CELL BIOLOGY AND METABOLISM:

p38 Mitogen-activated Protein Kinase

Pathway Promotes Skeletal Muscle
Differentiation: PARTICIPATION OF
THE MEF2C TRANSCRIPTION
FACTOR

Anna Zetser, Eran Gredinger and Eyal Bengal

J. Biol. Chem. 1999, 274:5193-5200.
doi: 10.1074/jbc.274.8.5193

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THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
p38 Mitogen-activated Protein Kinase Pathway Promotes Skeletal
Muscle Differentiation
PARTICIPATION OF THE MEF2C TRANSCRIPTION FACTOR*
(Received for publication, June 10, 1998, and in revised form, November 6, 1998)
Anna Zetser‡, Eran Gredinger‡, and Eyal Bengal§
From the Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine,
Technion-Israel Institute of Technology, Haifa 31096, Israel
Differentiation of muscle cells is regulated by extra-
cellular growth factors that transmit largely unknown
signals into the cells. Some of these growth factors in-
duce mitogen-activated protein kinase (MAPK) cascades
within muscle cells. In this work we show that the ki-
nase activity of p38 MAPK is induced early during ter-
minal differentiation of L8 cells. Addition of a specific
p38 inhibitor SB 203580 to myoblasts blocked their fu-
sion to multinucleated myotubes and prevented the ex-
pression of MyoD and MEF2 family members and myo-
sin light chain 2. The expression of MKK6, a direct
activator of p38, or of p38 itself enhanced the activity of
MyoD in converting 10T1/2 fibroblasts to muscle,
whereas treatment with SB 203580 inhibited MyoD. Sev-
eral lines of evidence suggesting that the involvement of
p38 in MyoD activity is mediated via its co-activator
MEF2C, a known substrate of p38, are presented. In
these experiments we show that MEF2C protein and
MEF2-binding sites are necessary for the p38 MAPK
pathway to regulate the transcription of muscle crea-
tine kinase reporter gene. Our results indicate that the
p38 MAPK pathway promotes skeletal muscle differen-
tiation at least in part via activation of MEF2C.
The development of skeletal muscle is a multistep process in
which pluripotent mesodermal cells give rise to myoblasts that
subsequently withdraw from the cell cycle and differentiate
into myotubes. These stages are driven by the expression and
activity of myogenic transcription factors from the MyoD fam-
ily (1). Two members of the MyoD family, Myf5 and MyoD, are
expressed in dividing myoblasts that are committed to the
myogenic lineage. However, these proteins are not functional in
myoblasts, and their activities are induced only at a subse-
quent stage to allow withdrawal from the cell cycle and termi-
nal differentiation (2). Induction of the activity of MyoD and
Myf5 proteins may be a result of the activity of extracellular
growth factors like insulin and insulin-like growth factors
known to promote terminal differentiation of myoblasts (3, 4).
Insulin and insulin-like growth factors are involved in the
* This work was supported by a United States-Israel Binational
Foundation grant, by an Israel Cancer Association grant, and by funds
from the Rappaport Foundation for Medical Research and the Founda-
tion for Promotion of Research in the Technion, Israel Institute of
Technology. The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
‡ These authors contributed equally to the work.
§ To whom correspondence should be addressed: Dept. of Biochemis-
try, Faculty of Medicine, Technion-Israel Inst. of Technology, P.O. Box
9649, Haifa 31096, Israel. Tel.: 972-4-8295-287; Fax: 972-4-8535-773;
E-mail: Bengal@tx.technion.ac.il.
This paper is available on line at http://www.jbc.org
Vol. 274, No. 8, Issue of February 19, pp. 5193–5200, 1999
Printed in U.S.A.
activation of phosphatidylinositol 3-kinases and mitogen-acti-
vated protein kinases (MAPK)1 via tyrosine kinase receptors
within many cell types including muscle (5, 6). One MAPK
induced by insulin is p38 MAPK (6). p38 is also activated by
exposure of cells to environmental stress or by treatment of
cells with pro-inflammatory cytokines (7, 8). The affectors reg-
ulating p38 are only partly explored. However, the direct in-
tracellular activators of p38 are MKK3 and MKK6 (MAPK
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kinase) (9, 10). The role of p38 in skeletal muscle differentia-
tion is not known. Several recent findings suggest that p38
MAPK may be involved in skeletal muscle differentiation: 1)
Transcripts of MKK6 gene are most abundant in skeletal mus-
cle (10). 2) Insulin that promotes skeletal muscle differentia-
tion also induces p38 activity (6). 3) p38 pathway regulates the
expression of glucose transporters in skeletal muscle cells (11).
4) p38 activates directly the transcription factor MEF2C in
inflammation (12). Together with the MyoD family, members of
the MEF2 family are necessary for the differentiation of myo-
blasts (13). 5) p38 pathway plays a role in specific gene expres-
sion and cell hypertrophy of the related cardiac muscle lineage
(14). In this report we show that 1) the p38 pathway plays an
essential role in the in vitro differentiation of myoblasts and 2)
that this role may be mediated via the MEF2C transcription
factor.
EXPERIMENTAL PROCEDURES
Materials
SB 203580 was a product of Calbiochem. Goat polyclonal antibody
raised against amino acids 341–360 of mouse p38 was obtained from
Santa Cruz Biotechnology. Rabbit polyclonal antibody that detects p38
MAPK only when activated by dual phosphorylation at Thr180 and
Tyr182 was purchased from New England Biolabs. Antibodies to MEF2A
and C proteins and to GAL4 (amino acids 94 –147) were purchased from
Santa Cruz Biotechnology. Protein A-Sepharose was supplied by Sigma.
