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Division of Critical Care Medicine, Children’s Hospital Medical Center, Cincinnati, OH 45229
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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expression. Activation of PP2A
inhibited JNK activity and JNK coprecipitated with the regulatory
subunit, PP2A-A
, supporting the conclusion that PP2A is a key
regulator of JNK in the context of an inflammatory
stimulus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; Ref. 9) and adhesion molecules
(e.g., ICAM-1; Ref. 10). There exist various regulatory mechanisms of AP-1 activity including transcriptional activation of subunits (to increase the abundance of AP-1 proteins), as well as posttranscriptional modification (e.g., phosphorylation of the proteins) to increase their activity (6). Although c-Jun is constitutively present in cells in an inactive form, activation of the cell by LPS induces phosphorylation of two serine residues (serine 63 and 73) within the transactivation domain of c-Jun (11). This phosphorylation is mediated by p46 and p54 isoforms of the c-Jun N-terminal kinase (JNK)3, a member of the mitogen-activated protein (MAP) kinase family, and serves to increase both the stability (12) and transcriptional activity (13) of c-Jun via phosphorylation.
It has long been postulated that the regulation of any phosphorylated
protein is in part balanced by the activity of kinases and
phosphatases. Recent work in TCR signaling has supported the simple but
eloquent hypothesis that phosphatases directly regulate associated
kinases. For example, the proline-enriched protein tyrosine phosphatase
was shown to dephosphorylate and inactivate the Src-related kinases
after T cell activation (14). Similarly, the I
B kinase
was shown to be regulated by the serine phosphatase, PP2A
(15). We hypothesized that in the context of an
inflammatory stimulus, such as LPS, JNK might similarly be a target of
regulation by phosphatases. In these studies, it is demonstrated that
inhibition of serine/threonine phosphatases by okadaic acid (OA)
resulted in increased JNK kinase activity that was associated with
increased phosphorylation of c-Jun, increased nuclear translocation of
AP-1 and AP-1-driven transcriptional activity, as well as increased
IL-1 expression. In coprecipitation studies, the regulatory subunit
of PP2A, PP2A-A, coprecipitated with JNK. Specific pharmacologic
inhibition of PP1 by phosphatidic acid (PA) had no effect on JNK
activity, whereas activation of PP2A by a high dose of the same agent
decreased JNK activity. Together these data support the hypothesis that
PP2A is a key endogenous regulator of AP-1 and as such may be a valid
therapeutic target in the setting of acute inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human acute monocytic leukemia cell line, THP-1, was
purchased from American Type Culture Collection (Manassas, VA). Cells
were cultured in RPMI 1640 medium containing 10% heat-inactivated FBS,
kanamycin, 2-ME, and 2% glutamine pH 7.35 at a density of 0.5–1
x 106 cells/ml at 37°C in humidified 5%
CO2 in air. Studies were performed in T25 flasks
(Becton Dickinson, Mountain View, CA) at a density of 5–10 x
106 cells/5 ml after a 3-h differentiation step
with IFN-
(100 U/ml). Stimulation was performed with 1 µg/ml LPS
(Escherichia coli, serotype O55:B5; Sigma, St. Louis, MO).
The pretreatment time for OA (Sigma) or PA
(1,2-dioleoyl-sn-glycero-3-phosphate; Avanti, Alabaster, AL) was 60 min
in all experiments. PA required dissolving in a 1:1 mixture of 100%
ethanol/sterile water with 30 min of ultrasonication. At the highest
dose used (5 mM), the dilution of PA into media was 1:1000.
Unstimulated and LPS-stimulated cells received a similar amount of 1:1
EtOH/H2O for drug vehicle control (1:1000).
