(Received for publication, August 31, 1995)
From the
In human platelets a proline-directed kinase distinct from the
ERK MAP kinases is stimulated by both thrombin and the thrombin
receptor agonist peptide SFLLRN and may be involved in the activation
of Ca-dependent cytosolic phospholipase A
(Kramer, R. M., Roberts, E. F., Hyslop, P. A., Utterback, B. G.,
Hui, K. Y., and Jakubowski, J. A.(1995) J. Biol. Chem. 270,
14816-14823). Here we show that this kinase is identical with or
closely related to p38 (the mammalian homolog of HOG1 from yeast), a
recently discovered protein kinase typically activated by inflammatory
cytokines and environmental stress. Further, we demonstrate that
activation of this kinase by thrombin is transient (with maximal
stimulation at 1 min), is accompanied by tyrosine phosphorylation, and
precedes the activation of the ERK kinases. This is the first report to
show that p38 kinase is activated by thrombin and to suggest a role for
this MAP kinase in the thrombin-mediated signaling events during
platelet activation.
We have recently shown that thrombin stimulates the activity of
the MAP ()kinases ERK1 and ERK2 but also activates another
proline-directed kinase that is distinguishable from ERK1/2 based on
its strong binding to anion exchange resin and the lack of reactivity
with anti-ERK1/2 antibodies (1) . We further noted that this
kinase readily phosphorylates cPLA
but not the S505A mutant
of cPLA
. This observation indicated that the serine
residing within the MAP kinase consensus sequence (i.e. Pro-Leu-Ser
-Pro) is the target phosphorylation site
for the kinase. Significantly, the thrombin receptor agonist peptide
SFLLRN also activated this proline-directed kinase but completely
failed to stimulate ERK1/2. Nonetheless SFLLRN, like thrombin, mediated
activation of cPLA
by phosphorylation, and we reasoned that
this unidentified kinase could play a role in the signal transduction
pathways activated through the thrombin receptor. We therefore further
characterized the kinase with the goal to determine its identity and
define its role in the thrombin-induced signaling events during
platelet activation.
In human platelets thrombin activates several kinases that
readily phosphorylate the Thr peptide derived from the
epidermal growth factor receptor(1) . Based upon the distinct
chromatographic and immunological characteristics, these kinases could
be distinguished and found to consist of the MAP kinases ERK1/2, as
well as another unidentified proline-directed kinase. When extracts
from control and thrombin-stimulated platelets were applied to MonoQ in
buffer containing 150 mM NaCl, ERK1/2 flowed through the
column. The unknown thrombin-activated Thr
kinase, on the
other hand, bound tightly to the column and eluted at
350 mM NaCl (Fig. 1A). Consequently, the isoelectric
point (IEP) of this kinase had to be significantly lower than that of
ERK1/2 (
6.8). For example, the IEP of cPLA
that binds
similarly to MonoQ eluting with
400 mM NaCl is
5.1(4) . Only three recently identified proline-directed
kinases exhibit calculated IEPs in agreement with the observed
chromatographic behavior of the platelet Thr
kinase.
These include JNK2 (p54
)(5) , p38
kinase(6) , and an ERK3 homolog (referred to as
p97
) (7) with IEPs of 5.7, 5.6, and 4.8,
respectively.
Figure 1:
Partial
purification of thrombin-stimulated Thr kinase activity.
Soluble extracts derived from 7.5
10
platelets
incubated for 2 min at 1.25
10
/ml in the absence or
presence of thrombin (5 units/ml) were passed through a MonoQ column in
buffer containing 150 mM NaCl as detailed under
``Experimental Procedures.'' The column was developed with a
30-ml linear gradient from 150 to 500 mM NaCl collecting
0.5-ml fractions. A, determination of kinase activity in
8.3-µl aliquots of column fractions as described under
``Experimental Procedures'' using 1 mM Thr
peptide substrate. B, aliquots (10 µl) of loaded
extracts (C), flow-through (FT) and selected MonoQ
fractions (as indicated) of control platelets (left panels, lanes 1-11), and thrombin-stimulated platelets (right panels, lanes 12-22) were subjected to
SDS-PAGE/immunoblotting probing with anti-p38 (C-20,
p38)
antibodies (upper panels) and anti-phosphotyrosine
(
PTyr) antibodies (lower panels) as detailed in Fig. 2. Molecular mass markers are indicated on the left; * designates the fractions with highest kinase
activity.