Plasmids
pEMSV-MyoD was described by Tapscott et al. (15). The 4R-tk-CAT
reporter gene was described by Weintraub et al. (16). pe1AT-CAT and
p(1enh110)-80MCK-CAT were described by Buskin and Hauschka (17).
For MEF2– 80MCK-CAT, three copies of a double-stranded oligonucleo-
tide of the MEF2 site (30 base pairs) from the MCK enhancer were
inserted upstream to a minimal MCK promoter (280 to 17 relative to
the transcription start site). pGEX-ATF2 plasmid was a gift from Dr.
Ami Aronheim and Dr. Michael Karin. The wild type (MKK6) and
activated (MKK6b(E)) alleles of MKK6 were described by Han et al.
(10). The MEF2C expression vector (pCDNAI-MEF2C) was a generous
gift of Dr. E. Olson (18). The inactive form of MEF2C was generated by
deleting a BglII fragment that removed amino acids 202–321 of
1
The abbreviations used are: MAPK, mitogen-activated protein ki-
nase; MCK, muscle creatine kinase; ER, estrogen receptor; ERK, extra-
cellular signal-regulated protein kinase; DM, differentiation medium;
CAT, chloramphenicol acetyltransferase; GST, glutathione S-transfer-
ase; PAGE, polyacrylamide gel electrophoresis; MHC, myosin heavy
chain.
5193
5194 Role of p38 Pathway in Myogenesis
MEF2C. GAL4-MEF2C constructs were described by Han et al. (12),
and GAL4-MyoD constructs were described by Weintraub et al. (19).
pGEX-MEF2C was constructed by inserting a polymerase chain reac-
tion fragment encoding amino acids 128 – 467 of MEF2C into the
pGEX-2T vector.
Cell Culture
L8 cells were a gift of Dr. David Yaffe (20). 10T1/2 cells were obtained
from ATCC. 10T1/2 cells that expressed the MyoD-estrogen receptor
(ER) chimera protein were described by Hollenberg et al. (21). Cell lines
were maintained in Dulbecco’s modified Eagle’s medium supplemented
with 15% calf serum (Hyclone), penicillin, and streptomycin (growth
medium). To induce differentiation, we used Dulbecco’s modified Ea-
gle’s medium supplemented with 10 mg of insulin/ml and 10 mg of
transferrin/ml (differentiation medium, DM). Differentiation of 10T1/2
cells that expressed MyoD-ER protein was induced by the addition of
DM that contained 1027 M estradiol.
Growth of Cells in the Presence of SB 203580
SB 203580 was dissolved in Me2SO to a concentration of 10 mM and
was added directly to the differentiation medium to a final concentra-
tion of 10 –20 mM as indicated. Control cells were incubated with the
same volumes of Me2SO without SB 203580. The medium was replaced
every 12 h with medium containing fresh SB 203580.
Transfections
Transfections were performed by calcium phosphate precipitation as
described (22). Cells in 6-cm TC dishes (Corning) were transfected for
12 h with a total amount of 10 mg of the following plasmid DNA: 1 mg of
pCMV-LacZ, 3 mg of CAT reporter gene, 3 mg of MyoD expression
plasmid, 3 mg of MEF2C expression vector, and 3 mg of expression
vector of wt MKK6 or activated form of MKK6b (MKK6b(E)). Following
transfection, the medium was replaced with either growth medium or
DM for another 24 – 48 h. The efficiency of transfections were tested in
soluble 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside assays as de-
scribed (23), and the amount of extracts used for the CAT assays were
adjusted accordingly (24).
Immunohistochemical Staining
Cells were fixed and immunostained as described (25). The primary
antibodies used were monoclonal anti-MyoD (5.8A) and polyclonal anti-
MHC (Sigma). The immunochemically stained cells were viewed at
3200 magnification in a fluorescence microscope (Olympus, model
BX50).
In Vitro Kinase Assays
Expression of GST Fusion Proteins—The GST-ATF2 and GST-
MEF2C (128 – 467) proteins were expressed from the bacterial expres-
sion vectors in BL21 strain of Escherichia coli and purified from ex-
tracts on glutathione beads as described (26).
Preparation of Cell Extracts—Cell extracts were prepared as de-
scribed (25).
In Vitro Kinase Assay for p38 —Cells were extracted in lysis buffer
(described above). Equal amounts of protein from each time point of
differentiation were rotated with 6 ml of anti-p38 antibody (Santa Cruz)
for 2.5 h at 4 °C. The immunoprecipitation procedure and the kinase
assay were performed as described for ERK (25). However, the sub-
strate in the present assay was GST-ATF2 (20 mg).
In Vitro Kinase Activity of p38 Using GST-MEF2C as a Substrate—
Equal amounts of cell extracts were collected at different times after the
initiation of cell differentiation and were mixed with GSH-agarose
beads to which GST or GST-MEF2C proteins were bound. The mixtures
were processed, and the kinase assay was performed as described by
Hibi and colleagues for the JNK kinase assay (26). Results were quan-
tified by PhosphorImager.
RNA Analysis
RNA was extracted and analyzed by Western blotting as described
(25). Blots were hybridized with probes for MEF2C (pEMSV-MEF2C),
MyoD (pEMSV-MyoD), myogenin (pEMSV-myogenin), MLC2 (PV-
ZLC2), p21 (pCDNA-Waf1), and GAPDH (pMGAP).
