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Plays a Central Role in Activation of the p42/44 Mitogen-Activated Protein Kinase by Endotoxin in Alveolar Macrophages1
Department of Medicine, University of Iowa College of Medicine and Veterans Administration Medical Center, Iowa City, IA
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, in LPS-induced activation of the ERK kinase
pathway. Kinase activity assays showed that LPS activates PKC ,
mitogen-activated protein/ERK kinase (MEK, the upstream activator of
ERK), and ERK. LPS did not activate Raf-1, the classic activator of
MEK. Pseudosubstrate-specific peptides with attached myristic acid are
cell permeable and can be used to block the activity of specific PKC
isoforms in vivo. We found that a peptide specific for PKC
partially blocked activation of both MEK and ERK by LPS. We also found
that this peptide blocked in vivo phosphorylation of MEK after LPS
treatment. In addition, we found that LPS caused PKC to bind to MEK
in vivo. These observations suggest that MEK is an LPS-directed target
of PKC . PKC has been shown in other systems to be
phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase.
We found that LPS activates PI 3-kinase and causes the formation of a
PKC /PI 3-kinase-dependent kinase complex. These data implicate the
PI 3-kinase pathway as an integral part of the LPS-induced PKC
activation. Taken as a whole, these studies suggest that LPS activates
ERK kinase, in part, through activation of an atypical PKC isoform, PKC
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alveolar macrophages play a central role in the response of the lung to bacterial infection. They are specialized cells, which respond to infection by phagocytosis and killing of bacteria (6, 7, 8). They also respond to LPS by releasing large amounts of inflammatory mediators (5, 9). We and others have shown that activation of various mitogen-activated protein kinase (MAP kinase) pathways is necessary for expression of cytokine genes in alveolar macrophages in response to LPS (10, 11).
Three major MAP kinase cascades have been described in mammalian cells,
the extracellular signal-related kinase (ERK), p38, and c-Jun
N-terminal kinase pathways. All three of these pathways have been
linked to activation by LPS and subsequent cytokine gene expression. In
addition to LPS, the ERK pathway is activated by growth and
differentiation signals (12). Activation of the p38 and
c-Jun N-terminal kinase pathways is often linked to cell stress
(13, 14). In this study, we focus on pathways that are
immediately upstream of LPS-induced ERK activation. The best described
pathway leading to ERK activation is the Ras-Raf-1-mitogen-activated
protein/ERK kinase (MEK)-ERK kinase cascade (12). Another
possible activator of MEK is the "atypical" protein kinase C (PKC)
isoform, PKC . PKC is one of a large family of serine/threonine
kinases and is characterized by a lack of Ca2+
dependence or 1,2-diacylglycerol responsiveness. It has been linked to
a number of mitogenic signals (15).
In previous studies from this laboratory, we evaluated some of the
early signaling events associated with LPS activation of ERK in
alveolar macrophages. We showed that LPS activates a
phosphatidylcholine-specific phospholipase (PC-PLC), resulting in the
generation of ceramide. We also showed that LPS activates PKC and
that an inhibitor of PC-PLC inhibits activation of PKC and ERK.
These studies, however, did not establish a direct link between
activation of PKC and activation of ERK kinase. In this study, we
show that LPS activates ERK without any demonstrable activation of
Raf-1. LPS activates PKC in a time-dependent manner, which matches
the time course of LPS-induced ERK activation. We also show that PKC
can be linked to MEK/ERK activation in two ways. First, LPS induces
formation of a complex between PKC and MEK. In addition, a PKC
-specific inhibitory peptide blocks LPS-induced MEK and ERK
activation and the in vivo phosphorylation of MEK. We also demonstrate
LPS-induced activation of the phosphatidylinositol 3-kinase (PI
3-kinase) pathway and PKC /PI 3-kinase-dependent kinase (PDK-1)
complex formation. PDK-1 (downstream of PI 3-kinase), a constitutively
active kinase, will, when in proximity to PKC , phosphorylate
Thr410 in the activation loop of PKC . This
initial activation event is thought to allow autophosphorylation of
Thr560, resulting in an active kinase
(15, 16, 17). These studies clearly demonstrate that PKC
is important for LPS-induced activation of ERK in human alveolar
macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alveolar macrophages were obtained from bronchoalveolar lavage as previously described (18). Briefly, normal volunteers with a lifetime nonsmoking history, no acute or chronic illness, and no current medications underwent bronchoalveolar lavage. The lavage procedure used five 20-ml aliquots of sterile, warmed saline in each of three segments of the lung. The lavage fluid was filtered through two layers of gauze and centrifuged at 1500 x g for 5 min. The cell pellet was washed twice in HBSS without Ca2+ and Mg2+ and suspended in complete medium, RPMI tissue culture medium (Life Technologies, Gaithersburg, MD) with 100 ng/ml LBP (a gift from Peter Tobias, The Scripps Research Institute, La Jolla, CA), and added gentamicin (80 µg/ml). Differential cell counts were determined with a Wright-Giemsa-stained cytocentrifuge preparation. All cell preparations had between 90 and 100% alveolar macrophages. This study was approved by the Committee for Investigations Involving Human Subjects at the University of Iowa.