Figure 2:
Immunological identification of
thrombin-stimulated Thr kinase. Soluble extracts (C) from thrombin-stimulated platelets (10 µl), MonoQ
fractions containing the Thr
kinase (K) (10
µl) (as in Fig. 1A), and standard proteins (St) were subjected to SDS-PAGE/immunoblotting probing with
different antibodies as detailed under ``Experimental
Procedures.'' A, anti-p38 (C-20) polyclonal antibody at
0.1 µg/ml (Santa-Cruz Biotechnology) (lanes 1 and 2), anti-phosphotyrosine (
P-Y) monoclonal
antibodies 4G10 (Upstate Biotechnology) plus PY20 (ICN) at 1 µg/ml
each (lanes 3 and 4), and anti-ERK1/2 polyclonal
antibody erk1-CT (Upstate Biotechnology) at 1 µg/ml (lanes 5 and 6). B, anti-JNK2 polyclonal
antibody (Santa-Cruz Biotechnology) at 0.1 µg/ml (lanes
7-9) and anti-ERK3 antibody (Transduction Laboratories) at 1
µg/ml (lanes 10-12). The ability of anti-JNK2 and
anti-ERK3 antibodies to recognize JNK2 and ERK3, respectively, was
verified with purified human JNK2 (Santa-Cruz Biotechnology) (lane
7) and human fibroblast ERK3 (Transduction Laboratories) (lane
10). C, anti-p38(N-20) antibodies (Santa-Cruz
Biotechnology) in the absence (lane 13) and presence of
immunizing peptide N-20 (lane 14), and anti-p38 (C-20)
antibody in the absence (lane 15) and presence of immunizing
peptide C-20 (lane 16). Migration position of molecular mass
marker is indicated on the right.
We therefore subjected extracts from
thrombin-stimulated platelets and the most active Thr kinase MonoQ fractions 34 and 35 (see Fig. 1A) to
SDS-PAGE/immunoblotting, probing with antibodies against JNK2, p38, and
ERK3. As shown in Fig. 2A anti-p38 antibodies
specifically recognized a protein of
40 kDa that was enriched in
the MonoQ fractions (lane 2) compared with the loaded platelet
extract (lane 1). The same
40-kDa protein strongly
reacted with anti-phosphotyrosine antibodies (lane 4).
Although ERK1 and ERK2 could be readily detected in extracts (lane
5), they were absent in the MonoQ fractions containing the
Thr
kinase activity (lane 6). As demonstrated in Fig. 2B, no immunoreactivity could be detected in
extracts or active MonoQ fractions when probing with anti-JNK
antibodies (lanes 8 and 9) or anti-ERK3 antibodies (lanes 11 and 12). By comparison both kinases could
be readily seen when standard proteins were tested (lane 7 and lane 10). We confirmed the specificity of two anti-p38
antibodies (anti-p38N and anti-p38C) for the
40-kDa protein by
competition experiments with the respective C- and N-terminal peptides
of p38 used for immunization. As demonstrated in Fig. 2C, the reactivity with the
40-kDa protein
was significantly decreased when the immunizing peptides were present
during immunoblotting, including the anti-p38N (lane 14 versus lane
13) and the anti-p38C (lane 16 versus lane 15)
antibodies. We further examined whether the kinase activity eluting
from the MonoQ column (Fig. 1A) paralleled the
immunoreactivity of the eluting kinase protein probing with both
anti-p38 and anti-phosphotyrosine antibodies. As shown in Fig. 1B (upper right panel), the MonoQ elution
of the
40-kDa protein recognized by the anti-p38 antibodies
correlated with the thrombin-induced Thr
kinase activity.
Likewise, coincident with the peak of kinase activity we detected
thrombin-induced tyrosine phosphorylation of the same
40-kDa
protein (Fig. 1B, lower panel). Taken
together, these data indicate that the proline-directed kinase
activated by thrombin is identical with, or closely related to, the p38
MAP kinase.
We determined the kinetics of thrombin-mediated
activation of the p38 kinase, resolving it from ERK1/2 by MonoQ
chromatography. As shown in Fig. 3A, thrombin induced a
transient stimulation of p38 kinase activity that reached a maximum at
1 min and was still detected at the latest time point measured (5 min).
