(Received for publication, August 8, 1996, and in revised form, October 16, 1996)
From the Division of Cell Biology, Research
Institute, The Hospital for Sick Children and the
Department of
Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada
and the ¶ Department of Medicine, University of British
Columbia, and Kinetek Pharmaceuticals Inc., Vancouver,
British Columbia V5Z 1A1, Canada
Activation of polymorphonuclear leukocytes (PMN) by chemotactic peptides initiates a series of functional responses that serve to eliminate pathogens. The intermediate steps that link engagement of the chemoattractant receptor to the microbicidal responses involve protein kinases that have yet to be identified. In this study we detected in human PMN the presence of p38 mitogen-activated protein kinase (MAPK), which became rapidly tyrosine phosphorylated and activated in response to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). Pretreatment of PMN with wortmannin, a phosphatidylinositol 3-kinase inhibitor, or bis-indolylmaleimide, a protein kinase C antagonist, resulted in partial inhibition of p38 phosphorylation upon fMLP stimulation. Similarly, phosphorylation of p38 was only partially inhibited when the fMLP-induced cytosolic calcium transient was prevented. Stimulation of PMN by the chemoattractant also resulted in the rapid phosphorylation and activation of MAPK-activated protein kinase-2 (MAPKAPK-2), which was completely inhibited by the specific p38 inhibitor, SB203580. The physical interaction of p38 with MAPKAPK-2 was studied by coimmunoprecipitation. These two kinases were found to be associated in unstimulated PMN but dissociated upon activation of the cells by fMLP. Together these findings demonstrate the activation of p38 by chemotactic peptides in human PMN by a process involving phosphatidylinositol 3-kinase, protein kinase C, and calcium. p38, in turn, is an upstream activator of MAPKAPK-2.
Polymorphonuclear leukocytes (PMN)1 respond rapidly to invading microorganisms or tissue injury by activation of numerous effectors, including the generation of superoxide anions, secretion of lytic enzymes, and phagocytosis of particles. These responses serve to neutralize and destroy the invading pathogens (1). The recruitment of PMN to sites of bacterial infection and their subsequent activation are initiated by binding of chemoattractants to specific cell surface receptors, which are coupled to heterotrimeric G proteins (2). Receptor engagement triggers a complex cascade of biochemical events which culminates in the activation of the microbicidal responses. Many of these intervening steps have yet to be defined.
Increased phosphorylation of several proteins has been found to correlate with the stimulation of PMN effectors, thereby suggesting a causal role in the activation process (3, 4, 5, 6). Additional support for a central role of phosphorylation was provided by the finding that pharmacological agents that interfere with protein kinases and phosphatases are also potent modulators of PMN responsiveness (7, 8, 9, 10). Indeed, there is abundant evidence that protein kinase C isoforms are essential to the microbicidal response (11, 12), and activation of tyrosine phosphorylation seems to be equally important (7, 9). Studies in PMN stimulated with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) led to the identification of Erk-1 and Erk-2 as major targets of tyrosine phosphorylation (13, 14, 15). These Ser/Thr kinases are members of a family of mitogen-activated protein kinases (MAPK) which are characterized by dual phosphorylation on tyrosine and threonine residues during activation. Other members of the MAPK family include p38 (16, 17) and JNK (18, 19, 20), which can be activated by physico-chemical stress, lipopolysaccharides, and by some cytokines (21). These kinases were identified more recently, and their presence and regulation in PMN have not been explored.
p38 was originally cloned from Saccharomyces cerevisiae (termed HOG1) (22), but homologs were subsequently found in Xenopus (mpk2) (23), murine (p38) (16), and human tissues (CSBP1 and CSBP2) (17, 24). In view of its conservation and wide distribution, it would appear that p38 serves an important function in cellular responses. Indeed, the development of an inhibitor of p38 has pointed to its involvement in the synthesis of interleukin-1 and tumor necrosis factor in monocytes (17), interleukin-8 in peripheral blood mononuclear cells (25), and in platelet aggregation and secretion (26). Phosphorylation of a downstream substrate is believed to mediate the aforementioned effects of p38, but the identity of these putative target(s) remains unclear. One possible effector may be MAPK-activating protein kinase-2 (MAPKAPK-2), a Ser/Thr kinase that possesses the ability to phosphorylate the small heat shock protein HSP27 (23, 27). HSP27 has, in turn, been suggested to modulate actin microfilament dynamics and cellular thermoresistance (28, 29).