Immunoprecipitation
Alveolar macrophages were cultured in complete medium with or
without LPS (1 µg/ml, Sigma, St. Louis, MO). In some cases,
inhibitory peptides (PKC : myr-SIYRRGARRWRKL-OH; PKC 
ß:
myr-RFARKGALRQKNV-OH; nonsense: myr-LRISRAGRYRANWYRKR-OH; the
myristate on the N terminus of these peptides allows for membrane
permeability) were added 30 min before the LPS. After culture, cells
were lysed on ice for 20 min in 500 µl lysis buffer (0.05 M Tris (pH
7.4), 0.15 M NaCl, 1% Nonidet P-40, 0.5 M PMSF, 50 µg/ml aprotinin,
10 µg/ml leupeptin, 50 µg/ml pepstatin, 0.4 mM sodium
orthovanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate,
all from Boehringer Mannheim, Indianapolis, IN). The lysates were then
sonicated for 20 s and spun at 15,000 x g for 10
min, and the supernatant was saved. Protein was measured, and 200–600
µg from each sample were removed for immunoprecipitation. The samples
were cleared by incubating for 2 h with 1 µg/sample rabbit IgG
and 10 µl/sample GammaBind Sepharose (Pharmacia, Piscataway, NJ).
After centrifugation, the supernatants were transferred to a tube
containing 3 µg/sample kinase Ab (all of the Abs used in this paper,
except for the phospho-ERK Ab (Sigma), were obtained from Santa Cruz
Biotechnology, Santa Cruz, CA) bound to GammaBind Sepharose, and
rotated at 4oC overnight. The beads were
subsequently washed three times with high salt buffer (0.5 M Tris (pH
7.4), 0.50 M NaCl, and 1% Nonidet P-40) and three times with lysis
buffer without protease inhibitors. The immunoprecipitated complexes
were either released with 2x sample buffer for Western analysis or
used to determine kinase activity. In the case of Western analysis, the
samples were sometimes divided, and two separate gels were run to
evaluate the formation of protein complexes.
Kinase activity assay
After immunoprecipitating the relevant kinase from alveolar
macrophages, the protein-containing pellet was washed twice with kinase
buffer (20 mM MgCl2, 25 mM HEPES, 20 mM
ß-glycerophosphate, 20 mM p-nitrophenylphosphate, 20 mM
sodium orthovanadate, and 2 mM DTT). The pellet was then suspended in
20 µl kinase buffer, and the following was added: 20 µM ATP, 5
µCi [
-32P]ATP (BLU 002Z, DuPont/NEN,
Boston MA) and 10 µg myelin basic protein (MBP, Sigma) or 5 µg
MEK-1 or 5 µg ERK (polyhistidine-tagged fusion proteins from Santa
Cruz Biotechnology). The total volume of sample plus additions at
this point was 25 µl. The reaction was continued for 15 min to 1
h, depending on the kinase, at 25°C, and then stopped by the addition
of 25 µl/sample 2x sample buffer. The samples were
boiled for 5 min and run on a 12% SDS-PAGE gel. The gel was dried, and
autoradiography was performed to visualize the
32P-labeled MBP, MEK-1, or ERK. Densitometry was
performed on films and fold increase calculated as experimental
sample/control sample.
Western analysis
Western analysis (all Abs from Santa Cruz Biotechnology) was
performed on three different types of samples: 1) whole cell protein,
for the phosphorylated form of ERK; 2) immunoprecipitated proteins from
the kinase activity assay to determine equal loading of the proteins in
the assay; and 3) samples divided into two fractions after
immunoprecipitation to monitor intracellular complex formation. For
straight Westerns, 50–100 µg of protein were mixed 1:1 with 2x
sample buffer (20% glycerol, 4% SDS, 10% 2-ME, 0.05% bromphenol
blue, and 1.25 M Tris (pH 6.8); all chemicals from Sigma), loaded onto
a 10% SDS-PAGE gel, and run at 30 mA for 3 h. Cell proteins were
transferred to nitrocellulose (ECL, Amersham, Arlington Heights, IL)
overnight at 30 V. The nitrocellulose was then blocked with 5% milk in
TTBS (Tris-buffered saline with 0.1% Tween 20) for 1 h, washed,
and then incubated with the primary Ab (anti-phosphorylated ERK,
Sigma) overnight. The blots were washed four times with TTBS and
incubated for 1 h with HRP-conjugated anti-rabbit IgG Ab
(Amersham, at 1:20,000 dilution). Immunoreactive bands were developed
using a chemiluminescent substrate (SuperSignal West Femto; Pierce,
Rockford, IL). To determine equal loading in the kinase activity
assays, 20% of the total immunoprecipitated protein was mixed 1:1 with
2x sample buffer and Western analysis performed as described above.