The amount of p38 protein purified by MonoQ chromatography was the same
for all time points examined, as verified by SDS-PAGE immunoblotting (Fig. 3B). The appearance and disappearance of p38
kinase activity in thrombin-stimulated platelets temporally coincided
with the tyrosine phosphorylation of p38 (Fig. 3C). By
comparison, activation of the ERKs was delayed with maximal stimulation
at 2 min following thrombin stimulation, as shown by the ability of
ERK1/2 to phosphorylate the Thr peptide substrate (Fig. 4A) and the decreased electrophoretic mobility of
the ERK proteins (Fig. 3C and Fig. 4B),
indicative of activation(8) . The data in Fig. 4also
reveal that activation by thrombin of p38 is more prominent than that
of ERK1/2. A similar robust activation of p38 and delayed stimulation
of ERK1/2 were observed in aspirinized platelets (where the synthesis
of endogenous thromboxane A
is inhibited), demonstrating
that p38 kinase is the target of thrombin and not of the secondary
agonist thromboxane A
that is released from activated
platelets.
Figure 3:
Time course of thrombin-mediated
activation of p38 kinase. Platelets (0.6 ml at 1.25
10
/ml) were incubated at 37 °C in the absence
(-THR) and presence of 5 units/ml of thrombin
(+THR) as described under ``Experimental
Procedures.'' After addition of 150 µl of Triton X-100
stopping mixture and ultracentrifugation, the extracts were diluted
with 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 100
µM Na
VO
, and 50 mM
-glycerophosphate, pH 7.5 and subjected to MonoQ
chromatography as described under ``Experimental
Procedures.'' A, determination of kinase activity in
pooled MonoQ fractions containing the p38 kinase using 2 mM
Thr
peptide substrate as detailed under
``Experimental Procedures.'' B,
SDS-PAGE/immunoblotting verifying that equal amounts of p38 kinase
protein were present in the pooled active MonoQ fractions derived from
platelets incubated without thrombin (-THR) for 30 s to
5 min (lanes 1-4) and with thrombin (+THR)
for 0-5 min (lanes 5-10). C,
SDS-PAGE/immunoblotting of solubilized lysates (10 µl) from
platelets incubated with thrombin (5 units/ml) for 0-5 min as
indicated, probing for the presence of tyrosine-phosphorylated proteins
(
P-Y, lanes 1-5), p38 kinase (lanes
6-10), and ERK1/2 (lanes 11-14) using the
antibodies as described in Fig. 2. The data shown are
representative of two independent experiments yielding similar results,
and the values shown in A are means ± range of
duplicate incubations.
Figure 4:
Differential activation of p38 and ERK
kinases by thrombin. Platelets (0.6 ml at 1.25
10
/ml) were incubated at 37 °C with 5 units/ml
thrombin, and the reaction was quenched as described under
``Experimental Procedures.'' The solubilized lysates were
cleared by ultracentrifugation, diluted with 1 mM EGTA, 1
mM DTT, 100 µM Na
VO
, and
50 mM
-glycerophosphate, pH 7.5 (buffer A), and passed
over a MonoQ column equilibrated in buffer A containing 50 mM NaCl. ERK1/2 and p38 were eluted with a step salt gradient and
recovered in 1.5 ml of buffer A containing 250 mM NaCl and 1.5
ml of buffer A containing 450 mM NaCl, respectively, as
described under ``Experimental Procedures.'' A,
determination of kinase activity in 8.3 µl of the 1.5-ml MonoQ
pools using 2 mM Thr
peptide substrate as
detailed under ``Experimental Procedures.'' B,
SDS-PAGE/immunoblotting demonstrating the presence of ERK1/2 (lanes
5-8) and p38 (lanes 1-4) in the respective
1.5-ml MonoQ pools probing with the antibodies described in Fig. 2. The data shown are representative of two independent
experiments yielding similar results, and the values shown in A are means ± range of duplicate incubations, each assayed in
duplicate.
The p38 kinase belongs to a new subfamily of
stress-activated MAP kinases related to the HOG1 gene product, a kinase
required for adaptation to osmotic stress in Saccharomyces
cerevisiae(9) , and has only recently been identified in
mammalian cells. Thus, Han et al.(10) first described
this novel kinase of apparent molecular mass of 38 kDa (therefore
referred to as p38) in cells of monocytic lineage, observing that it is
rapidly phosphorylated on tyrosine residues in response to endotoxin.