In the current study we investigated whether p38 and/or MAPKAPK-2 is involved in the signaling process of human PMN activated by fMLP. Using immunoprecipitation, fractionation, and in vitro kinase assays we found that these enzymes are present as an inactive complex in unstimulated cells. Chemotactic stimulation promoted the activation and dissociation of p38 and MAPKAPK-2 by a pathway involving phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC).
fMLP, phorbol 12-myristate 13-acetate (PMA), and
thapsigargin were purchased from Sigma. The acetoxymethyl ester of
BAPTA was from Molecular Probes. Ficoll-Paque, dextran T-500, protein A-Sepharose, and Mono Q were from Pharmacia Biotech Inc.
(Québec, Canada). Bis-indolylmaleimide or GF 109203X
(BIM) was from BioMol. [-32P]ATP (4,500 Ci/mmol) was
from ICN. Glutathione S-transferase-tagged ATF-2 was
obtained for Santa Cruz Biotechnologies (Santa Cruz, CA), and
recombinant human HSP27 was from StressGen (Victoria, BC, Canada).
Monoclonal phosphotyrosine antibody (clone 4G10) was purchased from
Upstate Biotechnology Inc. (Lake Placid, NY). Rabbit polyclonal p38
antibody was kindly provided by Dr. Brent Zanke (Ontario Cancer
Institute, Princess Margaret Hospital, Toronto, Canada). Rabbit
polyclonal MAPKAPK-2 antibodies were raised against a peptide encoding
the COOH-terminal domain (SRVLKEDKERWEDVKGC). The p38 inhibitor
SB203580 was generously given by SmithKline Beecham (King of Prussia,
PA).
Human PMN were isolated from blood freshly drawn by venipuncture from healthy donors essentially as described previously (30). Briefly, whole blood was sedimented on dextran T-500 to remove red cells, and the resulting supernatant was overlaid onto Ficoll-Paque cushions and centrifuged. Contaminating red cells in the pellet were removed by hypotonic lysis. The purified PMN were resuspended in HEPES-buffered (25 mM, pH 7.4) bicarbonate-free RPMI 1640 medium at 107 cells/ml and stored at room temperature on a rotator until use. For immunoprecipitation and cell fractionation experiments, the cell suspension was treated with 2.5 mM diisopropyl fluorophosphate for 15 min at room temperature, sedimented, and resuspended in fresh HEPES-buffered RPMI 1640 before use. For the experiments, PMN were resuspended in HEPES-buffered saline (140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 3 mM KCl, 10 mM glucose, and 10 mM HEPES, pH 7.4). Unless indicated otherwise, all experiments were performed at 37 °C.
Ca2+ Depletion and BufferingWhere indicated, intracellular Ca2+ stores were depleted by treating a PMN suspension with 100 nM thapsigargin for 5 min at 37 °C in Ca2+-free HEPES-buffered saline. Alternatively, intracellular Ca2+ was buffered by treating suspended PMN with 10 µM BAPTA-acetoxymethyl ester for 30 min at 37 °C in regular (Ca2+-containing) HEPES buffer. Following this incubation period, the cells were resuspended in nominally Ca2+-free HEPES-buffered saline and incubated an additional 1 min before stimulation with fMLP.
Immunoprecipitation and ImmunoblottingFollowing stimulation of PMN, the incubation was stopped by adding of 2 volumes of ice-cold HEPES-buffered saline followed by a rapid sedimentation of the cells in a microcentrifuge. For immunoblotting of whole cell lysates, pellets containing 106 cells were immediately resuspended in boiling Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol) and subjected to analysis by SDS-PAGE. Immunoprecipitation of tyrosine-phosphorylated proteins was performed as described previously (13). For immunoprecipitation of p38 and MAPKAPK-2, the cell pellet (107 cells) was solubilized in 1 ml of ice-cold Nonidet P-40 buffer (1 mM EDTA, 1% (v/v) Nonidet P-40, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 mM sodium vanadate, 5 mM NaF, and 50 mM Tris-HCl, pH 8.0). After 15 min at 4 °C, the lysates were centrifuged at 14,000 × g for 5 min to remove insoluble debris, and the appropriate antibody (1:500 dilution for anti-p38 and 1:250 for anti-MAPKAPK-2) was added to the soluble fraction, which was then incubated for 1 h at 4 °C. Subsequently, 30 µl of a 50% slurry of protein A-Sepharose (preblocked overnight with 5% albumin) was added, and the lysates were incubated an additional 2 h at 4 °C. The immune complexes were then washed five times with Nonidet P-40 buffer and subjected to a kinase assay or resuspended in Laemmli sample buffer and subjected to SDS-PAGE.