For complex formation assays, immunoprecipitated samples were divided
in two after the last immunoprecipitation wash, and two gels were run.
After transfer, one blot was analyzed for the immunoprecipitated
protein (MEK or PKC ), and the other blot was analyzed for PKC
or PDK-1.
In vivo phosphorylation of MEK
Alveolar macrophages were labeled with 1.25 mCi 32Pi/group (NEN Life Science Products, Boston, MA) in phosphate-free RPMI without serum for 3 h at 37°C. The cells were harvested and placed in RPMI with 100 ng/ml LBP and treated with peptides for 30 min. After the peptide incubation, the cells were stimulated with LPS for 15 min at 37°C. The cells were harvested, resuspended in lysis buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M Na3PO4 (pH 7.2), 2 mM Na3VO4, 1 µM okadaic acid, 100 µg/ml PMSF, 50 µg/ml aprotinin, 10 µg/ml leupeptin, and 50 µg/ml pepstatin, all from Boehringer Mannheim), and sonicated. MEK was immunoprecipitated from the lysate, and the sample separated on a 10% SDS-PAGE discontinuous gel as described above.
PI 3-kinase activity assay
After culture, whole cell lysates were obtained and PI 3-kinase
was immunoprecipitated using an Ab to the p85-regulatory subunit of PI
3-kinase (Santa Cruz Biotechnology). Activity was assayed by measuring
the formation of PI 3-[32P]phosphate (19, 20). After overnight incubation with Ab-coated beads (see
Immunoprecipitation), the bound protein was washed three
times with buffer I (PBS containing 1% Nonidet P-40 and 100 µM
Na3VO4), three times with
buffer II (100 mM Tris-HCl (pH 7.5), 500 mM LiCl, and 100 µM
Na3VO4), and finally three
times with buffer III (Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA and
100 µM Na3VO4). After
washing, immunoprecipitates were resuspended in 50 µl buffer III with
the addition of 10 µl 100 mM MgCl2 and 10 µl
PI (2 µg/ml). The samples sat at room temperature for 5 min before
the addition of 10 µl ATP (ATP 440 µM with 30 µCi/10 µl
[-32P]ATP). The samples were then shaken at
room temperature for 10 min. The reaction was stopped by the addition
of 20 µl 8 N HCl and 160 µl chloroform-methanol (1:1). The lipids
were extracted by standard methods, dried down, resuspended in 20 µl
chloroform-methanol (1:1), and separated on thin layer silica gel
plates (pretreated with 10% w/v potassium oxalate) in a solvent system
of chloroform-methanol-water-NH4OH (60:47:11:2.2,
v/v/v/v). Incorporation of 32P into PI
3-phosphate was detected by autoradiography, and activity was
quantified on a Bio-Rad Molecular Imager FX.
Statistical analysis
Statistical analysis of the densitometric data was performed by determining the fold increase of all the samples as they relate to the control. Statistical comparisons were performed using a paired t test with a probability value of p < 0.05 considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially wanted to confirm that LPS activated ERK in alveolar
macrophages. To do this, we cultured alveolar macrophages in complete
RPMI for various time points. At the end of the experiment, whole cell
lysates were obtained, and Western analysis was performed. The blots
were probed with an Ab specific for the phosphorylated form of ERK
(phospho-Thr183 and Tyr185)
and one for total ERK. We found that LPS caused a time-dependent
increase in the amount of phosphorylated ERK in alveolar macrophages
(Fig. 1
). The best described pathway for
ERK activation is the Ras-Raf-1-MEK-ERK phosphorylation cascade. The
next two experiments evaluate the feasibility of this scenario in
LPS-induced ERK activation.
