(Received for publication, October 8, 1996, and in revised form, November 18, 1996)
From the Molecular Biology and Virology Laboratory, The Salk Institute, La Jolla, California 92037
Ternary complex factors (TCFs) bind to the serum response element in the c-fos promoter and mediate its activation by many extracellular stimuli. Some of these stimuli activate the ERK subclass of mitogen-activated protein kinases (MAPKs) that target the TCF Sap-1a. We show that Sap-1a is also phosphorylated by the stress-activated JNK subclass of MAPKs leading to stimulation of both c-fos serum response element and E74-site-dependent transcription in RK13 cells. Several JNK-1 phosphorylation sites were mapped within Sap-1a, and mutation of these sites affected the transactivation mediated by Sap-1a and JNK-1. The impact of these phosphorylation sites varied at different promoters and was dependent on whether Sap-1a was stimulated by ERK-1 or JNK-1. Additionally, a comparison of Sap-1a with another TCF, Elk-1, revealed that these proteins behaved differently to stimulation by ERK-1 and JNK-1. Furthermore, activation of Sap-1a by JNK-1 was inhibited by the p38MAPK in RK13 cells, possibly by competition for a common upstream activator. Altogether, our data suggest that Sap-1a plays an important role in the nuclear response elicited by cellular stress.
The c-fos proto-oncogene encodes a transcription factor whose mutation or overexpression can lead to the development of bone tumors (1). In addition, the c-Fos protein is involved in the malignant progression of skin tumors (2), is required for the generation of osteoclasts (3), and may participate, as part of the AP-1 transcription factor, in the regulation of a variety of genes (1).
The c-fos gene is shut down in quiescent cells, and gene transcription starts within minutes after stimulation of cells with growth factors, phorbol esters, neurotransmitters, cytokines, oxidants, ultraviolet light, or Ca2+ (4). Three major promoter elements have been identified conferring this promiscuous inducibility of c-fos: the sis-inducible element, the serum response element (SRE),1 and the cAMP response element. However, the majority of the signals enters the c-fos promoter via the SRE, and the responses via the other elements are dependent on the presence of the SRE, which identifies the SRE as the pivotal control element within the c-fos promoter (5-7).
Before, during, and after induction of cells the c-fos SRE is occupied by a protein complex consisting of the serum response factor (SRF) and a ternary complex factor (TCF) (8), which belongs to the ETS transcription factor family (9). TCFs require protein-protein interactions with SRF in order to bind stably to the c-fos SRE, while the SRF protein can bind independently of TCF. However, TCFs also interact with and function in the absence of SRF at high affinity ETS binding sites (10).
Presently, three TCFs have been identified: Elk-1, Sap-1a, and Sap-2/Erp/Net (4). Phosphorylation of a TCF by the ERK subclass of mitogen-activated protein kinases (MAPKs) induces c-fos gene transcription in response to serum or growth factors (10-14). In addition, it has been shown that Elk-1 is phosphorylated by the stress-activated JNK subclass of MAPKs (15-18).
In this report, we demonstrate that the TCF Sap-1a is a target for JNK-1. Phosphorylation occurs at multiple sites, and most of them contribute to Sap-1a-mediated transactivation in a promoter-dependent manner. However, in comparison to Elk-1, Sap-1a is less potently activated by JNK-1, while the opposite appears to be true for activation by ERK-1 at the c-fos SRE. Furthermore, we show that JNK activation of Sap-1a can be negatively interfered with by the p38MAPK, which belongs to a third class of mammalian MAPKs (19).
Expression vectors for Sap-1a and point mutants thereof, Sap-1a-(1-267), Elk-1, and GAL4 fusions, have been described before (10, 12). GST (glutathione S-transferase) fusion protein expression vectors were constructed by isolating the EcoRI/BamHI inserts from the respective G/S268-431 clones (10), ligating them into EcoRI/BamHI-cut pBluescript KS+ (Stratagene), isolating the EcoRI/SacI inserts, and ligating them into pGEX2T-6His-PL2 (kindly provided by R. A. Hipskind), which was linearized with EcoRI and SacI. BXB and hemagglutinin (HA)-tagged ERK-1 mammalian expression vectors have been described in the literature (20, 21). The luciferase reporter plasmids were derived from the tk80-luc vector by cloning either two copies of the c-fos SRE or three copies of the E74 binding site in front of a herpes simplex virus thymidine kinase basal promoter and the firefly luciferase cDNA (12).