In vitro kinase assay for JNK activity
JNK activity was determined as previously reported
(16). Approximately 5–10 x
106 cells in suspension were washed with 0.9%
NaCl, centrifuged for 5 min at 3000 rpm at 4°C, and the cell pellet
was lysed at 4°C with a lysis buffer containing 50 mM Tris, 250 mM
NaCl, 3 mM EDTA, 3 mM EGTA, 1% Triton X-100, 0.5% Nonidet P-40, 10%
glycerol, 2 mM DTT, 1 mM PMSF, 0.1 mM sodium orthovanadate, 2 mM
para-nitro phenylphosphate, and 0.3 U/ml aprotinin. The cell lysate was
centrifuged for 10 min at 10,000 x g at 4°C and
stored at -80°C for kinase assay. Measurement of supernatant protein
concentration was performed by the Bradford assay using the Bio-Rad
(Hercules, CA) reagent. For immunoprecipitation, 800 µg of sample
protein, adjusted to a final volume of 300 µl with lysis buffer, was
mixed on a rocker at 4°C for 60 min with either 5 µl polyclonal
anti-JNK Ab (cat. no. sc-474; Santa Cruz Biotechnology, Santa Cruz,
CA) or 5 µl anti-MKP-2 (Santa Cruz). After the sample-Ab mixing,
the NaCl concentration was adjusted to 400 mM with 1 M NaCl, and 30
µl of protein G-Sepharose beads (Amersham-Pharmacia, Piscataway, NJ),
equilibrated with lysis buffer, were mixed at 4°C overnight. The
pellet was washed twice with the lysis buffer, followed by washing with
a kinase assay buffer containing 25 mM HEPES, 300 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM
DTT, 20 mM -glycerophosphate, 0.1 mM
Na3VO4, 2 µg/ml
leupeptin, 1 mM PMSF, 0.3 U/ml aprotinin, and 2 mM para-nitro
phenylphosphate, and the pellet was resuspended in kinase assay buffer.
For jun-kinase activity, the resuspended pellet was mixed with 20 µg
GST-c-Jun or GST protein devoid of c-Jun as a result of not placing the
c-Jun expression sequence into the GST expression vector
(Amersham-Pharmacia GST gene-fusion kit), 5 µCi of
[32P]ATP (New England Nuclear, Boston, MA), in
40 µM ATP, and incubated at 30°C for 30 min. The reaction was
stopped by the addition of 2x Laemmli buffer, the sample was boiled,
centrifuged, and separated on a 10% Tris-glycine gel using a Novex
Mini Cell electrophoresis system (San Diego, CA). The gels were washed,
dried, and exposed on a phosphor-imager screen for 16 h, followed
by scanning on a Molecular Dynamics Storm system (Sunnyvale, CA), and
analyzed using Image-Quant (Molecular Dynamics) image analysis
software.
Phosphatase activity determination
A nonradioactive, malachite green-based phosphatase assay kit
(Upstate Biotechnology, Lake Placid, NY) used phosphate release to
measure phosphatase activity. THP-1 cells were either pretreated with
OA (60 min) or left untreated and stimulated with LPS as above for
1 h. Total cellular proteins were then extracted in RIPA buffer
with no additive agents. Protein concentrations were determined using a
Bio-Rad bicinchoninic acid assay. Assays were run in 96-well plates
according to the manufacturer’s instructions. Briefly, 5 µg cellular
protein were incubated with 4.5 µl (
175 µM) of the substrate,
phosphoprotein (amino acid sequence: KRpTIRR), and PP2A buffer (20 mM
MOPS, pH 7.5, 60 mM 2-ME, 0.1 M NaCl, and 0.1 mg/ml serum albumin) in
total volume of 25 µl. Reactions were started with the addition of
the phosphoprotein substrate and conducted for 10 min in room
temperature. The reactions were terminated by the addition of 100 µl
malachite green solution and color developed for 15 min before reading
the plate at 650 nm. All determinations were performed in duplicate,
and the absorbance of the reactions was corrected by determining the
absorbance from duplicate reactions not provided the phoshoprotein
substrate. The amount of phosphate released (pmol) was then calculated
from a standard curve (0–2000 pmol). Although the reaction buffer and
phosphorylated substrate used in the assay kit are designed to detect
specific activity of a given serine-threonine phosphatases, use of this
assay cannot fully differentiate between PP2A and PP1 activity.
Furthermore, although the assay substrate is designed for
serine-threonine phosphatase activity, it cannot fully exclude
involvement of additional dual-specific phosphatases.