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To evaluate the activation of Raf-1 in LPS-treated alveolar
macrophages, we performed kinase activity assays using
immunoprecipitated ERK and Raf-1 from LPS-treated cells. In Fig. 2, we show that LPS strongly activates
ERK kinase, while having little effect on the activity of Raf-1 in
alveolar macrophages. In addition, we show composite data from three
experiments, using densitometry to calculate fold increase. Control
values are designated as 1. To ensure that we were not missing possible
Raf-1 activation, we performed a kinase activity assay on Raf-1
compared with ERK immunoprecipitated from various time points (Fig. 3). This experiment demonstrates that at
multiple time points when there is demonstrable ERK activation, there
is no Raf-1 activity. To validate our experimental system,
we performed one more experiment. Alveolar macrophages were treated
with LPS or PMA, and then kinase activity assays were performed for ERK
2 or Raf-1. Fig. 4 shows that LPS
activated ERK without activating Raf-1. PMA, however, a strong
activator of the conventional and novel PKCs known to activate Raf-1,
does activate Raf-1 in alveolar macrophages.
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, MEK, and ERK
Our previous work demonstrated that LPS can activate an atypical
PKC isoform, , and that this is linked to LPS-induced activation of
a PC-PLC. We also showed that LPS generates ceramide in alveolar
macrophages. Because of this finding and previous work by other authors
showing that PKC could be activated by ceramide and phosphatidic
acid, we evaluated the hypothesis that PKC might be the upstream
kinase responsible for MEK activation in alveolar macrophages
(21, 22). In Fig. 5, kinase
activity assays, using relevant substrates (MEK for PKC , ERK for
MEK, and MBP for ERK), show that LPS causes a substantial activation of
PKC , MEK, and ERK. In Fig. 6, we show
that LPS activates PKC over an extended time course. We also
evaluated the response of PKC to various amounts of LPS and found that
PKC was activated by a range of LPS doses (10 ng/ml to 1 µg/ml)
(data not shown). These experiments show that LPS activates PKC and
that the results of activation assays are consistent with a PKC
-MEK-ERK pathway.
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-specific
peptide
An important step in the activation of PKC is the removal of
the pseudosubstrate region from the kinase core (Fig. 7A). In these experiments, we
use a myristolated peptide specific for the pseudosubstrate region
to inhibit PKC activity in vivo. Fig. 8 shows that the PKC -specific peptide
blocks LPS-induced ERK activation, whereas a peptide specific for PKC
and ß has no effect. Fig. 8 (bottom) shows data from
three separate experiments, confirming the kinase activity assay shown.
In Fig. 9, using a kinase activity assay,
we show that the PKC -specific peptide also blocks MEK activation,
whereas the - and ß-specific peptide and a peptide made from
scrambled -amino acids had no effect. These experiments directly
link LPS-induced ERK activation to PKC , by showing that a PKC
-specific inhibitor can inhibit ERK activation by LPS.
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Along with activation by phosphorylation, one mechanism, which
regulates kinase activity, is the formation of signaling complexes. For
activation to occur, kinases must be brought into proximity with each
other. We investigated the effect of LPS on PKC /MEK complex
formation. Alveolar macrophages were treated with LPS and then MEK was
immunoprecipitated from the lysates. After immunoprecipitation, the
sample was divided in half and Western analysis for MEK (equal loading)
and PKC (association) was performed. Fig. 10 shows that LPS causes an increase in
MEK/PKC association.
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-specific peptide
To further evaluate the link between PKC and MEK, we performed
the following assay. Alveolar macrophages were phosphate loaded with
32P and then treated with a PKC -specific
peptide or a nonsense peptide before activation with LPS. The cells
were then lysed and MEK was immunoprecipitated from the lysate. A 10%
SDS gel was run and dried, and an autoradiograph was obtained. Fig. 11 shows that LPS causes an increased
phosphorylation of MEK that is blocked by the PKC -specific peptide
and not by the nonsense peptide. This experiment places PKC
upstream of MEK in LPS-activated alveolar macrophages.
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Activation of PI 3-kinase results in the conversion of PI
4-phosphate and PI 4,5-bisphosphate to PI 3,4-bisphosphate and PI
3,4,5-trisphosphate, respectively. These lipid species interact with
the pleckstrin homology domains on a number of proteins bringing them
to the membrane where they become activated (16, 17). The
initial downstream event after PI 3-kinase activation is activation of
the kinase PDK-1. This kinase is known to phosphorylate a threonine in
the activation loop of a number of kinases. Phosphorylation by PDK-1 is
the initial event in a sequence of phosphorylations of these kinases
that result in activation. PDK-1 has been shown to phosphorylate
protein kinase A, Akt (protein kinase B), PKC (on
Thr410; see Fig. 7
B), and some of the
"novel" PKCs (15, 16, 17, 23). One defining feature of
PDK-1 is that it is a constitutively active kinase, which is regulated
by binding to its pleckstrin homology domain and proximity to
substrate. To investigate the role of PI 3-kinase on PKC activation
in alveolar macrophages, we performed the following experiments.
