of retinoids, isoproterenol, and thiazolidinedione is mediated by
a multipartite response element. J. Biol. Chem. 275:31722–31732.
16. Del Mar Gonzalez-Barroso, M., D. Ricquier, and A. M. Cassard-Doulcier.
2000. The human uncoupling protein-1 gene (UCP1): present status and
perspectives in obesity research. Obes. Rev. 1:61–72.
17. Derijard, B., J. Raingeaud, T. Barrett, I. H. Wu, J. Han, R. J. Ulevitch, and
VOL. 25, 2005PKA ACTIVATION OF MKK3 AND p38? MAPK5477
on June 13, 2014 by guest
R. J. Davis. 1995. Independent human MAP-kinase signal transduction path-
ways defined by MEK and MKK isoforms. Science 267:682–685.
18. Enslen, H., J. Raingeaud, and R. J. Davis. 1998. Selective activation of p38
mitogen-activated protein (MAP) kinase isoforms by the MAP kinase ki-
nases MKK3 and MKK6. J. Biol. Chem. 273:1741–1748.
19. Franke, B., J. W. Akkerman, and J. L. Bos. 1997. Rapid Ca2?-mediated
activation of Rap1 in human platelets. EMBO J. 16:252–259.
20. Galitzky, J., M. Reverte, M. Portillo, C. Carpene, M. Lafontan, and M.
Berlan. 1993. Coexistence of ?1-,?2-, and ?3-adrenoceptors in dog fat cells
and their differential activation by catecholamines. Am. J. Physiol. 264:
21. Garrington, T. P., and G. L. Johnson. 1999. Organization and regulation of
mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell Biol.
22. Garruti, G., and D. Ricquier. 1992. Analysis of uncoupling protein and its
mRNA in adipose tissue deposits of adult humans. Int. J. Obesity 16:383–390.
23. Goedert, M., A. Cuenda, M. Craxton, R. Jakes, and P. Cohen. 1997. Acti-
vation of the novel stress-activated protein kinase SAPK4 by cytokines and
cellular stresses is mediated by SKK3 (MKK6); comparison of its substrate
specificity with that of other SAP kinases. EMBO J. 16:3563–3571.
24. Gonzalez-Barroso, M., C. Fleury, F. Bouillaud, D. Nicholls, and E. Rial.
1998. The uncoupling protein UCP1 does not increase the proton conduc-
tance of the inner mitochondrial membrane by functioning as a fatty acid
anion transporter. J. Biol. Chem. 273:15528–15532.
25. Gonzalez-Barroso, M. M., C. Fleury, I. Arechaga, P. Zaragoza, C. Levi-
Meyrueis, S. Raimbault, D. Ricquier, F. Bouillaud, and E. Rial. 1996. Acti-
vation of the uncoupling protein by fatty acids is modulated by mutations in
the C-terminal region of the protein. Eur. J. Biochem. 239:445–450.
26. Han, J., J. D. Lee, L. Bibbs, and R. J. Ulevitch. 1994. A MAP kinase targeted
by endotoxin and hyperosmolarity in mammalian cells. Science 265:808–811.
27. Han, J., J. D. Lee, Y. Jiang, Z. Li, L. Feng, and R. J. Ulevitch. 1996.
Characterization of the structure and function of a novel MAP kinase kinase
(MKK6). J. Biol. Chem. 271:2886–2891.
28. Herzig, S., F. Long, U. S. Jhala, S. Hedrick, R. Quinn, A. Bauer, D. Rudolph,
G. Schutz, C. Yoon, P. Puigserver, B. Spiegelman, and M. Montminy. 2001.
CREB regulates hepatic gluconeogenesis through the coactivator PGC-1.
29. Himms-Hagen, J., A. Melnyk, M. C. Zingaretti, E. Ceresi, G. Barbatelli, and
S. Cinti. 2000. Multilocular fat cells in WAT of CL-316243-treated rats
derive directly from white adipocytes. Am. J. Physiol. Cell Physiol. 279:
30. Huang, C. C., J. L. You, M. Y. Wu, and K. S. Hsu. 2004. Rap1-induced p38
mitogen-activated protein kinase activation facilitates AMPA receptor traf-
ficking via the GDI.Rab5 complex. Potential role in (S)-3,5-dihydroxyphe-
nylglycene-induced long term depression. J. Biol. Chem. 279:12286–12292.