Cytoplasmic and nuclear extracts and determination of AP-1 activation by EMSA
EMSA was performed as previously reported (17). After stimulation with LPS in the presence or absence of OA, cells were collected on ice by gentle agitation and centrifuged (1500 x g, 10 min, 4°C). Cell pellets were washed twice with ice-cold PBS and then treated with 50 µl ice-cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF and centrifuged (1500 x g, 10 min, 4°C). The supernatant was aspirated off and the pellet resuspended in 50 µl buffer A + 0.1% Nonidet P-40. This suspension was incubated on ice for 5 min, then centrifuged (14,000 rpm, 10 min at 4°C). The supernatant containing the cytoplasmic portion was saved. The crude nuclear pellet was resuspended in 200 µl buffer C (20 mM HEPES, pH 7.9, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 25% glycerol, v/v) and incubated on ice for 15 min. This fraction was centrifuged (14,000 rpm, 15 min at 4°C) and the supernatant containing the nuclear protein extracts was collected. Protein concentrations were determined using a Bio-Rad Bradford assay mentioned above. Proteins were assessed either by EMSA (nuclear) or by Western blot analysis (cytoplasmic). Purity of nuclear extracts was confirmed by absence of cytoplasmic protein contamination (data not shown).
EMSAs used a double-stranded AP-1 consensus oligonucleotide probe
(5'-CGC TTG ATG AGT CAG CCG GAA-3') (Promega, Madison, WI) that was
end-labeled with [-32P]ATP (3000 Ci/mmol at
10 mCi/ml; Amersham, Arlington Heights, IL). Binding reactions
containing 35 fmol (1 x 105 dpm) of
oligonucleotide and 1 µg of nuclear protein, were performed for 30
min at room temperature in binding buffer (50 mM Tris-HCl, pH 7.5, 250
mM NaCl, 2.5 mM DTT, 20% glycerol (v/v), and 0.5 µg poly(dI:dC)
(Pharmacia)). Reaction volumes were held constant at 10 µl. Unlabeled
competitive oligonucleotide (AP-1) or irrelevant oligonucleotide
(Oct-1) was added 30 min before the addition of radiolabeled probe. The
binding reactions were stopped after 1 h with the addition of 1
µl 10x gel loading buffer (250 mM Tris-HCl, pH 7.5, 0.2%
bromophenol blue, 0.2% xylene cyanol, 40% glycerol (v/v)). The
reaction was run on a nondenaturing, 4% polyacrylamide electrophoretic
gel in 0.5x TBE buffer (45 mM Tris-borate, pH 8.0, 1 mM EDTA) at 100
V/15 mA for 2 h. The EMSA gel was vacuum dried and visualized with
Kodak X-OMAT film (Rochester, NY) exposed to the gel at -70°C for
3 h.
Western blot analysis
Cytoplasmic protein extracts were subjected to SDS-PAGE (10%)
according to the method of Laemmli (18). The separated
proteins were transblotted to nitrocellulose (0.45 µm; Novex, San
Diego, CA) for 2 h at 30 V. After transfer, the membrane was
blocked with 20 mM Tris-HCl, pH 7.5; 500 mM NaCl; and 0.05% TBST (v/v)
containing 5% dry milk for 2 h at room temperature. Blots were
incubated overnight at 4°C with primary Abs: anti-c-Jun (H-79;
Santa Cruz Biotechnology), anti-ser-63, and anti-ser-73
phospho-specific c-Jun (New England BioLabs, Beverly, MA); or
anti-PP2A-A (C-20, cat. no. sc-6112), anti-PP2A-C (cat. no.
sc-6110), and anti-PP2A-B (cat. no. sc-6114) (Santa Cruz
Biotechnology) in TBST with 5% BSA (1:1000 dilution). After washing,
secondary Ab (goat anti-rabbit IgG alkaline phosphatase;
Calbiochem, San Diego, CA) was added at a final dilution of 1:5000 in
TBST and incubated for 30 min. After washing, the membrane was
developed by the addition of alkaline phosphatase substrate solution
(5-bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium in 10 mM
Tris, pH 9.5). Molecular weight markers (Amersham) were used to
estimate the size of the immunoreactive bands.
Determination of AP-1 transcriptional activity
To determine the transcriptional activity of AP-1, transient
transfections of THP-1s were performed using a luciferase-linked,
tandem AP-1 promoter construct, 3xAP1Luc (19), provided by
Dr. Roland Schmid (University of Ulm, Ulm, Germany). Transient
transfections were performed as previously reported (20).