Production of GST Fusion ProteinsEscherichia
coli BL21 bacteria, which were transformed with a GST fusion
protein expression vector, were used to inoculate a 100-ml culture and
were grown to an optical density of 0.8 measured at 600 nm. Then,
bacteria were induced with 0.5 mM
isopropyl--D-thiogalactopyranoside for 3 h and
harvested by centrifugation. After a wash with phosphate-buffered saline, bacteria were suspended in 10 ml of 6 M guanidine
HCl, 0.1 M sodium phosphate, pH 8, frozen for 10 min at
80 °C, and shaken for 1 h at room temperature. Debris was
removed by centrifugation, and the supernatant was incubated with 0.5 ml of Ni2+-nitrilotriacetic acid-agarose (Qiagen) for 3-12
h. The slurry was poured into a column, and after extensive washing
with 6 M guanidine HCl, 0.1 M sodium phosphate,
pH 8, bound proteins were eluted with 4 × 0.5 ml of 6 M guanidine HCl, 0.1 M sodium phosphate pH 4. Proteins were renatured by stepwise dialysis against 25 mM
Hepes, pH 7.5, 100 mM NaCl, 10% glycerol, 5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 8 M, 4 M, 2 M, 1 M, or no
urea at 4 °C. After the removal of precipitated material, renatured
proteins were frozen in liquid nitrogen and stored at
70 °C.
HA-tagged JNK-1 or HA-tagged ERK-1
was transiently produced in transfected rabbit kidney epithelial-like
RK13 cells grown on 6-cm dishes. 36 h after transfection, RK13
cells were washed once with phosphate-buffered saline and then lysed
with 600 µl of lysis buffer (10 mM Tris, 30 mM Na4P2O7, pH 7.1, 50 mM NaCl, 50 mM NaF, 1% Triton X-100, 0.2 mM dithiothreitol, 20 µg/µl aprotinin, 20 µg/µl
leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM Na3VO4) at 4 °C for 5 min.
After scraping and transfer to a microcentrifuge tube, the lysate was
briefly vortexed and tumbled for 20 min, and debris was removed by
centrifugation. 1 µl of monoclonal 12CA5 antibody (Boehringer
Mannheim) was added to the supernatant, and 15 µl of agarose beads
with coupled protein A (RepliGen) after 1 h of incubation. After
one more hour, the beads were pelleted by centrifugation for 20 s,
washed three times with 500 µl of the above described lysis buffer,
and then twice with 500 µl of kinase buffer (15 mM Mops,
pH 7.2, 12 mM MgCl2, 15 mM -glycerophosphate, 0.5 mM EGTA, 0.2 mM
dithiothreitol, 0.5 mM Na3VO4, 10 µM ATP). Finally, the beads were suspended in 50 µl of
kinase buffer, and a typical in vitro kinase reaction was
set up with 10 µl of this slurry plus 1 µg of purified protein (in a volume of approximately 1 µl) plus 0.05 µM
[
-32P]ATP (3000 Ci/mmol). The reaction was allowed to
proceed for 30 min at 30 °C and then stopped by boiling in SDS-PAGE
sample buffer. Phosphorylated proteins were resolved on a 10% SDS-PAGE gel, and the gel was, after drying, exposed to an x-ray film.