31. Jaeschke, A., M. P. Czech, and R. J. Davis. 2004. An essential role of the
JIP1 scaffold protein for JNK activation in adipose tissue. Genes Dev. 18:
32. Jiang, Y., C. Chen, Z. Li, W. Guo, J. A. Gegner, S. Lin, and J. Han. 1996.
Characterization of the structure and function of a new mitogen-activated
protein kinase (p38beta). J. Biol. Chem. 271:17920–17926.
33. Kawasaki, H., G. M. Springett, N. Mochizuki, S. Toki, M. Nakaya, M.
Matsuda, D. E. Housman, and A. M. Graybiel. 1998. A family of cAMP-
binding proteins that directly activate Rap1. Science 282:2275–2279.
34. Kelkar, N., C. L. Standen, and R. J. Davis. 2005. Role of the JIP4 scaffold
protein in the regulation of mitogen-activated protein kinase signaling path-
ways. Mol. Cell. Biol. 25:2733–2743.
35. Klingenberg, M., K. S. Echtay, M. Bienengraeber, E. Winkler, and S. G.
Huang. 1999. Structure-function relationship in UCP1. Int. J. Obes.
36. Konrad, D., P. J. Bilan, Z. Nawaz, G. Sweeney, W. Niu, Z. Liu, C. N.
Antonescu, A. Rudich, and A. Klip. 2002. Need for GLUT4 activation to
reach maximum effect of insulin-mediated glucose uptake in brown adipo-
cytes isolated from GLUT4myc-expressing mice. Diabetes 51:2719–2726.
37. Kortelainen, M.-L., G. Pelletier, D. Ricquier, and L. J. Bukowiecki. 1993.
Immunohistochemical detection of human brown adipose tissue uncoupling
protein in an autopsy series. J. Histochem. Cytochem. 41:759–764.
38. Kozak, U. C., J. Kopecky, J. Teisinger, S. Enerback, B. Boyer, and L. P.
Kozak. 1994. An upstream enhancer regulating brown-fat-specific expression
of the mitochondrial uncoupling protein gene. Mol. Cell. Biol. 14:59–67.
39. Lafontan, M., and M. Berlan. 1993. Fat cell adrenergic receptors and the
control of white and brown fat cell function. J. Lipid Res. 34:1057–1091.
40. Lee, C. M., D. Onesime, C. D. Reddy, N. Dhanasekaran, and E. P. Reddy.
2002. JLP: a scaffolding protein that tethers JNK/p38MAPK signaling mod-
ules and transcription factors. Proc. Natl. Acad. Sci. USA 99:14189–14194.
41. Lee, J. C., J. T. Laydon, P. C. McDonnell, T. F. Gallagher, S. Kumar, D.
Green, D. McNulty, M. J. Blumenthal, J. R. Heys, S. W. Landvatter, et al.
1994. A protein kinase involved in the regulation of inflammatory cytokine
biosynthesis. Nature 372:739–746.
42. Lever, J. D., R. T. Jung, J. O. Nnodim, P. J. Leslie, and D. Symons. 1986.
Demonstration of a catecholaminergic innervation in human perirenal
brown adipose tissue at various ages in the adult. Anat. Rec. 215:227–229.
43. Li, Z., Y. Jiang, R. J. Ulevitch, and J. Han. 1996. The primary structure of
p38 gamma: a new member of p38 group of MAP kinases. Biochem. Biophys.
Res. Commun. 228:334–340.
44. Lin, A., A. Minden, H. Martinetto, F. X. Claret, C. Lange-Carter, F. Mer-
curio, G. L. Johnson, and M. Karin. 1995. Identification of a dual specificity
kinase that activates the Jun kinases and p38-Mpk2. Science 268:286–290.