Briefly, cells were centrifuged (1100 rpm, 10 min) and washed once in
warmed sterile TBS buffer. Cells were then resuspended in STBS buffer
in a concentration of 2 x 106/ml followed
by the addition of 10 µg/ml DEAE dextran (Sigma). Cells were
separated into 6-ml aliquots, and 6 µg of the 3xAP1Luc construct with
a -galactosidase-Luc reporter construct were added. Cell/dextran/DNA
mixes were incubated at 37°C for 10–15 min until trypan blue
inclusion reached 20%. At this time, transfected cells were
centrifuged (1100 rpm, 10 min), washed once with warmed STBS, and then
resuspended in 10 ml fresh RPMI 1640 media and placed in T25 flasks (48
h, 37°C, 5% CO2) before being subjected to
experimental conditions. For transient transfection assays, because no
differentiation step occurred, cells were stimulated with 10 µg/ml of
LPS as previously reported (20). Pretreatment with OA or
PA was for 60 min before LPS stimulation. Cell lysates were harvested
in 100 µl luciferase lysis buffer (Promega), subjected to a
freeze-thaw cycle, and centrifuged (4000 rpm, 5 min) before measuring
luciferase activity according to the manufacturer’s instructions
(Promega) using a Berthold AutoLumat LB953 luminometer. Luciferase
activity is reported as light units corrected for total cellular
protein.
Measurement of IL-1
Cell culture supernatant levels of IL-1 were measured by
ELISA (BioSource International, Camarillo, CA) according to the
manufacturer’s instructions. The minimal detectable level for IL-1
was 1 pg/ml. Samples values were determined in duplicate from four to
eight wells per condition.
Statistics
Values are expressed as mean ± SEM. Intergroup comparisons were made using unpaired, two-tailed Student’s t test. Differences were accepted as significant for p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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THP-1 cells were differentiated by pretreatment with IFN- for
30 min and stimulated with LPS (1 µg/ml media). Cell extracts were
harvested over time and subjected to in vitro kinase activity
determination. As shown in Fig. 1
A, JNK activity increased
significantly over time with peak activation at 60 min. Control lanes
(e, empty GST-vector) were run in the absence of c-Jun-GST
to confirm the specificity of the kinase reactions. For subsequent
studies, the 60-min time point was used. Similarly, a dose-response
curve for LPS activation of JNK at 60 min was performed as shown in
Fig. 1B. Although as little as 10 ng/ml LPS stimulated JNK
activity compared with control cells (0 ng/ml lane), the highest
baseline-to-signal ratio was achieved with 1 µg/ml LPS (1000 ng/ml
lane). Thus, this dose was used for subsequent studies. Activation of
JNK was associated with a similar increase over time of c-Jun
expression (Fig. 1C) via a well described, autoamplification
process (21, 22) supporting the biological relevance of
the measured kinase activity. These results established a working in
vitro model of inflammation for examining the regulation of JNK by
phosphatases.
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Because JNK is phosphorylated on a threonine residue
(6), we aimed to determine whether JNK was regulated by
serine/threonine phosphatases using the inhibitor, OA. Treatment with
OA before LPS stimulation resulted in a dose-dependent and significant
increase in JNK activity compared with LPS-stimulated cells (Fig. 2A). Of note, OA added
directly to the kinase reaction after immunoprecipitation did not
effect JNK activity (data not shown), suggesting that there was no
direct effect of OA on JNK. Interestingly, OA alone was sufficient to
augment JNK activity as shown in Fig. 2B, although there
remained a significant effect on LPS-mediated JNK activation (compare
lanes 2 and 3 to lanes 6 and
7, Fig. 2B). This OA-induced increase in JNK
activity correlated to a dose-dependent inhibition of serine/threonine
phosphatase activity (Fig. 3). The effect
of increased JNK activity induced by OA was also associated with
hyperphosphorylation of c-Jun as demonstrated by Western blot analysis
(Fig. 4). These results demonstrated that
OA inhibition of serine/threonine phosphatases substantially increased
activation of JNK resulting in c-Jun hyperphosphorylation.