Western Analysis
Cells were lyzed as described for the kinase assays, and equal
amounts of extracted proteins were loaded and separated by SDS-
PAGE and transferred to nitrocellulose filters. Immunoblotting was
conducted with the following two antibodies: anti-p38 (Santa Cruz)
1:100 and anti-phospho-p38 antibody (New England Biolabs) 1:100.
Proteins were visualized using the enhanced chemiluminescence kit of
Amersham Pharmacia Biotech.
Metabolic Labeling of Cells and Immunoprecipitation of MEF2C
Labeling with [35S]Methionine—Cells were incubated in methionine-
free Dulbecco’s modified Eagle’s medium and dialyzed calf serum for 40
min and then incubated with 100 mCi/ml [35S]methionine for 3 h before
proteins were extracted in RIPA buffer (50 mM Tris, pH 7.9, 150 mM
NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5 mM dithiothreitol, 0.1 mM
Na3VO4, 2 mg/ml leupeptin, 20 mM p-nitrophenyl phosphate, and 100
mg/ml phenylmethylsulfonyl fluoride).
Labeling with [32P]Orthophosphate—L8 cells were incubated in Dul-
becco’s modified Eagle’s medium without sodium phosphate to which
[32P]orthophosphate was added at 0.75 mCi/ml for 3 h before proteins
were extracted in RIPA buffer.
Immunoprecipitation—Equal amounts of labeled proteins were incu-
bated with antibody for 2 h and then for an additional hour with protein
A-Sepharose. The protein A-Sepharose beads were washed four times
with RIPA containing 0.5 M NaCl and once in RIPA.
RESULTS
p38 Activity Is Induced during Differentiation of L8 Cells—
The in vitro kinase activity of p38 was studied using extracts of
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L8 muscle cells from different stages of differentiation. Kinase
activity of p38 was analyzed by immunoprecipitation of p38,
which was used to phosphorylate its substrate, ATF2 (Fig. 1A).
The kinase activity of p38 was present in dividing myoblasts
and was gradually induced during the differentiation to
multinucleated myotubes. Kinase activity was specific; it was
induced by UV irradiation and inhibited by the p38-specific
inhibitor SB 203580 added to L8 cells (Fig. 1A). The amount of
the phosphorylated form of p38 that reflects the active kinase
was studied in L8 cells. An antibody that recognizes the active
dual phosphorylated form of p38 was used with the same ex-
tracts in a Western analysis. Phosphorylated forms of p38
accumulated during the differentiation of cells in DM (Fig. 1B).
The level of total p38 protein in differentiating cells was not
constant; however, the calculated percentage of phosphorylated
p38 in cells growing in DM for 48 h was 2–3-fold higher than
that of dividing myoblasts (Fig. 1B). Next, we assayed p38
kinase activity with a second substrate, GST-MEF2C. p38 was
recently demonstrated to associate and directly phosphorylate
MEF2C (12). In addition, MEF2C is an essential transcription
activator of muscle-specific genes (13). Phosphorylation of GST-
MEF2C substrate was increased during the differentiation of
L8 myoblasts to multinucleated myotubes (Fig. 1C). p38 was
probably the kinase that phosphorylated GST-MEF2C because
kinase activity was significantly inhibited in cells treated with
SB 203580 (Fig. 1C, lanes 3 and 4). The increased activity of
p38 during muscle differentiation suggests that it might have a
role in this process. Four isoforms of p38 (a, b, g, and d) are
presently known. To study the relative contributions of p38
isoforms in the phosphorylation of MEF2C, each isoform was
immunodepleted from muscle extracts. Differential depletion of
p38 isoforms affected the phosphorylation of MEF2C (Fig. 1C,
lanes 6 –11). p38a contributed most of p38 kinase activity (40%
inhibition), whereas p38b and d were less active (25 and 10%
inhibition, respectively). Depletion of p38g did not inhibit the
phosphorylation of MEF2C (Fig. 1C, lane 10).
Treatment of L8 Cells with SB 203580 Inhibits Their Differ-
entiation—To evaluate the role of p38 in myogenesis, we used
the p38 inhibitor SB 203580 (27) that was added to L8 cells at
concentrations that blocked most of the p38 activity (Fig. 1A).
The inhibitor was added concomitantly with the induction of
differentiation (differentiation medium, see “Experimental
Procedures”). After 36 h in that medium, cells not treated with
the drug fused to form developed myotubes, whereas cells
treated with the drug did not form myotubes (Fig. 2). Moreover,
 Role of p38 Pathway in Myogenesis
FIG. 1. p38 kinase activity is induced during differentiation of
L8 cells. A, the in vitro kinase activity of p38 was studied using
extracts of L8 muscle cells grown in differentiation medium for different
time periods as indicated. Kinase activity of p38 was analyzed by
immunoprecipitation of p38. The immunocomplex was used to phospho-
rylate the substrate, GST-ATF2 protein, as described under “Experi-
mental Procedures.” Proteins were separated over SDS-PAGE, and the
amount of phosphorylated GST-ATF2 was quantified using the Phos-
phorImager and plotted in the histogram. Maximal kinase activity was
set to 100 units. Average results from two independent experiments are
presented. Bars represent standard errors. B, the same protein extracts
from L8 cells as in A were separated using SDS-PAGE, and phospho-
rylated forms of p38 were identified by Western blotting with an anti-
body that recognized the dual phosphorylated form of p38 (upper
bands). The same blot was exposed also to an antibody that recognized
all forms of p38 (lower bands). The control lanes (Cont.): 2, extract of C6
cells; 1, extract of anisomycine-treated C6 cells. The asterisk represents
a nonspecific band. C, GST-MEF2C protein was phosphorylated in vitro
as described under “Experimental Procedures” using extracts of L8 cells
grown in differentiation medium for different time periods as indicated.