For immune complex kinase assays, immunoprecipitates were resuspended
in 25 µl of kinase buffer (20 mM MgCl2, 1 mM sodium vanadate, 5 mM NaF, 20 mM
-glycerophosphate, 20 mM
p-nitrophenylphosphate, 2 mM dithiothreitol, 20 µM ATP, 5 µCi of [
-32P]ATP, and 50 mM HEPES, pH 7.4) and 3 µg of either glutathione S-transferase-tagged ATF-2 or HSP27. Following an incubation
of 20 min at 30 °C, an equal volume of 2 × Laemmli sample
buffer was added, and samples were boiled for 5 min (except for
MAPKAPK-2 immunoprecipitation experiments, where samples were kept on
ice until analyzed by SDS-PAGE). Immunoprecipitates were subjected to
SDS-PAGE (10% acrylamide) and then electrotransferred onto polyvinylidene difluoride (PVDF) membranes. The blots were incubated with the appropriate antibody (1:2,000 dilution for p38 and MAPKAPK-2) followed by a horseradish peroxidase-coupled anti-rabbit or anti-mouse antibody (1:5,000 dilution), which was detected by enhanced
chemiluminescence (ECL; Amersham). For kinase assays, phosphorylated
substrates were detected with a PhosphorImager (Molecular Dynamics
Inc.). Tyrosine phosphorylation was quantitated by densitometric
scanning of the x-ray films using a Protein Database Inc. (Huntington
Station, NY) model DNA 35 scanner with the Discovery series
one-dimensional gel analysis software version 2.1.
PMN (5 × 107) were
treated with or without 100 nM fMLP for 1 or 2 min at
37 °C. The incubation was terminated by adding 2 volumes of ice-cold
HEPES-buffered saline and rapidly sedimenting the cells in a
microcentrifuge. The cell pellet was solubilized in Nonidet P-40 buffer
and the debris removed by centrifugation at 14,000 × g
for 5 min. The supernatant (1 mg of protein) was fractionated on a
Mono Q column (1-ml bed volume) equilibrated with buffer A (12.5 mM MOPS, pH 7.2, 12.5 mM
-glycerophosphate,
0.5 mM EGTA, 7.5 mM MgCl2, and 1 mM dithiothreitol). The column was eluted with a 10-ml
linear 0-0.8 M NaCl gradient in buffer A using the Pharmacia fast protein liquid chromatography system. Fractions (250 µl) were collected and assessed for HSP27 kinase activity and
analyzed by SDS-PAGE and immunoblotting.
Immunoblotting of lysates of human PMN with a p38-specific
polyclonal antibody revealed a single band of approximately 42 kDa (not
shown). To determine whether treatment with fMLP induces activation of
p38, PMN were stimulated for increasing lengths of time, and the kinase
was immunoprecipitated. As shown in Fig. 1A,
fMLP induced a time-dependent, transient tyrosine
phosphorylation of p38. Phosphotyrosine accumulation was apparent at
the earliest time analyzed, 0.5 min, attained maximal level at 1 min,
and returned to near basal levels between 5 and 10 min. Fig.
1B demonstrates that equal amounts of the MAPK were
immunoprecipitated at all times. The phosphorylation of p38 was also
demonstrated by immunoprecipitating tyrosine-phosphorylated proteins
from PMN lysates. Blotting these immunoprecipitates with a p38-specific
antibody also revealed a 42-kDa protein in stimulated, but not in
resting (control) cells (Fig. 1C). We next determined
whether the kinase activity of p38 was also stimulated by the
chemotactic peptide. Only a marginal amount of ATF-2 phosphorylation
was detected in p38 immunoprecipitates obtained from unstimulated PMN.
By contrast, a sizable activity was consistently observed in
precipitates from fMLP-stimulated cells (Fig. 1D). Together
these results demonstrate the presence p38 in human PMN and the ability
of fMLP to phosphorylate and activate this kinase.
Role of PI 3-kinase in p38 Activation
We next proceeded to
investigate the signaling pathway leading to the activation of p38.