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The biological relevance of OA-induced activation of JNK was
supported by the associated findings of increased AP-1 nuclear
translocation and binding as detected by EMSA (Fig. 5A). In this study,
pretreatment with escalating doses of OA resulted in a significant
increase in the signal for AP-1. This signal was competed off by
nonradiolabeled AP-1 but not Oct-1. To test the functional consequence
of this finding, AP-1 promoter-driven expression of luciferase was
determined using the 3xAP1Luc construct in transient transfection
assays (Fig. 5B). These results showed that OA treatment
resulted in a dose-dependent augmentation of AP-1-driven luciferase
expression measured 5 h after LPS stimulation. The finding of
decreased luciferase expression at the highest dose (500 nM) was likely
a result of cytotoxicity from OA. In preliminary toxicity studies, cell
viability remained >95% (by MTT assay) at 2 h after 1000 nM OA
and there was minimal LDH release, whereas by 4 h cell viability
decreased by 50% and LDH was detected in cell culture supernatants
(data not shown). The presence of a signal for AP-1 and a high level of
constitutive AP-1 luciferase activity in unstimulated cells was likely
the result of serum activation of this pathway as has been demonstrated
by others (23, 24).
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after LPS stimulation in
human monocytes (9), we measured cell culture supernatant
levels of IL-1 in our model system. Supernatants were harvested
4 h after stimulation with LPS either with (1 h pretreatment) or
without OA. As shown in Fig. 6, OA before
LPS resulted in a significant increase in IL-1 at the 500- and
1000-nM doses. In unstimulated cells and those treated with 10, 100, or
500 nM OA alone, no IL-1 was detected (data not shown). However,
1000 nM OA alone did result in an increase in IL-1 expression from
THP-1 cells independent of LPS stimulation, supporting the observation
that OA likely effects the serum-induced activation of AP-1. Taken
together, these data supported the hypothesis that serine/threonine
phosphatase inhibition by OA results in a biologically relevant
activation of JNK resulting in increased proinflammatory cytokine
expression.
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with JNK
Because previous data had demonstrated physical associations
between unrelated phosphatases and the kinases that they regulated, we
hypothesized that there may be a similar association between JNK and
one of the serine/threonine phosphatases. Western blot analysis of
whole-cell lysates showed the presence of all regulatory subunits for
PP2A whose levels were generally unaffected during the 30 min after LPS
stimulation (Fig. 7A). To test
the hypothesis that one of the regulatory subunits of PP2A was
physically associated with JNK, lysates from either unstimulated or
LPS-stimulated cells were immunoprecipitated with either anti-JNK
Ab or an irrelevant, isotype-matched -MKP-2 Ab. PP2A-A
(molecular mass = 65 kDa) was the only subunit that demonstrated a
specific interaction in the anti-JNK immunoprecipitated product
(Fig. 7B), and suggested that this PP2A subunit shared a
physical association with JNK. It was also observed that LPS
stimulation consistently resulted in a diminished PP2A-A signal
after JNK immunoprecipitation (Fig. 7B, lane 4 vs
6) that was confirmed by an analysis of the kinetics of this
after LPS stimulation (Fig. 7C). Although it is intriguing
to speculate that the inflammatory stimulus somehow dissociates
PP2A-A from JNK thus favoring kinase activity, this hypothesis
remains to be fully tested.
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Although the above data suggested that PP2A was the key regulatory
phosphatase, OA at the doses used may have inhibitory effects on both
PP2A and PP1. Recently the PA, 1,2-dioleoyl-sn-glycero-3-phosphate, has
been shown to be both a selective inhibitor of PP1 and an activator of
PP2A at higher doses (25). We used this agent to confirm
that PP2A was the crucial regulator of JNK activity. Treatment with PA
either alone (5 µM) or at lower doses (100 and 500 mM) resulted in no
change in JNK activity as measured by the kinase assay (Fig. 8) in stark contrast to the findings with
OA (compare with Fig. 2
). However, after LPS activation of JNK, PA at a
dose (5 µM) described to activate PP2A, substantially reduced the JNK
activity. Of note, pretreatment with PA did not alter the observed
dissociation of PP2A and JNK as determined by immunoprecipitation
studies (data not shown). These results provided further support for
the conclusion that PP2A can actively regulate LPS-induced JNK
activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data presented provide evidence that JNK is regulated by PP2A in
the context of an inflammatory stimulus. Pharmacological inhibition of
PP2A by OA resulted in substantial increases in JNK activity. Although
thought to be a nonspecific PP1/PP2A inhibitor, others had demonstrated
that the doses of OA used only inhibited PP2A similar to our findings
(30). Nevertheless, the availability of a PP1 selective
inhibitor, a PA derivative, allowed us to confirm the specificity of
these findings (25). PA had no effect on JNK until a
higher dose, which also activates PP2A, was used. This dose resulted in
an inhibition of JNK activity in a manner consistent with our
hypothesis. The data presented are also consistent with previous
observations that described an effect of OA on inducing IL-1
expression via a mechanism related to the presence of an AP-1-sensitive
binding site on a chloramphenicol acetyltransferase reporter plasmid
(31). Induction of increased chloramphenicol
acetyltransferase activity was hypothesized to involve the effect of OA
on protein phosphorylation. This effect of OA had also been observed to
be related to increased transcriptional activation of the jun
proto-oncogene in PMA-stimulated Jurkat cells (32). This
study concluded that the protein phosphatases may have been negative
regulators of the AP-1 transcription factor. Our data are consistent
with both these previous studies and support the hypothesis that OA
regulation of AP-1 activity is related to a negative regulatory effect
of PP2A on JNK activity.