In one case, L8 cells were grown for 48 h in DM in the presence of 20 mM
SB 203580. Isoforms of p38 were depleted from extracts in the following
way: antibodies to the different isoforms of p38 were added to extracts
followed by an immunoprecipitation procedure. p38-depleted extracts
were used to phosphorylate GST-MEF2C. Control lane, GST protein
was used in a kinase assay with extract of L8 cells grown for 48 h in DM.
the expression of muscle specific genes like myogenin and
MLC2 was completely inhibited in cells treated with the drug
(Fig. 3A). The expression of MEF2 that participates with the
MyoD family in the differentiation process was also abolished
in the drug-treated cells (Fig. 3A). Induction of the cyclin/
cyclin-dependent kinase inhibitor p21 (Waf-1) participating in
the exit of myoblasts from the cell cycle was inhibited in SB
203580-treated cells. Consequently, the drug blocked differen-
tiation completely. We noticed dead cells in both untreated and
treated cells grown in the serum-free medium (DM), which
probably underwent programmed cell death under conditions
5195
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FIG. 2. The p38 inhibitor, SB 203580, prevents the fusion of L8
myoblasts to multinucleated myotubes. The p38 inhibitor, SB
203580 (20 mM), was added to L8 cells immediately after the addition of
DM when cells were about 80% confluent. SB 203580 was replaced with
fresh drug every 12 h, and cells were grown under these conditions for
36 h (middle panel). In a control plate, L8 cells were grown only in the
presence of DM for 36 h (top panel). In a third plate, cells were treated
for 36 h with DM and SB 203580 and for an additional period of 24 h
with DM only (bottom panel). Arrowheads point at multinucleated
myotubes. Cells were photographed at 2003 magnification.
of mitogen deprivation (Fig. 2) (28). However, the drug was
most probably not toxic to cells because the percentage of living
cells was not changed in drug-treated culture, and these cells
recovered and differentiated following removal of the drug
(Figs. 2 and 3B).
Activation of p38 with MKK6 Induces Muscle Differentiation
by MyoD—The dramatic effect of p38 inhibitor on the expres-
sion of MyoD and MEF2 family members prompted us to in-
vestigate whether p38 affected also the activity of the myogenic
regulator MyoD. The ectopic expression of MyoD converts
10T1/2 fibroblasts to muscle (29). The effect of p38 on MyoD
activity was studied by transiently transfecting 10T1/2 fibro-
blasts with expression vectors of MyoD and wild type MKK6,
the direct activator of p38. Most 10T1/2 fibroblasts transfected
with MyoD expression vector also expressed the differentiation
marker MHC when grown for 24 h in differentiation medium
(Fig. 4). However, if the transfected cells were continuously
grown in high serum-containing medium (15% bovine calf se-
rum), only 28% of the cells that expressed MyoD expressed also
MHC (Fig. 4). Under these conditions, cells that were co-trans-
fected with MKK6 exhibited a significantly higher proportion of
MHC staining in the cytoplasm of MyoD-expressing cells (69%
of the cells) (Fig. 4). On the other hand, addition of SB 203580
reversed the effect of MKK6 and completely inhibited MyoD,
i.e. only about 5% of the cells that expressed MyoD also ex-
pressed MHC. In a parallel experiment, we studied the effects
5196 Role of p38 Pathway in Myogenesis
FIG. 3. SB 203580 inhibits the expression of muscle-specific
markers in L8 cells. A, L8 cells were grown in DM and in the presence
or absence of SB 203580 for different periods of time as indicated. Total
RNA was extracted from cells, separated over an agarose gel, and
blotted onto a filter. Specific transcripts were determined by hybridiz-
ing the filter to different labeled DNA probes as indicated. Hybridiza-
tion to GAPDH was used to control for loading of RNA on the gel. B, L8
cells were grown in DM in the presence or absence of SB 203580 as
indicated. In one plate, cells were treated for 48 h with DM and SB
203580 and for an additional period of 48 h with DM only (lane 4). Total
RNA was extracted and processed as described for A.
of MKK6 and SB 203580 on the in vitro kinase activity of p38
in extracts from transfected 10T1/2 cells (Fig. 4C). As expected,
MKK6 induced whereas SB 203580 inhibited p38 kinase activ-
ity. Like MKK6, ectopic expression of p38 isoforms, mainly
p38g and d, in 10T1/2 cells strongly induced the ability of MyoD
to activate endogenous MHC (data not shown). Therefore we
conclude that activation of p38 MAPK pathway appears to
contradict the inhibitory effects of serum on the function of
MyoD as judged by the activation of endogenous MHC
expression.