Initially, we found that LPS activated PI 3-kinase in a time-dependent
manner (Fig. 12). LPS-treated alveolar
macrophages were lysed at various time points, and PI 3-kinase was
immunoprecipitated. Kinase activity was determined using PI as a
substrate and then separating the phosphorylated species on a TLC
plate. We then evaluated whether or not PDK-1 might be involved in PKC
activation by examining complex formation. We found that LPS caused
a significant increase in the association of PDK-1 with PKC (Fig. 13). These experiments suggest that LPS
activates PKC via activation of PI 3-kinase and PKC /PDK-1
complex formation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in LPS activation of ERK.
LPS causes a rapid and time-dependent activation of ERK. This is not
matched by an increase in the activation of Raf-1, the best described
activator of MEK (the MAP kinase kinase for ERK). Based on studies in
the literature and previous studies in our laboratory, we evaluated the
hypothesis that PKC , rather than Raf-1, was the MAP kinase kinase
involved in MEK activation in LPS-treated alveolar macrophages. We
demonstrated that this was so in a number of ways. First we showed
activation of PKC by LPS. To link this to MEK activation, we used a
PKC pseudosubstrate-specific peptide to show that in vivo blocking
of PKC resulted in decreased MEK and ERK activation. Finally, we
showed that LPS induced the physical association of MEK and PKC and
that in vivo phosphorylation of MEK by LPS was blocked by a PKC
-specific peptide. As further support for the activation of PKC
by LPS, we showed that LPS activated the lipid kinase, PI 3-kinase, and
caused an association between PKC and PDK-1 (an activation loop
kinase). The various kinase activity assays shown in this study show
slightly different time frames. Because of the difficulties in using
primary cells and in freezing cellular activity at short time frames,
we do not make any conclusions about the relative time frames of these
events. We have shown, however, that ERK, PKC , and PI 3-kinase are
all activated shortly after endotoxin exposure. In conclusion, these
studies show that LPS activates ERK in alveolar macrophages, at least
in part, through a Raf-1-independent pathway involving PKC (Fig. 14).
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. Insulin also has been shown to activate ERK in a Raf-1-independent
manner (26). Zheng et al. (27) showed in
Swiss 3T3 cells that EGF activated ERK without any demonstrable Raf-1
activation. More recently, Kartha et al. (28) have shown
that in myocytes, platelet-derived growth factor stimulates a MEK
kinase that is distinct from all members of the Raf family. In an
inflammation model, IL-8 has been shown to activate ERK independently
of Ras and Raf-1 (29). Directly applicable to our
hypothesis is a study by Takeda et al. (30), who showed
that lysophosphatidic acid activated ERK in a Ras-independent manner
and that ERK activation could be blocked by inhibitors of PKC and
PI 3-kinase.
Previous work in our laboratory suggests that ceramide is involved in
PKC activation in LPS-treated cells (31). We found
that LPS increased amounts of ceramide in alveolar macrophages and that
the addition of exogenous ceramide resulted in ERK activation. Studies
in other laboratories have also found that PKC is activated by
ceramide (22, 32). A number of studies have documented
that LPS induces ceramide and that increases in ceramide can be linked
to a Raf-1-independent activation of ERK (33, 34, 35).
Further, a recent study demonstrated that ceramide inhibited Raf-1
activity (36). A study by Muller et al. (37)
also showed that whereas TNF and ceramide both increased Ras-Raf-1
complexes, the ceramide bound to the catalytic domain of Raf-1,
preventing activity.
Ceramide may play a second role in LPS activation of ERK, because it
has been linked to PKC activation. Ceramide has been shown to bind
and activate PKC in a number of studies. Recently, Wang et al.
(32) have shown that PKC activation by ceramide is
very concentration dependent; low doses activate and high doses
inhibit. Varying amounts of ceramide induction by LPS might be one way
alveolar macrophages regulate the magnitude of their response to
LPS.
Another possible pathway involved in the activation of PKC and
subsequently ERK by LPS in alveolar macrophages is the PI 3-kinase
pathway. PDK-1, a kinase that is downstream of PI 3-kinase is known to
phosphorylate PKC in the activation loop
(Thr410) (38, 39). Sajan et al.
(40) have linked PDK-1 and PKC to ERK activation by
LPS in rat adipocytes. In a model closer to ours, Herrara-Velit et al.
