(Received for publication, November 29, 1994; and in revised form, January 27, 1995)
From the
Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro-inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro-inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group.
Several MAP ()kinase signal transduction pathways
have been molecularly characterized(1) . At least four
genetically distinct signaling pathways have been defined in the yeast Saccharomyces cerevisiae(2) . One pathway leads to
activation of the FUS3 and KSS1 MAP kinases and is required for the
response to mating pheromone(3) . A second MAP kinase pathway
(MPK1) functions during cell wall biosynthesis(4, 5) .
A third genetically defined MAP kinase pathway (HOG1) is involved in
osmoregulation(6) . The fourth MAP kinase pathway (SMK1) is
required for the control of sporulation(7) . Significantly,
these MAP kinase pathways appear to function independently because
mutations that disrupt one pathway do not alter signal transduction
mediated by the other pathways(2) . This independent function
may arise from the substrate specificity of the MAP kinase cascades. In
addition, it has been established that there is an important role for
tethering proteins (e.g. STE5) that bind multiple components
of the MAP kinase cascade to create a functional signal transduction
module(8, 9) .
Although detailed information is available for yeast, the organization of MAP kinase pathways in mammals is more poorly understood. The ERK group of MAP kinases is activated by growth factors via a Ras-dependent signal transduction pathway(10) . In contrast, the JNK group of MAP kinases (also designated SAPK) is activated by pro-inflammatory cytokines and environmental stress(11, 12, 13, 14, 15, 16, 17) . JNK activation is also observed during co-stimulation of T lymphocytes(18) . Importantly, the signal transduction pathways that lead to ERK and JNK activation are biochemically and functionally distinct(11) .
Recently, a novel mammalian MAP kinase (p38) was identified by Han et al.(19) . This MAP kinase isoform has been implicated in the mechanism of activation of MAPKAP kinase-2 (20, 21) and the expression of pro-inflammatory cytokines(22) . Homologs of p38 MAP kinase (CSBP1 and CSBP2) have been identified in human tissues(22) . A p38 MAP kinase homolog (MPK2) has also been identified in Xenopus laevis(20) . The purpose of this study was to examine the mechanism of p38 activation and to establish the relationship of the p38 MAP kinase pathway to the ERK and JNK signal transduction pathways.
The plasmid pCMV-Flag-JNK1 (11) and
the expression vectors for human MKP-1 (CL100) and PAC-1 (29) have been described. The plasmid pCMV-Flag-p38 MAP kinase
was prepared using the expression vector pCMV5 (30) and the
p38 cDNA. The Flag epitope (-Asp-Tyr-Lys-Asp-Asp-Asp-Aps-Lys-; Immunex
Corp.) was inserted between codons 1 and 2 of p38 by insertional
overlapping polymerase chain reaction(31) . A similar
polymerase chain reaction procedure was employed to replace Thr and Tyr
with Ala and Phe, respectively. The
sequence of all plasmids was confirmed by automated sequencing using an
Applied Biosystems model 373A machine.
Control experiments were performed to assess the specificity of the observed immunofluorescence. No fluorescence was detected when the transfected cells were stained in the absence of the primary M2 monoclonal antibody. In addition, we did not observe fluorescence in experiments using mock-transfected cells. Together, these data demonstrate that the immunofluorescence observed detects the epitope-tagged p38 MAP kinase.
Figure 1:
Substrate specificity of
p38 MAP kinase. Panel A, substrate phosphorylation by p38 MAP
kinase was examined by incubation of bacterially-expressed p38 MAP
kinase with different proteins and [-
P]ATP.