GST-Sap proteins were
phosphorylated in vitro as described above with the
following modifications: the ATP concentration was 1 µM
in the kinase buffer, and the amount of radioactivity was increased to
0.5 µM [-32P]ATP (3000 Ci/mmol). After
SDS-PAGE, a gel slice with the protein of interest was cut out of the
dried gel, rehydrated with 500 µl of freshly made 50 mM
NH4HCO3, and then ground up. Another 500 µl
of 50 mM NH4HCO3, 10 µl of
-mercaptoethanol, and SDS to a final concentration of 0.1% were
added, and the mixture was boiled for 3 min. After 5 h of
extraction, another extraction with such a volume of 50 mM
NH4HCO3 was performed so that the combined
supernatants had a volume of 1300 µl. After a final clear spin, 20 µg of boiled RNase A and 300 µl of ice-cold trichloracetic acid
were added, and proteins were precipitated for 1 h at 4 °C. After centrifugation, the pellet was washed once in ice-cold ethanol, air-dried, and taken up in 100 µl of 50 mM
NH4HCO3 supplemented with 5 µg of
chymotrypsin. Proteolytic digestion was performed for a total of 6 h with another 5 µg of chymotrypsin added after 3 h. 500 µl of
water were added, and the sample was lyophilized, relyophilized after
addition of 300 µl of water, and finally dissolved in 400 µl of pH
1.9 buffer (88% (w/v) formic acid:glacial acetic acid:water
(50:156:1794)). After centrifugation, the supernatant was lyophilized
and the pellet was dissolved in pH 1.9 buffer in a volume adequate for
two-dimensional phosphopeptide mapping. This was performed by spotting
up to 5 µl of the phosphopeptide mixture onto cellulose thin-layer
plates (Merck Darmstadt, No. 5716), electrophoresis in pH 1.9 buffer
(1.3 kV; 25 min), and ascending chromatography in
1-butanol:pyridine:glacial acetic acid:water (75:50:15:60) in the
second dimension (22).
RK13 cells were transiently
transfected by the calcium phosphate coprecipitation method with
indicated luciferase reporter gene constructs and protein expression
plasmids (12). Luciferase activity was determined 36 h after
transfection and normalized to transfection efficiency with the help of
a cotransfected -galactosidase expression vector (10). Typically,
the results are given as the mean (± S.E.) of three experiments.
To investigate whether the
ETS family transcription factor Sap-1a can be phosphorylated by JNK-1,
HA-tagged JNK-1 was transiently expressed in RK13 cells,
immunoprecipitated with an anti-HA antibody, and then employed in an
in vitro kinase assay with a GST fusion protein encompassing
the C-terminal transactivation domain of Sap-1a. As shown in Fig.
1A (upper left panel, lane
7), JNK-1 alone was unable to elicit a significant degree of
phosphorylation of GST-Sap. However, coexpression of an upstream
activator of JNK-1, a constitutively activated form of a MAPK kinase
kinase (MEKKc), resulted in considerable phosphorylation of GST-Sap
(lane 6). As a control, the GST moiety itself was not
phosphorylated by JNK-1 (Fig. 1A, lower left
panel). We then compared the phosphorylation of GST-Sap by JNK-1
and by immunoprecipitated HA-tagged ERK-1. Already ERK-1 expression on
its own led to some phosphorylation of GST-Sap, and coexpression of the
ERK-1 upstream activator BXB, which is a constitutively active Raf-1
kinase (20), potentiated this phosphorylation (Fig. 1A,
upper left panel, lanes 2 and 3). Since the amounts of HA-tagged JNK-1 present in the immunoprecipitates were even slightly higher than those for HA-tagged ERK-1 as judged by
Western blotting utilizing an anti-HA antibody (Fig. 1B),
GST-Sap appears to be a better substrate for ERK-1 than for JNK-1
in vitro, while the opposite holds true for the Jun
transcription factor (Fig. 1A, upper right panel)
that is the prototypical JNK substrate. Likewise GST-Sap, the GST
fusion protein containing the C-terminal activation domain of Elk-1 was
more readily phosphorylated by ERK-1 than by JNK-1 in vitro
(Fig. 1A, lower right panel), although this
difference was not as pronounced as with GST-Sap.
