Vol. 14, No.

10

MOLECULAR AND CELLULAR BIOLOGY, OCt. 1994, p. 6683-6688

0270-7306/94/$04.00+0
Copyright C) 1994, American Society for Microbiology

c-Jun N-Terminal Phosphorylation Correlates with Activation of

the JNK Subgroup but Not the ERK Subgroup of
Mitogen-Activated Protein Kinases
AUDREY MINDEN,1 ANNING LIN,1 TOD SMEAL,1 BENOIT DERIJARD,2 MELANIE COBB,3
ROGER DAVIS,2 AND MICHAEL KARIN"*

Department of Pharmacology, Program in Biomedical Sciences, Center for Molecular Genetics, University of
Califomia, San Diego, School of Medicine, La Jolla, Califomia 92093-06361; Department of Pharmacology,
Southwestem Medical Center, University of Texas, Dallas, Texas 75235-9041; and Howard Hughes
Medical Institute and Program in Molecular Medicine, University of Massachusetts
Medical School, Worcester, Massachusetts 018052
Received 23 May 1994/Returned for modification 5 July 1994/Accepted 12 July 1994
c-Jun transcriptional activity is stimulated by phosphorylation at two N-terminal sites: Ser-63 and -73.
Phosphorylation of these sites is enhanced in response to a variety of extracellular stimuli, including growth
factors, cytokines, and UV irradiation. New members of the mitogen-activated protein (MAP) kinase group of
signal-transducing enzymes, termed JNKs, bind to the activation domain of c-Jun and specifically phosphorylate these sites. However, the N-terminal sites of c-Jun were also suggested to be phosphorylated by two other
MAP kinases, ERK1 and ERK2. Despite these reports, we find that unlike the JNKs, ERK1 and ERK2 do not
phosphorylate the N-terminal sites of c-Jun in vitro; instead they phosphorylate an inhibitory C-terminal site.
Furthermore, the phosphorylation of c-Jun in vivo at the N-terminal sites correlates with activation of the
JNKs but not the ERKs. The ERKs are probably involved in the induction of c-fos expression and thereby
contribute to the stimulation of AP-1 activity. Our study suggests that two different branches of the MAP kinase
group are involved in the stimulation of AP-1 activity through two different mechanisms.

which MAP kinase is responsible for phosphorylation of the

N-terminal sites of c-Jun, we compared the pattern of c-Jun
phosphorylation with that of JNK and ERK activation in
response to various extracellular stimuli. We found that in
living cells the phosphorylation of the N-terminal sites of c-Jun
parallels the activation of JNK and does not correlate with
ERK activation. Therefore, ERK1 and ERK2 are unlikely to
be responsible for stimulation of c-Jun transcriptional activity,
although they can contribute to induction of AP-1 activity by
another mechanism based on c-fos induction (19). Although
some extracellular stimuli activate both ERKs and JNKs, other
stimuli preferentially activate only one MAP kinase subgroup.

The DNA binding activity of c-Jun, a component of transcription factor AP-1 (5), is inhibited by phosphorylation at Ser
and Thr residues located next to its C-terminal DNA binding
domain (8, 10, 21). On the other hand, the transcriptional
activity of c-Jun is stimulated by phosphorylation at Ser-63 and
-73 within its N-terminal activation domain (6, 24, 28, 29). It
was reported that the N-terminal sites of c-Jun are phosphorylated in vitro by the mitogen-activated protein (MAP) kinases
ERK1 and ERK2 (23, 24). However, other investigators found
that the same kinases preferentially phosphorylate one of the
inhibitory C-terminal sites, Ser-243 (3, 10). A novel serine/
threonine kinase activity, termed JNK, which binds to c-Jun
and specifically phosphorylates its N-terminal sites in response
to UV irradiation and Ha-Ras expression, was recently identified (18). Most cells express two isoforms of JNK, 46 and 55
kDa in size and termed JNK1 and JNK2, that are highly similar
in their modes of regulation (18, 30). Recently, a cDNA clone
encoding the 46-kDa isoform, JNK1, was isolated (11). Sequence analysis of JNK1 identified it as a novel and distant
member of the MAP kinase group of signal-transducing enzymes (11). Like activation of ERK1 and ERK2 (1), activation
of JNK1 requires its phosphorylation on adjacent Thr and Tyr
residues (11). Although the JNKs are rapidly and strongly
activated following UV irradiation (11, 14, 18), a very effective
stimulator of c-Jun N-terminal phosphorylation (13), it was
also reported that UV-mediated stimulation of c-Jun Nterminal phosphorylation may correlate with activation of
ERK1 and ERK2 (25). To solve the controversy regarding