45. Miyamoto, N. G. 1987. Nucleotide sequence of the human beta-actin pro-
moter 5? flanking region. Nucleic Acids Res. 15:9095.
46. Moriguchi, T., N. Kuroyanagi, K. Yamaguchi, Y. Gotoh, K. Irie, T. Kano, K.
Shirakabe, Y. Muro, H. Shibuya, K. Matsumoto, E. Nishida, and M. Hagi-
wara. 1996. A novel kinase cascade mediated by mitogen-activated protein
kinase kinase 6 and MKK3. J. Biol. Chem. 271:13675–13679.
47. Morrison, D. K., and R. J. Davis. 2003. Regulation of MAP kinase signaling
modules by scaffold proteins in mammals. Annu. Rev. Cell Dev. Biol. 19:
48. Nedergaard, J., V. Golozoubova, A. Matthias, A. Asadi, A. Jacobsson, and B.
Cannon. 2001. UCP1: the only protein able to mediate adaptive non-shiv-
ering thermogenesis and metabolic inefficiency. Biochim. Biophys. Acta
49. Nicholls, D. G., and R. M. Locke. 1984. Thermogenic mechanisms in brown
fat. Physiol. Rev. 64:1–64.
50. Nnodim, J. O., and J. D. Lever. 1988. Neural and vascular provisions of rat
interscapular brown adipose tissue. Am. J. Anat. 182:283–293.
51. Pecqueur, C., E. Couplan, F. Bouillaud, and D. Ricquier. 2001. Genetic and
physiological analysis of the role of uncoupling proteins in human energy
homeostasis. J. Mol. Med. 79:48–56.
52. Raingeaud, J., A. J. Whitmarsh, T. Barrett, B. Derijard, and R. J. Davis.
1996. MKK3- and MKK6-regulated gene expression is mediated by the p38
mitogen-activated protein kinase signal transduction pathway. Mol. Cell.
53. Ricquier, D., and F. Bouillaud. 2000. Mitochondrial uncoupling proteins:
from mitochondria to the regulation of energy balance. J. Physiol. 529:3–10.
54. Ricquier, D., and F. Bouillaud. 2000. The uncoupling protein homologues:
UCP1, UCP2, UCP3, StUCP and AtUCP. Biochem. J. 345:161–179.
55. Robidoux, J., T. L. Martin, and S. Collins. 2004. Beta-adrenergic receptors
and regulation of energy expenditure: a family affair. Annu. Rev. Pharmacol.
56. Rohlfs, E. M., K. W. Daniel, R. T. Premont, L. P. Kozak, and S. Collins.
1995. Regulation of the uncoupling protein gene (Ucp) by ?1, ?2, ?3-adren-
ergic receptor subtypes in immortalized brown adipose cell lines. J. Biol.
57. Ross, S. R., L. Choy, R. A. Graves, N. Fox, V. Solevjeva, S. Klaus, D.
Ricquier, and B. M. Spiegelman. 1992. Hibernoma formation in transgenic
mice and isolation of a brown adipocyte cell line expressing the uncoupling
protein gene. Proc. Natl. Acad. Sci. USA 89:7561–7565.
58. Rothwell, N. J., and M. J. Stock. 1979. A role for brown adipose tissue in
diet-induced thermogenesis. Nature 281:31–35.
59. Sawada, Y., K. Nakamura, K. Doi, K. Takeda, K. Tobiume, M. Saitoh, K.
Morita, I. Komuro, K. De Vos, M. Sheetz, and H. Ichijo. 2001. Rap1 is
involved in cell stretching modulation of p38 but not ERK or JNK MAP
kinase. J. Cell Sci. 114:1221–1227.
60. Shabalina, I. G., A. Jacobsson, B. Cannon, and J. Nedergaard. 2004. Native
UCP1 displays simple competitive kinetics between the regulators purine
nucleotides and fatty acids. J. Biol. Chem. 279:38236–38248.
61. Stein, B., H. Brady, M. X. Yang, D. B. Young, and M. S. Barbosa. 1996.
Cloning and characterization of MEK6, a novel member of the mitogen-
activated protein kinase kinase cascade. J. Biol. Chem. 271:11427–11433.