Cloning of the p38 kinase revealed that its predicted sequence is 52%
identical to the yeast kinase HOG1 (6) and shares with HOG1 the
unique sequence TGY comprising the dual phosphorylation site typical of
MAP kinases. Lee et al.(11) identified the new kinase
CSBP, a target of cytokine synthesis inhibitors, that was found to be
identical with p38 kinase. Furthermore, Rouse et al.(12) discovered a stress-activated kinase recognized by
antibodies against the Xenopus kinase Mpk2, a kinase closely
related to HOG1 from yeast, and Freshney et al.(13) purified an interleukin-1-stimulated kinase from
human epidermal carcinoma cells whose biochemical properties closely
resembled those of the p38 kinase. Recent studies by Raingeaud et
al.(14) showed that p38 kinase is activated not only by
osmotic stress and endotoxin but is also stimulated by inflammatory
cytokines, particularly tumor necrosis factor, and exposure to UV
radiation. In contrast, the p38 kinase was only poorly activated by
growth factors, interferon- and phorbol
ester(6, 10, 14) . Here, we report that the
serine protease thrombin known to activate a heterotrimeric G
protein-coupled receptor causes a marked activation of the p38 kinase.
The stimulation of p38 kinase by thrombin not only precedes that of the
ERKs but is also more pronounced than that of the ERKs. This suggests
that p38, rather than the ERKs, may be involved in early
proline-directed phosphorylation events during thrombin-mediated
platelet activation. The difference in the temporal pattern of
activation is consistent with the notion that the p38 and ERK MAP
kinases are independently regulated by distinct signaling pathways (15) .
The sequential kinase cascade leading to the
activation of the ERK MAP kinases lies downstream of Ras and consists
of two protein kinases (Raf and MAP kinase kinase) acting sequentially
to activate the ERKs(15) . In contrast, the upstream regulatory
mechanisms and protein kinases involved in the activation of p38 are
not yet fully elucidated. Activation of p38 kinase requires dual
phosphorylation on Thr and
Tyr
(14) . Recently, a MAP kinase kinase referred
to as JNKK (16) or MKK4 (17) was identified that
activates the p38 and also the JNK kinases. Another kinase, called
MKK3, cloned as the human homolog of the yeast MAP kinase kinase PBS2,
activated only the p38 kinase and not the ERK and JNK MAP
kinases(17) . Thus, MKK3 may be a component of an independent
signaling pathway that specifically activates the p38 kinase. Recently,
the small GTP-binding proteins Rac1 and Cdc42 were shown to be
efficient activators of the signaling cascade affecting the p38 kinase (18) .
Our studies show that the p38 kinase is present in
human platelets, where it is transiently and potently stimulated by
thrombin. Although the nature of the signaling pathways that
participate in stimulation of platelets upon activation of the thrombin
receptor has not been completely defined, it is known that the
activated thrombin receptor couples to phosphatidylinositol biphosphate
metabolism and inhibition of adenylate cyclase via the G proteins
G and G
, respectively(19) . Receptors
coupled to heterotrimeric G proteins are thought to activate the ERK
MAP kinase pathway via activated
and
G protein
subunits(20) . The thrombin receptor agonist peptide SFLLRN
also stimulates the proline-directed kinase, now identified as p38,
but, unlike thrombin, does not activate ERK1/2(1) . These
findings suggest the existence of parallel pathways leading to the
activation of either p38 or the ERKs. While both pathways can be
stimulated by thrombin, SFLLRN activates solely the signaling pathway
causing p38 stimulation. It thus appears that, at least in
SFLLRN-stimulated platelets, the p38 kinase pathway is responsible for
regulation of cPLA
. While other physiological functions of
the p38 kinase remain to be elucidated, p38 is likely to be an integral
part of a signaling pathway utilized by the thrombin receptor in
platelets, and it will be of great interest to further investigate the
role of p38 in platelet function.
Thrombin rapidly and potently stimulates the p38 kinase in
human platelets, demonstrating that extracellular stimuli other than
stress-related events and proinflammatory cytokines can activate this
proline-directed kinase. Taken together with our previous findings (1) these observations suggest that in platelets (i) thrombin
activates two distinct signaling pathways that result in the activation
of either the p38 or the ERK MAP kinases, (ii) the thrombin receptor
agonist peptide SFLLRN exclusively signals through the p38 pathway, and
(iii) cPLA appears to be one of the downstream targets of
p38 kinase.