Since we wanted to employ BXB and MEKKc as selective activators of ERK-1 and JNK-1, respectively, we had to demonstrate that BXB does not activate JNK-1 and, conversely, that ERK-1 was not activated by MEKKc. Therefore, BXB and JNK-1 or alternatively MEKKc and ERK-1 were coexpressed, and the ability of JNK-1 and ERK-1 to phosphorylate GST-Sap, GST-Elk, and histidine-tagged Jun in vitro was assessed. No activation of JNK-1 by BXB or of ERK-1 by MEKKc was observed (Fig. 1A); rather, as can be best seen with the in vitro phosphorylation of GST-Elk, a small inhibitory effect of BXB on JNK-1 (compare lanes 7 and 8) and of MEKKc on ERK-1 (compare lanes 3 and 4) was noticeable. This is not due to an effect of BXB or MEKKc on the level of JNK-1 or ERK-1 expression, since protein levels were fairly comparable under these conditions (Fig. 1B). Thus, cross-activation between the BXB-activated ERK pathway and the MEKKc-activated JNK pathway does not occur in RK13 cells under our experimental conditions.
Stimulation of Sap-1a Activity by JNK-1Previously, Sap-1a
was shown to stimulate transcription upon phosphorylation by ERKs (10,
14). This was reproduced here with a c-fos SRE-driven
luciferase construct (Fig. 2A). Expression of
ERK-1 alone had little effect on c-fos SRE-driven luciferase activity, indicating that the basal activity of ERK-1 as observed in
our in vitro kinase assay (see Fig. 1A) was
insufficient to trigger gene transcription. However, the joint
expression of BXB and ERK-1 stimulated transcription by nearly 30-fold,
while BXB alone was 4 times less stimulatory presumably due to the
levels of endogenous ERKs being insufficient. As a control, the
Sap-1a-(1-267) molecule was employed, which lacks the C-terminal
activation domain but is still capable of binding to DNA and
interacting with the SRF protein (10). No activation of transcription
was observable with Sap-1a-(1-267) (Fig. 2A), proving once
more that the C terminus of Sap-1a is required for transactivation.
Also, Sap-1a was unable to elicit an activation of a reporter construct
devoid of a c-fos SRE (Fig. 2C), indicating that
the observed effects were binding site-specific.
Expression of JNK-1 had no effect on Sap-1a-mediated c-fos SRE stimulation, but expression of MEKKc led to 5-fold activation of luciferase activity (Fig. 2A). This degree of activation could be enhanced to 16-fold by coexpression of JNK-1, indicating that JNK-1 and MEKKc synergize to activate Sap-1a. Again, Sap-1a-(1-267) was unresponsive to stimulation, and the effects were binding site-specific (see Fig. 2C). Consistent with the in vitro phosphorylation data, MEKKc was unable to cooperate with ERK-1 nor was BXB able to do so with JNK-1, confirming that the ERK and JNK pathways are not stimulated by the same upstream activator in RK13 cells.
Sap-1a is not only capable of forming a ternary complex with the SRF protein at the c-fos SRE but can also, independently of SRF, activate transcription from an ETS binding site such as the E74 site (10). Thus, we tested whether Sap-1a was able to mediate activation of transcription via the E74 site upon expression of MEKKc and JNK-1. As shown in Fig. 2B, Sap-1a-dependent transcription was stimulated by both MEKKc/JNK-1 and BXB/ERK-1. Thus, Sap-1a is not only a target for JNK-1 at the c-fos promoter but also at ETS binding sites without a juxtaposed SRF binding site.
Next, we investigated whether the C-terminal transactivation domain of
Sap-1a is capable of responding to MEKKc/JNK-1 when fused to the DNA
binding domain of the yeast protein GAL4. As shown in Fig.