To study the regulation of JNK activity, we used an in vitro model of
macrophage immunostimulation with LPS as was used by others
(33). Subsequent to this observation, there are have been
several levels of kinase-dependent cell signaling that have been
implicated that result in JNK activation by immunostimulation. Various
models have demonstrated activation of protein tyrosine kinases
(33, 34), stress-activated protein kinase kinases
(35), and the MAP kinases (eg., extracellular
signal-related kinase (ERK) 1/2; Ref. 36). Based on our
data, we are unable to exclude the possibility of an effect of PP2A
inhibition on an upstream kinase that is necessary for JNK activation.
Although PP2A has been demonstrated to inhibit constitutive protein
kinase C (37) and ERK2 (38), these studies
focused on regulation of cellular entry into the growth cycle, whereas
our focus was on the state of JNK regulation in the context of an
inflammatory stimulus. The methodology that could be used to define
this direct regulation would include co-overexpression of JNK with a
biologically active form of PP2A, or more specifically, PP2A-A,
which has proven to be technically difficult (39).
Successful overexpression of the regulatory elements of PP2A has been
limited to the catalytic subunit (40), although this
molecular strategy remains a focus of our ongoing investigations.
The strongest evidence for PP2A directly regulating JNK is derived from
the immunoprecipitation studies, which clearly demonstrate a physical
association between PP2A-A and JNK. This association consistently
seemed to be less after LPS treatment and raises the intriguing
possibility that immunostimulation somehow results in the physical
dissociation of PP2A-A from JNK, although this hypothesis remains to
be tested. There exist other examples of regulated phosphatase-kinase
pairs that share a physical association. For example, ERK2 has been
shown to bind the MAP kinase phosphatase-3 (MKP-3) resulting in
regulation of MAP kinase activation (41), whereas PP1 has
been shown to bind the A-kinase anchoring protein AKAP220
(42). Physical associations between various subunits of
the ubiquitous PP2A and kinase species do not seem unique to JNK
because these have been described for the catalytic subunit, PP2A-C,
binding to both the calmodulin-dependent protein kinase IV
(43), the 70-kDa S6 kinase (44), and the
p21-activated kinase (45). Alternatively, the regulatory
subunit of PP2A may simply serve as a scaffolding protein for a larger
signal transduction complex comprised of JNK and other upstream members
of the MAP kinase family that could be regulated by other, OA-sensitive
phosphatases. Determination of such protein-protein interactions
remains the focus of ongoing studies. Together with our data, these
observations remain consistent with the hypothesis that regulation of
key phosphorylation events is governed by the proximity of kinases and
phosphatases close to their substrate (26). Therefore,
phosphatases may be a target of therapeutic interventions in an attempt
to regulate inflammatory cell signaling.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas P. Shanley, Division of Critical Care Medicine, OSB-5, Children’s Hospital Medical Center, Cincinnati, OH 45229-3039.
3 Abbreviations used in this paper: JNK, c-jun N-terminal kinase; MAP, mitogen-activated protein; OA, okadaic acid; PA, phosphatidic acid; ERK, extracellular signal-related kinase.