MATERIALS AND METHODS

Cells. HeLa and FR3T3 cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine
serum (FBS). MRC5 cells were grown in modified Eagle's
medium with 10% FBS. PC12 cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% FBS and
5% donor horse serum.
Two-dimensional phosphopeptide mapping. Recombinant
c-Jun was phosphorylated by ERK1 and ERK2 that was
purified from rat la HIRc B cells. Glutathione S-transferase
(GST)-c-Jun(1-223) was phosphorylated in vitro by the solidstate kinase assay described below. The phosphorylated proteins were separated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE), digested with trypsin, and
applied to thin-layer cellulose plates. Proteins were then
resolved horizontally by electrophoresis at pH 1.9 and vertically by ascending chromatography as described previously (6,
8), and the plates were exposed to X-ray film. For analysis of
c-Jun phosphorylation in vivo, cells were metabolically labeled
with 32Pi as previously described (6, 8). c-Jun was isolated by

Corresponding author. Mailing address: Department of Pharmacology, Program in Biomedical Sciences, Center for Molecular Genetics, University of California at San Diego, School of Medicine, 9500
Gilman Dr., La Jolla, CA 92093-0636. Phone: (619) 534-1361. Fax:
(619) 534-8158.
*

6683

6684

MOL. CELL. BIOL.

MINDEN ET AL.
in vivo

ERK 1,2 + in vivo

ERK 1.2

4. Q.

.:

*aA;

9b
A

FIG. 1. Tryptic peptide maps of c-Jun phosphorylated in vitro and in vivo. Recombinant c-Jun (100 ng) was phosphorylated in vitro with
purified ERK1 and ERK2 (a mixture of both enzymes purified from insulin-stimulated rat la HIRc B cells), with [y-32P]ATP, for 15 min as
described previously (21). c-Jun protein labeled with 32P in vivo was immunopurified from Ha-ras-transformed FR3T3 cells (28, 29). The
phosphorylated proteins were digested with trypsin. Digests of c-Jun phosphorylated in vitro (380 cpm) or in vivo (450 cpm) and a 2:1 mixture of
both were applied to thin-layer cellulose plates and resolved horizontally by electrophoresis at pH 1.9 and vertically by ascending chromatography.
The plates were exposed to X-ray film for 4 days at - 70C. The origin is indicated by an arrowhead. X and Y are the phosphopeptides that contain
Ser-73 and -63, respectively. X' is an alkylated derivative of X. a, b, and c are phosphoisomers of the tryptic peptide that contains the C-terminal
sites. T1 and T2 are phosphoisomers of the peptide that contains Thr-91, -93, and -95. Identical results were obtained with ERK1 or ERK2 that
was homogeneously purified to >90% purity.