Another approach was used to investigate whether p38 af-
fected the activity of MyoD. We used a cell line expressing an
estrogen receptor-MyoD chimera protein (21). The chimera pro-
tein remains inactive in the cytoplasm of cells. Treatment of
cells with estradiol induces the chimera protein that migrates
to the nucleus and activates muscle-specific transcription. In
this cell line, myogenesis is initiated by the activity of the
chimera protein. Therefore, inhibition of MyoD in these cells is
expected to abolish myogenesis. Activity of the chimera protein
was induced by the addition of estradiol to cells that were
grown in the presence or absence of SB 203580. Cells were
grown in the presence of estradiol for 24 h, at which time
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FIG. 4. MyoD activity is up-regulated by MKK6 and down-
regulated by treatment of cells with SB 203580. A, 10T1/2 fibro-
blasts were transiently transfected either with MyoD expression vector
or with expression vectors of MyoD and MKK6. In one case, cells that
were transfected with MyoD and MKK6 were treated with 20 mM of SB
203580. Transfected cells were grown in the presence of high serum
(growth medium) for 24 h after which they were fixed and double-
stained for MHC (cytoplasmic staining) and for MyoD (nuclear stain-
ing). B, quantification of the results presented in A. Transfected cells
were grown as described in A, except in one case in which cells were
grown in DM for 24 h. The percentage of differentiation was calculated
by dividing the number of double-stained cells (MyoD and MHC) by the
total number of MyoD-stained cells (single- and double-stained). Each
bar in the histogram represents the results of counting of about 100
transfected cells. C, the in vitro kinase activity of p38 was analyzed
using extracts from transfected cells as described under “Experimental
Procedures.”
mRNA levels of several muscle-specific genes were induced to
significant levels (Fig. 5, lane 3) (30). If cells were treated with
SB 203580 during that period, the induction of MEF2, myoge-
nin, and MLC2 expression was significantly inhibited, al-
though expression of chimeric MyoD-ER remained unchanged
(Fig. 5, lanes 4 and 5). Therefore, the inhibitor either inacti-
vated the chimera MyoD protein or affected other transcription
factors or co-activators that may function in concert with MyoD
to initiate myogenesis.
MKK6 Augments Transcription of MCK via the MEF2-bind-
ing Site—To further analyze the effect of MKK6 on the tran-
scriptional activity of MyoD, expression vectors of these pro-
teins were co-transfected with a reporter gene that contained a
minimal promoter and four MyoD-binding sites (4R-tk-CAT).
Surprisingly, the activity of MyoD in the induction of this
reporter gene was almost not affected by co-transfection of
MKK6 (Fig. 6A, lanes 9 –12). However, MyoD activity was
increased more significantly by MKK6 in activating the tran-
scription of a reporter gene whose expression was driven by
MCK regulatory sequences (pe1AT-CAT) (Fig. 6A, lanes 1– 4).
This reporter gene contained two MEF2-binding sites within
the MCK enhancer element that could contribute to the effect
of MKK6 (12). Co-expression of MEF2C did not further poten-
tiate the transcriptional activity of MyoD on this promoter (not
shown). These results may be explained by previous results
 Role of p38 Pathway in Myogenesis
FIG. 5. Treatment with SB 203580 prevents muscle differenti-
ation of cells expressing a conditional MyoD-ER chimera pro-
tein. 10T1/2 cells that constitutively express a chimera protein of MyoD
and estrogen receptor hormone-binding domain were studied. MyoD
activity was induced by the addition of estradiol and differentiation
medium to cells. Some cells were treated with SB 203580 at different
concentrations added together with estradiol and DM, and RNA was
extracted 24 h later. RNA was separated over agarose gel and analyzed
by Northern blotting. Blot was repeatedly hybridized with labeled
probes of MyoD, MEF2, myogenin, MLC2, and GAPDH, which was used
to control for loading of RNA. RNA was underloaded in lane 2 as can be
noticed by the appearance of a weak GAPDH band.
that indicated that endogenous MEF2 expression was stimu-
lated by the expression of myogenin in fibroblast cells (31). For
this reason, we did not co-transfect a MEF2C expression vector
in the subsequent experiments that analyzed MCK regulatory
sequences. To study the possible role of the MEF2 sites in the
stimulation of MyoD activity by MKK6, a similar transfection
experiment was done with an MCK reporter gene that con-
tained the MyoD-binding sites and only one MEF2 site (p110-
MCK-CAT) (17). Induction of this reporter gene by MyoD was
only mildly affected by the co-expression of MKK6 (Fig. 6A,
lanes 5– 8). To prove MKK6 activated MCK via the MEF2 site,
we generated a reporter gene that contained multiple MEF2
sites of the MCK enhancer and a basal promoter of MCK
(MEF2-MCK-CAT). Indeed, this promoter was induced by the
expression of MEF2C and was further induced by MKK6 (Fig.
6A, lanes 13–15). Expression of MKK6 alone was sufficient to
induce this promoter in a significant fashion (Fig. 6A, lane 16).