(41) have shown that in LPS-treated monocytes, PKC
activation is PDK-1 dependent. PKC activation requires
autophosphorylation, phosphorylation of Thr402 in
the activation loop (probably by PDK-1), removal of the pseudosubstrate
from the catalytic domain, and subcellular localization near a
substrate (42). It is possible that both ceramide and PI
3-kinase play a role in the activation of PKC by LPS. Additional
studies will be necessary to delineate exactly how PKC is activated
in alveolar macrophages. These studies, however, clearly show that PKC
plays a central role in ERK activation in LPS-stimulated human
alveolar macrophages.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martha M. Monick, Division of Pulmonary, Critical Care, and Occupational Medicine, Room 100, Eckstein Medical Research Building, University of Iowa Hospitals and Clinic, Iowa City, IA 52242.
3 Abbreviations used in this paper: LBP, LPS-binding protein; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; MEK, mitogen-activated protein/ERK kinase (the upstream activator of ERK); PKC, protein kinase C; PC-PLC, phosphatidylcholine-specific phospholipase; MBP, myelin basic protein; PI 3-kinase, phosphatidylinositol 3-kinase; PDK-1, PI 3-kinase-dependent kinase.
Received for publication April 3, 2000. Accepted for publication July 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activation and cytokine release in human alveolar macrophages is PKC-independent and TK- and PC-PLC-dependent. Am. J. Respir. Cell Mol. Biol. 18:384. receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn. J. Exp. Med. 191:669. and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3-kinase. J. Biol. Chem. 274:12722., bifunctionally regulated by ceramide and arachidonic acid. EMBO J. 14:1961.[Medline]
rapidly activates the mitogen-activated protein kinase (MAPK) cascade in a MAPK kinase kinase-dependent, c-Raf-1-independent fashion in mouse macrophages. Proc. Natl. Acad. Sci. USA 92:1614. and protein kinase C-zeta mediate RAS-independent activation of MAP kinase by a Gi protein-coupled receptor. EMBO J. 18:386.[Medline]
is activated by ceramide, resulting in coactivation of NF-B/JNK kinase and cell survival. J. Neurosci. Res. 55:293.[Medline]
production by human monocytes involves the raf-1/MEK1-MEK2/ERK1-ERK2 pathway. Infect. Immun. 67:3824.. J. Biol. Chem. 274:8117. and phosphoinositide-dependent protein kinase-1 are required for insulin-induced activation of ERK in rat adipocytes. J. Biol. Chem. 274:30495. in bacterial lipopolysaccharide-treated human monocytes. J. Biol. Chem. 272:16445.This article has been cited by other articles:
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D. P. Sester, K. Brion, A. Trieu, H. S. Goodridge, T. L. Roberts, J. Dunn, D. A. Hume, K. J. Stacey, and M. J. Sweet CpG DNA Activates Survival in Murine Macrophages through TLR9 and the Phosphatidylinositol 3-Kinase-Akt Pathway J. Immunol., October 1, 2006; 177(7): 4473 - 4480. [Abstract] [Full Text] [PDF]
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M. M. Monick, L. S. Powers, T. J. Gross, D. M. Flaherty, C. W. Barrett, and G. W. Hunninghake Active ERK Contributes to Protein Translation by Preventing JNK-Dependent Inhibition of Protein Phosphatase 1 J. Immunol., August 1, 2006; 177(3): 1636 - 1645. [Abstract] [Full Text] [PDF]
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D. MacGlashan Jr. and N. Vilarino Nonspecific Desensitization, Functional Memory, and the Characteristics of SHIP Phosphorylation following IgE-Mediated Stimulation of Human Basophils J. Immunol., July 15, 2006; 177(2): 1040 - 1051. [Abstract] [Full Text] [PDF]
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E. Sanchez-Tillo, M. Comalada, C. Farrera, A. F. Valledor, J. Lloberas, and A. Celada Macrophage-Colony-Stimulating Factor-Induced Proliferation and Lipopolysaccharide-Dependent Activation of Macrophages Requires Raf-1 Phosphorylation to Induce Mitogen Kinase Phosphatase-1 Expression. J. Immunol., June 1, 2006; 176(11): 6594 - 6602. [Abstract] [Full Text] [PDF]