immunoprecipitation, separated by SDS-PAGE, and subjected

to two-dimensional phosphopeptide mapping (6, 8).
Protein kinase assays. JNK activity was measured by the
solid-state kinase assay with GST-c-Jun(1-223) as a ligand and
a substrate as described previously (18). Briefly, cell extracts
were mixed with glutathione (GSH)-agarose beads to which
GST-c-Jun(1-223) was bound. After incubation at 4C for
several hours, the beads were washed extensively and incubated in kinase buffer (20 mM N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid [HEPES] [pH 7.6], 20 mM MgCl2, 10
mM ,-glycerophosphate, 20 mM p-nitrophenylphosphate, 0.5
mM Na3VO4, 2 mM dithiothreitol, 50 puM ATP) containing 5
,uCi of [-y-32P]ATP at 30C for 20 min. The activity of transiently expressed JNK1 was determined as described previously (11). Myelin basic protein (MBP) kinase activity was
measured by incubating 3.5 jig of whole-cell extract with 4 ,ug
of MBP in kinase buffer containing [_y-32P]ATP at 30C for 15
min. ERK2 activity was measured by immunoprecipitation of
whole-cell extracts with anti-ERK2 antibodies (TR2, a gift
from M. Weber, recognizes ERK2 in rodent cells and was used
for FR3T3 and PC12 cells; A249, which recognizes ERK2 in
human cells, was used for HeLa and MRC5 cells) by standard
procedures (6). The immune complexes were incubated in
kinase buffer (10 mM MgCl2, 10 mM HEPES [pH 7.5], 10 ,uM
ATP) containing [_y-32P]ATP and 8 p.g of MBP for 15 min at
30C. In all assays, the phosphorylated proteins were resolved
by SDS-PAGE and visualized by autoradiography. In addition,
we analyzed JNK activity by in-gel kinase assays with GST-cJun(1-79) as a substrate as described previously (18).
Ras activation assays. PC12 cells were metabolically labeled
with 32p; as previously described (6, 8). Two minutes after cell
stimulation with nerve growth factor (NGF) (50 ng/ml) or
tumor necrosis factor alpha (TNF-ot) (10 ng/ml), whole-cell
extracts were prepared and Ha-Ras was isolated by immunoprecipitation with anti-Ha-Ras antibodies [v-H-ras(259)] from
Santa Cruz Biologicals. The bound guanine nucleotides were
eluted, and the GTP and GDP content was determined by
ascending chromatography as described previously (16).

RESULTS
ERKI and ERK2 do not phosphorylate the stimulatory sites
of c-Jun. We examined the phosphorylation of c-Jun with a
mixture of purified ERK1 and ERK2, also known as pp42 and
pp44 MAP kinases, isolated from insulin-stimulated rat la
HIRc B cells (7, 27). Although c-Jun was efficiently phosphorylated by ERK1 and ERK2, phosphopeptide mapping indicated that phosphorylation occurred at a C-terminal site(s)
rather than at Ser-73 or Ser-63 (Fig. 1). Previous studies
indicated that tryptic phosphopeptides a, b, and c correspond
to the C-terminal phosphorylation sites (8, 21), while phosphopeptides X and Y correspond to the N-terminal phosphorylation sites, Ser-73 and -63, respectively (24, 28). Identical
results were obtained with homogeneously purified ERK1 and
ERK2 that were greater than 90% pure (20a). ERK1 and
ERK2 also phosphorylated c-Jun at two minor sites, represented by phosphopeptides Ti and T2. These sites are phosphorylated to a very low extent in vivo (13) and correspond to
Thr-91, -93, or -95 (18). So far, no function can be ascribed to
phosphorylation of these residues (lOa). Using manual Edman
degradation, we confirmed previous findings (3, 10) that the
major ERK1 and ERK2 phosphorylation site on c-Jun is
Ser-243 (20a). These results differ from those of Pulverer et al.
(23, 24), who found that purified ERK1 and ERK2 phosphorylate c-Jun only at Ser-63 and -73. Despite much effort, we
failed to observe any considerable phosphorylation of c-Jun by
either ERK1 or ERK2 or a mixture of both enzymes at either
of the two N-terminal sites. By contrast, Derijard et al. (11)
have shown that immunopurified JNK1 specifically phosphorylates c-Jun at Ser-63 and -73. Although JNK1 also phosphorylates Thr-91, -93, or -95 to a lower extent, it does not
phosphorylate Ser-243, the major ERK1 and ERK2 site. The
same results were obtained with a more crude preparation
containing both forms of JNK (18). The JNKs also differ from
ERK1 and 2 by their ability to phosphorylate GST-c-Jun
fusion proteins, containing the N-terminal activation domain
of c-Jun, much more efficiently than MBP. ERK1 and ERK2,