Activation of PI 3-kinase, which catalyzes the formation of
phosphatidylinositol 3,4,5-phosphate, is one of the earliest responses
of PMN to chemoattractants (31). Although the activation of PI 3-kinase
in human PMN has been associated with functional events (32, 33, 34), the
downstream events have yet to be defined. We therefore considered the
possibility that p38 may be a target of the activation of PI 3-kinase.
We tested the effects of two structurally different inhibitors of PI
3-kinase on the activation of p38 by fMLP. As shown in Fig. 2A, pretreatment of PMN for 10 min with 100 nM wortmannin resulted in a partial inhibition (60%) of
the tyrosine phosphorylation of p38 in response to fMLP. Unexpectedly,
a similar pretreatment of PMN with 100 µM LY294002, a
second antagonist of PI 3-kinase, failed to inhibit the fMLP-induced
phosphorylation of p38. In fact, pretreatment with this antagonist
alone, in the absence of fMLP, induced a pronounced activation of p38,
which was enhanced by the subsequent addition of the chemoattractant
(Fig. 2A). Identical conclusions were reached when the
kinase activity of p38 was measured using ATF-2 as a substrate (Fig.
2B). To determine whether this effect of LY294002 was
specific to p38, PMN were pretreated with either wortmannin or
LY294002, and the phosphorylation of Erk-1 and -2 in response to fMLP
was assessed using an antibody that recognizes only the phosphorylated
form of these kinases. As shown in Fig. 2C, both wortmannin
and LY294002 inhibited the phosphorylation of these MAPKs to a similar
extent. Therefore, the stimulatory effect of LY294002 is unique to p38.
Although the underlying mechanism remains obscure, it is clear that
LY294002 should be used with caution when assessing the effects of PI
3-kinase in PMN.
Fig. 2D illustrates that, contrary to what was proposed
earlier (13, 14, 15), Erk-1 and -2 are not the main 40-42-kDa tyrosine-phosphorylated proteins in stimulated PMN. In this experiment, cells were pretreated with wortmannin or LY294002 and then stimulated with fMLP. Immunoblotting of whole cell lysates with a
phosphotyrosine-specific antibody shows that the fMLP-induced tyrosine
phosphorylation of the 42-kDa band was reduced by wortmannin but not by
LY294002, which in fact increased phosphorylation even in the absence
of the chemoattractant. This profile parallels the effects of LY294002 on p38 and differs from its effects on Erk (cf. Fig. 2,
A and C).
Several isoforms of PKC coexist in PMN and are known
to be activated by chemotactic peptides. To evaluate whether PKC
isoforms mediate the effect of fMLP on p38, we used a reasonably
specific inhibitor, namely BIM (35). Pretreatment of PMN with 5 µM BIM for 10 min had little effect by itself but
resulted in partial inhibition (65%) of the tyrosine phosphorylation
of p38 induced by fMLP (Fig. 3A). To ensure
that BIM was effectively inhibiting PKC, this kinase was stimulated
directly with phorbol ester. Incubation of PMN with 100 nM
PMA for 5 min induced a low tyrosine phosphorylation of p38. This
PMA-induced tyrosine phosphorylation of p38 was fully inhibited by 5 µM BIM. These results suggest that the concentration of
BIM used sufficed to inhibit PKC thoroughly. Because BIM inhibited p38
activation by fMLP only partially, and since maximally stimulatory doses of PMA yielded a response smaller than that elicited by the
chemoattractant, we conclude that the response to fMLP is only
partially dependent on PKC.
The activation of PMN by fMLP triggers an increase in cytosolic
Ca2+ concentration ([Ca2+]i) which
originates both from release of intracellular stores and from
extracellular Ca2+ influx. The role of the cation in the
activation of p38 was investigated next, using two different strategies
aimed at minimizing the elevation in [Ca2+]i. In
the first series of experiments, PMN were stimulated with 100 nM fMLP in the absence of extracellular Ca2+.
As shown in Fig. 3B, tyrosine phosphorylation of p38
proceeded normally under these conditions. Because release from
intracellular stores is the predominant component of the early response
of [Ca2+]i to fMLP, omission of external
Ca2+ has comparatively small effects on this response. To
analyze more effectively the role of [Ca2+]i,
experiments were undertaken using cells that had their internal stores
depleted prior to addition of the chemoattractant. The stores were
depleted using thapsigargin, an inhibitor of the endomembrane
Ca2+-ATPase. Parallel experiments using indo-1 demonstrated
that, following pretreatment with 100 nM thapsigargin for 5 min in Ca2+-free medium, stimulation with fMLP failed to
increase [Ca2+]i (not illustrated; see Ref. 36).