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 L. Servillo, C. Balestrieri, A. Giovane, P. Pari, D. Palma, G. Giannattasio, M. Triggiani, and M. L. Balestrieri Lysophospholipid Transacetylase in the Regulation of Paf Levels in Human Monocytes and Macrophages FASEB J, May 1, 2006; 20(7): 1015 - 1017. [Abstract] [Full Text] [PDF]
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N. Vilarino, K. Miura, and D. W. MacGlashan Jr Acute IL-3 Priming Up-Regulates the Stimulus-Induced Raf-1-Mek-Erk Cascade Independently of IL-3-Induced Activation of Erk J. Immunol., September 1, 2005; 175(5): 3006 - 3014. [Abstract] [Full Text] [PDF]
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 N. Ledirac, S. Antherieu, A. D. d'Uby, J.-C. Caron, and R. Rahmani Effects of Organochlorine Insecticides on MAP Kinase Pathways in Human HaCaT Keratinocytes: Key Role of Reactive Oxygen Species Toxicol. Sci., August 1, 2005; 86(2): 444 - 452. [Abstract] [Full Text] [PDF]
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A. G. Granja, M. L. Nogal, C. Hurtado, V. Vila, A. L. Carrascosa, M. L. Salas, M. Fresno, and Y. Revilla The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through a Nuclear Factor of Activated T Cell-dependent Transactivation Pathway J. Biol. Chem., December 17, 2004; 279(51): 53736 - 53746. [Abstract] [Full Text] [PDF]
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 W. Wu, R. D. Mosteller, and D. Broek Sphingosine Kinase Protects Lipopolysaccharide-Activated Macrophages from Apoptosis Mol. Cell. Biol., September 1, 2004; 24(17): 7359 - 7369. [Abstract] [Full Text] [PDF]
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A. Punturieri, R. S. Alviani, T. Polak, P. Copper, J. Sonstein, and J. L. Curtis Specific Engagement of TLR4 or TLR3 Does Not Lead to IFN-{beta}-Mediated Innate Signal Amplification and STAT1 Phosphorylation in Resident Murine Alveolar Macrophages J. Immunol., July 15, 2004; 173(2): 1033 - 1042. [Abstract] [Full Text] [PDF]
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 P. Robin, I. Boulven, C. Bole-Feysot, Z. Tanfin, and D. Leiber Contribution of PKC-dependent and -independent processes in temporal ERK regulation by ET-1, PDGF, and EGF in rat myometrial cells Am J Physiol Cell Physiol, April 1, 2004; 286(4): C798 - C806. [Abstract] [Full Text] [PDF]
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J. S. Lee, Z. Hmama, A. Mui, and N. E. Reiner Stable Gene Silencing in Human Monocytic Cell Lines Using Lentiviral-delivered Small Interference RNA: SILENCING OF THE p110{alpha} ISOFORM OF PHOSPHOINOSITIDE 3-KINASE REVEALS DIFFERENTIAL REGULATION OF ADHERENCE INDUCED BY 1{alpha},25-DIHYDROXYCHOLECALCIFEROL AND BACTERIAL LIPOPOLYSACCHARIDE J. Biol. Chem., March 5, 2004; 279(10): 9379 - 9388. [Abstract] [Full Text] [PDF]
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S. D. Savkovic, A. Koutsouris, and G. Hecht PKC{zeta} participates in activation of inflammatory response induced by enteropathogenic E. coli Am J Physiol Cell Physiol, September 1, 2003; 285(3): C512 - C521. [Abstract] [Full Text] [PDF]
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M. M. Monick, L. S. Powers, N. S. Butler, and G. W. Hunninghake Inhibition of Rho Family GTPases Results in Increased TNF-{alpha} Production After Lipopolysaccharide Exposure J. Immunol., September 1, 2003; 171(5): 2625 - 2630. [Abstract] [Full Text] [PDF]
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 E. Mazzoni, A. Adam, E. Bal de Kier Joffe, and J. A. Aguirre-Ghiso Immortalized Mammary Epithelial Cells Overexpressing Protein Kinase C {gamma} Acquire a Malignant Phenotype and Become Tumorigenic in Vivo Mol. Cancer Res., August 1, 2003; 1(10): 776 - 787. [Abstract] [Full Text] [PDF]
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M. B. Hock and M. A. Brown Nuclear Factor of Activated T Cells 2 Transactivation in Mast Cells: A NOVEL ISOFORM-SPECIFIC TRANSACTIVATION DOMAIN CONFERS UNIQUE Fc{epsilon}RI RESPONSIVENESS J. Biol. Chem., July 11, 2003; 278(29): 26695 - 26703. [Abstract] [Full Text] [PDF]
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 T. Kikuchi, Y. Yoshikai, J. Miyoshi, M. Katsuki, T. Musikacharoen, A. Mitani, S. Tanaka, T. Noguchi, and T. Matsuguchi Cot/Tpl2 is Essential for RANKL Induction by Lipid A in Osteoblasts Journal of Dental Research, July 1, 2003; 82(7): 546 - 550. [Abstract] [Full Text] [PDF]