VOL14
N-TERMINAL
194c-Jun
VOL.14, 1994

PHOSPHORYLATION AND JNK ACTIVATION

however, phosphorylate MBP much more efficiently than they

6685

He-a

MoC6
A
do GST-c-Jun fusion proteins (11, 18).
0 15' 30' 60' 90'
0
15' 30 60 90
Differential regulation of ERK and JNK. We compared the
of
ERKi
and
of
with
the
JNK
regulation
activity
regulation
ERK2 in response to a variety of extracellular stimuli in several
40- G-cJ
wikqu
cell lines. HeLa cells, human MRC5 fibroblasts, and rat FR3T3
fibroblasts were stimulated with UV light, epidermal growth
factor (EGF), or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or were left untreated. Following stimuEGF - -qm
lation, the activities of JNK, ERK1, and ERK2 in cell extracts
were compared (Fig. 2 and 3). JNK activity was assayed by a
uv
solid-state kinase assay (18) employing GST-c-Jun(1-223),
.4-G-cJ
containing the N-terminal phosphorylation sites of c-Jun, as
Tasfce
both the ligand and substrate (18). ERK activity was assayedBUVtol
with MBP, an efficient substrate for these enzymes (2). In all
three cell lines, UV irradiation was the strongest inducer of
Transtected
B Control
JNK activity, followed by EGF. TPA, however, was a relatively
I C UVI 'C TPA TNFa EGF UV
inefficient activator of JNK in HeLa and MRC5 cells and
2A and 3A).
slightly inhibited JNK activity in FR3T3 cells (Fig.TPA
(JNK showed a somewhat stronger response to in T in COS
lymphocells [11] and a considerably stronger response
cytes [30].) Phosphopeptide mapping of in vitro-phosphorylated GST-c-Jun confirmed that JNK activation correlated
UV
UV
TPAEGF C
c
C
with increased phosphorylation of Ser-63 and -73 (Fig. 3B). In
153015' 15'
fact, the ratio of phosphate incorporation into Ser-73 (phosto that into Ser-63 (phosphopeptide Y) correphopeptide X) with
*-MBP
opwo eV WO
MRC5
the extent of JNK activation measured by
lates very well
the total amount of phosphate incorporation into GST-c-Jun
(Fig. 3A). We also examined the regulation of JNK activity by
an in-gel kinase assay with GST-c-Jun(1-223) as a substrate
C TPA EGF U V (-)
As expected, UV irradiation activated both forms of JNK
(18).
15
15' 15
and a more modest activation was obtained in response to
EGF, while TPA did not significantly activate either form of 4F33
A
4*-MBP
Hela 2.
o
JNK (data not shown). In addition, we transfected an expresHeLa
cells
sion vector encoding epitope-tagged JNK1 (11) into
and examined the activation of JNK1 in response to various
FIG. 2. Activation of JNK and MBP kinases in human fibroblasts
stimuli by using an immune complex kinase assay. The results
and epithelial cells. (A) Human MRC5 fibroblasts or HeLa cells were
in medium containing 0.5% FBS for 48 or 12 h, respectively,
verified that UV irradiation was
of these experiments (Fig.
kept
m and
then were left untreated, exposed to UV light (40 J of UV-C per
the most effective activator of JNK1 of the stimuli that were