Using a comparable protocol, the effect of Ca2+ depletion
on the phosphorylation of p38 was assessed by immunoprecipitation and
blotting. The pretreatment with thapsigargin reduced, but did not
eliminate, the tyrosine phosphorylation of p38 (see Fig. 5). It is
noteworthy that the transient elevation in
[Ca2+]i which accompanies inhibition of the
ATPase by thapsigargin in Ca2+-free medium had no effect on
p38 phosphorylation.
The role of [Ca2+]i was also analyzed in cells loaded with BAPTA, an effective Ca2+-chelating agent. Under the conditions used, BAPTA virtually eliminates the [Ca2+]i transient elicited by fMLP, as measured with indo-1 (not shown). As in the case of thapsigargin, obliteration of the [Ca2+]i transient with BAPTA only partially inhibited (70%) the chemoattractant-induced tyrosine phosphorylation of p38. Together, these observations indicate that elevated [Ca2+]i is not sufficient, nor is it absolutely necessary, to activate p38.
Activation of MAPKAPK-2 by fMLP in Human PMNp38 was proposed
to be an upstream activator of MAPKAPK-2. This suggestion is based on
the ability of p38 to reactivate dephosphorylated, inactive MAPKAPK-2
(27). Because p38 was found to be activated by fMLP, we next determined
whether MAPKAPK-2 is also present and activated by chemoattractants in
PMN. A single highly immunoreactive band of 50 kDa was detected in PMN
lysates by the MAPKAPK-2 antibody (Fig. 4). Upon
stimulation with fMLP, a progressive decrease in the electrophoretic
mobility of MAPKAPK-2 was noticed. The shift was detectable within
30 s, reached maximal levels around 1 min, and persisted for up to
10 min.
To define whether MAPKAPK-2 is indeed phosphorylated and activated upon
fMLP stimulation of PMN, the kinase was immunoprecipitated from cells
treated with or without the chemoattractant. The immune complexes were
tested for kinase activity using HSP27 as a substrate. Fig.
5A illustrates that stimulation of the cells
with fMLP induced a marked increase in the ability of MAPKAPK-2 to
phosphorylate HSP27. A second phosphorylated band of 50 kDa was also
noticeable in fMLP-stimulated samples, which became clearly apparent
upon longer exposure of the radiograms (Fig. 5B). Although
fainter than the HSP27 band, the 50-kDa band was consistently observed and likely represents autophosphorylated MAPKAPK-2 (37).
Independent evidence that MAPKAPK-2 is activated by fMLP and is responsible for the HSP27 kinase activity in stimulated PMN was obtained by subjecting cell lysates to Mono Q chromatography. In chemoattractant-stimulated PMN, an increase in HSP27 kinase activity was detected in fractions 21-23 (Fig. 5C). The collected fractions were also analyzed by SDS-PAGE, and the distribution of MAPKAPK-2 was tested by immunoblotting. Using the polyclonal MAPKAPK-2-specific antibody, immunoreactive bands were observed in the same fractions that displayed stimulated HSP27 kinase activity. These results thus demonstrate the presence of a 50-kDa MAPKAPK-2 in human PMN which possesses HSP27-kinase activity, which is stimulated by fMLP.
Because p38 activity was affected differentially by wortmannin and
LY294002, we used these agents to define whether this kinase or Erk is
responsible for the phosphorylation and activation of MAPKAPK-2. As
shown in Fig. 6A, pretreatment of PMN with
wortmannin partially inhibited the electrophoretic mobility of
MAPKAPK-2 induced by fMLP. Moreover, as observed for p38 in Fig.
3A, pretreatment with LY294002 alone induced the
electrophoretic mobility shift of MAPKAPK-2. Subsequent stimulation
with fMLP further potentiated the band shift. That the mobility shift
was an indication of an active MAPKAPK-2 was confirmed with a kinase
assay. Fig. 6, B and C, shows that the
autophosphorylation of MAPKAPK-2 and the phosphorylation of HSP27,
respectively, were inhibited similarly by wortmannin and stimulated by
LY294002, as observed previously for p38. These results indicate that
the regulation of MAPKAPK-2 by PI 3-kinase closely resembles that of
p38 and clearly differs from those of Erk-1 and -2.