The mutated ATF2 protein (mATF2) was created by substitution
of the phosphorylation sites Thr-69 and Thr-71 with Ala. The
phosphorylation reaction was terminated after 30 min by addition of
Laemmli sample buffer. The phosphorylated proteins were resolved by
SDS-PAGE and detected by autoradiography. The rate phosphorylation of
the substrate proteins was quantitated by PhosphorImager analysis. The
relative phosphorylation of ATF2, myelin basic protein (MPB),
EGF-R, and I
B was 1.0, 0.23, 0.04, and 0.001, respectively. Panel B, cell extracts expressing epitope-tagged JNK1 and p38
MAP kinase were incubated with a GST fusion protein containing the
activation domain of ATF2 (residues 1-109) immobilized on
gluthathione-agarose. The supernatant was removed and the agarose was
washed extensively. Western blot analysis of the supernantant and
agarose-bound fractions with the M2 monoclonal antibody was used to
detect the protein kinases by enhanced chemiluminescence detection.
Control experiments were performed using immobilized
GST.
Although the ERK substrates myelin basic protein and
the EGF-R were phosphorylated by p38, it was unclear whether these
proteins represent preferred substrates for this protein kinase. We
therefore tested several additional proteins as potential substrates
for p38. This analysis demonstrated a low level of phosphorylation of
IB (Fig. 1A). However, the transcription factor
ATF2 was found to be an excellent p38 substrate. Phosphorylation of
ATF2 caused by p38 resulted in an electrophoretic mobility shift during
polyacrylamide gel electrophoresis. The site(s) of phosphorylation were
mapped to the NH
-terminal activation domain of ATF2 by
deletion analysis (data not shown). Interestingly, JNK phosphorylates
ATF2 on Thr-69 and Thr-71(25) . We therefore tested the
hypothesis that p38 phosphorylates ATF2 on the same sites. It was found
that the replacement of Thr-69 and Thr-71 with Ala residues blocked the
phosphorylation of ATF2 caused by p38 (Fig. 1A). We
conclude that p38 phosphorylates ATF2 within the
NH
-terminal activation domain on Thr-69 and Thr-71.
Significantly, the phosphorylation of ATF2 on these sites causes
increased transcriptional activity(25) . Thus, the
transcription factor ATF2 is a potential target of signal transduction
by p38 MAP kinase and JNK.
It is known that JNK binds to the
activation domain of the substrate
c-Jun(11, 12, 38, 39, 40) .
By analogy to JNK, it is possible that p38 MAP kinase binds to ATF2. To
test this hypothesis, we incubated cell extracts with immobilized GST
or GST-ATF2 (activation domain; residues 1-109). The complexes
were extensively washed and the bound protein kinases were detected by
Western blotting. This analysis demonstrated that both p38 MAP kinase
and JNK bind to the ATF2 activation domain ()(Fig. 1B).
Figure 2:
Phorbol
ester weakly activates p38 MAP kinase. The activity of p38 MAP kinase
and JNK was measured in immunecomplex protein kinase assays using
[-
P]ATP and ATF2 as substrates. The
phosphorylated ATF2 was detected after SDS-PAGE by autoradiography. The
figure shows the effect of treatment of HeLa cells with 10 nM phorbol myristate acetate. The rate of phosphorylation was
quantitated by PhosphorImager analysis and is presented as the JNK and
p38 protein kinase activity relative to control cells treated without
agonist (1.0).
Figure 3:
EGF weakly activates p38 MAP kinase. The
activity of p38 MAP kinase and JNK was measured in immunecomplex
protein kinase assays using [-
P]ATP and
ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE
by autoradiography. The figure shows the effect of treatment of HeLa
cells with 10 nM EGF. The rate of phosphorylation was
quantitated by PhosphorImager analysis and is presented as the JNK and
p38 protein kinase activity relative to control cells treated without
agonist (1.0).
Figure 4:
UV radiation activates p38 MAP kinase. The
activity of p38 MAP kinase and JNK was measured in immunecomplex
protein kinase assays using [-
P]ATP and
ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE
by autoradiography. The figure shows the effect of treatment of HeLa
cells with 40 J/m
UV-C. The rate of phosphorylation was
quantitated by PhosphorImager analysis and is presented as the JNK and
p38 protein kinase activity relative to control cells treated without
agonist (1.0).