The cells
or incubated with either EGF (15
or TPA (50
tested, followed by EGF. TPA treatment had only a marginal were
stimulation,
after
at
the
harvested
indicated
times
minutes)
(in
cells.
effect on JNK1 activity in these
and whole-cell extracts were prepared. Approximately 40 of protein
By contrast, using MBP kinase assays, we foundbutthat
mixed with equal amounts of GSH-agarose beads loaded with
was
were
only
were efficiently activated by both EGF and TPA
After 3 h at
the beads were extensively washed
GST-c-Jun(1-223).
The same
weakly activated by UV irradiation (Fig. 2C and
and the solid-state phosphorylation assay was performed. After 15
results were found by an immune complex kinase assay with the proteins were resolved by SDS-PAGE. The migration position of
anti-ERK2 antibodies (Fig. 3D for FR3T3 cells and data not
GST-c-Jun is indicated (G-cJ). (B) HeLa cells were transfected with a
JNK1 expression vector (14
shown for HeLa and MRC5 cells).
plate) encoding the epitope60 h, the cells were subjected to no further
N-terminal
tagged enzyme. After
phosphorylation measured in vivo paral- treatment
(laneC); incubated with 50 ng of TPA per ml, 15 ng of EGF
lels JNK activation. To examine whether JNK or ERK activaml, or 10 ng of TNF-ox per ml; or exposed to 40 J of UV-C per
per
tion parallels the phosphorylation of Ser-63 and -73 of c-Jun,
After 15 min, the cells were harvested and JNK1 activity was deterwe treated FR3T3 cells with the agents described above and
mined by an immune complex kinase assay with the M2 monoclonal
analyzed the phosphorylation patterns of c-Jun. Phosphopepas a substrate. Extracts of nonstimuantibody and GST-c-Jun(1-79)
FR3T3 cells
tide maps of c-Jun isolated
lated (lane C) and UV-C-irradiated mock-transfected cells were used
N-terminal
indicated that UV was a very efficient inducer of
as a control. (C) MRC5 or HeLa cells were treated as described above
(indicated by the similar intensities of the X for the indicated times. Approximately 3.5 of whole-cell extract wasat
phosphorylation
incubated with 4 of MBP in kinase buffer containing
and Y spots) and EGF had a slightly weaker effect, while TPA
was ineffective (Fig. 4). In addition, both TPA and EGF 30C for 15 min. In lane (-), unstimulated whole-cell extract was
incubated without MBP. The proteins were resolved by SDS-PAGE.
enhanced the intensity of phosphopeptide b, a result that could
The migration positions of MBP are indicated.
be attributed to partial dephosphorylation of the C-terminal
sites (8), enhanced phosphorylation of Ser-243 (see above), or
a combination of both effects. These results are consistent with
previous results indicating that in HeLa cells andofhuman
ciently activated by TPA but not by UV, are unlikely to
fibroblasts, UV light is a very efficient stimulator c-Jun phosphorylate the N-terminal sites of c-Jun in these cells. On
-