We conclude that MEF2 sites probably play a role in the stim-
ulation of MCK transcription observed in the presence of
MKK6. Addition of the p38 inhibitor, SB 203580, significantly
blocked the transcriptional activity of MyoD in the induction of
the two MCK enhancer reporter genes, (pe1AT-CAT and p110-
MCK-CAT) (Fig. 6B, lanes 1– 4), and more modestly affected its
activity on the minimal promoter driven by E boxes (4R-tk-
CAT) (Fig. 6B, lanes 5 and 6). The transcriptional activity of
MEF2 on a basal promoter driven by MEF2 sites was blocked
by SB203580 (Fig. 6B, lanes 9 and 10). All in all, these results
suggest that p38 modulates the transcriptional activity of the
5197
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FIG. 6. p38 affects the transcription of MCK reporter genes via
the MEF2-binding sites. A, 10T1/2 fibroblasts were transiently trans-
fected with expression vectors of MyoD or MEF2C with or without the
direct activator of p38, MKK6. Several reporter genes were used;
pe1AT-CAT carried a regulatory sequence of MCK enhancer that con-
tained two MEF2-binding sites and MCK promoter; p110 MCK CAT
carried a smaller MCK enhancer that contained one MEF2-binding site
and MCK promoter; 4R-tk-CAT that contained four MyoD-binding sites
in the promoter region and MEF2-MCK-CAT that contained three
MEF2-binding sites and MCK promoter. All reporter genes contained
the CAT reading frame. Protein extracts of the transfected cells were
used in a CAT assay according to the transfection efficiency that was
measured as described under “Experimental Procedures.” The chloram-
phenicol products were separated by thin layer chromatography and
quantified by phosphor imaging (Fuji). For each reporter gene, the
maximal CAT activity was set to 100 units. Average results from three
independent experiments are presented. Error bars represent standard
errors. B, the same constructs described in A were used to transfect
10T1/2 cells with the difference that some plates were treated immedi-
ately after transfection with 10 mM SB 203580. Also, the expression
vector of GAL4-VP16 was co-transfected with the pGAL-CAT reporter
gene. For each reporter gene, the maximal CAT activity was set to 100
units. Average results from three independent experiments are pre-
sented. Error bars represent standard errors.
MCK reporter gene via the MEF2-binding sites.
A Transcriptionally Inactive MEF2C Protein Abrogates the
Effects of MKK6 on the MCK Enhancer—To study the role of
MEF2 in mediating the effects of p38 on the transcription of the
MCK reporter gene, we generated a transcriptionally inactive
MEF2C protein (see “Experimental Procedures”). This protein
did not contain a large part of its transactivation domain that
included two phosphorylation sites of p38 (MEF2C-D) (12). A
similar MEF2A protein that functioned as a dominant negative
was recently described by Ornatsky and colleagues (32). By
itself, MEF2C-D retained minimal transcriptional activity com-
pared with the wild type protein (Fig. 7A, compare lanes 2 and
6). When co-expressed with wild type MEF2C, the mutant
protein was able to repress the transcriptional activity of the
5198 Role of p38 Pathway in Myogenesis
FIG. 7. A transcriptionally inactive MEF2C protein abrogates
the effects of MKK6 on the MCK enhancer. A, MEF2C-D represses
the transcriptional activity of wild type MEF2C protein. 10T1/2 fibro-
blasts were transfected with different expression vectors and reporter
genes as indicated. Reporter genes: Pe1AT-CAT contains enhancer and
promoter elements of MCK, 4R-tk-CAT contains four MyoD-binding
sites and the basal tk promoter, and pGAL-CAT contains five GAL4-
binding sites and a basal tk promoter. MEF2C-D expression vector was
transfected at 2 mg (31), 4 mg (32), and 8 mg (34). Cells were lyzed 48 h
after transfection, and protein extracts of the transfected cells were
used in a CAT assay according to the transfection efficiency that was
measured as described under “Experimental Procedures.” The chloram-
phenicol products were separated by thin layer chromatography and
quantified by phosphor imaging (Fuji). For each reporter gene, the
maximal CAT activity was set to 100 units. Average results from two
independent experiments are presented. B, MEF2C-D represses the
induction of MCK transcription mediated by MyoD and MKK6. 10T1/2
fibroblasts were transfected with different plasmid DNA as indicated.
Reporter gene and expression vectors are described in A. MEF2C-D
expression vector was transfected at 2 mg (31) and 4 mg (32). Trans-
fection, CAT assay and processing of the results were done as described
for A. Average results from three independent experiments are pre-
sented. Error bars represent standard errors.
former protein (Fig. 7A, lanes 2–5). However, the mutant
MEF2C protein did not repress activation by MyoD (lanes 8
and 9) or GAL4-VP16 (lanes 11 and 12) and therefore was
specific to wild type MEF2C. To find out whether the mutant
protein could block the effect of p38 on the MCK regulatory
sequences, we transfected the different expression plasmids
with the MCK reporter gene (pe1AT-CAT). The transcription-
ally inactive MEF2C protein inhibited the induced expression
of MCK reporter mediated by MyoD (Fig. 7B, lanes 1– 4). More-
over, it also abolished the additional activity contributed by
MKK6 (Fig. 7B, lanes 5–7). The inhibition was specific to the
MCK enhancer that contained the MEF2-binding site, because
the inactive MEF2C protein did not inhibit MyoD activation of
reporter gene containing only MyoD-binding sites (4R-tk-CAT-
)(Fig. 7A, lanes 8 and 9). We conclude that MKK6 induced the
transcription of MCK via MEF2 protein(s) and that this induc-
tion was blocked by competition of the transcriptionally inac-
tive MEF2C protein.
FIG. 8. Transcriptional activity of GAL4-MEF2C protein is in-
duced, whereas activities of GAL4-MyoD proteins are not af-
Downloaded from http://www.jbc.org/ by guest on November 24, 2014
fected by MKK6. 10T1/2 fibroblasts were transfected with different
expression constructs as indicated and the pGAL4-CAT reporter gene.
Cells were lyzed 48 h after transfection, and protein extracts of the
transfected cells were used in a CAT assay according to the transfection
efficiency that was measured as described under “Experimental Proce-
dures.” The chloramphenicol products were separated by thin layer
chromatography and quantified by phosphor imaging (Fuji). Average
results from three independent experiments are presented. Error bars
represent standard errors.