Figure 5:
Osmotic stress activates p38 MAP kinase.
The activity of p38 MAP kinase and JNK was measured in immunecomplex
protein kinase assays using [-
P]ATP and
ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE
by autoradiography. The figure shows the effect of treatment of HeLa
cells with 300 mM sorbitol. The rate of phosphorylation was
quantitated by PhosphorImager analysis and is presented as the JNK and
p38 protein kinase activity relative to control cells treated without
agonist (1.0).
Figure 6:
Interleukin-1 activates p38 MAP kinase.
The activity of p38 MAP kinase and JNK was measured in immunecomplex
protein kinase assays using [-
P]ATP and
ATF2 as substrates. The phosphorylated ATF2 was detected after SDS-PAGE
by autoradiography. The figure shows the effect of treatment of HeLa
cells with 10 ng/ml interleukin-1. The rate of phosphorylation was
quantitated by PhosphorImager analysis and is presented as the JNK and
p38 protein kinase activity relative to control cells treated without
agonist (1.0).
Figure 7:
Tumor necrosis factor activates p38 MAP
kinase. The activity of p38 MAP kinase and JNK was measured in
immunecomplex protein kinase assays using
[-
P]ATP and ATF2 as substrates. The
phosphorylated ATF2 was detected after SDS-PAGE by autoradiography. The
figure shows the effect of treatment of HeLa cells with 10 ng/ml tumor
necrosis factor
. The rate of phosphorylation was quantitated by
PhosphorImager analysis and is presented as the JNK and p38 protein
kinase activity relative to control cells treated without agonist
(1.0).
Figure 8:
LPS
activates p38 MAP kinase. The activity of p38 MAP kinase and JNK1 was
examined using Chinese hamster ovary cells that express human CD14. The
effect of treatment of the cells with 10 ng/ml LPS is presented. The
protein kinase activity was measured in immunecomplex protein kinase
assays using [-
P]ATP and ATF2 as
substrates. The phosphorylated ATF2 was detected after SDS-PAGE by
autoradiography. The rate of phosphorylation was quantitated by
PhosphorImager analysis and is presented as the JNK and p38 protein
kinase activity relative to control cells treated without agonist
(1.0).
Figure 9:
Dual
phosphorylation on Thr and Tyr is required for p38 MAP kinase
activation. Panel A, COS-1 cells expressing wild-type
(Thr-Gly-Tyr
) or mutated
(Ala
-Gly-Phe
) p38 MAP kinase were treated
without and with UV-C (40 J/m
). The cells were harvested 30
min following exposure to UV-C radiation. Control experiments were
performed using mock-transfected cells. The level of expression of
epitope-tagged p38 MAP kinase and the state of Tyr phosphorylation of
p38 MAP kinase was examined by Western blot analysis using the M2
monoclonal antibody and the phosphotyrosine monoclonal antibody PY20.
Immune complexes were detected by enhanced chemiluminescence. Panel
B, the p38 MAP kinase was isolated from cells
metabolically-labeled with [
P]phosphate by
immunoprecipitation with the M2 monoclonal antibody and SDS-PAGE. The
p38 MAP kinase phosphorylation was examined by phosphoamino acid
analysis. Panel C, the p38 MAP kinase was isolated from the
COS-1 cells by immunoprecipitation. Protein kinase activity was
measured in the immune complex using
[
-
P]ATP and GST-ATF2 as substrates. The
phosphorylated GST-ATF2 was detected after SDS-PAGE by
autoradiography.
Figure 10:
MAP kinase phosphatase inhibits p38 MAP
kinase activation. The effect of expression of human MKP1 and PAC1 on
p38 MAP kinase activity is presented. The cells were treated without
and with 40 J/m UV-C. Control experiments were performed
using mock-transfected cells (control) and cells transfected with the
catalytically inactive mutated phosphatase mPAC1
(Cys
/Ser). The activity of p38 MAP kinase was measured
with an immunecomplex protein kinase assay employing
[
-
P]ATP and GST-ATF2 as
substrates.