--X

2B)

ng/ml)

ERKs

4C,

3C).

ng/ml).
,ug

2),

min,

,ug/100-mm

c-Jun

mi2.

from 32P-labeled

,ug

N-terminal phosphorylation (13) whereas TPA has a negligible

effect on phosphorylation of Ser-63 and -73 (8). Collectively,
these results indicate that ERKi and ERK2, which are effi-

,ug

[-y-32P]ATP

the other hand, there is a very good correlation between the

stimulation of N-terminal c-Jun phosphorylation in both HeLa
and FR3T3 cells and the activation of JNK.

MOL. CELL. BIOL.

MINDEN ET AL.

6686
A

TPA

EGF

15' 30' 60'

4- G-cJ

T2

i
C

TPA

Differential activation of JNK and ERK by Ras-dependent

and -independent stimuli. Originally, phosphorylation of the
N-terminal sites of c-Jun was found to be stimulated in
response to expression of oncogenic Ha-Ras (6, 28, 29). This
suggested that the c-Jun N-terminal kinase is also stimulated in
response to Ras activation. Indeed, JNK activity was found to
be elevated in Ha-ras-transformed cells (18), and JNK1 was
shown to be activated in response to acute Ha-Ras expression
(11). In addition, UV irradiation of HeLa cells was shown to
activate Ha-Ras (13). However, activation of Ha-Ras is also
known to result in ERK1 and ERK2 activation (15, 26, 31, 34).
Such results may have contributed to the confusion regarding
the role of ERK1 and ERK2 in c-Jun phosphorylation. To
further examine the relationships between activation of HaRas and the stimulation of JNK and ERK activity in response
to extracellular stimuli, we used PC12 pheochromocytoma
cells. As expected, incubation of these cells with either EGF
(data not shown) or NGF resulted in activation of Ha-Ras, as
evidenced by the increase in the amount of GTP bound to this
protein (Fig. 5A). Incubation with TNF-ot, however, did not
result in Ha-Ras activation at any of the time points tested
(Fig. 5A and data not shown). Consistent with these results,
treatment with EGF (data not shown) or NGF resulted in
activation of ERK2, as indicated by an immune complex kinase
assay, while TNF-ax had only a minor effect on ERK2 activity
(Fig. SB). JNK activity, on the other hand, was strongly
activated by TNF-ot and more modestly by NGF (Fig. 5B). The
response of JNK to EGF was of a magnitude similar to that of
its response to NGF (data not shown). These results suggest
that while ERKI and ERK2 activation is coupled mostly to
activation of Ha-Ras in these cells, the JNKs can be activated
in response to both Ras-dependent and -independent signals.

90

NWP~ -4-- G-cJ

bb
.

*--G-cJ

...........

TPA
..

EGF

UV

DISCUSSION

15'

30'

60'

Our results indicate that c-Jun N-terminal phosphorylation

and JNK activity are regulated very similarly, suggesting that
the JNKs are responsible for the stimulation of c-Jun transcriptional activity. This conclusion is further supported by the
ability of the JNKs to bind to the c-Jun activation domain and
the requirement of this binding for stimulation of c-Jun
transcriptional activity (18). We have recently shown that
recombinant JNK1 specifically and efficiently phosphorylates
the N-terminal sites of c-Jun without affecting any of its
inhibitory C-terminal sites (11). Collectively, these results
indicate that either JNK1 or JNK2 (the 55-kDa form) is
responsible for stimulating the transcriptional activity of c-Jun
in vivo. Despite earlier reports that the more classical members
of the MAP kinase group, ERK1 and ERK2, can also phosphorylate the stimulatory N-terminal sites of c-Jun (23, 24), we

90'

_._.

MBP

E.
,,
__

'l-I
EGF__

UV

D
C

4-. MBP

NRS

EGF TPA

MBP

aERK2
C EGF TPA

aERK2
C
UV

_
v

4--MBP

FIG. 3. Regulation of JNK and ERK activities in FR3T3 cells. (A)

Rat FR3T3 cells were kept in medium containing 0.5% FBS for 12 h
and subjected to the treatments described in the legend to Fig. 2. At
the indicated time points, cells were harvested, and extracts were used
to phosphorylate GST-c-Jun(1-223) (G-cJ) as described above. (B) In

vitro-phosphorylated GST-c-Jun(1-223) from the 15-min time point

was eluted from SDS-PAGE gels and digested with trypsin as described previously (8). The digests were applied to thin-layer cellulose
plates and resolved as described in the legend to Fig. 1. See the legend
to Fig. 1 for definitions of the abbreviations. (C) FR3T3 cells were
treated as described above for the indicated times or left in growth
medium containing 0.5% FBS, and extracts were prepared. Extracts
were used to phosphorylate MBP with the kinase buffer described in
the legend to Fig. 2C. (D) Cells were treated as described above for 15
min or were left untreated (lane C). ERK2 was then immunoprecipitated from whole-cell extracts with a specific antibody (a gift from M.
Weber). Normal rabbit serum (NRS) was used as a control. The
immune complexes were incubated with kinase buffer containing
[y-32P]ATP and 8 ,ug of MBP for 15 minutes at 30C.

TPA

Control

NGF

TNFcx

*x

GTP-_
:::.:.
.....

.:fkf:

.,..:. :.........

...

.. ;.d.

-i

,.:.,,, | S d '".W:.:'we.5 .F

*. : :. .. IS Fa.! I =;$W.i.i:. . .::.

'...,,,., ." |

l E.l'.W,^ ..-,'.

FM

r;

..

uv

EGF

i-V

FIG. 4. Regulation of c-Jun phosphorylation in FR3T3 cells.