That p38, and not Erk, is the upstream activator of MAPKAPK-2 was
confirmed by pretreating PMN with a specific inhibitor of p38, namely
SB203580 (17). Pretreatment of PMN for 20 min with 10 µM
SB203580 completely eliminated the electrophoretic mobility shift of
MAPKAPK-2 upon stimulation with fMLP (Fig.
7A). More direct evidence of the effect of
SB203580 on the activity of MAPKAPK-2 was obtained by quantifying its
autophosphorylation (Fig. 7B) and its ability to
phosphorylate HSP27 in vitro (Fig. 7C). SB203580 consistently eliminated the activation of MAPKAPK-2 in PMN stimulated by fMLP.
Association of p38 with MAPKAPK-2 in Human PMN
Since the
phosphorylation and activation of MAPKAPK-2 and p38 occur in parallel,
we explored the possibility that these two kinases might physically
associate in vivo. In the first series of experiments,
MAPKAPK-2 was immunoprecipitated, and the presence of p38 in the immune
complexes was assessed. The results of one such experiment are
illustrated in Fig. 8A (left panel). An
immunoreactive band of 42 kDa was detected in both untreated and
fMLP-stimulated PMN, suggesting an association between MAPKAPK-2 and
p38. Of note, lower amounts of p38 were consistently detected in the
immune complexes following stimulation of cells with fMLP. This
observation suggests that dissociation of the complex occurred upon
activation of p38 and/or MAPKAPK-2. However, at least part of the
active p38 remains associated with MAPKAPK-2, since clear
immunoreactivity at 42 kDa was detectable when MAPKAPK-2 precipitates
were blotted with anti-phosphotyrosine antibodies (Fig.
8A, right panel). Accordingly, MAPKAPK-2 precipitates obtained from fMLP-stimulated cells displayed kinase activity toward ATF-2, indicative of associated p38 activity and
p38 immunoprecipitates contained HSP27 kinase activity (Fig. 8B). The interaction between the two kinases was also probed
using the reverse protocol, i.e. immunoprecipitation of p38
followed by detection of MAPKAPK-2. However, immunoblotting of such
precipitates with anti-MAPKAPK-2 antibody did not yield conclusive
results because of the close proximity of the immunoglobulin heavy
chain (53 kDa) with MAPKAPK-2 (not shown).
Stimulation of PMN by chemotactic peptides leads to the phosphorylation of several intracellular proteins that are believed to participate in the activation of the microbicidal function of these cells. In the present study we described the presence of p38 and MAPKAPK-2 in human PMN and their activation by fMLP which was found to be rapid and transient. This result contrasts with the much slower activation of p38 induced by cytokines or environmental stresses, which has been found to peak after 15-30 min (21). Moreover, we provided evidence that these kinases are associated with each other in quiescent cells and that their dissociation is facilitated by chemotactic stimulation.
The kinase(s) directly responsible for the phosphorylation and
activation of p38 in human PMN have yet to be determined. At least
three dual specificity kinases, MKK3 (37), MKK4 (37, 38), and MKK6
(39), possess the ability to phosphorylate p38. However, their presence
and role toward p38 activation in PMN remain to be defined. Recent
studies have shown that p38 can be activated in cells transfected with
constitutively active Rac or Cdc42 (40, 41, 42) possibly via a
21-
ctivated
inase (PAK) (40).
This notion stems from the demonstration that overexpression of a
dominant-negative PAK prevented the activation of p38 induced by Rac or
Cdc42. It is therefore attractive to suggest that one pathway leading
to p38 activation by fMLP in PMN involves Rac and/or Cdc42, followed by
PAK. Indeed, two isoforms of PAK have been detected in human PMN, where
they can be activated by fMLP (43). Moreover, Knaus et al.