3, the respective GAL4-Sap-(268-431) fusion protein was
stimulated more than 200-fold by MEKKc/JNK-1, and a similar degree of
activation was obtained upon coexpression of BXB/ERK-1. MEKKc or BXB
alone were again less active than in combination with the respective
MAPK, and expression of ERK-1 or JNK-1 alone revealed no significant
effect on transactivation (data not shown). Similarly, the
GAL4-Elk-(307-428) fusion protein was activated more than 200-fold by
MEKKc/JNK-1, while induction by BXB/ERK-1 was 3-fold less effective. In
contrast, the GAL4 moiety on its own was only slightly inducible, which
demonstrates that the activation of transcription mediated by the GAL4
fusion proteins is due to the C-terminal activation domains of Sap-1a
and Elk-1.
Finally, we compared the two TCFs Sap-1a and Elk-1 under conditions
where both factors are expressed to an equivalent level (10). Sap-1a
was more sensitive to activation by ERK-1 than by JNK-1 (Fig.
4), and while activation levels at the c-fos
SRE and the E74 site were comparable upon ERK-1 activation, the E74 site was only stimulated about half as well as the c-fos SRE
upon JNK-1 activation. On the contrary, Elk-1 activated
c-fos SRE-dependent transcription more upon
JNK-1 than upon ERK-1 stimulation, while the opposite held true for E74
site-dependent transcription (Fig. 4). Also, Elk-1 was more
active at the E74 site than at the c-fos SRE. Furthermore,
while Elk-1 was more active than Sap-1a on the E74 site, Sap-1a was
2.6-fold more inducible at the c-fos SRE upon ERK-1
stimulation but ~35% less inducible than Elk-1 upon JNK-1
stimulation. Altogether, Sap-1a and Elk-1 do not behave equivalently in
response to activation of the ERK and JNK MAPK pathways.
Identification of Phosphorylation Sites
To identify JNK-1
phosphorylation sites within Sap-1a, GST fusion proteins of wild-type
Sap-1a and mutations thereof at potential MAPK sites were
phosphorylated in vitro by JNK-1 and digested with
chymotrypsin, and the resulting phosphopeptides were separated in two
dimensions on cellulose thin-layer plates (Fig. 5).
Phosphopeptides 3-5 vanished upon mutation of serines 381 and 387 to
alanines, and phosphopeptides d-f vanished upon mutation of threonine
420 and serine 425, indicating that these sites are targeted by JNK-1. In addition, mutation of threonines 361, 366, and 420 and serines 381, 387, and 425 led to the disappearance of phosphopeptides-(3-5) and c-f, indicating that phosphopeptide c is due to phosphorylation at
threonines 361 and 366. However, more JNK-1 phosphorylation sites exist
in Sap-1a, since the phosphopeptides a and b were still observable with
the latter mutant. In conclusion, Sap-1a is phosphorylated by JNK-1 at
several sites, including residues 361, 366, 381, 387, 420, and 425.
We next assessed the importance of these phosphorylation sites for the
activity of Sap-1a. To that end, alanine mutations of Sap-1a were
compared with the wild-type molecule for their ability to activate
c-fos SRE and E74 site-dependent transcription (Fig. 6A). The alanine double mutant
T361A/T366A displayed a reduced transactivation potential, behaved
similarly in response to BXB/ERK-1 and MEKKc/JNK-1 stimulation, and was
less active on the c-fos SRE than at the E74 site. The
single alanine mutants S381A and S387A were only 50% as active as
wild-type Sap-1a upon MEKKc/JNK-1 stimulation, but they had nearly
wild-type activity upon BXB/ERK-1 stimulation on the c-fos
SRE. Consistently, the S381A/S387A double mutant reduced
transactivation to a level of ~10% of wild-type Sap-1a at the
c-fos SRE upon MEKKc/JNK-1 stimulation compared with only
25% upon BXB/ERK-1 stimulation. No such difference was observable at
the E74 site where the response to both ERK-1 and JNK-1 was reduced
equally. The T420A/S425A mutant was approximately 30% less active than
the wild-type with the exception of BXB/ERK-1 stimulation at the
c-fos SRE where it was ~40% more active than wild-type
Sap-1a. Finally, mutation at all six aforementioned sites resulted in a
protein that was only ~5% as active as the wild-type, indicating
that phosphorylation at several sites, especially at threonines 361 and
366 and serines 381 and 387, cooperatively stimulates Sap-1a-mediated
transactivation.