The p38 Pathway Affects the MEF2C Protein but Not the
MyoD Protein—Although the results presented in Fig. 6 sug-
gest that the p38 pathway operates via MEF2 sites, we could
still notice an effect on MyoD (Fig. 6, A, lanes 9 –12, and B,
lanes 5 and 6). This effect on MyoD may result from its asso-
ciation with MEF2, which is known to function as a co-activator
of MyoD (18). To study the effect of p38 on MyoD independently
of MEF2, we used the GAL4 activator/reporter system. The
transcription activators in this system were composed of MyoD
fragments that were fused to the DNA-binding domain of yeast
transcription factor GAL4. The chimeric activators are ex-
pected to bind to the GAL4 DNA-binding sites of a reporter
gene and activate transcription via the transactivation domain
of MyoD. MEF2 was suggested to interact with the DNA-
binding domain of MyoD-E12 heterodimers (18). Two MyoD
proteins that were not expected to interact with MEF2 were
used; one that did not contain the HLH domain (GAL-DHLH
MyoD) and another that contained a substituted DNA-binding
domain from the Drosophila acheate-scute protein (GAL-
T4basic MyoD). The transcription mediated by GAL4-MyoD
proteins was not affected by co-expression of MKK6 (Fig. 8,
lanes 7–12). However, MKK6 augmented the activity of a chi-
meric GAL4-MEF2C transcription factor (Fig. 8, lanes 1 and 2).
The activity of two MEF2C mutants that did not contain their
p38 phosphorylation sites (293, 300A and 387A) was not sig-
nificantly increased by the expression of MKK6 (Fig. 8, lanes
3– 6). Therefore, we conclude that the transcriptional activity of
GAL4-MyoD was not affected, whereas the activity of GAL4-
MEF2C was affected by MKK6.
MEF2C Is Phosphorylated by p38 in Muscle Cells—To dem-
onstrate that GAL4-MEF2C protein is phosphorylated in cells
by p38, 293 cells were transfected with expression vector of
GAL4-MEF2C alone or GAL4-MEF2C with MKK6 (Fig. 9A). In
a duplicate set of transfected plates, the cells were treated with
SB 203580. Proteins were metabolically labeled with [35S]me-
thionine, and GAL4-MEF2C was immunoprecipitated. The mo-
bility of the GAL4-MEF2C protein in the gel indicated its
 Role of p38 Pathway in Myogenesis
FIG. 9. Phosphorylation of MEF2 proteins in cells. A, 293 cells
transfected with GAL4 (lanes 8 and 9) or GAL4-MEF2C (lanes 1–7)
expression vector alone or with MKK6 were labeled with [35S]methi-
onine. As indicated, some transfected cells were treated with SB 203580
(20 mM) for 36 h before proteins were extracted. GAL4 proteins were
immunoprecipitated from protein extracts and separated using SDS-
PAGE. Lanes 5–7, immunoprecipitates were treated with alkaline phos-
phatase. The asterisks represent truncated forms of GAL4-MEF2C. B,
dividing myoblasts (0 h in DM) and differentiating myotubes (48 h in
DM) were metabolically labeled with [35S]methionine (lanes 1– 4) or
with [32P]orthophosphate (lanes 5–9). MEF2A and MEF2C proteins
were immunoprecipitated and separated over SDS-PAGE. In lanes 3
and 7, a competitive peptide against which the MEF2 antibody was
made was added to extracts prior to the immunoprecipitation proce-
dure. Lanes 4 and 9 are control lanes; MEF2 antibody was not added to
the immunoprecipitation reaction.
phosphorylation status. MEF2C was phosphorylated by p38 in
cells because it migrated faster in cells treated with SB 203580
(Fig. 9A, compare lanes 1 and 2). Ectopic expression of MKK6
in the transfected cells resulted in a smear of slower migrating
GAL-MEF2C species (Fig. 9A, lane 3). Phosphorylation of
MEF2C was confirmed by treatment of the immunoprecipitates
with alkaline phosphatase that compressed the smear to a
tight faster migrating band (Fig. 9A, lanes 5–7). Unlike GAL4-
MEF2C, the migration of transfected GAL4 protein was not
affected by the co-expression of MKK6 (Fig. 9A, lanes 8 and 9).
Therefore, both the expression of MKK6 and treatment with
SB 203580 affected the phosphorylation state of transfected
GAL4-MEF2C protein in cells.
To find out if MEF2 proteins are phosphorylated in muscle
cells, we immunoprecipitated endogenous MEF2 proteins from
L8 cells (Fig. 9B). MEF2 proteins were detected mainly in
differentiated myotubes (Fig. 9B, lanes 1 and 2). Differentiated
cells were phosphate-labeled, and MEF2 proteins were isolated
to learn whether these proteins were phosphorylated in myo-
tubes. MEF2 proteins are phosphorylated in muscle cells (Fig.
9B, lane 6). Phosphorylation of MEF2 proteins is partly inhib-
5199
ited by treatment of cells with SB 203580 (Fig. 9B, lane 8).
Consequently, MEF2 proteins are phosphorylated by p38 in L8
muscle cells.
DISCUSSION
In this report we suggest for the first time the possible
involvement of p38 in skeletal muscle differentiation. We show
that the activity of p38 was induced in L8 myoblasts during the
formation of multinucleated myotubes. Interference with p38
activity by the specific inhibitor SB 203580 completely abol-
ished muscle fusion and expression of all myogenic markers
that were tested. We studied the effects of p38 MAPK on
muscle-specific transcription and found that its activator,
MKK6, stimulated the expression of muscle-specific genes, and
the effect was mediated by MEF2C. The latter was demon-
strated before to be an essential transcription activator in
myogenesis (13, 33, 34).