Figure 11:
Subcellular distribution of p38 MAP
kinase. Epitope-tagged p38 MAP kinase was expressed in COS cells. The
cells were treated without (Panel A) or with (Panel
B) 40 J/m UV radiation and then incubated for 60 min
at 37 °C. The p38 MAP kinase was detected by indirect
immunofluorescence using the M2 monoclonal antibody. The images were
acquired by digital imaging microscopy and processed for image
restoration.
The requirement of dual phosphorylation for activation
establishes that p38 is a member of the MAP kinase group of signal
transducing proteins(1) . However, the absence of detectable
phosphorylation of cPLA, c-Myc, and c-Jun together with the
strong phosphorylation of ATF2 indicates that the substrate specificity
of p38 differs from both the JNK(11, 12, 13, 14, 16, 17, 25) and
ERK(41, 42) subgroups of MAP kinase. It is therefore
likely that the p38 MAP kinase signal transduction pathway has a
distinct function in the cell. Indeed, it has recently been established
that p38 may function in a signal transduction pathway that leads to
phosphorylation of small heat shock proteins (20, 21) and increased expression of inflammatory
cytokines(22) .
EGF and phorbol ester are potent activators of the ERK signal transduction pathway(10) . However, we found that these treatments did not cause a marked increase in p38 protein kinase activity. These data indicate that the mechanism of activation of p38 is not identical to the ERK group of MAP kinases. In contrast, the pattern of activation of p38 was found to be similar to JNK, a MAP kinase that is potently activated by pro-inflammatory cytokines and environmental stress(1) . Thus p38, like JNK, may be regulated, in part, by a stress-activated signal transduction pathway (Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8). This conclusion is consistent with the observation that both p38 and JNK1 are able to complement a defect in the expression of the HOG1 stress-activated MAP kinase in yeast(15, 19) . Although p38 and JNK both appear to be activated by a stress-induced signal transduction pathway, a significant question remains concerning the organization of these pathways.
The comparison of the regulation of p38 and JNK activation reveals a marked similarity between these protein kinases, but differences in the time course and extent of activation were also observed (Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8). These differences indicate that the p38 and JNK pathways may be distinct. Indeed, p38 and JNK could represent parallel stress-activated signal transduction pathways(49) . Alternatively, it is possible that p38 and JNK are activated by a common pathway. A rigorous test of these hypotheses requires the molecular cloning of the dual specificity kinase kinases that activate p38 and JNK. Recently, two MAP kinase kinases (MKK3 and MKK4) that activate p38 MAP kinase have been identified(49) . MKK3 is specific for p38 MAP kinase(49) . In contrast, MKK4 activates both JNK (49, 50) and p38 MAP kinase(49) . Thus, p38 and JNK are activated by related MAP kinase kinases (Fig. 12).
Figure 12: Mammalian MAP kinases form an integrated network of signal transduction pathways. The major stimuli that activate JNK and p38 MAP kinases are pro-inflammatory cytokines and environmental stress. In contrast, the ERK group of MAP kinases are activated in cells treated with EGF or phorbol ester. This difference is accounted for, in part, by the substrate specificities of the MAP kinase kinases MEK1(53) , MEK2(53) , MKK3(49) , and MKK4 (49, 50) which activate ERK, p38, and JNK. These pathways are illustrated schematically.
The original identification of p38 demonstrated that this protein is tyrosine phosphorylated in LPS-treated cells(19, 51) . This study demonstrates that p38 is a member of the MAP kinase group that is activated by dual phosphorylation on Tyr and Thr by a stress-induced signal transduction pathway. Endotoxic LPS, an activator of the p38 MAP kinase pathway, can therefore be considered to be a form of environmental stress that elicits septic shock(52) .