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K. C. Corbit, N. Trakul, E. M. Eves, B. Diaz, M. Marshall, and M. R. Rosner Activation of Raf-1 Signaling by Protein Kinase C through a Mechanism Involving Raf Kinase Inhibitory Protein J. Biol. Chem., April 4, 2003; 278(15): 13061 - 13068. [Abstract] [Full Text] [PDF]
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 J Berkes, V K Viswanathan, S D Savkovic, and G Hecht Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation Gut, March 1, 2003; 52(3): 439 - 451. [Abstract] [Full Text] [PDF]
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T. Hirai and K. Chida Protein Kinase C{zeta} (PKC{zeta}): Activation Mechanisms and Cellular Functions J. Biochem., January 1, 2003; 133(1): 1 - 7. [Abstract] [Full Text] [PDF]
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M. M. Monick, P. K. Robeff, N. S. Butler, D. M. Flaherty, A. B. Carter, M. W. Peterson, and G. W. Hunninghake Phosphatidylinositol 3-Kinase Activity Negatively Regulates Stability of Cyclooxygenase 2 mRNA J. Biol. Chem., August 30, 2002; 277(36): 32992 - 33000. [Abstract] [Full Text] [PDF]
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 M.M. Monick and G.W. Hunninghake Activation of second messenger pathways in alveolar macrophages by endotoxin Eur. Respir. J., July 1, 2002; 20(1): 210 - 222. [Abstract] [Full Text] [PDF]
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P. Robin, I. Boulven, C. Desmyter, S. Harbon, and D. Leiber ET-1 stimulates ERK signaling pathway through sequential activation of PKC and Src in rat myometrial cells Am J Physiol Cell Physiol, July 1, 2002; 283(1): C251 - C260. [Abstract] [Full Text] [PDF]
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L. Samavati, M. M. Monick, S. Sanlioglu, G. R. Buettner, L. W. Oberley, and G. W. Hunninghake Mitochondrial KATP channel openers activate the ERK kinase by an oxidant-dependent mechanism Am J Physiol Cell Physiol, July 1, 2002; 283(1): C273 - C281. [Abstract] [Full Text] [PDF]
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Y. Ouadrhiri, C. Pilette, R. C. Monteiro, J.-P. Vaerman, and Y. Sibille Effect of IgA on Respiratory Burst and Cytokine Release by Human Alveolar Macrophages . Role of ERK1/2 Mitogen-Activated Protein Kinases and NF-kappa B Am. J. Respir. Cell Mol. Biol., March 1, 2002; 26(3): 315 - 332. [Abstract] [Full Text] [PDF]
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M. M. Monick, R. K. Mallampalli, A. B. Carter, D. M. Flaherty, D. McCoy, P. K. Robeff, M. W. Peterson, and G. W. Hunninghake Ceramide Regulates Lipopolysaccharide-Induced Phosphatidylinositol 3-Kinase and Akt Activity in Human Alveolar Macrophages J. Immunol., November 15, 2001; 167(10): 5977 - 5985. [Abstract] [Full Text] [PDF]
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M. M. Monick, A. B. Carter, P. K. Robeff, D. M. Flaherty, M. W. Peterson, and G. W. Hunninghake Lipopolysaccharide Activates Akt in Human Alveolar Macrophages Resulting in Nuclear Accumulation and Transcriptional Activity of {{beta}}-Catenin J. Immunol., April 1, 2001; 166(7): 4713 - 4720. [Abstract] [Full Text] [PDF]
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I. Suzuma, K. Suzuma, K. Ueki, Y. Hata, E. P. Feener, G. L. King, and L. P. Aiello Stretch-induced Retinal Vascular Endothelial Growth Factor Expression Is Mediated by Phosphatidylinositol 3-Kinase and Protein Kinase C (PKC)-zeta but Not by Stretch-induced ERK1/2, Akt, Ras, or Classical/Novel PKC Pathways J. Biol. Chem., January 4, 2002; 277(2): 1047 - 1057. [Abstract] [Full Text] [PDF]
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P. Rojnuckarin, Y. Miyakawa, N. E. Fox, J. Deou, G. Daum, and K. Kaushansky The Roles of Phosphatidylinositol 3-Kinase and Protein Kinase Czeta for Thrombopoietin-induced Mitogen-activated Protein Kinase Activation in Primary Murine Megakaryocytes J. Biol. Chem., October 26, 2001; 276(44): 41014 - 41022. [Abstract] [Full Text] [PDF]
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