(43) noted that PAK activation by fMLP includes a PI
3-kinase-dependent step, which may explain the inhibitory
effect of wortmannin on p38 reported in the present study. It is clear,
however, that an additional pathway(s) must contribute to the
activation of p38, since only partial inhibition was obtained with a
concentration of wortmannin known to block PI 3-kinase completely. That
an additional pathway may involve PKC is suggested by the following
observations. First, the activation of p38 was partially prevented by
BIM, a PKC inhibitor. Second, PMA, a direct activator of PKC, also
induced partial activation of p38. Finally, obliteration of the
cytosolic Ca2+ transient triggered by fMLP resulted in a
degree of inhibition comparable to that observed with BIM. It is thus
tempting to speculate that Ca2+-sensitive isoforms of PKC,
such as the
or
isoenzymes, may be involved in the activation of
p38. These two conventional isoforms of PKC have, in fact, been
detected in human PMN (44, 45, 46). That the PKC-dependent
pathway of p38 activation is independent of PAK is suggested by the
fact that PMA is unable to activate PAK in human PMN (43).
Interestingly, the inhibitory effects of wortmannin and BIM on p38
activation are not additive (results not shown), suggesting convergence
of these pathways and the existence of additional activating
signals.
The results we obtained with two distinct inhibitors of PI 3-kinase
yielded an apparent discrepancy. Although wortmannin partially blocked
the activation of p38, LY294002 had little inhibitory activity and, in
fact, markedly stimulated the kinase even in the absence of
chemoattractants. The mechanisms of inhibition of these agents are
different: wortmannin is an alkylating agent, producing irreversible
inhibition of PI 3-kinase (47), whereas LY294002 is a competitive
antagonist of the ATP binding site of the lipid kinase (48). Another
possible target of wortmannin and LY294002 may be found in
apamycin
nd
KBP12
arget (RAFT, the mammalian homolog of the yeast TOR),
which has homology to the lipid kinase domain of PI 3-kinase and PI
4-kinase (49). It is tempting to speculate that RAFT might be upstream
of p38 and that LY294002 and wortmannin modulate differentially the
kinase activity of RAFT, through its lipid kinase domain. However, the presence of RAFT in human PMN and its regulation have yet to be uncovered. Since both PI 3-kinase inhibitors were comparably effective in suppressing the fMLP-induced activation of Erk-1 and -2, it is
likely that PI 3-kinase was inhibited in both instances, as expected
from the concentrations of the antagonists used, based on earlier
literature (33). It is therefore reasonable to assume that LY294002 has
an additional effect on PMN, independent of its inhibition of PI
3-kinase. Alternatively, LY294002 may be able to activate purinergic
receptors on the surface of PMN (50), which may in turn lead to
activation of p38. Also, the effect of the comparatively large (100 µM) doses of LY294002 may be stress-related; indeed,
unlike Erk-1 and -2, p38 is activated by several types of stress,
including chemical stress. Clearly, further studies are required to
establish the mechanism by which LY294002 stimulates p38.
MAPKAPK-2 is activated in vitro by Erk-1 and Erk-2 (51) and also by p38 (23, 27). However, it has become increasingly apparent that in vivo only p38 is able to phosphorylate and activate MAPKAPK-2 (23, 27, 52). Our experiments revealed the presence of MAPKAPK-2 in PMN and established fMLP as a potent activator of this kinase. Because Erk-1 and -2 are also stimulated by fMLP, the possibility existed that MAPKAPK-2 was phosphorylated by these kinases instead of, or in addition to, p38. However, several lines of evidence argue against this possibility. First, LY294002, which inhibited Erk-1 and 2 but not p38, failed to inhibit MAPKAPK-2. Second, SB203580, which has no discernible effects on Erk-1 or -2 (17, 52), prevented the phosphorylation of MAPKAPK-2. Additionally, although we observed a constitutive association between p38 and MAPKAPK-2 in vivo, the latter was never detected to interact with Erk-1 and -2 in either untreated or fMLP-treated PMN (results not shown).
The ability of a member of the MAPK family to form heterodimeric complexes with other kinases in vivo is not without precedent. Hsiao et al. (53) noted that inactive Erk-2 associates with p90rsk in Xenopus oocytes; upon activation the complex was found to dissociate. Moreover, McLaughlin et al. (54) observed that in its inactive form, MAPKAPK-3 associates with p38 and that the interaction is disrupted upon exposure of the cells to hypertonic sorbitol, a treatment known to activate stress kinases. These interactions resemble the association between MAPKAPK-2 and p38 reported here. In other systems multiple kinases appear to be maintained in elaborate complexes by specific docking proteins (55). It will be of interest to determine whether additional kinases and/or docking proteins are found complexed with MAPKAPK-2 and p38 in PMN. Identification of the constituents of such putative complexes may help us understand the role of p38 and MAPKAPK-2 in the functional responses of PMN.