Received for publication November 24, 1999. Accepted for publication October 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1 in rat liver macrophages. Hepatology 22:613.[Medline]
promoter via fibrinogen engagement of the CD18 integrin receptor. Am. J. Respir. Cell. Mol. Biol. 20:1059. B. Int. J. Mol. Med. 4:223.[Medline]
leads to intercellular adhesion molecule-1 expression. J. Biol. Chem. 274:28978.B kinase that activates the transcription factor NF-B. Nature 388:548.[Medline]
B and preservation of IB by IL-10 and IL-13. J. Clin. Invest. 100:2443.[Medline]
B. J. Clin. Invest. 101:1163.[Medline]
promoter in human monocytic cells: regulation by Egr-1, c-Jun, and NF-B transcription factors. J. Biol. Chem. 272:17795.-actin promoter in ventricular myocytes. J. Biol. Chem. 271:10827. expression via an AP-1-mediated mechanism. Scand. J. Immunol. 38:570.[Medline]
production by human monocytes involves the raf-1/MEK1-MEK2/ERK1-ERK2 pathway. Infect. Immun. 67:3824.
signaling targeted by SV40 small t to promote cell growth and NF-B activation. EMBO J. 16:5662.[Medline]
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 K.-J. Yin, C. Y. Hsu, X.-Y. Hu, H. Chen, S.-W. Chen, J. Xu, and J.-M. Lee Protein phosphatase 2A regulates bim expression via the Akt/FKHRL1 signaling pathway in amyloid-beta peptide-induced cerebrovascular endothelial cell death. J. Neurosci., February 22, 2006; 26(8): 2290 - 2299. [Abstract] [Full Text] [PDF]
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M. Zhao, A. Wimmer, K. Trieu, R. G. DiScipio, and I. U. Schraufstatter Arrestin Regulates MAPK Activation and Prevents NADPH Oxidase-dependent Death of Cells Expressing CXCR2 J. Biol. Chem., November 19, 2004; 279(47): 49259 - 49267. [Abstract] [Full Text] [PDF]
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P. Iribarren, Y.-H. Cui, Y. Le, G. Ying, X. Zhang, W. Gong, and J. M. Wang IL-4 Down-Regulates Lipopolysaccharide-Induced Formyl Peptide Receptor 2 in Murine Microglial Cells by Inhibiting the Activation of Mitogen-Activated Protein Kinases J. Immunol., November 15, 2003; 171(10): 5482 - 5488. [Abstract] [Full Text] [PDF]
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 S. Kins, P. Kurosinski, R. M. Nitsch, and J. Gotz Activation of the ERK and JNK Signaling Pathways Caused by Neuron-Specific Inhibition of PP2A in Transgenic Mice Am. J. Pathol., September 1, 2003; 163(3): 833 - 843. [Abstract] [Full Text] [PDF]
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T. L. Andreone, M. O'Connor, A. Denenberg, P. W. Hake, and B. Zingarelli Poly(ADP-Ribose) Polymerase-1 Regulates Activation of Activator Protein-1 in Murine Fibroblasts J. Immunol., February 15, 2003; 170(4): 2113 - 2120. [Abstract] [Full Text] [PDF]
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S. Mitsuhashi, H. Shima, N. Tanuma, N. Matsuura, M. Takekawa, T. Urano, T. Kataoka, M. Ubukata, and K. Kikuchi Usage of Tautomycetin, a Novel Inhibitor of Protein Phosphatase 1 (PP1), Reveals That PP1 Is a Positive Regulator of Raf-1 in Vivo J. Biol. Chem., January 3, 2003; 278(1): 82 - 88. [Abstract] [Full Text] [PDF]
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K. Bshesh, B. Zhao, D. Spight, I. Biaggioni, I. Feokistov, A. Denenberg, H. R. Wong, and T. P. Shanley The A2A receptor mediates an endogenous regulatory pathway of cytokine expression in THP-1 cells J. Leukoc. Biol., November 1, 2002; 72(5): 1027 - 1036. [Abstract] [Full Text] [PDF]
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N. J. Avdi, K. C. Malcolm, J. A. Nick, and G. S. Worthen A Role for Protein Phosphatase-2A in p38 Mitogen-activated Protein Kinase-mediated Regulation of the c-Jun NH2-terminal Kinase Pathway in Human Neutrophils J. Biol. Chem., October 18, 2002; 277(43): 40687 - 40696. [Abstract] [Full Text] [PDF]
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,
p < 0.05; 
500 nM) were associated with lower luciferase
expression that was likely the result of toxicity over the course of
the experiment (5 h). Values were determined in duplicate and are
expressed as mean ± SEM (n = 8 wells per
condition) standardized to protein content of samples. Results are
representative of three separate experiments.