Members of the MEF2 and MyoD families act within a reg-
ulatory network that establishes the differentiated phenotype
of skeletal muscle (13). MyoD and MEF2 family members work
in concert to activate the expression of many muscle-specific
genes including their own. Therefore, inhibition of the activity
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of either MEF2 or MyoD family members results in complete
inhibition of skeletal muscle differentiation. p38 MAPK is a
potential activator of MEF2C and as part of the MEF2-MyoD
circuit it is expected to control muscle differentiation.
That MEF2 family members played a part in mediating p38
function in the activation of MCK transcription and myogen-
esis was suggested by the following. 1) Induction of MCK re-
porter gene by MyoD was augmented by MKK6 only if MEF2
sites were present at the regulatory sequences. MKK6 did not
significantly affect the activation of MyoD from promoters that
did not contain MEF2 site (Fig. 6). 2) The effect of MKK6 was
specifically abolished by the expression of a transcriptionally
inactive MEF2C protein (Fig. 7). 3) The transcriptional activity
of GAL4-MEF2C fusion proteins was induced, whereas the
activity of GAL4-MyoD proteins was not changed by MKK6
(Fig. 8).
MEF2C is not the only member of the family that is ex-
pressed in muscle. In fact MEF2A protein accumulates before
MEF2C, which is expressed later during the differentiation of
myoblasts (35, 36). For that reason we should expect that other
members of the family may be similarly regulated by p38.
Interestingly, MEF2A contains a serine at the position equiv-
alent to Ser387 found in MEF2C (37), suggesting that it may be
regulated similarly. Indeed, recent studies suggest that
MEF2A is phosphorylated by p38.2
We noticed that inhibition of p38 in muscle cells also abol-
ished the expression of MEF2 (Figs. 3A and 5). Inhibition of
MEF2 expression may be a result of the repression of MEF2C
that functions to induce its own transcription and/or the re-
pression of other transcription factors such as MyoD or Myf5.
Our results do not rule out the possibility that MyoD was
directly affected by p38, because the reporter gene regulated by
MyoD only, 4R-tk-CAT, was mildly affected by MKK6 or SB
203580 (Fig. 6). Other transcription factors activated by p38,
like ATF2, CREB, and Elk-1 may participate in the differenti-
ation process induced by p38.
Four isoforms of p38 are known (a, b, g, and d). We suggest
that p38a and b are the major isoforms involved in differenti-
ation of L8 cells because 1) The inhibitor, SB 203580, that is
specific to the a and b isoforms (38 – 40) inhibited the differen-
tiation of L8 cells (Figs. 2 and 3), and 2) immunodepletion of the
a and b but not of g and d isoforms from L8 extracts signifi-
cantly inhibited the phosphorylation of GST-MEF2C (Fig. 1C).
2
J. McDermott, personal communication.
5200 Role of p38 Pathway in Myogenesis
Interestingly, our preliminary studies suggest that g and d
isoforms are more efficient than a and b isoforms in the induc-
tion of muscle differentiation when transfected with MyoD.
Farther studies will elucidate the role of p38 isoforms in muscle
differentiation.
Recently, we reported that ERK MAPK activity was induced
during the differentiation of C2 cells and that this activity
promoted muscle differentiation (25). The similarities in the
pattern of activities of p38 and ERK in muscle cells imply that
these two distinct pathways have similar effects on muscle
differentiation. However, our data suggest that the two path-
ways also exhibit distinct activities. Firstly, inhibition of ERK
with PD 098059 only partially prevented the fusion of myo-
blasts and did not inhibit the expression of muscle-specific
markers, whereas inhibition of p38 with SB 203580 prevented
fusion of myoblasts and expression of muscle-specific markers.
Secondly, the activities of ERK and p38 are differentially in-
duced; p38 is induced earlier than ERK in cells grown in
differentiation medium (Ref. 25 and present work). These dif-
ferences suggest that the two pathways perform distinct func-
tions and that their combined activity may be required for the
differentiation process.
Studies of different cell lineages suggest that a distinct bal-
ance between the different MAPK pathways is essential for the
survival of these cells. During maturation of HL-60 cells to the
neutrophil phenotype, the activity of JNK was reduced,
whereas the activity of p38 was induced (41). In rat pheochro-
mocytoma PC-12 cells that serve as a model for neuronal dif-
ferentiation, induced activity of ERK MAPK and reduced ac-
tivities of JNK and p38 pathways were critical for the survival
of these cells (42). The induction of p38 in myocardial cells
served to protect them from apoptosis in one study (43) or to
increase apoptosis in another study (44). In these cellular mod-
els, cell survival or programmed cell death happens as a result
of a balance between the activities of ERK, JNK, and p38
MAPKs. Therefore, it is possible that the induction of ERK and
p38 MAPKs during skeletal muscle differentiation plays a role
in preventing programmed cell death as well as in the activa-
tion of muscle-specific genes.
Acknowledgments—We thank Dr. Uri Nudel and Dr. David Yaffe for
L8 cells. We thank Dr. Stephen J. Tapscott for the 10T1/2 MyoD-ER cell
line and muscle reporter genes. We thank Dr. Jiahuai Han for MKK6
and GAL4-MEF2C constructs and the antibodies to different isoforms of
p38. We thank Dr. Ami Aronheim and Dr. Michael Karin for offering us
the pGEX-ATF2 plasmid. We thank Dr. Eric Olson for the MEF2C
expression vector. We thank Dr. Bianca Raikhlin-Eisenkraft for critical
reading of the manuscript.
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