FR3T3 cells were kept in growth medium containing 0.5% FBS for 12
h and were then metabolically labeled with 32p; for 3 h. Equal numbers
of cells were exposed to EGF (15 ng/ml), TPA (50 ng/l), UV (40 J/m2),
or no further treatment (Control). After 15 min, the cells were lysed
and c-Jun was immunoprecipitated, separated by SDS-PAGE, and
subjected to phosphopeptide mapping as described in the legend to
Fig. 1. See the legend to Fig. 1 for definitions of the abbreviations.

find that these protein kinases phosphorylate an inhibitory

C-terminal site rather than the N-terminal sites that are
phosphorylated by JNK. Furthermore, we find no correlation
between the activation of ERK1 and ERK2 and the stimulation of N-terminal c-Jun phosphorylation. While both ERK1
and ERK2 and JNK are activated by growth factors, JNK is
preferentially activated in response to UV irradiation and
TNF-a, which have little effect on ERK activity. Although
Radler-Pohl et al. (25) detected activation of ERK1 and ERK2
in response to UV irradiation and postulated it to have a role
in stimulation of c-Jun phosphorylation and transcriptional
activity, we note that the level of ERK activation observed by
those authors was low and very similar to the rather weak
activation reported here. On the other hand, ERK activity is
efficiently stimulated by TPA, which does not stimulate c-Jun
N-terminal phosphorylation and is a weak activator or even an
inhibitor of JNK in fibroblasts and epithelial cells.
The results from this work suggest that JNK is regulated by
a signal transduction pathway that is different from the one
which regulates ERK1 and ERK2. Activation of JNK parallels
c-jun transcription (5). Yet activation of ERK1 and ERK2
parallels the induction of c-fos, and serum-activated protein
kinases with sizes identical to those of ERK1 and ERK2

1:

6687

NGF TNFa

GDP-_W
Y

X,!r.

c-Jun N-TERMINAL PHOSPHORYLATION AND JNK ACTIVATION

VOL. 14, 1994

4W

*-G-cJ

-*

MBP

FIG. 5. Activation of JNK involves Ras-dependent and Ras-independent pathways. (A) PC12 cells were metabolically labeled with 32p;
for 3 h and stimulated with NGF (50 ng/ml) or TNF-a (10 ng/ml) for
2 min or given no further treatment (lanes C). Following stimulation,
cell extracts were prepared and Ras GTP content was measured as
described previously (16). Eluted GTP and GDP are indicated. (B)
PC12 cells were stimulated with NGF or TNF-ot for 15 min or were left
untreated (lane C), and whole-cell extracts were prepared. JNK
activity was measured by the solid-state kinase assay with GST-c-Jun
(G-cJ) as a substrate (18). ERK activity was measured by an immune
complex kinase assay with MBP as a substrate.

phosphorylate and stimulate the activity of TCF/Elk-1, a

transcription factor that activates the c-fos promoter (17, 22,
32). Furthermore, dominant negative ERK1 and ERK2 mutants inhibit c-fos promoter activation (20, 33). Previous studies have shown that TPA induces c-fos more efficiently than it
does c-jun (4), whereas UV light and TNF-ao induce c-jun more
efficiently than they do c-fos (9, 12). The induction of the two
components of the AP-1 complex through different and distinctly regulated members of the MAP kinase group could
ensure that AP-1 activity responds to a large spectrum of
extracellular stimuli. It is also possible that through the divergent activation of JNK and ERK, different extracellular stimuli
induce different types of AP-1 complexes (e.g., Jun-Jun homodimers versus Jun-Fos heterodimers) and thereby lead to
activation of overlapping yet different sets of target genes.
ACKNOWLEDGMENTS
We thank M. Weber for the anti-ERK2 antibodies and Y. BenNeriah for his critical comments on the manuscript.
A.M. and A.L. were supported by postdoctoral fellowships from the
Tobacco Related Disease Research Program (TRDRP). This work was
supported by grants from the National Institutes of Health, the
American Cancer Society, the Council for Tobacco Research, and the
TRDRP.
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MINDEN ET AL.

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