Sap-1a and Elk-1 are very similar to each other at the C terminus and possess homologous potential MAPK phosphorylation sites (Fig. 6B). Thus, we tested whether alanine mutants of Elk-1 would display a phenotype comparable with the homologous Sap-1a mutants. The Elk-1 S383A mutation caused a drastic phenotype, especially with regard to activation of the E74 site upon BXB/ERK-1 stimulation, but surprisingly had much less effect on MEKKc/JNK-1 stimulation at the E74 site, a behavior clearly distinct from the homologous S381A mutant of Sap-1a. Mutation of serine 389 in Elk-1 resulted in a protein that responded differently to BXB/ERK-1 and MEKKc/JNK-1 on the E74 site; either ~50% less active than the wild-type or ~30% more active, respectively. Again, this behavior contrasts that of the homologous Sap-1a mutant S387A. Finally, while the T420A/S425A mutant of Sap-1a, as well as the single T420A and S425A mutants (data not shown), behaved similarly to the wild-type, mutation of the homologous sites in Elk-1 at positions 417 or 422 reduced transactivation at least 3-fold under all conditions tested. Thus, the homologous phosphorylation sites in Elk-1 and Sap-1a affect the function of these two TCFs differently.
p38MAPK Blocks JNK-1 ActivationHaving
established that two of the MAPK subclasses target Sap-1a, we tried to
detect an activation of Sap-1a by the p38 subclass of MAPKs in RK13
cells but failed to do so, although it can occur in other cell
types.2 Rather, we observed that
application of the specific p38MAPK inhibitor SB202190 (23)
even promoted slightly the Sap-1a/JNK-1-mediated activation of the
c-fos SRE-driven luciferase reporter (Fig.
7A), which may indicate that endogenous
p38MAPK inhibits JNK-1. Indeed, exogenous
p38MAPK was able to reduce MEKKc/JNK-1-mediated
transactivation of Sap-1a (Fig. 7A).
These results prompted us to investigate whether p38MAPK could inhibit JNK-1 activation. To that end, increasing amounts of p38MAPK were coexpressed with JNK-1/MEKKc, and JNK-1 activity was measured in an in vitro kinase assay after immunoprecipitation. As shown in Fig. 7B, p38MAPK drastically reduced the activity of JNK-1 in vitro without significantly affecting the protein levels. Less inhibition was observable when the cells had been incubated with the SB202190 compound (data not shown). As a control, ERK-1 activation by BXB was not inhibited by p38MAPK. One possible reason for the inhibitory effect of p38MAPK on JNK-1 could be the sequestration of a common upstream activator. Indeed, coexpression of JNK kinase (JNKK), which can activate both JNKs and p38MAPK (24), reduced the inhibitory effect of p38MAPK on JNK-1 activation (Fig. 7B). Altogether, these data suggest that p38MAPK can interfere with the activation of JNK-1 in RK13 cells.