62. Tanaka, N., M. Kamanaka, H. Enslen, C. Dong, M. Wysk, R. J. Davis, and
R. A. Flavell. 2002. Differential involvement of p38 mitogen-activated pro-
tein kinase kinases MKK3 and MKK6 in T-cell apoptosis. EMBO Rep. 3:
63. Thomas, S. A., and R. D. Palmiter. 1997. Thermoregulatory and metabolic
phenotypes of mice lacking noradrenaline and adrenaline. Nature 397:94–97.
64. van Liefde, I., A. van Witzenberg, and G. Vauquelin. 1992. Multiple beta
adrenergic receptor subclasses mediate the I-isoproteronol-induced lipolytic
response in rat adipocytes. J. Pharmacol. Exp. Ther. 262:552–558.
65. Vossler, M. R., H. Yao, R. D. York, M. G. Pan, C. S. Rim, and P. J. Stork.
1997. cAMP activates MAP kinase and Elk-1 through a B-Raf- and Rap1-
dependent pathway. Cell 89:73–82.
66. Wang, X. S., K. Diener, C. L. Manthey, S. Wang, B. Rosenzweig, J. Bray, J.
Delaney, C. N. Cole, P. Y. Chan-Hui, N. Mantlo, H. S. Lichenstein, M.
Zukowski, and Z. Yao. 1997. Molecular cloning and characterization of a
novel p38 mitogen-activated protein kinase. J. Biol. Chem. 272:23668–23674.
67. Wang, Y., B. Su, V. P. Sah, J. H. Brown, J. Han, and K. R. Chien. 1998.
Cardiac hypertrophy induced by mitogen-activated protein kinase kinase 7, a
specific activator for c-Jun NH2-terminal kinase in ventricular muscle cells.
J. Biol. Chem. 273:5423–5426.
68. Wu, Z., P. Puigserver, U. Andersson, C. Zhang, G. Adelmant, V. Mootha, A.
Troy, S. Cinti, B. Lowell, R. C. Scarpulla, and B. M. Spiegelman. 1999.
Mechanisms controlling mitochondrial biogenesis and respiration through
the thermogenic coactivator PGC-1. Cell 98:115–124.
69. Wysk, M., D. D. Yang, H. T. Lu, R. A. Flavell, and R. J. Davis. 1999.
5478ROBIDOUX ET AL.MOL. CELL. BIOL.
on June 13, 2014 by guest
Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for tu- Download full-text
mor necrosis factor-induced cytokine expression. Proc. Natl. Acad. Sci. USA
70. Yao, Z., K. Diener, X. S. Wang, M. Zukowski, G. Matsumoto, G. Zhou, R.
Mo, T. Sasaki, H. Nishina, C. C. Hui, T. H. Tan, J. P. Woodgett, and J. M.
Penninger. 1997. Activation of stress-activated protein kinases/c-Jun N-ter-
minal protein kinases (SAPKs/JNKs) by a novel mitogen-activated protein
kinase kinase. J. Biol. Chem. 272:32378–32383.
71. Yasuda, J., A. J. Whitmarsh, J. Cavanagh, M. Sharma, and R. J. Davis. 1999.
The JIP group of mitogen-activated protein kinase scaffold proteins. Mol.
Cell. Biol. 19:7245–7254.
72. Young, J. B., E. Saville, N. J. Rothwell, M. J. Stock, and L. Landsberg. 1982.
Effect of diet and cold exposure on norepinephrine turnover in brown adi-
pose tissue of the rat. J. Clin. Investig. 69:1061–1071.
73. Yu, J., D. Bian, C. Mahanivong, R. K. Cheng, W. Zhou, and S. Huang. 2004.
p38 mitogen-activated protein kinase regulation of endothelial cell migration
depends on urokinase plasminogen activator expression. J. Biol. Chem. 279:
VOL. 25, 2005 PKA ACTIVATION OF MKK3 AND p38? MAPK5479
on June 13, 2014 by guest