In this report, we have shown that the transcription factor Sap-1a is phosphorylated by JNK-1 at multiple sites and thus activated to stimulate transcription from either the c-fos SRE or an ETS binding site. Phosphorylation at the different serine and threonine residues in the C-terminal activation domain appears to cooperate in enhancing the transactivation potential of Sap-1a, since the more these amino acids were mutated to alanine, the less active Sap-1a was. The phosphorylation sites in Sap-1a that are recognized by JNK-1 are also targeted by ERKs (10, 14), but we observed that they may have different importance for signaling via ERK-1 and JNK-1. For instance, alanine instead of serine at position 387 reduced Sap-1a activation of the c-fos SRE by one-half upon JNK-1 stimulation, but had no impact on stimulation by ERK-1. This hints at the fact that the stoichiometry of phosphorylation at a particular site is different upon ERK-1 and JNK-1 phosphorylation, and ERK-1 and JNK-1 indeed appear to have a different site preference in the Sap-1a molecule.3
Interestingly, alanine mutants of Sap-1a had a different quantitative impact on Sap-1a-mediated transactivation measured with a c-fos SRE or an E74-site-driven luciferase reporter construct. Since the employed reporter plasmids were identical except for the SRE or E74 binding site, this is not an effect due to different core promoters. Rather, one possible explanation is that the surface of Sap-1a at these binding sites is different; at the c-fos SRE, but not at the E74 site, Sap-1a is interacting with SRF, which may alter its conformation. Thus, phosphorylation sites would be exposed differently at the c-fos SRE and the E74 site and could therefore contribute differently to the interaction with its coactivator CBP (25, 26) or components of the basal transcription machinery. Our results highlight the fact that alterations of protein conformation by either inter- or intramolecular protein-protein interactions may lead to different effects of phosphorylation at a particular site with regard to the activity of a protein. Since intramolecular interactions between N- and C-terminal regions of TCFs cannot be excluded (27), this may cast doubts on studies using fusions of the C terminus to a heterologous DNA binding domain such as that from the yeast transcription factor GAL4 to study the impact of phosphorylation. However, our study proves that full-length Elk-1 is also targeted and activated by JNK-1, as shown previously solely with fusion proteins (15-17).
A comparison of Sap-1a with Elk-1 revealed that these two TCFs respond differently to ERK and JNK activation depending on the promoter context. It appears that Elk-1 is in general more responsive to JNK-1 stimulation, while ERK-1 activates Sap-1a more than Elk-1 at the c-fos SRE and vice versa at the E74 site. Since TCFs are expressed in a cell-type-specific manner (13), the molar ratio of Elk-1 to Sap-1a could determine the strength of gene activation in response to ERKs and JNKs. However, only p46 JNK-1 was employed in this study, and at least 10 different JNK isoforms encoded by three genes exist (28). These isoforms appear to have preferences in the targeting of different transcription factors (28), which leaves the possibility that one or more of them phosphorylates and thus activates Sap-1a more than Elk-1 in contrast to p46 JNK-1. Thus, in addition to the molar ratio of TCFs expressed in a certain cell type, the spectrum of JNKs present would also determine the degree of activation of a TCF-regulated gene.
Surprisingly, we found that p38MAPK can inhibit the stimulation of JNK-1 in RK13 cells. Since expression of the common upstream activator JNKK (24) alleviated this repression, this effect is, at least in part, due to the competition for a limiting upstream activator. Thus, cells with a high load of p38MAPK may not be able to activate the JNK pathway efficiently. Interestingly, application of the p38MAPK inhibitor SB202190 led to a smaller degree of JNK-1 inhibition. This may be due to a lower affinity of SB202190-complexed p38MAPK for JNKK or may indicate that enzymatic activity of p38MAPK is also contributing to repression of JNK-1 activity, for instance by phosphorylation and activation of JNK phosphatases or by an inhibitory phosphorylation of JNK kinases.
Extracellular stimuli leading to the activation of JNKs may take
different routes within the cell. Some of these stress signals, such as
ultraviolet light or heat, may lead to the activation of the Rac and
Cdc42 small G-proteins (29), while tumor necrosis factor or changes
in osmolarity lead to the activation of the Ca2+-induced
Pyk2 protein kinase (30). But finally, these different routes converge
on the JNKs, and our study has identified Sap-1a as a novel effector of
this subclass of MAPKs that may therefore play an important role in the
nuclear response to stress. Often, stress induces apoptosis of cells,
and it has recently been shown that JNK activation is required for
ceramide-initiated apoptosis (31). This suggests that Sap-1a may also
be involved in programmed cell death, consistent with the fact that
c-fos is induced prior to apoptosis in vivo
(32).
We thank R. Davis for providing p38MAPK expression plasmid, D. Bohmann for the 6His-Jun bacterial expression vector, and M. Karin for SB202190 as well as the MEKKc, HA-JNK-1, and JNKK expression vectors.